EP4627060A1

EP4627060A1 - Modified t cells

Info

Publication number: EP4627060A1
Application number: EP23822076.8A
Authority: EP
Inventors: Omer DUSHEK
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2022-12-02
Filing date: 2023-12-01
Publication date: 2025-10-08
Also published as: CN120603932A; WO2024115919A1; GB202218144D0

Abstract

The invention relates to enhancing target pMHC discrimination of T cells by modulating the amount of expression and/or activity of factors associated with T cell receptor (TCR) kinetic proofreading.

Description

MODIFIED T CELLS

Field of invention

The invention relates to modified T cells, methods of preparing modified T cells, and uses thereof.

Background of the invention

T cells play a key role in the adaptive immune response by discriminating between healthy and abnormal cells via their T cell receptors (TCRs). For instance, viral or tumour specific peptides presented on major histocompatibility complex class I or II (MHC-I or MHC-II) molecules on the surface of infected or cancerous cells can be recognized by the TCR. Following antigen recognition, T cells become activated and produce an immune response.

T cells must be able to recognize any foreign peptide they encounter, but there is a physical limit on the number of T cells any organism can have. Therefore, cross-reactivity is an essential feature of the TCR. It has been reported that each T cell recognises ~10⁶ peptides to achieve full immune coverage (1).

Antigen discrimination can be quantified by analysing the potency of different affinity ligand antigens. A high discrimination power allows a small change in ligand affinity to be amplified into a larger change in potency, or the concentration of ligand required to elicit a response (2). Reference 2 reported the capacity of TCRs to discriminate cognate ligands to be enhanced compared to conventional surface receptors such as GPCRs.

Many T cell therapies rely on introduction of heterologous TCRs or modified autologous TCRs into a patient to exploit novel, high affinity binding interactions between the TCR and a pathological peptide presented via the major histocompatibility complex. For example, in adoptive T cell transfer therapy T cells are engineered ex vivo to express a TCR that targets a specific tumour antigen of interest and are then re-infused back into the patient. These TCRs are optimised, validated and screened for positive target and negative off-target binding in vitro. Testing in animal models is rare, since TCRs are designed with human immunological compatibility in mind, so the first in vivo administration of therapeutic TCRs or therapeutic T cells bearing said TCRs is usually in human clinical trials.

Exogenously introduced therapeutic TCRs into patients bypass thymic selection and functional central tolerance processes, increasing the risk of off-target interactions with self-antigens. Although these off-target interactions with self-antigens are expected to be of lower affinity, it has recently been shown that TCR antigen discrimination based on affinity is imperfect: primary human T cells can respond to pMHC with affinities as low as KD ~ 1 mM (2). Therefore, although the affinity between introduced TCRs and off-target self-antigens is expected to be low, it may be functionally significant in vivo and can result in serious, and even fatal, side effects. For example, fatalities in the introduced A3A TCR clinical trial, whose on-target antigen was MAGE- A3, have been attributed to off-target binding of the A3A TCR to the self-antigen Titin (3,4), which were not identified in extensive in vitro safety screens due to a combination of low affinity binding and lack of surface expression on in vitro cultured cells.

Strategies exist to minimise the risk of self-antigen interaction, but these have limitations. In vitro screens for low affinity off-target binding are labour-intensive and expensive, and do not achieve full coverage of tissue-, differentiation- and patient-specific self-peptide repertoires. Computational prediction and artificial intelligence (Al)-based strategies can be employed, but the small and variable number of residue contact points between the TCR (at the CDR3a and CDR30 loops) and antigen render this highly complex. Furthermore, existing strategies focus on modification of the TCR itself and have neglected the role of the intracellular signalling cascade in ligand discrimination.

It is an object of the invention to provide further or improved T cells with enhanced discrimination for high-affinity on-target antigens relative to lower-affinity off-target antigens.

It is also an object of the invention to provide further or improved T cells with minimised risk of cross-reactivities in adoptive T cell transfer therapies.

Summary of the invention

The inventors found a way to alter the ability of T cells to discriminate their high- affinity on-target antigens from low-affinity off-target antigens, without loss of sensitivity for the high-affinity target. This is achieved by exploiting the membrane-proximal intracellular events that initiate TCR signalling and T cell activation.

In particular, the inventors altered T cell discrimination capacity by modulating the kinetic proofreading (KP) mechanism in T cells. The KP mechanism is a succession of reversible biochemical steps initiated at the TCR following binding of the cognate MHC- presented peptide (pMHC). It is only when the final biochemical step is complete that a TCR activated state is reached (see Figure 2). The incremental, multi-step progression towards a TCR activated state ensures a time delay between TCR-pMHC binding and TCR signalling (5). For TCR signalling to be triggered, the pMHC must remain bound to the TCR throughout the entire multi-step process. If the pMHC dissociates during any step, TCR signalling will not occur and the TCR will be reset to a TCR resting state such that the KP progression can begin again upon re-engagement of the TCR. Thus, higher affinity ligands and/or ligands at higher local concentration are more likely to lead to TCR signalling. Equally, small differences in the off-rate (k_off) of a TCR-pMHC interaction are amplified into larger differences in T cell activation.

The inventors found that reducing the amount and/or activity of a factor which promotes progression through the KP mechanism (i.e. a factor which progresses the T cell towards the TCR activated state) enhances ligand discrimination. Conversely, increasing the amount and/or activity of a factor which counteracts propagation through the KP mechanism (i.e. a factor which progresses the T cell towards the TCR resting state) would also enhance ligand discrimination.

For example, Examples 1, 7 and 4 show that modified T cells with reduced expression and/or activity of CD8 and Lek, respectively, provided enhanced ligand discrimination compared to wild-type T cells. Interestingly, T cells modified to reduce expression and/or activity of factors that are known to be involved in T cell antigen recognition, but not involved in the KP mechanism, e.g. CD43 and CD2, do not provide enhanced ligand discrimination compared to wild-type T cells (see Examples 2 and 3). Surprisingly, as shown in the Examples 1 and 4, the modified T cells exhibited enhanced T cell discrimination for its target antigen without loss of sensitivity for said target antigen, i.e. without any change in on-target potency. Surprisingly, Example 9 shows that overexpression of CD4 in pMHC-I restricted T cells provides enhanced ligand discrimination compared to the wild-type T cells. This further confirms the effects of modulating KP pathway mechanism in discrimination enhancement, since expression of CD4 competes with CD8 for binding to Lek. Overexpression of CD4 sequesters Lek away from CD8, effectively reducing the amount of Lek. Hence, overexpression of CD4 progresses pMHC-I restricted T cells towards the TCR resting state.

Example 10 shows that discrimination can be enhanced still further by combining the modulations described herein. Knocking out CD8 and overexpressing CD4 in pMHC-I restricted T cells results in improved enhancement of discrimination.

Accordingly, the invention provides a modified T cell characterised in that the function of a factor associated with T cell receptor (TCR) kinetic proofreading is modulated.

The invention also provides an inhibitor of a factor which is associated with TCR kinetic proofreading in a T cell and which progresses the T cell towards the TCR activated state, optionally wherein the inhibitor is a shRNA.

The invention also provides a sgRNA for knocking out a factor which is associated with TCR kinetic proofreading in a T cell and which progresses the T cell towards the TCR activated state, optionally wherein the sgRNA comprises one or more sequences comprising 5 to 35 consecutive nucleotides of the gene encoding the factor.

The invention also provides a vector comprising a shRNA molecule described herein or one or more sgRNAs described herein.

The invention also provides a pharmaceutical composition comprising an inhibitor described herein, one or more sgRNAs described herein, or a vector described herein.

The invention also provides a method of preparing a modified T cell, comprising modulating the function of a factor associated with TCR kinetic proofreading in a T cell.

The invention also provides a modified T cell obtainable or obtained by any of the methods described herein.

The invention also provides a method of enhancing target pMHC discrimination of a T cell, comprising preparing a modified T cell according to any of the methods described herein. The invention also provides a method of preparing a population of modified T cells for adoptive cell therapy, the method comprising culturing a modified T cell described herein.

The invention also provides a population of modified T cells produced by any of the methods described herein.

The invention also provides a method of treating cancer, an infection or an inflammatory disease comprising administering a modified T cell described herein, a population of T cells described herein, an inhibitor described herein, a sgRNA described herein, a vector described herein, or a pharmaceutical composition described herein to a patient in need thereof, optionally wherein the infection is a chronic infection or the inflammatory disease is an autoimmune disease.

The invention also provides a modified T cell described herein, a population of T cells described herein, an inhibitor described herein, a sgRNA described herein, a vector described herein, or a pharmaceutical composition described herein for use as a medicament.

The invention also provides a modified T cell described herein, a population of T cells described herein, an inhibitor described herein, a sgRNA described herein, a vector described herein, or a pharmaceutical composition described herein for use in a method of treating cancer, an infection or an inflammatory disease, optionally wherein the infection is a chronic infection or the inflammatory disease is an autoimmune disease.

Brief description of the figures

Figure 1. Schematic of TCR signalling pathway (6). T Cell Receptor (TCR) activation promotes a number of signalling cascades that ultimately determine cellular responses through regulating cytokine production, cell survival, proliferation, differentiation, and target cell killing. Examples of factors involved in these signalling pathways are provided in Figure 1.

Figure 2. Schematic of kinetic proofreading mechanism (7). Free pMHC complexes (denoted ‘P’) can bind to free T cell receptors (TCRs; denoted ‘T’) to form a TCR-pMHC complex that may undergo a series of ‘N’ biochemical modifications (denoted as complexes CO, Cl, C2 and CN) with a rate of k_p. ‘CN’ is the signalling- competent TCR state, also referred herein to as the TCR activated state. k_on = on rate; k_off= off rate.

Figure 3. CD8 alpha negative primary human T cells were generated. CD8 alpha was knocked-out in primary human CD8+ T cells by electroporation of Cas9 protein and synthetic guide RNA. T cells were stained with Anti-CD8 alpha (BV-421) antibody and CD8 alpha surface expression was measured by flow cytometry.

Figure 4. Measuring c259 TCR/pMHC affinities using SPR at 37°C. Steady-state binding affinity for the selected 8-peptide panel. Bar plot represents mean KD with SD.

Figure 5. CD8 alpha negative primary human T cells exhibit enhanced antigen discrimination compared to wild-type primary human T cells. CD8 alpha was knocked-out in primary human CD8+ T cells by electroporation of Cas9 protein and synthetic guide RNA. The T cells were subsequently lentivirally transduced with the c259 TCR. Wild-type T cells (Circles) and CD8 alpha negative T cells (Squares) were stimulated by U87 target cells loaded with the indicated concentration of antigen (x-axis) and surface 4 IBB expression was measured by flow cytometry after 20 hours (y-axis). Representative data is shown from 1 out of 3 experiments. The affinity of each peptide to the c259 TCR is shown.

Figure 6. CD8 alpha negative primary human T cells require a higher concentration of lower-affinity but not higher-affinity antigens to induce T cell activation compared to wild-type cells. (A) The concentration of antigen required to activate 15% of T cells (Pl 5) is plotted over the TCR/pMHC affinity measured using SPR (Figure 4). P15 values were calculated from dose response curves of T cell activation assayed by: (4 IBB) measuring 41BB surface expression by flow cytometry, (Nluc) measuring U87 target cell killing and (IL2) by measuring secretion of IL2 in the supernatant. (B) The fold-change in P15 between CD8 alpha negative cells and wild-type cells from panel A is plotted over the TCR/pMHC affinity. F test for non-zero slope. Each point represents mean P15, error bars represent SD.

Figure 7. Lek was knocked-down in primary human T cells by shRNA. Human primary T cells were lentivirally transduced with a vector encoding the c259 TCR and an shRNA molecule. Cells were transduced with either a scramble control shRNA sequence or with an anti-Lck shRNA sequence are shown in squares. T cells were intracellularly stained with Anti-Lck (AF647) antibody and Lek intracellular expression was measured by flow cytometry.

Figure 8. Lek knock-down primary human T cells exhibit enhanced antigen discrimination compared to wild-type primary human T cells. Human primary T cells were lentivirally transduced with a vector encoding the c259 TCR and an shRNA molecule. Cells transduced with a scramble control shRNA sequence are shown in circles and cells transduced with an anti-Lck shRNA sequence are shown in squares. T cells were stimulated by U87 target cells loaded with the indicated concentration of antigen (x-axis) and target cell killing was measured after 20 hours (y-axis). Representative data is shown from 1 out of 3 experiments. The affinity of each peptide to the c259 TCR is shown.

Figure 9. Lek knock-down primary human T cells require a higher concentration of lower-affinity but not higher-affinity antigens to induce T cell activation compared to wildtype cells. (A) The concentration of antigen required to activate 15% of T cells (Pl 5) is plotted over the TCR/pMHC affinity measured using SPR (Figure 4). T cell activation was assayed by measuring U87 target cell killing (B) The fold-change in P15 between Lek knockdown cells and wild-type cells from panel A is plotted over the TCR/pMHC affinity. F test for non-zero slope. Each point represents mean P15 from 3 independent biological experiments, error bars represent SD.

Figure 10. The knock-out of a T cell factor involved in kinetic proofreading enhances the discriminatory power of T cells. Representative dose response curves comparing the activation of unmodified T cells (solid line) or knock-out T cells (dashed line) against a high affinity ligand (circles) or a low affinity ligand (squares). T cells were stimulated by U87 target cells loaded with the indicated concentration of antigen (x-axis) and 4 IBB surface expression was measured after 4 hours (y-axis). In unmodified cells, the concentration of antigen required to activate 15% of T cells (Pl 5) is higher for the low affinity ligand than for the high affinity ligand. The KO reduces the P15 of the low affinity ligand while maintaining the same P15 of the high affinity ligand.

Figure 11. CD2 negative primary human T cells were generated. CD2 was knocked-out in primary human CD8+ T cells by electroporation of Cas9 protein and synthetic guide RNA. T cells were stained with Anti-CD2 (PE) antibody and CD2 surface expression was measured by flow cytometry. Figure 12. CD2 primary human T cells exhibit decreased antigen sensitivity compared to wild-type primary human T cells. CD2 was knocked-out in primary human CD8+ T cells by electroporation of Cas9 protein and synthetic guide RNA. The T cells were subsequently lentivirally transduced with the c259 TCR. Wild-type T cells (Circles) and CD2 negative T cells (Squares) were stimulated by U87 target cells loaded with the indicated concentration of antigen (x-axis) and surface 4 IBB expression was measured by flow cytometry after 20 hours (y-axis). Representative data is shown from 1 out of 3 experiments. The affinity of each peptide to the c259 TCR is shown.

Figure 13. CD2 negative primary human T cells require a higher concentration of lower-affinity and higher-affinity antigens to induce T cell activation compared to wildtype cells. (A) The concentration of antigen required to activate 15% of T cells (Pl 5) is plotted over the TCR/pMHC affinity measured using SPR (Figure 4). P15 values were calculated from dose response curves of T cell activation assayed by: (4 IBB) measuring 41BB surface expression by flow cytometry, (Nluc) measuring U87 target cell killing, (IL2) by measuring secretion of IL2 in the supernatant or (IFNg) by measuring secretion of IFN gamma in the supernatant (B) The fold-change in P15 between CD2 negative cells and wild-type cells from panel A is plotted over the TCR/pMHC affinity. F test for non-zero slope. Each point represents mean P15, error bars represent SD.

