Modified t cells

Abstract

The present invention relates to enhancing target pMHC differentiation of T cells by modulating the expression and / or activity of factors related to T cell receptor (TCR) kinetics correction.

Description

Technical Field

[0001] The present invention relates to modified T cells, methods for preparing modified T cells, and uses thereof. Background Art

[0002] T cells play a key role in adaptive immune responses by distinguishing healthy cells from abnormal cells through their T cell receptors (TCRs). For example, TCRs can recognize virus- or tumor-specific peptides presented on major histocompatibility complex class I or class II (MHC-I or MHC-II) molecules on the surface of infected or cancerous cells. After antigen recognition, T cells are activated and produce an immune response.

[0003] T cells must be able to recognize any foreign peptide they encounter, but there are physical limits on the number of T cells any organism can possess. Therefore, cross-reactivity is a fundamental characteristic of TCRs. It has been reported that each T cell recognizes approximately 10 6 peptides to achieve complete immune coverage (1).

[0004] Antigen discrimination can be quantified by analyzing the potency of different affinity ligands for the antigen. High discrimination allows small changes in ligand affinity to be amplified into larger changes in potency or into larger changes in the concentration of ligand required to elicit a response (2). Reference 2 reported that TCRs have an enhanced ability to discriminate between cognate ligands compared to conventional surface receptors such as GPCRs.

[0005] Many T cell therapies rely on introducing a heterologous TCR or modified autologous TCR into the patient to exploit novel high-affinity binding interactions between the TCR and pathological peptides presented through the major histocompatibility complex. For example, in adoptive T cell transfer therapy, T cells are engineered ex vivo to express TCRs that target specific target tumor antigens and then infused back into the patient. These TCRs are optimized, validated, and screened in vitro for positive target binding and negative off-target binding. Testing in animal models is rare because TCRs are designed with human immunocompatibility in mind, so the first in vivo administration of a therapeutic TCR or therapeutic T cells bearing said TCR is usually in human clinical trials.

[0006] 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 low affinity, recent studies have shown that affinity-based TCR antigen discrimination is imperfect: primary human T cells can respond to pMHC with a KD as low as approximately 1 mM (2). Therefore, although the affinity between the introduced TCR and off-target self-antigen is expected to be low, it may be functionally significant in vivo and may lead to serious or even fatal side effects. For example, deaths in the clinical trial of the introduced A3ATCR (whose on-target antigen was MAGE-A3) were attributed to off-target binding of the A3ATCR to the self-antigen titin (3,4), which was not identified in extensive in vitro safety screening due to a combination of low affinity binding and lack of surface expression on cultured cells.

[0007] There are strategies to minimize the risk of autoantigen interactions, but these strategies have limitations. In vitro screening for low-affinity off-target binding is labor-intensive and expensive, and does not fully cover tissue-specific, differentiation-specific, and patient-specific self-peptide libraries. Strategies based on computational prediction and artificial intelligence (AI) can be employed, but are highly complicated by the small and variable number of residue contact points between the TCR (at the CDR3α and CDR3β loops) and the antigen. In addition, existing strategies focus on modifying the TCR itself, while ignoring the role of intracellular signal transduction cascades in ligand discrimination.

[0008] It is an object of the present invention to provide further or improved T cells with enhanced discrimination for high affinity on-target antigens relative to lower affinity off-target antigens.

[0009] It is a further object of the present invention to provide further or improved T cells with minimal risk of cross-reactivity in adoptive T cell transfer therapy. Summary of the Invention

[0010] The inventors have discovered a method to alter the ability of T cells to distinguish between their high-affinity on-target antigens and low-affinity off-target antigens without losing sensitivity to high-affinity targets. This is achieved by exploiting membrane-proximal intracellular events that initiate TCR signaling and T cell activation.

[0011] Specifically, the inventors altered the discriminatory capacity of T cells by modulating the kinetic proofreading (KP) mechanism in T cells. The KP mechanism is a series of reversible biochemical steps that begin at the TCR after binding to a cognate MHC-presenting peptide (pMHC). Only when the final biochemical step is completed can the TCR be activated (see Figure 2 ). The multi-step progression towards the TCR activated state ensures a time delay between TCR-pMHC binding and TCR signal transduction (5). For TCR signal transduction to be triggered, pMHC must remain bound to the TCR throughout the multi-step process. If pMHC dissociates at any step, TCR signal transduction will not occur and the TCR will reset to the TCR resting state, allowing the KP progression to start again after reengagement of the TCR. Therefore, higher affinity ligands and / or higher local concentrations of ligands are more likely to cause TCR signal transduction. Similarly, the dissociation rate (k) of the TCR-pMHC interaction is off ) are magnified into larger differences in T cell activation.