Figure 14. CD43 negative primary human T cells were generated. CD43 was knocked-out in primary human CD8+ T cells by electroporation of Cas9 protein and synthetic guide RNA. T cells were stained with Anti-CD43 (PE) antibody and CD43 surface expression was measured by flow cytometry.

Figure 15. CD43 primary human T cells do not exhibit a difference in antigen sensitivity or discrimination compared to wild-type primary human T cells. CD43 was knocked-out in primary human CD8+ T cells by electroporation of Cas9 protein and synthetic guide RNA. The T cells were subsequently lentivirally transduced with the c259 TCR. Wild-type T cells (Circles) and CD43 negative T cells (Squares) were stimulated by U87 target cells loaded with the indicated concentration of antigen (x-axis) and target cell killing was measured after 20 hours (y-axis). Representative data is shown from 1 out of 3 experiments. The affinity of each peptide to the c259 TCR is shown. Figure 16. CD43 negative primary human T cells require the same concentration of lower-affinity and higher-affinity antigens to induce T cell activation compared to wildtype cells. (A) The concentration of antigen required to activate 15% of T cells (Pl 5) is plotted over the TCR/pMHC affinity measured using SPR (Figure 4). P15 values were calculated from dose response curves of T cell activation assayed by: (4 IBB) measuring 41BB surface expression by flow cytometry, (Nluc) measuring U87 target cell killing, (IL2) by measuring secretion of IL2 in the supernatant or (IFNg) by measuring secretion of IFN gamma in the supernatant (B) The fold-change in P15 between CD43 negative cells and wild-type cells from panel A is plotted over the TCR/pMHC affinity. F test for nonzero slope. Each point represents mean P15, error bars represent SD.

Figure 17. CD43 negative and CD2 negative primary human T cells show no difference in discrimination capacity relative to wild type primary human T cells.

Figure 18. Side-by-side comparison of antigen discrimination capability upon knockout of CD8 alpha, CD43 and CD2. The concentration of antigen required to activate 15% of T cells (Pl 5) is plotted over the TCR/pMHC affinity measured using SPR (Figure 4). The fold-change in P15 between CD8 alpha negative cells and wild-type cells (triangles), CD43 negative cells and wild-type cells (circles) and CD2 negative cells and wild-type cells (squares) is plotted over the TCR/pMHC affinity. P15 values were calculated from dose response curves of T cell activation assayed by: (A) (4 IBB) measuring 41BB surface expression by flow cytometry, (B) (Nluc) measuring U87 target cell killing and (C) (IL2) by measuring secretion of IL2 in the supernatant. F test for nonzero slope. Each point represents mean P15, error bars represent SD.

Figure 19. T cells treated with an Lek chemical inhibitor (A-770041) exhibit enhanced antigen discrimination compared to wild-type primary human T cells (CD69 surface expression). Human primary T cells were lentivirally transduced with a vector encoding the c259 TCR and treated with either 0 nM of the Lek inhibitor (circles) or 100 nM of the Lek inhibitor (squares) for 1 hour. T cells were stimulated by U87 target cells loaded with the indicated concentration of antigen (x-axis) and CD69 surface expression was measured after 4 hours (y-axis). Representative data is shown from 1 out of 3 experiments. Figure 20. T cells treated with an Lek chemical inhibitor (A-770041) respond to higher affinity antigens but do not activate against lower-affinity antigens. The concentration of antigen required to activate 15% of T cells (Pl 5) is plotted over the TCR/pMHC affinity measured using SPR (Figure 4). P15 values were calculated from dose response curves of T cell activation assayed by: (4 IBB) measuring 4 IBB surface expression by flow cytometry and (TNF alpha) by measuring secretion of TNF alpha in the supernatant. Each point represents an independent biological replicate. Dashed line indicated highest peptide concentration tested (100 pM). Peptides which did not activate at any of the tested concentrations are depicted as P15 >100 pM. Statistical significance was determined using a paired t-test ( * = p<0.05, ** = p<0.01, *** = p<0.001, **** = pO.0001).

Figure 21. CD8 alpha negative primary human T cells were generated. CD8 alpha was knocked-out in primary human CD8+ T cells by electroporation of Cas9 protein and synthetic guide RNA. T cells were stained with Anti-CD8 alpha (PE) antibody and CD8 alpha surface expression was measured by flow cytometry.

Figure 22. CD8 alpha negative primary human T cells transduced with the a3a TCR respond to MAGE- A3 but not titin presented on T2 cells (surface 4 IBB assay). Wildtype T cells (Circles) and CD8 alpha negative T cells (Squares) were stimulated by T2 target cells loaded with the indicated concentration of antigen (x-axis). (A and B) Surface 4 IBB expression was measured by flow cytometry after 20 hours (y-axis). Representative data is shown from 1 out of 3 experiments. (C) The concentration of antigen required to activate 50% of T cells (EC50) is plotted. Each point represents an independent biological experiment. ** = p<0.01.

Figure 23. CD8 alpha negative primary human T cells transduced with the a3a TCR respond to MAGE- A3 but not titin presented on T2 cells (killing assay). Wild-type T cells (Circles) and CD8 alpha negative T cells (Squares) were stimulated by T2 target cells loaded with the indicated concentration of antigen (x-axis). (A and B) Target killing was measured after 20 hours (y-axis). Representative data is shown from 1 out of 3 experiments. (C) The concentration of antigen required to activate 50% of T cells (EC50) is plotted. Each point represents an independent biological experiment. **** = p<0.0001. Figure 24. CD8 alpha negative primary human T cells transduced with the a3a TCR respond to MAGE-A3 but not titin presented on T2 cells (IFN gamma assay). Wildtype T cells (Circles) and CD8 alpha negative T cells (Squares) were stimulated by T2 target cells loaded with the indicated concentration of antigen (x-axis). (A and B) IFN gamma secretion was measured after 20 hours (y-axis). Representative data is shown from 1 out of 3 experiments. (C) The concentration of antigen required to activate 50% of T cells (EC50) is plotted. Each point represents an independent biological experiment. **** = p<0.0001.

Figure 25. CD8 alpha negative primary human T cells transduced with the a3a TCR respond to MAGE- A3 but not titin presented on T2 cells (TNF alpha assay). Wildtype T cells (Circles) and CD8 alpha negative T cells (Squares) were stimulated by T2 target cells loaded with the indicated concentration of antigen (x-axis). (A and B) TNF alpha secretion was measured after 20 hours (y-axis). Representative data is shown from 1 out of 3 experiments. (C) The concentration of antigen required to activate 50% of T cells (EC50) is plotted. Each point represents an independent biological experiment. * = p<0.05.

Figure 26. CD8 alpha negative primary human T cells transduced with the a3a TCR respond to MAGE- A3 but not titin presented on T2 cells (IL-2 assay). Wild-type T cells (Circles) and CD8 alpha negative T cells (Squares) were stimulated by T2 target cells loaded with the indicated concentration of antigen (x-axis). (A and B) IL-2 secretion was measured after 20 hours (y-axis). Representative data is shown from 1 out of 3 experiments. (C) The concentration of antigen required to activate 50% of T cells (EC50) is plotted. Each point represents an independent biological experiment. ** = p<0.01.

Figure 27. Overview of genetic engineering process. (A) CD8 is knocked out of cytotoxic wild-type T cells (which canonically express CD8, ‘CD8+’) to produce an engineered CD8 KO cytotoxic T cell. (B) CD4 is first overexpressed in cytotoxic wildtype T cells (which canonically do not express CD4). Then CD8 is knocked out of said engineered CD4+ cytotoxic T cells, such that the resulting engineered cytotoxic T cell is CD8 KO CD4+.

Figure 28. CD4 overexpressing primary human T cells were generated. T cells were transduced with a dual c259 and CD4 vector. T cells were stained with Anti-CD4 (PE) antibody and CD4 alpha surface expression was measured by flow cytometry. Figure 29. CD4 overexpressing primary human T cells require a higher concentration of lower-affinity but not higher-affinity antigens to induce T cell activation compared to wild-type cells. (A) The concentration of antigen required to activate 15% of T cells (Pl 5) is plotted over the TCR/pMHC affinity measured using SPR. P15 values were calculated from dose response curves of T cell activation assayed by measuring 4 IBB surface expression by flow cytometry. (B) The fold-change in P15 between CD4 overexpressing cells and wild-type cells from panel A is plotted over the TCR/pMHC affinity. F test for non-zero slope. Each point represents mean P15, error bars represent SD. **** = p = 0.0001.

Figure 30. Combining CD8 alpha knock-out with CD4 overexpressing leads to an additive enhancement of T cell ligand discrimination. The fold-change in P15 between CD4 overexpressing cells and wild-type cells or between CD8 KO CD4 overexpressing cells and wild-type cells is plotted over the TCR/pMHC affinity. F test for non-zero slope. Each point represents mean P15, error bars represent SD. **** = p=0.0001; ***** = pO.OOOl.

Detailed description of the invention

Modified T cells and factors

The invention relates to modulating the function (e.g. amount and/or activity) of a factor associated with T cell receptor (TCR) kinetic proofreading. One or more such factors may be modulated.

Hence, the invention also provides a modified T cell comprising a factor associated with T cell receptor (TCR) kinetic proofreading, wherein the function of the factor is modulated. The modified T cell may comprise one or more such modulated factors.

The inventors found that modifying a T cell to reduce the function of a factor which promotes progression through the KP mechanism enhanced said T cell’s discrimination for its cognate pMHC. Without wishing to be bound by theory, in an unmodified T cell, the longer a pMHC is bound to a TCR, the further through the KP mechanism the TCR complex will progress and, therefore, the more likely the TCR is to signal through the canonical TCR signalling cascade, thereby activating the T cell. In contrast, in a modified T cell of the invention, the function of a factor associated with the KP mechanism is modified such that the rate of progression through the KP mechanism is decreased. This means that pMHCs must remain bound to the T cell’s TCR for longer before the TCR fires. Thus, the likelihood of an off-target pMHC to trigger TCR signalling is decreased, and antigen discrimination is enhanced.

The rate of progression of the T cell through the KP mechanism may be decreased in various ways. For example, this may be achieved by reducing the function of factors which promotes the multi-step progression towards a TCR activated state. This may also be achieved by increasing the function of factors which counteract (e.g. opposes, inhibits or reverses) such multi-step progression, and hence restoring the T cell towards the TCR resting state. Methods of reducing or increasing the function of such factors are described further below. For example, activation by low affinity peptides is preferentially disrupted if co-receptor function is perturbed.

Hence, a factor useful with the invention may be a factor that promotes progression of the T cell through the KP mechanism. For example, the factor may contribute to the progression of the T cell from a TCR resting state towards a TCR activated state (e.g. from T to CN state in Figure 2). The factor may aid in phosphorylation of the cytoplasmic chains of the TCR-CD3 subunit (e.g. ITAMs on the cytoplasmic tail of the CD247 chains), ZAP70, and/or LAT (see Figure 1). The factor may be a kinase, a kinase-recruiter, a scaffold molecule, or a co-stimulatory molecule. For example, the factor may be CD8a, CD80, CD4, Lek, ZAP70 or LAT. The function of such a factor is typically reduced in a modified T cell of the invention. Function of said factor may be reduced directly (e.g. by modulating the expression and/or activity of the factor) or indirectly (e.g. by modulating a binding partner of the factor such that the expression and/or activity of the factor is modulated, such as overexpressing the binding partner resulting in the factor being sequestered).

A factor useful with the invention may be a factor that counteracts progression through the KP mechanism. For example, the factor may reset the TCR complex to a resting state upon dissociation of a pMHC from the TCR are factors which oppose (or reverse) progression through the KP mechanism. The function of such a factor is typically increased in a modified T cell of the invention. A modified T cell of the invention is a T lymphocyte. The T cell may be an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte. The T lymphocyte may be a CD4+ T lymphocyte. The T lymphocyte may be a CD8+ cytotoxic T lymphocyte.

The modified T cell may be a CD4+ T cell or a CD8+ T cell.

The modified T cell may additionally comprise a TCR specific to a target antigen of interest (also referred to herein as a TCR of interest). The modified T cell may have been selected for expression of said TCR of interest prior to modulation of the modulated factor, or the TCR of interest may have been transduced into the T cell prior to or following modulation of the modulated factor.

The modified T cell is preferably suitable for use in adoptive T cell transfer therapy (ACT). The modified T cell retains the T cell’s suitability for ACT.

The modified T cell exhibits enhanced discrimination for its target antigen relative to a reference T cell, e.g. a corresponding T cell in which the function of the relevant factor is not modulated.

The modified T cell maintains the sensitivity for its target antigen compared to a reference T cell, e.g. within >80%, >90%, >95%, or 100%. The reference T cell is typically a corresponding T cell in which the function of the relevant factor is not modulated.

Hence, the invention also provides a method of enhancing pMHC discrimination of a T cell. The invention also provides a method of enhancing pMHC discrimination of a T cell, whilst maintaining the sensitivity to the pMHC. Such methods comprise preparing a modified T cell according to any of the methods described herein. For example, the method may comprise modulating (e.g. reducing) the function (e.g. amount and/or activity) of a factor associated with T cell receptor (TCR) kinetic proofreading. The factor may be CD4, CD8, Lek, LAT and/or Zap70. For example, the factor may be CD8 and/or Lek. Methods for modulating the function of a factor are explained further below.

Also provided is a method, such as an ex vivo method, of preparing a population of modified T cells for adoptive cell therapy. The method may comprise culturing a modified T cell of the invention. The method may comprises preparing one or more modified T cells as provided herein and expanding said T cells. Also provided is a population of modified T cells produced according to methods herein.

The methods of the invention may additionally comprise a step of introducing a TCR of interest into the T cell. Said introduction may be by any method known in the art, for example by transduction. Said introduction may be performed before or after modulating the factor associated with the KP mechanism. Said introduction may be performed simultaneously with modulation of the factor, for example by introducing the TCR and a modulator (e.g. inhibitor) in the same transduction step, optionally in the same vector, optionally on the same expression construct.

For example, in some instances, a method of the invention may comprise: (i) modulating the amount and/or activity of a factor involved in the TCR KP mechanism in a T cell and then (ii) transducing a TCR of interest into the T cell. In other instances, a method of the invention may comprise: (i) transducing a TCR of interest into the T cell and then (ii) modulating the amount and/or activity of a factor involved in the TCR KP mechanism in a T cell. In other instances, a method of the invention may comprise: (i) modulating the amount and/or activity of a factor involved in the TCR KP mechanism in a T cell and, simultaneously, (ii) transducing a TCR of interest into the T cell. In some instances, methods of the invention may further comprise a step of (iii) modulating the amount and/or activity of a further factor involved in the TCR KP mechanism in the T cell. Step (iii) may be performed before, during or after any of steps (i) and (ii).