[0012] The inventors have found that reducing the amount and / or activity of factors that promote progression through the KP mechanism (i.e., factors that cause T cells to progress to a TCR-activated state) enhances ligand discrimination. Conversely, increasing the amount and / or activity of factors that resist propagation through the KP mechanism (i.e., factors that cause T cells to progress to a TCR-resting state) also enhances ligand discrimination.

[0013] For example, Examples 1, 7 and 4 show that, compared to wild-type T cells, modified T cells with reduced CD8 and Lck expression and / or activity, respectively, provide enhanced ligand discrimination. Interestingly, compared to wild-type T cells, T cells modified to reduce the expression and / or activity of factors 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 (see Examples 2 and 3). Surprisingly, as shown in Examples 1 and 4, modified T cells exhibit enhanced T cell discrimination to their target antigens without losing sensitivity to the target antigen, i.e., without any change in target efficacy.

[0014] Surprisingly, Example 9 shows that overexpression of CD4 in pMHC-I-restricted T cells provides enhanced ligand discrimination compared to wild-type T cells. This further confirms the role of regulatory KP pathway mechanisms in enhanced discrimination, as CD4 expression competes with CD8 for binding to Lck. Overexpression of CD4 sequesters Lck from CD8, effectively reducing the amount of Lck. Thus, overexpression of CD4 allows pMHC-I-restricted T cells to progress toward a TCR-quiescent state.

[0015] Example 10 shows that discrimination can be further enhanced by combining the modulation described herein. Knockout of CD8 and overexpression of CD4 in pMHC-I restricted T cells resulted in enhanced discrimination.

[0016] Therefore, the present invention provides a modified T cell, characterized in that the function of a factor associated with the correction of T cell receptor (TCR) dynamics is modulated.

[0017] The present invention also provides an inhibitor of a factor that is associated with the correction of TCR dynamics in T cells and causes T cells to progress toward a TCR activated state, optionally wherein the inhibitor is shRNA.

[0018] The present invention also provides an sgRNA for knocking out a factor, wherein the factor is associated with TCR dynamics correction in T cells and causes T cells to progress toward a TCR activation state, optionally wherein the sgRNA comprises one or more sequences of 5 to 35 consecutive nucleotides comprising a gene encoding the factor.

[0019] The present invention also provides a vector comprising the shRNA molecule described herein or one or more sgRNAs described herein.

[0020] The present invention also provides a pharmaceutical composition comprising the inhibitor described herein, one or more sgRNAs described herein, or the vector described herein.

[0021] The present invention also provides a method for preparing modified T cells, comprising regulating the function of factors associated with TCR kinetic correction in T cells.

[0022] The present invention also provides modified T cells obtainable or obtained by any of the methods described herein.

[0023] The present invention also provides a method for enhancing target pMHC discrimination of a T cell, comprising preparing a modified T cell according to any of the methods described herein.

[0024] The present invention also provides a method for preparing a modified T cell population for adoptive cell therapy, the method comprising culturing the modified T cells described herein.

[0025] The present invention also provides modified T cell populations produced by any of the methods described herein.

[0026] The present invention also provides a method for treating cancer, infection or inflammatory disease, comprising administering to a patient in need thereof the modified T cells described herein, the T cell population described herein, the inhibitor described herein, the sgRNA described herein, the vector described herein or the pharmaceutical composition described herein, optionally wherein the infection is a chronic infection or the inflammatory disease is an autoimmune disease.

[0027] The present invention also provides the modified T cells described herein, the T cell population described herein, the inhibitor described herein, the sgRNA described herein, the vector described herein, or the pharmaceutical composition described herein, for use as a drug.

[0028] The present invention also provides a modified T cell described herein, a T cell population described herein, an inhibitor described herein, an sgRNA described herein, a vector described herein, or a pharmaceutical composition described herein for use in a method for treating cancer, infection, or inflammatory disease, optionally wherein the infection is a chronic infection, or the inflammatory disease is an autoimmune disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the TCR signal transduction pathway (6). T cell receptor (TCR) activation initiates numerous signal transduction cascades that ultimately determine cellular responses by regulating cytokine production, cell survival, proliferation, differentiation, and target cell killing. Figure 1 Examples of factors involved in these signal transduction pathways are provided.

[0030] Figure 2 Schematic diagram of the kinetic correction mechanism (7). A free pMHC complex (denoted as "P") can bind to a free T cell receptor (TCR; denoted as "T") to form a TCR-pMHC complex, which can undergo a series of reactions with a rate of k p The "N" biochemical modification of the TCR is shown in Figure 2 (represented as complexes C0, C1, C2, and CN). "CN" is the TCR state with signal transduction ability, also referred to as the TCR activation state in this article. on = binding rate; k off = dissociation rate.

[0031] Figure 3 CD8α-negative primary human T cells were generated. CD8α was knocked out in primary human CD8+ T cells by electroporation with Cas9 protein and synthetic guide RNA. T cells were stained with anti-CD8α (BV-421) antibody, and CD8α surface expression was measured by flow cytometry.

[0032] Figure 4c259 TCR / pMHC affinity was measured using SPR at 37°C. Steady-state binding affinity for the selected 8-peptide panel. Bars represent mean K with SD. D .

[0033] Figure 5 CD8α-negative primary human T cells exhibit enhanced antigen discrimination compared to wild-type primary human T cells. CD8α was knocked out in primary human CD8+ T cells by electroporation of Cas9 protein and synthetic guide RNA. T cells were subsequently transduced with the c259 TCR via lentivirus. Wild-type T cells (circles) and CD8α-negative T cells (squares) were stimulated with U87 target cells loaded with the indicated concentrations of antigen (x-axis), and surface 41BB expression (y-axis) was measured by flow cytometry 20 hours later. Representative data are from 1 of 3 experiments. The affinity of each peptide for the c259 TCR is shown.

[0034] Figure 6 Compared to wild-type cells, CD8α-negative primary human T cells require higher concentrations of lower-affinity antigens rather than higher-affinity antigens to induce T cell activation. (A) The antigen concentration required to activate 15% of T cells (P15) was plotted against the TCR / pMHC affinity ( Figure 4 ) plot. P15 values ​​are calculated by a dose-response curve of T cell activation determined in the following manner: (41BB) is measured by flow cytometry for 41BB surface expression, (Nluc) for U87 target cell killing, and (IL2) for secretion of IL2 in the supernatant. (B) The fold change of P15 between CD8α negative cells and wild-type cells from Figure A is plotted relative to TCR / pMHC affinity. F-test for non-zero slopes. Each point represents mean P15, and error bars represent SD.

[0035] Figure 7 Lck knockdown in primary human T cells by shRNA. Human primary T cells were lentivirally transduced with vectors encoding the c259 TCR and shRNA molecules. Cells transduced with scrambled control shRNA sequences or anti-Lck shRNA sequences are shown in squares. T cells were intracellularly stained with anti-Lck (AF647) antibody, and Lck intracellular expression was measured by flow cytometry.

[0036] Figure 8.Lck knockdown primary human T cells exhibit enhanced antigen discrimination compared to wild-type primary human T cells. Human primary T cells were transduced with vectors encoding c259 TCR and shRNA molecules via lentivirus. Cells transduced with scramble control shRNA sequences are shown as circles, and cells transduced with anti-Lck shRNA sequences are shown as squares. T cells were stimulated with U87 target cells loaded with the indicated concentrations of antigen (x-axis), and target cell killing was measured after 20 hours (y-axis). Representative data are from 1 of 3 experiments. The affinity of each peptide for the c259 TCR is shown.

[0037] Figure 9 Compared to wild-type cells, Lck knockdown primary human T cells require higher concentrations of lower affinity antigens rather than higher affinity antigens to induce T cell activation. (A) The antigen concentration required to activate 15% of T cells (P15) was plotted against the TCR / pMHC affinity ( Figure 4 ) plot. T cell activation was determined by measuring U87 target cell killing. (B) The fold change of P15 between Lck knockdown cells and wild-type cells from Figure A was plotted relative to TCR / pMHC affinity. F-test for non-zero slope. Each point represents the average P15 from 3 independent biological experiments, and the error bars represent SD.

[0038] Figure 10 .Knockout of T cell factors involved in kinetic correction enhances T cell discrimination. Representative dose-response curves comparing activation of unmodified T cells (solid line) or knockout T cells (dashed line) against high-affinity ligands (circles) or low-affinity ligands (squares). T cells were stimulated with U87 target cells loaded with the indicated concentrations of antigen (x-axis), and 41BB surface expression was measured after 4 hours (y-axis). In unmodified cells, the antigen concentration required to activate 15% of T cells (P15) was higher for low-affinity ligands than for high-affinity ligands. KO reduced the P15 of low-affinity ligands while maintaining the same P15 of high-affinity ligands.

[0039] Figure 11 CD2-negative primary human T cells were generated. CD2 was knocked out in primary human CD8+ T cells by electroporation with Cas9 protein and synthetic guide RNA. T cells were stained with anti-CD2 (PE) antibody, and CD2 surface expression was measured by flow cytometry.