T cell discrimination capacity may be assessed by, for example, (a) exposing the T cell or a population of T cells to its high affinity target pMHC (i.e. the its cognate pMHC) and determining the concentration of pMHC required to achieve signalling; (b) exposing T cell or a population of T cells to one or more low affinity pMHCs and determining the concentration of these pMHCs required for signalling; (c) comparing the concentration of high affinity pMHC against the concentration of low affinity pMHC required to induce comparable signalling of cells expressing the pMHC-binding receptor, wherein a large difference in concentration indicates good discrimination. Weak or absent signalling induced by low affinity pMHC where the high affinity target pMHC induces signalling also indicates good discrimination. Signalling may be measured, for example, by induction of a molecule downstream in the TCR signalling cascade, for example, a cytokine (e.g. IL-2), or a surface molecule, (e.g. CD69 or 4-lBB(CD137)), or target cell killing (e.g. LDH release).

Function modulation

Modulation of the function of factors described herein comprises modulating the amount and/or activity of the factor in a T cell.

Reduction in the function (e.g. amount and/or activity) of a factor that promotes progression through the KP mechanism (e.g. CD8a, CD80, CD4, Lek, ZAP70 or LAT) enhances T cell discrimination, as shown in Examples 1 and 4. Hence, for embodiments of the invention relating to a factor that promotes progression through the KP mechanism, the function (e.g. amount and/or activity) of the factor is reduced. For example, the function (e.g. amount and/or activity) of one or more of CD8a, CD80, CD4, Lek, ZAP70 and LAT may be reduced. The function (e.g. amount and/or activity) of one or more of Lek, ZAP70 and LAT may be reduced.

Reduction may be complete or partial. For example, the amount of the factor may be reduced, e.g. by at least 10%, by at least 20%, by at least 30%, at least 40%, at least 50%, at least 60, at least 70%, at least 80%, at least 90%, or by 100%. The activity of the factor may be reduced, e.g. by at least 10%, by at least 20%, by at least 30%, at least 40%, at least 50%, at least 60, at least 70%, at least 80%, at least 90%, or by 100%.

The extent of reduction, e.g. complete reduction (e.g. knock out) or partial reduction (e.g. knock down), may be determined by a person skilled in the art. For example, the skilled person will appreciate that it may not be desirable for complete inhibition of the amount and/or activity of factors that carry out essential steps in the TCR signal transduction pathway (e.g. Lek, ZAP70 or LAT).

Hence, the invention comprises complete reduction in the amount of a factor (e.g. CD4 or CD8) surface expressed on the T cell.

The modified T cell may not express a functional form of a factor described herein.

For example, a modified T cell of the invention may not express a functional CD4. For example, a method of the invention may comprise deleting the CD4 gene from the genome of a T cell, such as a CD4+ T cell or an uncommitted CD4+CD8+ T cell. A method of the invention may comprise deleting the CD4 gene from the genome of an autologous or allogenic T cell. Hence, a modified T cell of the invention may be a CD4 knock out T cell, such as a CD4+ committed CD4 knock out, or a CD4+CD8+ uncommitted CD4 knock out. A modified T cell of the invention may be an autologous T cell or an allogenic T cell.

CD4 stabilises a TCR:pMHC-II complex and aids progression of the kinetic proofreading pathway towards the T cell activated state. Where a T cell recognises a pMHC-II antigen (i.e. the T cell comprises a TCR specific for a peptide presented via a MHC class II molecule), CD4 knock out enhances target antigen discrimination. Hence, a modified T cell of the invention may be a T cell which recognises pMHC-II, for example a T helper cell or an autologous or allogenic T cell engineered to express a TCR which recognises pMHC-II. In some embodiments a modified T cell of the invention recognises pMHC-II and the amount of CD4 has reduced, optionally completely reduced. In some embodiments a modified T cell of the invention recognises pMHC-II and CD4 has been knocked out. In some embodiments, a method of the invention may comprise deleting the CD4 gene from the genome of a T cell which recognises pMHC-II.

A modified T cell of the invention may not express a functional CD8. For example, a method of the invention may comprise deleting the CD8a and/or CD80 gene from the genome of a T cell, such as a CD8+ T cell or an uncommitted CD4+CD8+ T cell. A method of the invention may comprise deleting the CD8 gene from the genome of an autologous or allogenic T cell. Hence, a modified T cell of the invention may be a CD8 knock out T cell. The modified T cell may be a CD8+ committed CD8 knock out. The modified T cell may be a CD4+CD8+ uncommitted CD8 knock out. A modified T cell of the invention may be an autologous T cell or an allogenic T cell.

CD8 stabilises a TCR:pMHC-I complex and aids progression of the kinetic proofreading pathway towards the T cell activated state. Where a T cell recognises a pMHC-I antigen (i.e. the T cell comprises a TCR specific for a peptide presented via a MHC class I molecule), CD8a knock out enhances target antigen discrimination (as shown in Examples 1 and 7). Hence, a modified T cell of the invention may be a T cell which recognises pMHC-I, for example a T helper cell or an autologous or allogenic T cell engineered to express a TCR which recognises pMHC-I. In some embodiments a modified T cell of the invention recognises pMHC-I and the amount of CD8 protein has reduced, optionally completely reduced. In some embodiments a modified T cell of the invention recognises pMHC-I and the amount of CD8a polypeptide has reduced, optionally completely reduced. In some embodiments a modified T cell of the invention recognises pMHC-I and CD8 protein has been knocked out, optionally wherein CD8a polypeptide has been knocked out. In some embodiments, a method of the invention may comprise deleting the CD8a gene from the genome of a T cell which recognises pMHC-I.

The invention comprises partial reduction in the amount of a factor (e.g. Lek, ZAP70 or LAT) surface expressed on the T cell.

Means of reducing the amount and/or activity of the factor will be apparent to the skilled person. For example, the amount and/or activity of the factor may be reduced by impairing gene expression at the nucleic acid level and/or at the protein level, e.g. by modulating the gene encoding the factor or a component thereof, of expression of said gene, of the mRNA transcript, of translation of the mRNA transcript, of the protein or its activity. For example, reduction of amount and/or activity of the factor may be achieved by deleting the gene encoding the factor or a component thereof from the genome (i.e. knocking out), reducing expression of the factor or a component thereof, inhibiting the factor or a component thereof, introducing one or more mutations into the gene encoding the factor or a component thereof which compromises factor expression or function (e.g. binding activity and/or enzymatic activity), promoting degradation of the factor or a component thereof.

For example, means of knocking out, e.g. for CD4 or CD8a and/or CD80, will be apparent to the skilled person and include, for example, by CRISPR/Cas9 or CRISPR/Cas related systems, Zinc-fingers, TALENS, homologous recombination, nucleases.

Means of reducing the activity of a factor include inhibiting binding of the factor to its ligand or by inhibiting enzymatic activity. For example, binding of CD4 or CD8 to MHC may be inhibited, recruitment of Lek, ZAP70, or LAT may be inhibited, kinase activity of Lek, ZAP70 or LAT may be inhibited. Appropriate modulators, such as inhibitors, will be apparent to the skilled person and include small molecules, proteins, peptides, antibodies and fragments thereof, scFvs, VHHs, VNARs. Reduction of amount and/or activity of the factor may be achieved by a modulator, such as an inhibitor. The modulator (e.g. inhibitor) may be stably or transiently expressed. Hence, a modified T cell of the invention may further comprise a modulator (e.g. an inhibitor), or one or more genes encoding the modulator (e.g. the inhibitor). The modulator may be an inhibitor of gene expression of the factor (e.g. CRISPR/Cas-related systems, shRNA) (see Example 4). The modulator may be an inhibitor of the activity of the factor (e.g. a small molecule inhibitor) (see Example 5).

The modulator may be a small organic molecule, a small inorganic molecule, a nucleic acid, (e.g. an siRNA, a guide RNA, an antisense oligonucleotide), a peptide, a polypeptide, a protein, an antibody or an antigen-binding fragment thereof, a saccharide, a nucleic acid analog or derivative (e.g. an analog of NAADP), a phospholipid (e.g. an analog or derivative of phosphatidylinositol 3, 5 -bisphosphate), or the like. A small molecule is typically a molecule that contains several carbon to carbon bonds. A small molecule typically weights less than 5 kiloDaltons, such as less than 3 kDa, less than 2 kDa, less than 1 kDa, or less than 500 Daltons.

The modulator may bind directly to the factor and alter its function.

The inhibitor may, for instance, be a small molecule that specifically binds to the factor and acts as an antagonist of factor function. The inhibitor may be a nucleic acid agent that affects expression (transcription and/or translation) of the factor.

For example, the inhibitor may be a small-interfering RNA (siRNA). The design and production of siRNAs is within the common general knowledge of the skilled person, and are also available commercially (e.g. www.thermofisher.com).

The inhibitor may be a guide RNA (gRNA), such as for use with a CRISPR-type system. The gRNA may be designed such that expression of functional factor is prevented, for example by deleting a part of the genomic loci (e.g. an exon, start codon, promoter or ORF). The gRNA may be designed to delete a transcriptional repressor binding site on a promoter of the factor gene, such that expression of functional factor is enhanced. The design and production of gRNAs is within the common general knowledge of the skilled person, and such gRNAs are available commercially (e.g. www.genscript.com). The inhibitor may be an antisense oligonucleotide such as a morpholino. The design and production of morpholinos are within the common general knowledge of the skilled person, and are available commercially (e.g. www.gene-tools.com).

The inhibitor may be a small hairpin RNA (shRNA). The design and production of shRNAs are within the common general knowledge of the skilled person, and are available commercially (e.g. Reference 8).

The invention comprises increasing the amount and/or activity of first factor which competes for binding with a second factor involved in progressing the KP pathway towards the active state. For example, the invention comprises expression or overexpression of a factor which competes for binding with a factor involved in progressing the KP pathway towards the active state. Accordingly, increasing the amount of the first factor results in a decrease in the activity of the second factor. In some embodiments, the first factor is CD4 or CD8a and the second factor is Lek.

CD4 stabilises a TCR:pMHC-II complex; CD8 stabilises a TCR:pMHC-I complex. T cell signal transduction and activation is promoted via CD4 or CD8a interaction with the intracellular molecule Lek (see Figure 1 which shows CD4, but CD8 can be considered analogous). T cell activation by low affinity peptides is preferentially disrupted if coreceptor function is perturbed. Therefore, by increasing the amount of CD4 polypeptide in T cells which recognise peptide-MHC-I targets, CD4 competes with CD8a for Lek binding and effectively reduces the available amount of Lek in the cell. Equally, by increasing the amount of CD8a polypeptide (or the CD8 complex generally) in T cells which recognise peptide-MHC-II targets, CD8a competes with CD4 for Lek binding and effectively reduces the available amount of Lek in the cell.

For example, a modified T cell of the invention may comprise CD4. A modified T cell of the invention may comprise higher amounts of a CD4 polypeptide than a corresponding unmodified T cell. For example, a method of the invention may comprise increasing the amount of a CD4 polypeptide in a T cell, such as a CD8+ T cell, a CD4+ T cell, an uncommitted CD4+CD8+ T cell or an autologous T cell or an allogenic T cell. Hence, a modified T cell of the invention may be a CD8+ T cell, a CD4+ T cell, an uncommitted CD4+CD8+ T cell or an autologous or allogenic T cell, wherein the T cell comprises higher amounts of CD4 polypeptide than a corresponding unmodified T cell. Where a T cell recognises a pMHC-I antigen i.e. the T cell_comprises a TCR specific for a peptide presented via a MHC class I molecule), CD4 overexpression enhances target antigen discrimination (see Examples 9 and 10). Equally, where the T cell recognises a pMHC-II antigen (i.e. the T cell_comprises a TCR specific for a peptide presented via a MHC class II molecule), CD8 overexpression will enhance target antigen discrimination.

Hence, a modified T cell of the invention may be a T cell which recognises pMHC- I, for example a cytotoxic T cell or an autologous or allogenic T cell engineered to express a TCR which recognises pMHC-I. In some embodiments a modified T cell of the invention recognises pMHC-I and comprises higher amounts of a CD4 polypeptide than a corresponding unmodified T cell. For example, the T cell may have been engineered to express or overexpress CD4. In some embodiments, a method of the invention may comprise increasing the amount of CD4 polypeptide in a T cell, optionally in a T cell which recognises pMHC-I. Said method may comprise introducing a polynucleotide encoding CD4 into a T cell. For example, said method may comprise transducing a T cell with a polynucleotide encoding CD4, introducing a polynucleotide encoding CD4 into a T cell by electroporation, knock-in, transfection or any other method known to the skilled person. The method may comprise introducing into a T cell a polynucleotide encoding CD4 and a polynucleotide encoding a TCR which recognises a pMHC-I. For example, said method may comprise transducing a T cell with a polynucleotide encoding CD4 and a polynucleotide encoding a TCR which recognises a pMHC-I. Said transduction could be via the same or separate vector(s). Said introduction may be by any other method known to the skilled person, for example electroporation, knock-in, or transfection.

As another example, a modified T cell of the invention may comprise CD8, optionally CD8a. A modified T cell of the invention may comprise higher amounts of CD8 protein, optionally higher amounts of CD8a polypeptide than a corresponding unmodified T cell. For example, a method of the invention may comprise increasing the amount of CD8 protein optionally CD8a polypeptide in a T cell, such as a CD8+ T cell, a CD4+ T cell, an uncommitted CD4+CD8+ T cell or an autologous T cell or an allogenic T cell. Hence, a modified T cell of the invention may be a CD8+ T cell, a CD4+ T cell, an uncommitted CD4+CD8+ T cell or an autologous or allogenic T cell, wherein the modified T cell comprises higher amounts of CD8 protein (optionally CD8a protein) than a corresponding unmodified T cell.

Hence, a modified T cell of the invention may be a T cell which recognises pMHC- II, for example a helper T cell or an autologous or allogenic T cell engineered to express a TCR which recognises pMHC-II. In some embodiments a modified T cell of the invention recognises pMHC-II and comprises higher amounts of a CD8 protein, optionally a CD8a polypeptide than a corresponding unmodified T cell. For example, the T cell may have been engineered to express or overexpress CD8, optionally CD8a. In some embodiments, a method of the invention may comprise increasing the amount of CD8 protein optionally CD8a polypeptide in a T cell, optionally in a T cell which recognises pMHC-II. For example, said method may comprise introducing into a T cell a polynucleotide encoding CD8a, optionally also a polynucleotide encoding CD80. In another example, said method may comprise introducing into a T cell a polynucleotide encoding CD8a (optionally also a polynucleotide encoding CD80) and a polynucleotide encoding a TCR which recognises a pMHC-II. Said introduction could be via transduction, electroporation, knock-in or any other method known to the skilled person. For example, said method may comprise transducing a T cell with a polynucleotide encoding CD8a, optionally also with a polynucleotide encoding CD80. In another example, said method may comprise transducing a T cell with a polynucleotide encoding CD8a (optionally also with a polynucleotide encoding CD80) and a polynucleotide encoding a TCR which recognises a pMHC-II. Said transduction could be via the same or separate vector(s).

The amount of the first factor may be increased, e.g. by at least 10%, by at least 20%, by at least 30%, at least 40%, at least 50%, at least 60, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, or at least 500%. The first factor may be expressed in cells of the invention where it is not expressed in the normal cellular state. The first factor may be expressed where it was not previously expressed in the same cell. The first factor may be overexpressed, e.g. by at least 10%, by at least 20%, by at least 30%, at least 40%, at least 50%, at least 60, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, or at least 500%. The extent of increase may be determined by a person skilled in the art.