[0040] Figure 12CD2 primary human T cells exhibit reduced 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. T cells were subsequently transduced with c259 TCR via lentivirus. Wild-type T cells (circles) and CD2-negative T cells (squares) were stimulated with U87 target cells loaded with the indicated concentrations of antigen (x-axis), and surface 41BB expression (y-axis) was measured by flow cytometry 20 hours later. Representative data are from 1 of 3 experiments. The affinity of each peptide for the c259 TCR is shown.

[0041] Figure 13 CD2-negative primary human T cells require higher concentrations of lower-affinity and higher-affinity antigens to induce T cell activation compared to wild-type cells. (A) The antigen concentration required to activate 15% of T cells (P15) was plotted against the TCR / pMHC affinity ( Figure 4 ) plot. P15 values ​​are calculated by the dose-response curve of T cell activation determined in the following manner: (41BB) is measured by flow cytometry for 41BB surface expression, (Nluc) is measured for U87 target cell killing, (IL2) is measured for IL2 secretion in the supernatant, or (IFNg) is measured for IFN-γ secretion in the supernatant. (B) The fold change of P15 between CD2 negative cells and wild-type cells from Figure A is plotted relative to TCR / pMHC affinity. F-test for non-zero slope. Each point represents mean value P15, and error bars represent SD.

[0042] Figure 14 CD43-negative primary human T cells were generated. CD43 was knocked out in primary human CD8+ T cells by electroporation with Cas9 protein and synthetic guide RNA. T cells were stained with anti-CD43 (PE) antibody, and CD43 surface expression was measured by flow cytometry.

[0043] Figure 15 CD43 primary human T cells showed no differences 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 with Cas9 protein and synthetic guide RNA. T cells were subsequently lentivirally transduced with the c259 TCR. Wild-type T cells (circles) and CD43-negative T cells (squares) were stimulated with U87 target cells loaded with the indicated concentrations of antigen (x-axis), and target cell killing was measured 20 hours later (y-axis). Representative data from 1 of 3 experiments. Affinity of each peptide for the c259 TCR is shown.

[0044] Figure 16Compared to wild-type cells, CD43-negative primary human T cells require equal concentrations of lower-affinity and higher-affinity antigens to induce T cell activation. (A) The antigen concentration required to activate 15% of T cells (P15) was plotted against the TCR / pMHC affinity ( Figure 4 ) plot. P15 values ​​are calculated by the dose-response curve of T cell activation determined in the following manner: (41BB) is measured by flow cytometry for 41BB surface expression, (Nluc) is measured for U87 target cell killing, (IL2) is measured for IL2 secretion in the supernatant, or (IFNg) is measured for IFN-γ secretion in the supernatant. (B) The fold change of P15 between CD43 negative cells and wild-type cells from Figure A is plotted relative to TCR / pMHC affinity. F-test for non-zero slope. Each point represents mean value P15, and error bars represent SD.

[0045] Figure 17 There was no difference in the discriminatory ability of CD43-negative and CD2-negative primary human T cells relative to wild-type primary human T cells.

[0046] Figure 18 Side-by-side comparison of antigen discrimination after knockout of CD8α, CD43, and CD2. The antigen concentration required to activate 15% of T cells (P15) was plotted against the TCR / pMHC affinity measured using SPR ( Figure 4 ) plotting. The fold change of P15 between CD8α negative cells and wild-type cells (triangles), between CD43 negative cells and wild-type cells (circles), and between CD2 negative cells and wild-type cells (squares) is plotted relative to TCR / pMHC affinity. P15 values ​​are calculated by the dose-response curve of T cell activation determined in the following manner: (A) (41BB) measures 41BB surface expression by flow cytometry, (B) (Nluc) measures U87 target cell killing, and (C) (IL2) is by measuring the secretion of IL2 in the supernatant. F test for non-zero slope. Each point represents mean value P15, and error bars represent SD.

[0047] Figure 19 Compared to wild-type primary human T cells (CD69 surface expression), T cells treated with a Lck chemical inhibitor (A-770041) exhibit enhanced antigen discrimination. Human primary T cells were transduced with a vector encoding the c259 TCR via lentivirus and treated with 0 nM of the Lck inhibitor (circle) or 100 nM of the Lck inhibitor (square) for 1 hour. T cells were stimulated with U87 target cells loaded with the specified concentration of antigen (x-axis), and CD69 surface expression (y-axis) was measured 4 hours later. Representative data are from 1 of 3 experiments.