As explained herein, increase in the function (e.g. amount and/or activity) of a factor that counteracts (e.g. opposes, inhibits or reverses) progression through the KP mechanism may also enhance T cell discrimination. Hence, for embodiments of the invention relating to a factor that counteracts progression through the KP mechanism, the function (e.g. amount and/or activity) of the factor is increased. Means of increasing the amount and/or activity of the factor will be apparent to the skilled person.

Expression of the factor at the nucleic acid or protein level may be measured by any means known to the skilled person, for example flow cytometry, Western blotting, qRT-PCR, genomic sequencing, fluorescent labelling experiments, mass spectrometry and the like. Expression of the factor may be determined by any means known in the art, such a nucleic acid sequencing. The skilled person is able to determine the polypeptide sequence encoded by the factor gene and determine if the polypeptide would result in a functional factor using any of the methods commonly known in the art. Furthermore, the skilled person would understand that frameshift mutations and/or mutations leading to early termination codons would lead to a non-functional factor.

In some embodiments, the invention comprises modulating the function of one or more factors associated with the TCR KP pathway to increase the discrimination ability of a T cell. In some embodiments, the invention comprises modulating the function of two or more factors associated with the TCR KP pathway to increase the discrimination ability of a T cell. For example, two or more of CD8, CD4, Lek, ZAP70 and/or LAT may be modulated. In some embodiments, the invention comprises modulating the function of two, three, four, five or more factors associated with the TCR KP pathway.

For example, a modified T cell of the invention may comprise a TCR which recognises pMHC-I, wherein CD8a has been knocked out and wherein the amount of CD4 polypeptide has been increased (see Example 10). Correspondingly, a method of the invention may comprise (i) knocking out CD8a and (ii) increasing the amount of CD4 polypeptide in a T cell (see Figure 27). In some embodiments, the T cell is a pMHC-I restricted T cell (for example a cytotoxic T cell or an autologous or allogenic T cell comprising a TCR which recognises a pMHC-I). In some embodiments, the method comprises a further step (iii) of expressing a pMHC-I TCR in the T cell. Steps (i), (ii) and optionally (iii) may be performed at the same time or sequentially in any order. In some embodiments, step (i) is performed before step (ii).

For example, said method may comprise: (i) transducing a T cell with a sgRNA for knocking out CD8a; (ii) introducing a polynucleotide encoding CD4 into the T cell; and/or (iii) introducing a polynucleotide encoding a TCR which recognises a pMHC-I into the T cell. Said introduction may be by any other method known to the skilled person, for example transduction, electroporation, knock-in, or transfection.

For example, said method may comprise transducing a T cell with: (i) a sgRNA for knocking out CD8a; (ii) a polynucleotide encoding CD4; and/or (iii) a polynucleotide encoding a TCR which recognises a pMHC-I. Said sgRNA and polynucleotide(s) may be comprised within the same or separate vector(s).

Similarly, a modified T cell of the invention may comprise a TCR which recognises pMHC-II, wherein CD4 has been knocked out and wherein the amount of CD8a polypeptide and/or CD80 polypeptide has been increased. Correspondingly, a method of the invention may comprise (i) knocking out CD4 and (ii) increasing the amount of CD8a polypeptide and/or CD80 polypeptide in a T cell. In some embodiments, the T cell is a pMHC-II restricted T cell (for example a cytotoxic T cell or an autologous or allogenic T cell comprising a TCR which recognises a pMHC-II). In some embodiments, the method comprises a further step (iii) of expressing a pMHC-II TCR in the T cell. Steps (i), (ii) and optionally (iii) may be performed at the same time or sequentially in any order. In some embodiments, step (i) is performed before step (ii).

For example, said method may comprise: (i) transducing a T cell with a sgRNA for knocking out CD4; (ii) introducing one or more polynucleotide(s) encoding CD8a and/or CD8p into the T cell; and/or (iii) introducing a polynucleotide encoding a TCR which recognises a pMHC-II into the T cell. Said introduction may be by any other method known to the skilled person, for example transduction, electroporation, knock-in, or transfection.

For example, said method may comprise transducing a T cell with: (i) a sgRNA for knocking out CD4; (ii) one or more polynucleotide(s) encoding CD8a and/or CD80; and/or (iii) a polynucleotide encoding a TCR which recognises a pMHC-II. Said sgRNA and polynucleotide(s) may be comprised within the same or separate vector(s).

The modulation of the function (e.g. reduction in the amount and/or activity) of a factor may be measured relative to the corresponding factor’s function in a reference T cell. The reference T cell is typically a corresponding T cell in which the function of the relevant factor is not modulated. The reference T cell may be an unmodified T cell. The reference T cell may be a differentiated but immunologically naive T cell of the same class as the modified T cell. For example, where the modified T cell may be a CD4+ T cell, the reference T cell would also be a CD4+ T cell; if the modified T cell is a CD8+ T cell, the reference T cell is also a CD8+ T cell. The reference cell may bind the same antigen as the modified T cell i.e. may comprise the same TCR as the modified T cell.

As described herein, TCR restriction may dictate the modulation strategy employed to enhance T cell discrimination. For example, as described herein, where T cells comprise TCRs recognising pMHC-I, discrimination can be enhanced by reducing the amount and/or activity of CD8 and/or increasing the amount of CD4 relative to an unmodified T cell. Where T cells comprise TCR recognising pMHC-II, discrimination can be enhanced by reducing the amount and/or activity of CD4 and/or increasing the amount of CD8 relative to an unmodified T cell. Regardless of the restriction of a TCR, discrimination can be enhanced by reducing the amount and/or activity of factors which act downstream of CD4 or CD8, for example Lek or LAT.

Thus, in some embodiments, T cells of the invention comprise a TCR of interest which specifically recognises pMHC-I antigens. In other words, T cells of the invention may comprise a pMHC-I-restricted TCR of interest.

The TCR which specifically recognises a pMHC-I antigen may be c259. The c259 TCR is a well-studied receptor whose cognate peptide is the tumour-specific antigen NY- ESO-1 (SEQ ID NO: 13) (9,10). T cells of the invention may comprise a TCR which specifically recognises SEQ ID NO: 13, optionally presented via a MHC class I molecule or complex.

The TCR which specifically recognises a pMHC-I antigen may be a3a. The a3a TCR is a well-studied receptor whose cognate peptide is a MAGE -A3 derived peptide (SEQ ID NO: 30), presented via HLA-A*01:01. T cells of the invention may comprise a TCR which specifically recognises SEQ ID NO: 30, optionally presented via a MHC class I molecule or complex. Off-target binding of a3a to a low-affinity peptide has been associated with fatal autoimmune toxicity seen in clinical trials (4). Thus, by providing T cells having enhanced discrimination ability, herein is provided improved T cells, for example a3a T cells, with reduced off-target binding and an improved safety profile.

A TCR which recognises a pMHC-I is also referred to as a pMHC-I restricted TCR. Likewise, a pMHC-II restricted TCR means a TCR which recognises a pMHC-II. In some embodiments the T cell comprising a pMHC-I restricted TCR or a pMHC-II restricted TCR naturally expresses said TCR. In other embodiments, the T cell comprising a pMHC-I restricted TCR or a pMHC-II restricted TCR has been engineered to express said TCR. Said engineering can be by any method known in the art, for example transduction (such as viral transduction), electroporation, knock-in, gene editing or transfection.

Also provided herein is a method of identifying a T cell having enhanced discrimination for its cognate peptide. For example, the method may comprise: (i) modulating the amount and/or activity of a factor associated with TCR kinetic proofreading in a T cell; and (ii) identifying a T cell having a reduced sensitivity for one or more low affinity (or ‘off-target’) ligand(s) relative to the unmodulated T cell. Step (ii) may be performed by any method known to the skilled person, for example screening against said one or more low affinity ligand(s). For example, methods of identifying sensitivity to low affinity ligands may include flow cytometry, ELISA, affinity assays, cytokine screens, surface 4 IBB assays, IFN gamma assays, TNF alpha assays, IL 2 assays, killing assays. The invention also provides a T cell obtained or obtainable by said method.

T cell discrimination capacity may be assessed as described herein.

The T cell may comprise a TCR of interest, optionally wherein the TCR of interest recognises pMHC-I. In other embodiments the method may further comprise introducing a pMHC-I restricted TCR of interest into the T cell, wherein said introduction may be performed before or after the modulation step (i).

The T cell may comprise a TCR of interest which recognises pMHC-I. In other embodiments the method may further comprise introducing a pMHC-I restricted TCR of interest into the T cell, wherein said introduction may be performed before or after the modulation step (i). In some embodiments of the method of identifying a T cell, the T cell may be a population of T cells.

In some embodiments modulating the amount and/or activity of a factor is reducing the amount and/or activity of a factor. For example, the amount of a factor may be reduced by RNA interference or knockout, as described herein. The activity of a factor may be reduced by inhibition, as described herein. As described herein, the factor to be reduced may be CD8, CD4, Lek, ZAP70 or LAT, optionally CD8, CD4 or Lek. When the TCR of interest is a pMHC-I restricted TCR, the factor to be reduced is preferably CD8 (optionally CD8a) or Lek.

In some embodiments modulating the amount and/or activity of a factor is increasing the amount and/or activity of a factor. The amount of a factor may be increased by any method known to the skilled person, for example including transduction, transfection, electroporation, knock-in, and/or genetic engineering (such as genetic modification or gene editing). For example, the amount of a factor may be increased by transducing the gene encoding the factor into T cells. As another example, the amount of a factor may be increased by electroporation into T cells. The amount of a factor may be increased by overexpression. As described herein, the factor to be increased may be CD8 or CD4. When the TCR of interest is a pMHC-I restricted TCR.

Nucleic acids, vectors and host cells

Also provided herein are nucleic acids encoding or constituting modulators (e.g. inhibitors) described herein. The nucleic acid may be a DNA sequence. The nucleic acid may be an RNA sequence, such as mRNA.

The inhibitor may be a shRNA. The shRNA may comprise 5 to 35, 10 to 30 or 15 to 25 consecutive nucleotides of the gene encoding a factor described herein, such as CD4, CD8a, CD8p, ZAP70, LAT or Lek.

For example, a nucleic acid inhibitor, such as shRNA, for inhibiting Lek may comprise a polynucleotide sequence having > 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, or 100% sequence identity to any one of SEQ ID NOs: 5 to 8, and/or a polynucleotide sequence having > 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, or 100% sequence complementarity to any one of SEQ ID NOs: 5 to 8. The inhibitor, such as shRNA, may comprise SEQ ID NOs: 8 and 10. In the embodiment where the inhibitor is shRNA, the shRNA may additionally comprise a loop having > 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, or 100% sequence identity to SEQ ID NO: 9.

A nucleic acid inhibitor, such as shRNA, for inhibiting ZAP70 may comprise a polynucleotide sequence having > 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, or 100% sequence identity to any one of SEQ ID NOs: 21 to 24, and/or a polynucleotide sequence having > 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, or 100% sequence complementarity to any one of SEQ ID NOs: 21 to 24. In the embodiment where the inhibitor is a shRNA, the shRNA may additionally comprise a loop having > 80%, > 90%,

> 95%, > 96%, > 97%, > 98%, > 99%, or 100% sequence identity to SEQ ID NO: 9.

A nucleic acid inhibitor, such as shRNA, for inhibiting LAT may comprise a polynucleotide sequence having > 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, or 100% sequence identity to any one of SEQ ID NOs: 25 to 28, and/or a polynucleotide sequence having > 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, or 100% sequence complementarity to any one of SEQ ID NOs: 25 to 28. In the embodiment where the inhibitor is a shRNA, the shRNA may additionally comprise a loop having > 80%, > 90%,

> 95%, > 96%, > 97%, > 98%, > 99%, or 100% sequence identity to SEQ ID NO: 9.

The inhibitor may be based on a CRISPR/Cas system, e.g. a CRISPR-Cas9 system. The CRISPR/Cas system may comprise one or more (e.g. 1, 2, 3 or 4) synthetic guide RNAs (sgRNAs) for knocking out a factor, e.g. CD4, CD8a and/or CD80. The sgRNA may comprise one or more (e.g. 1, 2, 3 or 4) sequences comprising 5 to 35, 10 to 30 or 15 to 25 consecutive nucleotides of the gene encoding the factor. The CRISPR-Cas system may additionally comprise one or more Cas proteins or encoding polynucleotides, such as a Cas9 protein or Cas9-encoding polynucleotide, optionally suitable for knocking out CD4, CD8a and/or CD80. The one or more sgRNAs for knocking out CD8a may comprise a sequence having > 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, or 100% sequence identity to any of SEQ ID NOs: 1 to 4.

Also provided herein is a vector comprising the nucleic acid. The vector may be a viral vector. Conventional viral based expression systems could include retroviral, alpha- retroviral, lentivirus, adenoviral, adeno-associated (AAV) and herpes simplex virus (HSV) vectors for gene transfer. The vector may be a lentivirus vector. Non-viral transduction vectors include transposon based systems including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art. In some embodiments the vector is a dual vector which allows expression of both a modulator (e.g. shRNA) and a TCR of interest. In some embodiments the vector is a lentiviral vector, optionally a dual lentiviral vector.

The vector may be a cloning vector or an expression vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide of the invention.

The vector is preferably an RNA vector. Suitable RNA vectors include the RNA vectors as described in Schutsky, Keith, et al., Oncotarget 6.30 (2015): 28911 and Beatty, Gregory L., et al., Gastroenterology 155.1 (2018): 29-32.

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.

A nucleic acid may be provided in the form of an expression construct (or ‘expression cassette’), which includes control sequences operably linked to the inserted sequence, thus allowing for expression of modulators and/or factors as defined herein in vivo. Hence, also provided is one or more expression cassettes encoding the one or more nucleic acids that encode modulators and/or factors as defined herein. These expression cassettes, in turn, are typically provided within vectors (e.g. plasmids or recombinant viral vectors). Hence, also provided is a vector encoding one or more modulators and/or factors as defined herein. Further provided are vectors which collectively encode one or more modulators and/or factors as defined herein.

The vector may be a human artificial chromosome. Human artificial chromosomes are described in e.g. Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014).

The vector may be a non-viral delivery system, such as DNA plasmids, naked nucleic acid (e.g. naked RNA), and nucleic acid complexed with a delivery vehicle, such as a liposome. The nucleic acids, expression cassetes or vectors described herein may be introduced into a host cell, e.g. by transduction, or other means such as electroporation or transfection. Hence, also provided is a host cell comprising the one or more nucleic acids, expression cassettes or vectors of the invention. The nucleic acids, expression cassettes or vectors described herein may be introduced transiently or permanently into the host cell, allowing expression of an antibody from the one or more nucleic acids, 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.

Typically, the host cell is T cell of the invention. The nucleic acids, expression cassettes or vectors described herein may be introduced transiently or stable into the host cell.

A modified T cell of the invention may comprise one or more nucleic acids of the invention, one or more expression constructs of the invention, or one or more vectors of the invention.

Also provided is a kit suitable for modifying a T cell or a population of T cells to generate a modified T cell or population of modified T cells of the invention. The kit comprises one or more nucleic acids or vectors described herein. The kit may comprise further agents such as those discussed herein that improve transfection, transduction or transformation efficacy.

Pharmaceutical composition

Also provided is a composition comprising a modified T cell or population of modified T cells of the invention. The modified T cell or population of modified T cells may be at least 1% of the total cells in the composition, such as at least 5%, at least 10%, at least 15 at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.9% of the total cells in the composition. The total cells in the composition may consist or consist essentially of the modified T cell or population of modified T cells of the invention, i.e. no other cells are detectable in the composition.

The composition may be a pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers comprise aqueous carriers, diluents or excipients. Examples of suitable carriers include all aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers and solutes, which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorption agents and the like. It will be understood that compositions of the invention may also include other supplementary physiologically active agents.

The carrier is typically pharmaceutically “acceptable” in the sense of being compatible with the other ingredients in the composition and not injurious to the subject. Compositions include those suitable for parenteral administration, including subcutaneous, intramuscular, intravenous and intradermal administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include preparing the carrier for association with the isolated T cells. In general, the compositions are prepared by uniformly and intimately bringing into association any active ingredients with liquid carriers.

The composition may be suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration. Compositions suitable for parenteral administration include aqueous and non- aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes, which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

The composition described herein may be prepared in a manner known in the art and are those suitable for parenteral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition with one or more pharmaceutically acceptable carriers or diluents. The composition may comprise at least about IxlO⁶ to about IxlO¹² of the modified T cells of the invention.

The present disclosure also contemplates the combination of the composition described herein with other active agents and/or in addition to other treatment regimens or modalities such as radiation therapy or surgery. When the composition described herein is used in combination with known active agents, the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture.

Suitable anti-cancer agents will be known to persons skilled in the art.

Treatment in combination is also contemplated to encompass the treatment with either the composition of the invention followed by a known treatment, or treatment with a known agent followed by treatment with the composition of the invention, for example, as maintenance therapy.

For example, in the treatment of cancer it is contemplated that the composition of the present invention may be administered in combination with an alkylating agent (such as mechlorethamine, cyclophosphamide, chlorambucil, ifosfamidecysplatin, or platinum- containing alkylating agents such as cisplatin, carboplatin and oxaliplain), and antimetabolite (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), adenosine receptor agonists (such as CCPA, IB- MECA and CI-IB-MECA), a checkpoint inhibitor, including those of the PDL-1:PD-1 axis, nivolumab, pembrolizumab, atezolizumab, BMS-936559, MEDI4736, MPDL33280A or MSB0010718C), an inhibitor of the CTLA-4 pathway (such as ipilimumab and tremelimumab), an inhibitor of the TIM- 3 pathway or an agonist monoclonal antibody that is known to promote T cell function (including anti-OX40, such as MEDI6469; and anti-4- BB, such as PF-05082566).

The invention also provides a kit or article of manufacture including a pharmaceutical composition as described above.

The invention also provides a kit for use in a therapeutic application mentioned above, the kit comprising: (a) a container holding a polypeptide, nucleic acid, vector or pharmaceutical composition of the invention; and (b) a label or package insert with instructions for use.

Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a therapeutic composition which is effective for treating the condition and may have a sterile access port ( e.g, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the therapeutic composition is used for treating the condition of choice. In an embodiment, the label or package insert includes instructions for use and indicates that the therapeutic or prophylactic composition can be used to treat a cancer or other condition described herein.

The kit may further comprise a further container comprising a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, which would be known to persons skilled in the art, suitable examples of which include other buffers, diluents, filters, needles, and syringes.

Therapeutic uses

Also described here is use of a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention for enhancing T cell discrimination.

Also described herein is use of a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassete(s), or a pharmaceutical composition of the invention, in a method of treatment of the human or animal body by therapy, e.g. for use as a medicament.

For instance, also provided is a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an modified T cell, population of modified T cells of the invention. Hence, the invention also provides a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention for use in a method of treating cancer. The invention also provides the use of a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention for the manufacture of a medicament for the treatment of cancer. The invention also provides the use of a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein ), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention to treat cancer.

The cancer may be any cancer, such as a solid cancer. The cancer may be a malignancy listed in Table 1. The cancer may be haematological malignancy or B cell cancer.

Also provided is a method of treating or preventing an infection in a subject, the method comprising administering to the subject an effective amount of modified T cell, population of modified T cells of the invention. Hence, the invention also provides a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention for use in a method of treating or preventing an infection.

Also provided is a method of treating or preventing an inflammatory disease in a subject, the method comprising administering to the subject an effective amount of a modified T cell or population of modified T cells, of the invention. Hence, the invention also provides a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassete(s), or a pharmaceutical composition of the invention for use in a method of treating or preventing an inflammatory disease.

The invention also provides the use of a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention for the manufacture of a medicament for the treatment or prevention of an infection.

The invention also provides the use of a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention to treat or prevent an infection. The infection may be a chronic infection.

The invention also provides the use of a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention for the manufacture of a medicament for the treatment or prevention of an inflammatory disease.

The invention also provides the use of a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention to treat or prevent an inflammatory disease. The inflammatory disease may be an autoimmune disease.

Also provided is a method of performing adoptive cell therapy in a subject, the method comprising administering to the subject an effective amount of a modified T cell or a population of modified T cells of the invention.

Hence, the invention also provides a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition for use in adoptive cell therapy. The invention also provides the use of a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention for the manufacture of a medicament for adoptive cell therapy.

The invention also provides the use of a modified T cell, population of modified T cells, modulator (e.g. inhibitor such as shRNA or sgRNA described herein), polynucleotide(s), vector(s), expression cassette(s), or a pharmaceutical composition of the invention adoptive cell therapy.

The therapeutic uses and methods may comprise administering a therapeutically effective amount of the modified T cell or population of modified T cells.

Also provided is a method of formulating a composition for treating cancer, wherein said method comprises mixing a modified T cell or population of modified T cells of the invention with an acceptable carrier to prepare said composition.

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

The therapeutic methods and uses may comprise, prior to treatment with a modified T cell or population of modified T cells of the invention, determining whether the cancer expresses a target antigen specifically targeted by the modified T cell or population of modified T cells of the invention.

The method may comprise selecting a modified T cell or population of modified T cells based on the expression of the target antigen by the cancer, so that the modified T cell or population of modified T cells is specific for the cancer. The method may comprise transfecting or transforming a T cell with a nucleic acid of the invention in response to information on the expression of the target antigen by the cancer.

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%.

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.

The dose of the immune effector cell or population of immune effector cells may vary depending on the age and size of a subject, as well as on the disease, conditions and route of administration. The modified T cell or population of modified T cells may be administered at a dose of about IxlO⁶ to about IxlO¹² cells. The modified T cell or population of modified T cells may be administered at a dose of about IxlO⁵ cells/kg to about IxlO¹¹ cells/kg body weight.

The modified T cell or population of modified T cells may be administered as a single dose. The modified T cell or population of modified T cells 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.

The modified T cell or population of modified T cells may be administered intravenously.

The modified T cell or population of modified T cells may be administered with one or more additional therapy, such as one or more additional therapeutic agents. The additional therapeutic agent may be an anti-tumour agent. The additional therapeutic may be an additional immune effector cell.

Combined administration of the modified T cell or population of modified T cells 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 modified T cell or population of modified T cells of the invention may be administered before, after or concurrently with the additional therapeutic agent. The additional therapy may be chemotherapy, radiotherapy and/or surgery.

Prior to administration of the modified T cell or population of modified T cells of the invention, the subject may undergo lymphodepletion. Lymphodepletion may be achieved via administration to the subject with fluradabine, cyclophosphamide and/or bendamustine. Lymphodepletion may be carried out for at least about one day, such as about 2 days or about 3 days.

The biological activity and/or therapeutic efficacy of the administered modified T cell or population of modified T cells may be measured by known methods. For example, the method may comprise imaging, such as magnetic resonance imaging.

Table 1 - Examples of antigen-associated malignancies which may be addressed by the pMHC-binding receptors of the invention. pMHC-binding receptors of the invention may bind the peptides from said antigens presented on any MHC molecule, including MHC-I or

MHC-II and any allele variants thereof Embodiments of the Invention

1. A modified T cell characterised in that the function of a factor associated with T cell receptor (TCR) kinetic proofreading is modulated.

2. The modified T cell of embodiment 1, wherein the modified T cell is a CD4+ or a CD8+ T cell.

3. The modified T cell of embodiment 1 or 2, wherein the amount and/or activity of the factor is reduced.

4. The modified T cell of any one of the preceding embodiments, wherein the factor is a factor which progresses the T cell towards the TCR activated state.

5. The modified T cell of any one of the preceding embodiments, wherein the factor is a kinase, a kinase-recruiter, a scaffold molecule, or a co-stimulatory molecule.

6. The modified T cell of any one of the preceding embodiments, wherein the factor is CD8, CD4, Lek, ZAP70 or LAT.

7. The modified T cell of any one of the preceding embodiments, wherein the modified T cell further comprises a inhibitor for inhibiting the amount and/or activity of the factor.

8. The modified T cell of embodiment 7, wherein the inhibitor is a shRNA.

9. The modified T cell of any one of the preceding embodiments, wherein the amount and/or activity of the factor is partially reduced.

10. The modified T cell of embodiment 9, wherein the factor is Lek, ZAP70 or LAT.

11. The modified T cell of any one of embodiments 1 to 8, wherein the amount and/or activity of the factor is completely reduced.

12. The modified T cell of embodiment 11, wherein the factor is CD4.

13. The modified T cell of 1 to 8 and 11 to 12, wherein the modified T cell does not express a functional CD4, e.g. the modified T cell is a CD4 knock out. 14. The modified T cell of embodiment 11, wherein the factor is CD8.

15. The modified T cell of embodiment 1 to 8, 11 and 14, wherein the modified T cell does not express a functional CD8, e.g. the modified T cell is a CD8 knock out.

16. The modified T cell of embodiment 1 or 2, wherein the amount and/or activity of the factor is increased, optionally wherein the factor is a factor which progresses the T cell towards the TCR resting state.

17. The modified T cell of any one of the preceding embodiments, wherein the modified T cell exhibits enhanced discrimination for its target antigen relative to an unmodified T cell which binds the same antigen.

18. The modified T cell of any one of the preceding embodiments, wherein the modified T cell further comprises a TCR of interest.

19. The modified T cell of any one of the preceding embodiments, wherein the function of a further factor associated with T cell receptor (TCR) kinetic proofreading is modulated, optionally wherein the further factor is CD8, CD4, Lek, ZAP70 or LAT.

20. An inhibitor of a factor which is associated with TCR kinetic proofreading in a T cell and which progresses the T cell towards the TCR activated state, optionally wherein the inhibitor is a shRNA.

21. The inhibitor of embodiment 20, comprising (a) any of SEQ ID NOs: 5 to 8 and 21 to 28, and/or (b) a polynucleotide sequence complementary to SEQ ID NOs: 5 to 8 and 21 to 28.

22. A sgRNA for knocking out a factor which is associated with TCR kinetic proofreading in a T cell and which progresses the T cell towards the TCR activated state, optionally wherein the sgRNA comprises one or more sequences comprising 5 to 35 consecutive nucleotides of the gene encoding the factor.

23. The sgRNA of embodiment 22 for knocking out CD8a, wherein the sgRNA comprises SEQ ID NO: 1, 2, 3 or 4. 24. A vector comprising the shRNA molecule of embodiment 20 or 21 or one or more sgRNAs of embodiment 22 or 23.

25. The vector of embodiment 24, wherein the vector further comprises a polynucleotide encoding a TCR of interest.

26. A pharmaceutical composition comprising the inhibitor of embodiment 20 or 21, one or more sgRNAs of embodiment 22 or 23, or the vector of embodiment 24 or 25.

27. A method of preparing a modified T cell, comprising modulating the function of a factor associated with TCR kinetic proofreading in a T cell.

28. The method of embodiment 27, wherein the T cell is a CD4+ or a CD8+ T cell.

29. The method of embodiment 27 or embodiment 28, wherein modulating the function of the factor comprises reducing the amount and/or activity of the factor.

30. The method of embodiment 29, wherein the factor is a factor which progresses the T cell towards the TCR activated state, optionally wherein the factor is a kinase, a kinase- recruiter, a scaffold molecule, or a co-stimulatory molecule, such as CD8, CD4, Lek, ZAP70 or LAT.

31. The method of embodiment 29 or embodiment 30, wherein reducing the amount and/or activity of the factor comprises knocking out the factor, optionally wherein the factor is CD4 or CD 8.

32. The method of any one of embodiments 29 to embodiments 1 , wherein reducing the amount and/or activity of the factor comprises introducing one or more sgRNAs targeted to the gene encoding the factor into the T cell, optionally further comprising introducing a Cas9 protein or a polynucleotide encoding a Cas9 protein into the T cell.

33. The method of embodiment 29 or embodiment 30, reducing the amount and/or activity of the factor comprises partially reducing the factor, optionally wherein the factor is Lek, ZAP70 or LAT.

34. The method of any one of embodiments 29 to 33, wherein reducing the amount of the factor comprises introducing or expressing an inhibitor in the T cell. 35. The method of embodiment 33, wherein the inhibitor is as defined in embodiment 20 or 21, or is a small molecule inhibitor.

36. The method of embodiment 27 or embodiment 28, wherein modulating the function of the factor comprises increasing the amount and/or activity of the factor.

37. The method of embodiment 36, wherein the factor progresses the T cell towards the TCR resting state.

38. The method of any one of embodiments 27 to 37, wherein the method further comprises transducing a TCR of interest into the T cell.

39. The method of any one of embodiments 27 to 38, wherein the method enhances target antigen discrimination of the TCR of interest.

40. A modified T cell obtainable or obtained by any of the method according to embodiments 27 to 39.

41. A method of enhancing target pMHC discrimination of a T cell, comprising preparing a modified T cell according to the method of any of embodiments 27 to 39.

42. A method of preparing a population of modified T cells for adoptive cell therapy, the method comprising culturing the modified T cell of any one of embodiments 1 to 19 and 40.

43. A population of modified T cells produced by the method of embodiment 42.

44. A method of treating cancer, an infection or an inflammatory disease comprising administering a modified T cell according to any one of embodiments 1 to 19, a population of T cells according to embodiment 43, an inhibitor according to embodiment 20 or 21, a sgRNA according to embodiment 22 or 23, a vector according to embodiment 24 or 25, or a pharmaceutical composition according to embodiment 26 to a patient in need thereof, optionally wherein the infection is a chronic infection or the inflammatory disease is an autoimmune disease.

45. A modified T cell according to any one of embodiments 1 to 19, a population of T cells according to embodiment 43, an inhibitor according to embodiment 20 or 21, a sgRNA a according to embodiment 22 or 23, a vector according to embodiment 24 or 25, or a pharmaceutical composition according to embodiment 26 for use as a medicament.

46. A modified T cell according to any one of embodiments 1 to 19, a population of T cells according to embodiment 43, an inhibitor according to embodiment 20 or 21, a sgRNA according to embodiment 22 or 23, a vector according to embodiment 24 or 25, or a pharmaceutical composition according to embodiment 26 for use in a method of treating cancer, an infection or an inflammatory disease, optionally wherein the infection is a chronic infection or the inflammatory disease is an autoimmune disease.

Further Embodiments of the Invention

7. The modified T cell of any one of the preceding embodiments, wherein the modified T cell further comprises a inhibitor for inhibiting the amount and/or activity of the factor, such as a shRNA.

8. The modified T cell of any one of the preceding embodiments, wherein the amount and/or activity of the factor is partially reduced, optionally wherein the factor is Lek, ZAP70 or LAT. 9. The modified T cell of any one of embodiments 1 to 7, wherein the modified T cell does not express a functional CD4 or a functional CD8, e.g. the modified T cell is a CD4 knock out or a CD8 knock out.

10. The modified T cell of any one of the preceding embodiments, wherein the modified T cell further comprises a TCR of interest.

11. An inhibitor of a factor which is associated with TCR kinetic proofreading in a T cell and which progresses the T cell towards the TCR activated state, optionally wherein the inhibitor is a shRNA comprising (a) any of SEQ ID NOs: 5 to 8 and 21 to 28, and/or (b) a polynucleotide sequence complementary to SEQ ID NOs: 5 to 8 and 21 to 28.

12. A sgRNA for knocking out a factor which is associated with TCR kinetic proofreading in a T cell and which progresses the T cell towards the TCR activated state, optionally wherein the sgRNA comprises SEQ ID NO: 1, 2, 3 or 4.

13. A method of preparing a modified T cell, comprising modulating the function of a factor associated with TCR kinetic proofreading in a T cell.

14. The method of embodiment 13, wherein the T cell is a CD4+ or a CD8+ T cell.

15. The method of embodiment 13 or embodiment 14, wherein modulating the function of the factor comprises reducing the amount and/or activity of the factor.

16. The method of any one of embodiments 13 to 15, wherein the factor is a factor which progresses the T cell towards the TCR activated state, optionally wherein the factor is a kinase, a kinase-recruiter, a scaffold molecule, or a co-stimulatory molecule.

17. The method of any one of embodiments 13 to 16, wherein the factor is CD8, CD4, Lek, ZAP70 or LAT.

18. The method of any one of embodiments 15 to 17, wherein reducing the amount and/or activity of the factor comprises knocking out the factor, optionally wherein the factor is CD4 or CD 8.

19. The method of any one of embodiments 15 to 18, wherein reducing the amount and/or activity of the factor comprises introducing one or more sgRNAs targeted to the gene encoding the factor into the T cell, optionally further comprising introducing a Cas9 protein or a polynucleotide encoding a Cas9 protein into the T cell.

20. The method of any one of embodiments 15 to 17, wherein reducing the amount and/or activity of the factor comprises partially reducing the factor, optionally wherein the factor is Lek, ZAP70 or LAT.

21. The method of any one of embodiments 13 to 20, wherein reducing the amount of the factor comprises introducing or expressing an inhibitor in the T cell, optionally wherein the inhibitor is as defined in embodiment 13.

22. The method of any one of embodiments 13 to 21, wherein the method further comprises transducing a TCR of interest into the T cell.

23. A modified T cell obtainable or obtained by any of the method according to embodiments 13 to 22.

24. A modified T cell according to any one of embodiments 1 to 10 and 23, an inhibitor according to embodiment 11, or a sgRNA a according to embodiment 12 for use as a medicament.

25. A modified T cell according to any one of embodiments 1 to 10 and 23, an inhibitor according to embodiment 11, or a sgRNA a according to embodiment 12 for use in a method of treating cancer, an infection or an inflammatory disease, optionally wherein the infection is a chronic infection or the inflammatory disease is an autoimmune disease.

Other

It is to be understood that different applications of the disclosed modified T cells, methods 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.

In addition as used in this specification and the appended embodiments, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “a modified T cell” includes two or more “modified T cells”.

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.

For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotide or amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions /total number of positions in the reference sequence x 100).

Typically the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 3, SEQ ID NO: 3 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 3 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 3, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 3. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 3. If the sequence is shorter than SEQ ID NO: 3, the gaps or missing positions should be considered to be non-identical positions.

The skilled person is aware of different computer programs that are available to determine the identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In an embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Unless otherwise provided, factors described herein are human homologs.

The TCRs described herein may be humanised.

Reference herein to an ‘antigen’ in the context of TCR binding is to be understood as referring to a pMHC complex.

Reference herein to an “amount” of a factor is to be understood as referring to the mean amount of the factor in a population of cells. For example, reference herein to the amount of a protein or polypeptide in a T cell is to be understood as the mean amount of the protein or polypeptide in a population (e.g. 10⁶) of said T cells.

Reference herein to an “activity” of a factor is to be understood as referring to the mean level of activity of the factor in a population of cells. For example, reference herein to the activity of a protein or polypeptide in a T cell is to be understood as the mean level of activity of the protein or polypeptide in a population (e.g. 10⁶) of said T cells.

T cells modified according to the invention demonstrate enhanced discrimination for their target antigen relative to the corresponding unmodified T cell. A corresponding unmodified T cell is to be understood as a T cell which comprises the same TCR of interest as the modified T cell, but which does not comprise a factor which has been modulated as described herein.

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

The following examples illustrate the invention.

Example 1 - Knock-out of CD8 alpha enhances T cell ligand discrimination

In this Example, T cells were modified to knock out CD8a and the effect on ligand discrimination was investigated.

The CD8 co-receptor can be formed by a heterodimer formed by an alpha and a beta chain or a homodimer formed by two alpha chains. CD8a binds to MHC-I molecules at invariant domains, which stabilises the TCR-pMHC interaction. Additionally, the intracellular tail of CD8a binds Lek via a zinc clasp and recruits it to the proximity of the TCR, which enhances progression though the kinetic proofreading mechanism and improves signal transduction. Knocking out CD8a prevented formation of the CD8 coreceptor.

A CD8a negative T cell was population generated by Cas9/sgRNA electroporation as described in Example 6. The CD8a sequences targeted by sgRNA were SEQ ID NOs: 1-4. Knock-out efficiency of 95% was obtained. Subsequently, T cells were stained with an anti-CD8a PE-conjugated antibody and the remaining CD8a positive cells were depleted by pull-down with magnetic Anti-PE beads. A pure population of CD8a negative cells was obtained (Figure 3).

To quantify the ligand discrimination of CD8a negative T cells, T cells were transduced with the c259 TCR and co-cultured with U87 cells pulsed with a titration of the 8 peptide panel as described in Example 6. pMHC potency (Pl 5) was defined as the concentration of peptide that elicits 15% of the maximal response obtained.

The amount of T cell activation was indicated by measuring surface 4 IBB expression (Figure 5), T cell cytotoxic activity (Nluc; Figure 6) and cytokine secretion (IL2; Figure 6).

As shown in Figure 5, using surface 4 IBB expression as a measure of T cell activation, no significant difference was observed on T cell potency against the high- affinity 9V peptide. In CD8a negative T cells, potency to medium and lower affinity peptides was significantly reduced. These results show that CD8a deletion in T cells transduced with the affinity-matured c259 TCR lead to an increase in ligand discrimination without loss of on-target potency.

Similar results were observed when measuring T cell cytotoxic activity and cytokine secretion (Figure 6A). The fold-change in P15 between CD8 alpha negative cells and wild-type T cells significantly increased as ligand affinity is reduced (Figure 6B).

Therefore, CD8 alpha negative cells have higher ligand discrimination than wildtype T cells. This may be because activation to low affinity peptides, which have a stronger co-receptor dependence, was preferentially disrupted when co-receptor function (e.g. CD8) was perturbed.

Therefore, a modified T cell having reduced amount of expression and/or activity of CD8 alpha exhibits enhanced ligand discriminatory powers. Example 2 - Knock-out of CD43 has no impact on T cell activation

In this Example, T cells were modified to knock out CD43 and the effect on ligand discrimination was investigated.

CD43 is an abundant and heavily glycosylated protein expressed on the T cell surface . CD43 is known to be involved in T cell antigen recognition, e.g. by reducing T cell-APC interactions due to its negative charge or steric hindrance, or reducing the effective on-rate of the TCR/pMHC interaction due to its size and abundance on the cell surface. Furthermore, it has been reported that loss of CD43 produced increased T cell proliferation, T cell adhesion and T cell activation in mice.

A CD43 negative T cell was population generated by Cas9/sgRNA electroporation and CD43+ T cell depletion. The sgRNA sequences are set out in SEQ ID NOs: 18 to 20. Subsequently, T cells were stained with an anti-CD43 PE-conjugated antibody and a pure population of CD43 negative cells was obtained (Figure 14).

CD43 KO cells were transduced with the c259 TCR and expressed similar receptor levels to wild-type c259 cells.

Using a luciferase-based target cell cytotoxicity assay as a measure of T cell activation, we observed no difference in T cell activation to any of the tested peptides (Figure 15). Similar results were observed when cytokine secretion and 41BB surface expression (Figure 16) were measured. CD43 negative primary human T cells show no difference in discrimination capacity relative to wild type primary human T cells (Figure 17A).

In conclusion, genetic deletion of CD43 had no detectable impact on human T cell ligand sensitivity or discrimination in the cellular antigen presentation assay. Hence, a modified T cell having reduced amount of expression and/or activity of CD43 provides no effect on ligand discrimination. CD43 is not involved in the kinetic proofreading mechanism for TCR activation.

Example 3 - Knock-out of CD2 reduces TCR sensitivity but has no effect on ligand discrimination

In this Example, T cells were modified to knock out CD2 and the effect on ligand discrimination was investigated. The adhesion receptor CD2 is expressed on the T cell surface and it binds CD58 on the APC. CD2-CD58 binding stabilises the close contact at the immunological synapse and it has been demonstrated to increase T cell sensitivity by lowering the antigen threshold for activation. It had also been shown that the addition of CD58 increased T cell ligand discrimination in a reductionist assay with pMHCs immobilised on a plate. CD2 is not involved in the kinetic proofreading mechanism for TCR activation.

A CD2 negative T cell population was obtained by Cas9/sgRNA electroporation and CD2+ T cell depletion as described in Example 6. The sgRNA sequences are set out in SEQ ID NOs: 14 to 17. Subsequently, T cells were stained with an anti-CD2 PE- conjugated antibody and a pure population of CD2 negative cells was obtained (Figure 11).

CD2 KO cells were transduced with the c259 TCR and expressed similar receptor levels to wild-type c259 cells.

Using surface 4 IBB expression as a measure of T cell activation, a small defect in sensitivity across all peptides was observed (Figure 12). This loss of sensitivity was not amplified for lower affinity peptides, therefore, there was no effect on ligand discrimination. Similar results were observed when T cell cytotoxic activity and cytokine secretion (Figure 13) were measured. CD2 negative primary human T cells show no difference in discrimination capacity relative to wild type primary human T cells (Figure 17B).

Example 4 - Inhibition of Lek expression enhances T cell ligand discrimination

In this Example, T cells were modified to inhibit the expression of Lek and the effect on ligand discrimination was investigated.

Lek is a protein kinase that belongs to the Src family, which catalyses the phosphorylation of the IT AM domains in the cytoplasmic tails of the TCR-CD3 complex.. This biochemical step initiates the TCR signal transduction that results in T cell activation upon TCR/pMHC binding.

A dual lentiviral vector was constructed which expresses the c259 TCR (SEQ ID NO: 12) constitutively under the control of the EFl alpha promoter and also expresses an shRNA molecule constitutively under the control of the human U6 promoter. The shRNA is either scramble shRNA molecule (SEQ ID NO: 29) or (B) an anti- Lck shRNA molecule was constructed and transduced in human primary T cells. The target sequences of the shRNAs (and hence the sense portion of the shRNAs) are set out at SEQ ID NOs: 5 to 8. Each shRNA contained a loop portion, as set out at SEQ ID NO: 9. The antisense sense portion of each shRNA is the reverse complement of the sense strand, e.g. SEQ ID NO: 10 is the antisense portion for the sense portion as set out in SEQ ID NO: 5.T cells transduced with the dual vector encoding an anti-Lck shRNA molecule had a reduction in intracellular Lek staining compared with T cells transduced with a scramble (negative control) shRNA molecule (Figure 7).

To quantify the ligand discrimination of Lek knockdown T cells, T cells were cocultured with U87 cells pulsed with a titration of the 8 peptide panel as described in Example 6 (Figure 8). pMHC potency (Pl 5) was defined as the concentration of peptide that elicits 15% of the maximal response obtained.

Using target cell killing as a measure of T cell activation, no significant difference was observed on T cell potency against the high-affinity 9V peptide for Lek knockdown cells and wild-type cells. Potency to medium and lower affinity peptides was reduced (Figures 8 and 9A). Therefore, these results suggest that Lek knockdown in T cells can lead to an increase in ligand discrimination without loss of on-target potency.

The fold-change in P15 between Lek knockdown cells and wild-type T cells significantly increases as ligand affinity is reduced (Figure 9B).

Therefore, Lek knockdown cells have higher ligand discrimination than wild-type T cells. This may be because inhibition of Lek led to elongation of the kinetic proofreading time, thereby resulting in enhanced discrimination.

Therefore, a modified T cell having reduced amount of expression and/or activity of Lek exhibits enhanced ligand discriminatory powers.

Example 5 - Chemical inhibition of the Lek kinase enhances T cell ligand discrimination

Enhanced ligand discriminatory powers of Lek knockdown T cells were also observed when Lek was inhibited by a chemical inhibitor (A-770041). Results are shown in Figures 19 and 20. For Lek chemical inhibition assay: The A-770041 inhibitor was diluted in DMEM media. T cells were treated with the appropriate dose of A-770041 for 1 hour, then cocultured with peptide-pulsed U87 cells for 4 hours at 37°C, 5% CO2. Subsequently, supernatants were collected for ELISA analysis and cells were analysed by flow cytometry to measure surface CD69 expression.

Cellular co-culture assays: 50 000 U87 cells in 100 pl of DMEM were seeded in a 96-well Flat-bottom plate and incubated overnight. Peptides were diluted in DMEM to the appropriate concentration, added to each well containing U87 cells and incubated for 60 minutes at 37°C, 10% CO2. The media was discarded and 50 000 T cells were added to each well in 200 pl of RPMI media. Cells were incubated for 24 hours at 37°C, 5% CO2. Supernatants were collected for cytotoxicity and ELISA analysis. 25 pl of 100 mM EDTA PBS were added to each well containing the cells and samples were incubated for 5 minutes at 37°C, 5% CO2. Cells were detached by thoroughly pipetting each well and transferred to a 96-well V-bottom plate. Cells were stained for 20 minutes at 4°C, washed with PBS and analysed by flow cytometry.

Example 6 -Materials & Methods

This Example provides the materials and methods used in Examples 1 to 5. c259 TCR and affinity peptide panel

The c259 TCR is a well-studied receptor whose cognate peptide is the tumourspecific antigen NY-ESO-1 (SLLMWITQC, SEQ ID NO: 13) (9,10). The c259 TCR recognises the NY-ESO-1 peptide presented on HLA-A2, an MHC-I molecule. A library of peptides with different affinities for this receptor has been developed (Figure 4): 9V > 6V > 3V > 6T > 4D > 4A > 5Y.

Human primary CD8+ T cell blasts were transduced with the c259 TCR and costimulated with U87 cells pulsed with a titration of the selected peptide library. T cell activation was measured by different methods. (I) Flow cytometry analysis of cell surface expression of activation markers, such as 41BB. (II) ELISA analysis of secretion of cytokines, such as IL-2 (III) Measuring the cytotoxic activity of T cells with a luciferasebased assay. This platform was used in Examples 1 to 5 to quantify antigen discrimination using multiple T cell responses, with the aim of understanding how modulating different T cell molecules enhances antigen discrimination.

Cell culture

U87 and HEK 293T cell lines were used. Primary human T cells were isolated from leukocyte cones.

Lentivirus production

HEK 293T cells were seeded in a 6-well plate (Day 1) and incubated overnight to achieve approximately 80% confluency. Cells in each well were co-transfected (Day 2) using X-tremeGENE™ HP (Roche) with 0.8 pg of the appropriate lentiviral transfer plasmid encoding an antigen receptor (1G4 TCR or c259 TCR) and the lentiviral packaging plasmids: pRSV-Rev (0.25 pg), pMDLg/pRRE (0.53 pg), and pVSV-G (0.35 pg). The media was replaced 18 hours following transfection (Day 3). 24 hours after the media exchange, the supernatant from one well was harvested, filtered and used for the transduction of 1 Million human T cells (Day 4).

T cell blasts

T cells were isolated from leukocyte cones (Day 3) purchased from the NHS Blood Donor Centre at the John Radcliffe Hospital (Oxford University Hospitals). RosetteSep™ Human CD8+ or CD4+ T Cell Enrichment Cocktail (STEMCELL Technologies) was added at 150 pl/mL of sample and incubated at RT for 20 minutes. The sample was diluted with an equal volume of PBS and layered on Ficoll® Paque Plus (Cytiva) density gradient medium at a 0.8:1 ratio (Ficoll®:Sample).

The sample was centrifuged at 1200 g for 30 minutes (brake off). Cells at the interface of the Ficoll® media and plasma were collected (Buffy coat) and washed twice (Centrifuged at 500 g for 5 minutes). Cells were resuspended in complete RPMI media supplemented with IL2 (50 U/mL) at a density of 1 Million cells per mL. Dynabeads® Human T-Activator CD3/CD28 (Thermofisher) were added (1 Million beads per mL) and cells were incubated overnight. 1 Million cells were transduced with the filtered lentiviral supernatant (Day 4). On Day 6 and on Day 8, 1 mL of media was removed and replaced with 1 mL of fresh medium. On Day 9, Dynabeads® were removed using a magnetic stand (6 days following isolation). Cells were resuspended in fresh media every other day at a density of 1 Million per mL and used for co-culture experiments. 17 days following isolation T cells were discarded.

CRISPR/Cas9 knock-out of T cell proteins

Cas9 ribonucleoproteins (RNPs) were prepared by mixing 8.5 pg of TruCut Cas9 protein v2 (Thermofisher) with 150 pmol of sgRNA mix (Truguide synthetic gma, Thermofisher) and Opti-MEM (Gibco) to a final volume of 5 pl. The RNPs were incubated for 15 minutes at room temperature.

1 Million freshly isolated T cells were washed 3 times with Opti-MEM (Gibco) and re-suspended at a density of 20 Million per mL. The T cells were mixed with the RNPs and transferred into a BTX Cuvette Plus electroporation cuvette (2mm gap, Harvard Bioscience). The cells were electroporated using a BTX ECM 830 Square Wave Electroporation System (Harvard Bioscience) at 300 V, 2 ms. Immediately following electroporation, the cells were transferred to complete RPMI media supplemented with IL2 and Dynabeads® Human T-Activator CD3/CD28 (Thermofisher) were added.

Negative selection of T cell knock-out cells

The knock-out efficiency of the different targets was 75-90%. T cells with residual target protein expression were depleted by antibody staining and bead pull-down. T cells were re-suspended in MACS Buffer (PBS, 0.5% BSA, 2 mM EDTA) at a density of 10 Million cells per mL. Cells were stained with 5 pl of the corresponding PE-labelled antibody per million cells for 15 minutes at 4°C, washed with MACS and re-suspended at a density of 100 Million cells per mL. 1 pl of MojoSort anti-PE nanobeads (Biolegend) were added per million cells and incubated on ice for 15 minutes. The cells were washed with MACS and the beads were pulled-down magnetically. The supernatant containing the negatively selected cells was collected.

Cellular co-culture assays 50 000 U87 cells in 100 pl of DMEM were seeded in a 96-well Flat-botom plate and incubated overnight. Peptides were diluted in DMEM to the appropriate concentration, added to each well containing U87 cells and incubated for 60 minutes at 37°C, 10% CO2. The media was discarded and 50 000 T cells were added to each well in 200 pl of RPMI media. Cells were incubated for 24 hours at 37°C, 5% CO2. Supernatants were collected for cytotoxicity and ELISA analysis. 25 pl of 100 mM EDTA PBS were added to each well containing the cells and samples were incubated for 5 minutes at 37°C, 5% CO2. Cells were detached by thoroughly pipetting each well and transferred to a 96-well V-bottom plate. Cells were stained for 20 minutes at 4°C, washed with PBS and analysed by flow cytometry.

Flow Cytometry

All samples were analysed using a BD X-20 flow cytometer or Cytoflex LX Flow cytometer (Beckman Couter). Data was analysed using FlowJo vlO (BD Biosciences) and GraphPad Prism (GraphPad Software).

Table 2: Flow cytometry reagents

ELISAs

Invitrogen Human IFN gamma, IL2 or TNF-alpha Uncoated ELISA kits (Thermo Fisher Scientific) were used following the manufacturer’s protocol to quantify levels of cytokine in diluted T cell supernatant. A SpectraMax M3 microplate reader (Molecular Devices) was used to measure absorbance at 450 nm and 570 nm.

Cytotoxicity assay A Coelenterazine (CTZ) 2 mM stock solution was prepared in methanol, aliquoted and stored at -80°C. Supernatant from co-culture assays was mixed in a 1:1 ratio with PBS 10 pM CTZ and luminescence was read using a SpectraMax M3 microplate reader (Molecular Devices).

Measurements of c259 TCR/pMHC binding affinities and Surface Plasmon Resonance

The 3D binding affinities of the 8 peptide panel to the c259 TCR were determined by SPR at 37°C using an equilibrium binding assay. The summary mean equilibrium binding affinities are reported on Figure 4.

All SPR experiments were carried out in the SPR facility at the Sir William Dunn School of Pathology, Oxford, UK following the methods published on Pettmann et al. (2). Briefly, c259 TCR/pMHC steady-state binding affinities were measured on a Bioacore T200 (GE Healthcare) with a CAP chip using HBS-EP as running buffer. The CAP chip was saturated with streptavidin and biotinylated pMHCs were immobilised to the desired level. A titration of the TCR was flowed through at 37°C. CD58 was immobilised on a reference flow cell at matching levels to those of pMHCs on the remaining flow cells. The signal from the reference flow cell was subtracted (Single referencing) and the average signal from the closest buffer injection was subtracted (Double referencing). Steady-state binding affinity was calculated by fitting the one site-specific binding model on GraphPad Prism to double-referenced equilibrium RU values. The Bmax was constrained to the inferred Bmax from the empirical standard curve, relating maximal antibody binding to maximal TCR binding.

Example 7 - Knock-out of CD8 alpha abolishes a3a cross-reactivity to Titin

The a3a TCR is a well-studied receptor whose cognate peptide is a MAGE -A3 derived peptide (EVDPIGHLY, SEQ ID NO: 30). a3a recognises MAGE -A3 presented on HLA-A1, an MHC-I molecule. During clinical trials, when patients were administered T cells engineered with the a3a TCR they caused a fatal autoimmune toxicity against cardiac tissue (3). A subsequent study identified that the a3a TCR can recognise a peptide from the muscle protein Titin (ESDPTVAQY, SEQ ID NO: 31), which is the most likely cause of the fatal autoimmune reaction (4). Several studies have identified that the affinity of the a3a TCR against the off-target Titin peptide is lower than against its target MAGE-A3 peptide (4, 11).

Example 1 demonstrated that CD8 alpha knock-out can increase the ligand discrimination of T cells engineered with the c259 TCR. This example demonstrates that CD8 alpha knock-out can abolish the a3a TCR engineered T cells cross-reactivity to the lower affinity Titin peptide, whilst maintaining activation against its higher affinity target MAGE-A3 peptide.

Results

The CD8 co-receptor is a heterodimer formed by an alpha and a beta chain. CD8a binds to MHC-I molecules at invariant domains (12), which stabilises the TCR-pMHC interaction. Additionally, the intracellular tail of CD8a binds Lek via a zinc clasp and recruits it to the proximity of the TCR, which improves signal transduction (13). Without wishing to be bound by theory, it was hypothesised that activation by low affinity peptides, which have a stronger co-receptor dependence (14), would be preferentially disrupted if co-receptor function was perturbed. CD8a is required for CD80 expression on the cell surface (15). Therefore, it was predicted that knocking out CD8a by CRISPR/Cas9 would prevent formation of the CD8 co-receptor and generate T cells with enhanced ligand discriminatory powers. To test this prediction, a CD8a-negative T cell population was generated (Figure 21).

Human primary CD8+ T cell blasts were transduced with the a3a TCR and costimulated with T2 cells pulsed with a titration of either the MAGE -A3 or titin peptides. T cell activation was measured by different methods: (I) Flow cytometry analysis of cell surface expression of activation markers, such as 4 IBB; (II) ELISA analysis of secretion of cytokines, such as IL-2; and (III) Measuring the cytotoxic activity of T cells with a luciferase-based assay.

A protocol for the electroporation of human primary T cells with Cas9:sgRNA ribonucleoproteins (RNPs) and transduction with the a3a TCR was optimised. Knock-out efficiency of 95% was obtained. Subsequently, T cells were stained with an Anti-CD8a PE-conjugated antibody and the remaining CD8a positive cells were depleted by pull- down with magnetic Anti-PE beads. A pure population of CD8a negative cells was obtained (Figure 21).

T cells were co-cultured with T2 cells pulsed with a titration of the MAGE- A3 or titin peptides. Using surface 4 IBB expression as a measure of T cell activation, no significant difference on T cell potency against the high-affinity MAGE -A3 peptide was observed. No activation was detected against titin in the CD8 alpha KO T cells (Figure 22).

Similar results were observed when measuring T cell cytotoxic activity (Figure 23) and cytokine secretion (Figures 24-26).

Therefore, this example demonstrates that knocking out CD8a abolishes the activation of a3a TCR engineered T cells against the cross-reactive lower-affinity peptide titin, whilst maintaining a potent response to the higher affinity MAGE -A3 peptide.

Example 8 - Materials and Methods

This Example provides the materials and methods used in Example 7.

Cell culture

T2 cells were cultured at 37°C and 5% CO2 in RPMI 1640 (Sigma- Aldrich) supplemented with 10% FBS, 50 pg/mL Streptomycin and 50 units/mL Penicillin.

Primary human T cells were isolated from leukocyte cones and cultured at 37°C and 5% CO2 in RPMI 1640 (Sigma- Aldrich) supplemented with 10% FBS, 50 pg/mL Streptomycin, 50 units/mL Penicillin and 50 U/mL IL2. Every other day, cells were resuspended in fresh media at a density of 1 Million cells per mL.

Lentivirus production

HEK 293T cells were seeded in a 6-well plate (Day 1) and incubated overnight to achieve approximately 80% confluency. Cells in each well were co-transfected (Day 2) using X-tremeGENE™ HP (Roche) with 0.8 pg of the appropriate lentiviral transfer plasmid encoding an antigen receptor (a3a TCR) and the lentiviral packaging plasmids: pRSV-Rev (0.25 pg), pMDLg/pRRE (0.53 pg), and pVSV-G (0.35 pg). The media was replaced 18 hours following transfection (Day 3). 24 hours after the media exchange, the supernatant from one well was harvested, filtered and used for the transduction of 1 Million human T cells (Day 4).

T cell blasts

The sample was centrifuged at 1200 g for 30 minutes (brake off). Cells at the interface of the Ficoll® media and plasma were collected (Buffy coat) and washed twice (Centrifuged at 500 g for 5 minutes). Cells were resuspended in complete RPMI media supplemented with IL2 (50 U/mL) at a density of 1 Million cells per mL. Dynabeads® Human T-Activator CD3/CD28 (Thermofisher) were added (1 Million beads per mL) and cells were incubated overnight.

1 Million cells were transduced with the filtered lentiviral supernatant (Day 4). On Day 6 and on Day 8, 1 mL of media was removed and replaced with 1 mL of fresh medium. On Day 9, Dynabeads® were removed using a magnetic stand (6 days following isolation). Cells were resuspended in fresh media every other day at a density of 1 Million per mL and used for co-culture experiments. 17 days following isolation T cells were discarded.

CRISPR/Cas9 knock-out of T cell proteins

Negative selection of T cell knock-out cells

Cellular co-culture assays

100 000 T2 cells in 100 pl of DMEM were seeded in a 96-well Flat-bottom plate. Peptides were diluted in RPMI to the appropriate concentration, added to each well containing T2 cells and incubated for 120 minutes at 37°C, 5% CO2. The T2 cells were centrifuged at 500g for 5 minutes. The supernatant was discarded and 50 000 T cells were added to each well in 200 pl of RPMI media. Cells were incubated for 20 hours at 37°C, 5% CO2. Supernatants were collected for cytotoxicity and ELISA analysis. Cells were detached by thoroughly pipetting each well and transferred to a 96-well V-bottom plate. Cells were stained for 20 minutes at 4°C, washed with PBS and analysed by flow cytometry.

Flow Cytometry

Table 3: Flow cytometry reagents

ELISAs

Cytotoxicity assay

A Coelenterazine (CTZ) 2 mM stock solution was prepared in methanol, aliquoted and stored at -80°C. Supernatant from co-culture assays was mixed in a 1:1 ratio with PBS 10 pM CTZ and luminescence was read using a SpectraMax M3 microplate reader (Molecular Devices).

Example 9 - Overexpression of CD4 enhances the ligand discrimination of T cells engineered with an MHC-I restricted TCR

Canonically, cytotoxic CD8 positive T cells recognise peptides presented by MHC- I molecules whilst helper CD4 positive T cells recognise peptides presented by MHC-II molecules. By contrast, in adoptive T cell transfer therapy, both CD8 cytotoxic cells and CD4 helper cells are engineered with the same TCR (which recognises peptides presented via either a MHC-I molecule or a MHC-II molecule).

The CD8 co-receptor is a heterodimer formed by an alpha and a beta chain, whilst the CD4 co-receptor is formed by a single chain. CD8a binds to MHC-I molecules at invariant domains (12), whilst CD4 binds to MHC-II molecules at invariant domains. Additionally, the intracellular tails of CD8a and CD4 bind Lek via a zinc clasp and recruit it to the proximity of the TCR, which improves signal transduction (13). Without wishing to be bound by theory, it is hypothesised that activation by low affinity peptides is preferentially disrupted if co-receptor function is perturbed. Thus, it was predicted that, by overexpressing CD4 in T cells which recognise peptide-MHC-I targets (for example the c259 TCR and the a3a TCR), CD4 would compete against CD8 for Lek and thereby improve ligand discrimination. In other words, it was predicted that CD4 overexpression would increase the discrimination of any T cell that expresses a TCR recognising a peptide-MHC-I target.

To test this prediction, a cytotoxic CD4-overexpressing T cell population was generated (Figure 27).

A dual lentiviral vector encoding both the c259 TCR and CD4 was constructed. Human primary CD8+ T cells transduced with the dual vector expressed both the c259 TCR and CD4 proteins, as confirmed by flow cytometry (Figure 28).

To quantify the ligand discrimination of T cells overexpressing CD4, said transduced T cells were co-cultured with U87 cells pulsed with a titration of the 8 peptide panel. pMHC potency (Pl 5) was defined as the concentration of peptide that elicits 15% of the maximal response obtained. Using surface 4 IBB expression as a measure of T cell activation, no difference on T cell potency against the high-affinity 9V peptide was observed. Potency to medium and lower affinity peptides was reduced (Figure 29). Therefore, these results suggest that CD4 overexpression in T cells transduced with the c259 TCR can lead to an increase in ligand discrimination without loss of on-target potency. The fold-change in P15 between CD4 overexpressing cells and wild-type T cells significantly increased as ligand affinity was reduced (Figure 29B). Therefore, CD4 overexpressing cells have higher ligand discrimination than wild-type T cells.

Example 10 - Combining CD8 alpha knock-out with CD4 overexpression leads to an additive enhancement of T cell ligand discrimination

A population of CD8 alpha knock-out human primary T cells was generated as described in Example 1. The CD8 alpha knock-out T cells were transduced with the dual c259-CD4 lentiviral construct. The c259 TCR recognises peptides presented via pMHC-I.

To quantify the ligand discrimination of CD8 KO T cells overexpressing CD4, said transduced T cells were co-cultured with U87 cells pulsed with a titration of the 8 peptide panel. pMHC potency (Pl 5) was defined as the concentration of peptide that elicits 15% of the maximal response obtained. The fold-change in P15 between CD8 alpha negative CD4 overexpressing cells and wild-type T cells significantly increased as ligand affinity was reduced (Figure 30). Therefore, CD8 alpha negative cells CD4 have higher ligand discrimination than wild-type T cells. Additionally, the increase in discrimination observed was higher than with CD8 KO cells or CD4 overexpressing cells alone, demonstrating an additive effect (Figure 30).

Example 10 therefore demonstrates that CD8+ CD4+ c259 T cells have enhanced ligand discrimination compared to wild-type CD8+ c259 T cells (Figure 29). Furthermore, it was shown that the increase of discrimination provided by CD4 is additive to the previously observed increase of discrimination provided by CD8 alpha knock-out. Therefore, CD8 alpha KO CD4 overexpressing T cells demonstrated the highest increase in discrimination (Figure 30).

Example 11 - Materials and Methods

This Example provides the materials and methods used in Examples 9 and 10.

Cell culture

U87 and HEK 293T cell lines were cultured at 37°C and 10% CO2 in DMEM D6429 media (Sigma- Aldrich) supplemented with 10% FBS, 50 pg/mL Streptomycin and 50 units/mL Penicillin.

Lentivirus production

T cell blasts

CRISPR/Cas9 knock-out of T cell proteins

Cas9 ribonucleoproteins (RNPs) were prepared by mixing 8.5 pg of TruCut Cas9 protein v2 (Thermofisher) with 150 pmol of sgRNA mix (Truguide synthetic gma, Thermofisher) and Opti-MEM (Gibco) to a final volume of 5 pl. The RNPs were incubated for 15 minutes at room temperature. 1 Million freshly isolated T cells were washed 3 times with Opti-MEM (Gibco) and re-suspended at a density of 20 Million per mL. The T cells were mixed with the RNPs and transferred into a BTX Cuvette Plus electroporation cuvette (2mm gap, Harvard Bioscience). The cells were electroporated using a BTX ECM 830 Square Wave Electroporation System (Harvard Bioscience) at 300 V, 2 ms. Immediately following electroporation, the cells were transferred to complete RPMI media supplemented with IL2 and Dynabeads® Human T-Activator CD3/CD28 (Thermofisher) were added.

Negative selection of T cell knock-out cells

Cellular co-culture assays

50 000 U87 cells in 100 pl of DMEM were seeded in a 96-well Flat-bottom plate and incubated overnight. Peptides were diluted in DMEM to the appropriate concentration, added to each well containing U87 cells and incubated for 60 minutes at 37°C, 10% CO2. The media was discarded and 50 000 T cells were added to each well in 200 pl of RPMI media. Cells were incubated for 24 hours at 37°C, 5% CO2. Supernatants were collected for cytotoxicity and ELISA analysis. 25 pl of 100 mM EDTA PBS were added to each well containing the cells and samples were incubated for 5 minutes at 37°C, 5% CO2. Cells were detached by thoroughly pipetting each well and transferred to a 96-well V-bottom plate. Cells were stained for 20 minutes at 4°C, washed with PBS and analysed by flow cytometry.

Flow Cytometry All samples were analysed using a BD X-20 flow cytometer or Cytoflex LX Flow cytometer (Beckman Couter). Data was analysed using FlowJo vlO (BD Biosciences) and GraphPad Prism (GraphPad Software). Flow cytometry reagents were as presented in Table 4.

Table 4: Flow cytometry reagents

ELISAs

Cytotoxicity assay

Surface Plasmon Resonance

All SPR experiments were carried out in the Dunn School SPR facility following the methods published in Pettmann et al. (2). Briefly, c259 TCR/pMHC steady-state binding affinities were measured on a Bioacore T200 (GE Healthcare) with a CAP chip using HBS-EP as running buffer. The CAP chip was saturated with streptavidin and biotinylated pMHCs were immobilised to the desired level. A titration of the TCR was flowed through at 37°C. CD58 was immobilised on a reference flow cell at matching levels to those of pMHCs on the remaining flow cells. The signal from the reference flow cell was subtracted (Single referencing) and the average signal from the closest buffer injection was subtracted (Double referencing). Steady-state binding affinity was calculated by fitting the one site-specific binding model on GraphPad Prism to double-referenced equilibrium RU values. The Bmax was constrained to the inferred Bmax from the empirical standard curve, relating maximal antibody binding to maximal TCR binding.

References

1 Wooldridge et al., 2012, J Biol Chem., 287(2): 1168-77.

2 Pettmann, et al., 2021, eLife 10:e67092.

3 Linette et al., 2013, Blood, 122 (6):863-71.

4 Cameron et al., 2013, Science translational medicine, 5(197): 197ral03.

5 McKeithan, 1995, Proc Natl Acad Sci USA, 92(11):5042-6.

6 https://www.cellsignal.co.uk/pathways/t-cell-receptor-signaling.

7 Lever et al., 2014, Nat Rev Immunol, 14(9):619-29.

8 Moore et al., 2010, Methods Mol Biol., 629: 141-158.

9 Bethune et al., 2018, Proc Natl Acad Sci USA, 115(45):E10702-E10711

10 Linette, G.P. et al., 2013, Blood, 122(6), 863-871.

11 Zhao, X. et al., 2022, Science, 376(6589).

12 Gangadharan, D. and Cheroutre, H. (2004), Current Opinion in Immunology, 16(3), 264-270

13 Horkova, V., et al., 2020. Cell Reports, 30(5), pp.1504-1514. el.

14 Laugel et al., 2007, Journal Of Biological Chemistry, 282(33), 23799-23810.

15 Norment and Littman, 1988, The EMBO Journal, 7(11), 3433-3439.

Sequence listing

Claims

1. A modified T cell comprising a TCR of interest, characterised in that the function of a factor associated with T cell receptor (TCR) kinetic proofreading is modulated relative to an unmodified T cell comprising the TCR of interest.

2. The modified T cell of claim 1, wherein the TCR of interest is a pMHC-I-restricted TCR.

3. The modified T cell of claim 1 or 2, wherein the factor is CD4, CD8 or Lek.

4. The modified T cell of any one of the preceding claims, wherein the amount and/or activity of the factor is reduced relative to an unmodified T cell comprising the TCR of interest.

5. The modified T cell of any one of the preceding claims, wherein the factor is a factor which progresses the T cell towards the TCR activated state.

6. The modified T cell of any one of the preceding claims, wherein the amount and/or activity of the factor is at least partially reduced.

7. The modified T cell of claim 6, wherein the factor is CD8 or Lek.

8. The modified T cell of any one of the preceding claims, wherein the modified T cell further comprises an inhibitor for inhibiting the amount and/or activity of the factor.

9. The modified T cell of claim 8, wherein the inhibitor is a shRNA, optionally wherein the factor is Lek.

10. The modified T cell of any one of the preceding claims, wherein the amount and/or activity of the factor is completely reduced.

11. The modified T cell of claim 11 , wherein the factor is CD8, optionally CD8a.

12. The modified T cell of claim 12, wherein the modified T cell does not express a functional CD8a polypeptide, e.g. the modified T cell is a CD8a knock out.

13. The modified T cell of any one of claims 1 to 3, wherein the amount and/or activity of the factor is increased relative to an unmodified T cell comprising the TCR of interest, optionally wherein the factor is a factor which progresses the T cell towards the TCR resting state.

14. The modified T cell of claim 14, wherein the factor is CD4.

15. The modified T cell of claim 15, wherein the amount of a CD4 polypeptide is increased.

16. The modified T cell of any one of the preceding claims, wherein the modified T cell exhibits enhanced discrimination for its target antigen relative to an unmodified T cell which binds the same antigen.

17. The modified T cell of any one of the preceding claims, wherein the function of a further factor associated with T cell receptor (TCR) kinetic proofreading is modulated, optionally wherein the further factor is CD4, CD8, or Lek.

18. The modified T cell of any one of the preceding claims, wherein the amount of a CD4 polypeptide is increased and the modified T cell is a CD8a knock out.

19. The modified T cell of any one of the preceding claims, wherein the TCR of interest is a3a or c259.

21. The inhibitor of claim 19, comprising (a) any of SEQ ID NOs: 5 to 8 and 21 to 28, and/or (b) a polynucleotide sequence complementary to SEQ ID NOs: 5 to 8 and 21 to 28.

23. The sgRNA of claim 22 for knocking out CD8a, wherein the sgRNA comprises SEQ ID NO: 1, 2, 3 or 4.

24. A vector comprising the shRNA molecule of claim 20 or 21 or one or more sgRNAs of claim 22 or 23, optionally also comprising a polynucleotide encoding CD4.

25. The vector of claim 24, wherein the vector further comprises a polynucleotide encoding a TCR of interest, optionally c259 or a3a.

26. A pharmaceutical composition comprising the inhibitor of claim 20 or 21, one or more sgRNAs of claim 22 or 23, or the vector of claim 24 or 25.

27. A method of preparing a modified T cell which comprises a TCR of interest, comprising, in a T cell, modulating the function of a factor associated with TCR kinetic proofreading.

28. The method of claim 27, wherein the TCR of interest is a pMHC-I-restricted TCR.

29. The method of claim 27 or 28, wherein the T cell comprises the TCR of interest or wherein the method further comprises introducing the TCR of interest into the T cell.

30. The method of any one of claims 27 to 29, wherein the factor is CD4, CD8, or Lek.

31. The method of any one of claims 27 to 30, wherein modulating the function of the factor comprises reducing the amount and/or activity of the factor.

32. The method of claim 31 , wherein the factor is a factor which progresses the T cell towards the TCR activated state.

33. The method of claim 31 or 32, wherein reducing the amount and/or activity of the factor comprises at least partially reducing the factor, optionally wherein the factor is Lek or CD8.

34. The method of claim 33, wherein reducing the amount of the factor comprises introducing or expressing an inhibitor in the T cell.

35. The method of claim 34, wherein the inhibitor is as defined in claim 20 or 21, or is a small molecule inhibitor.

36. The method of any one of claims 31 to 33, wherein reducing the amount and/or activity of the factor comprises knocking out the factor, optionally wherein the factor is CD8, optionally CD8a.

37. The method of claim 36, wherein the method comprises knocking out CD8a.

38. The method of any one of claims 31 to 33 and 36 to 37, wherein reducing the amount and/or activity of the factor comprises introducing one or more sgRNAs targeted to the gene encoding the factor into the T cell, optionally further comprising introducing a Cas9 protein or a polynucleotide encoding a Cas9 protein into the T cell.

39. The method of claim 38, wherein the method also comprises transducing the TCR of interest into the T cell.

40. The method of any one of claims 27 to 30, wherein modulating the function of the factor comprises increasing the amount and/or activity of the factor.

41. The method of claim 40, wherein the factor progresses the T cell towards the TCR resting state.

42. The method of claim 41, wherein the method comprises overexpressing the factor, optionally wherein the factor is CD4.

43. The method of any one of claims 27 to 33 and 36 to 42, wherein the method comprises increasing the amount of a CD4 polypeptide in the T cell.

44. The method of any one of claims 27 to 33 and 36 to 43, wherein the method comprises transducing CD4 into the T cell, optionally wherein the method further comprises transducing the TCR of interest into the T cell.

45. The method of any one of claims 27 to 33 and 36 to 44, wherein the method comprises knocking out CD8a and increasing the amount of a CD4 polypeptide in the T cell.

46. The method of any one of claims 27 to 45, wherein the method enhances target antigen discrimination of the modified T cell.

47. The method of any one of claims 27 to 46, wherein the TCR of interest is a3a or c259.

48. A modified T cell obtainable or obtained by any of the method according to claims 27 to 47.

49. A method of enhancing target antigen discrimination of a T cell, comprising preparing a modified T cell according to the method of any of claims 27 to 47.

50. A method of identifying a modified T cell comprising a TCR of interest which has enhanced discrimination for its target antigen, wherein the method comprises (i) preparing a modified T cell according to the method of any one of claims 26 to 45; and (ii) screening the modified T cell for reduced sensitivity for one or more low affinity ligand(s) relative to an unmodified T cell comprising the TCR of interest, optionally wherein the TCR of interest is a pMHC-I-restricted TCR.

51. A method of preparing a population of modified T cells for adoptive cell therapy, the method comprising culturing the modified T cell of any one of claims 1 to 19 and 48.

52. A population of modified T cells produced by the method of claim 51.

53. A method of treating cancer, an infection or an inflammatory disease comprising administering a modified T cell according to any one of claims 1 to 19, a population of T cells according to claim 50, an inhibitor according to claim 20 or 21, a sgRNA according to claim 22 or 23, a vector according to claim 24 or 25, or a pharmaceutical composition according to claim 26 to a patient in need thereof, optionally wherein the infection is a chronic infection or the inflammatory disease is an autoimmune disease.

54. A modified T cell according to any one of claims 1 to 19, a population of T cells according to claim 50, an inhibitor according to claim 20 or 21, a sgRNA according to claim 22 or 23, a vector according to claim 24 or 25, or a pharmaceutical composition according to claim 26 for use as a medicament.

55. A modified T cell according to any one of claims 1 to 19, a population of T cells according to claim 50, an inhibitor according to claim 20 or 21, a sgRNA according to claim 22 or 23, a vector according to claim 24 or 25, or a pharmaceutical composition according to claim 26 for use in a method of treating cancer, an infection or an inflammatory disease, optionally wherein the infection is a chronic infection or the inflammatory disease is an autoimmune disease.