[0048] Figure 20 T cells treated with a chemical inhibitor of Lck (A-770041) responded to higher affinity antigens but were not activated against lower affinity antigens. The antigen concentration required to activate 15% of T cells (P15) was plotted against the TCR / pMHC affinity ( Figure 4 ) drawing. P15 value is calculated by the dose response curve of T cell activation determined in the following manner: (41BB) is measured by flow cytometry 41BB surface expression, (TNFα) is measured by secretion of TNFα in the supernatant. Each point represents an independent biological repeat. The dotted line represents the highest peptide concentration tested (100 μM). Peptides that were not activated at any test concentration are described as P15>100 μM. Statistical significance was determined using paired t-test (*=p<0.05, ***=p<0.01, ***=p<0.001, ***=p<0.0001).

[0049] Figure 21 CD8α-negative primary human T cells were generated. CD8α was knocked out in primary human CD8+ T cells by electroporation with Cas9 protein and synthetic guide RNA. T cells were stained with anti-CD8α (PE) antibody, and CD8α surface expression was measured by flow cytometry.

[0050] Figure 22 .CD8α-negative primary human T cells transduced with a3a TCR respond to MAGE-A3 but do not respond to titin presented on T2 cells (surface 41BB assay). Wild-type T cells (circles) and CD8α-negative T cells (squares) were stimulated by T2 target cells loaded with the specified concentration of antigen (x-axis). (A and B) After 20 hours, surface 41BB expression (y-axis) was measured by flow cytometry. Representative data are from 1 of 3 experiments. (C) Plot of the antigen concentration required to activate 50% of T cells (EC50). Each point represents an independent biological experiment. **=p<0.01.

[0051] Figure 23 CD8α-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 CD8α-negative T cells (squares) were stimulated with T2 target cells loaded with the indicated concentrations of antigen (x-axis). (A and B) Target killing was measured after 20 hours (y-axis). Representative data from one of three experiments. (C) Antigen concentration required to activate 50% of T cells (EC50) is plotted. Each point represents an independent biological experiment. **** = p < 0.0001.

[0052] Figure 24 CD8α-negative primary human T cells transduced with the a3a TCR respond to MAGE-A3 but not titin presented on T2 cells (IFN-γ assay). Wild-type T cells (circles) and CD8α-negative T cells (squares) were stimulated with T2 target cells loaded with the indicated concentrations of antigen (x-axis). (A and B) IFN-γ secretion was measured after 20 hours (y-axis). Representative data from one of three experiments. (C) Antigen concentration required to activate 50% of T cells (EC50) is plotted. Each point represents an independent biological experiment. **** = p < 0.0001.

[0053] Figure 25 CD8α-negative primary human T cells transduced with the a3a TCR respond to MAGE-A3 but not titin presented on T2 cells (TNF-α assay). Wild-type T cells (circles) and CD8α-negative T cells (squares) were stimulated with T2 target cells loaded with the indicated concentrations of antigen (x-axis). (A and B) TNF-α secretion was measured 20 hours later (y-axis). Representative data from 1 of 3 experiments. (C) Antigen concentration required to activate 50% of T cells (EC50) is plotted. Each point represents an independent biological experiment. * = p < 0.05.

[0054] Figure 26 CD8α-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 CD8α-negative T cells (squares) were stimulated with T2 target cells loaded with the indicated concentrations of antigen (x-axis). (A and B) IL-2 secretion was measured 20 hours later (y-axis). Representative data from 1 of 3 experiments. (C) Antigen concentration required to activate 50% of T cells (EC50) is plotted. Each point represents an independent biological experiment. ** = p < 0.01.

[0055] Figure 27 Overview of the genetic engineering process. (A) CD8 is knocked out from cytotoxic wild-type T cells (which normally express CD8, 'CD8+') to generate engineered CD8 KO cytotoxic T cells. (B) CD4 is first overexpressed in cytotoxic wild-type T cells (which normally do not express CD4). CD8 is then knocked out from the engineered CD4+ cytotoxic T cells, resulting in engineered cytotoxic T cells that are CD8 KO CD4+.

[0056] Figure 28Primary human T cells overexpressing CD4 were generated. T cells were transduced with dual c259 and CD4 vectors. T cells were stained with anti-CD4 (PE) antibody, and CD4α surface expression was measured by flow cytometry.

[0057] Figure 29 Compared to wild-type cells, primary human T cells overexpressing CD4 require higher concentrations of lower-affinity antigens rather than higher-affinity antigens to induce T cell activation. (A) The antigen concentration required to activate 15% of T cells (P15) is plotted against TCR / pMHC affinity measured using SPR. P15 values ​​are calculated from a dose-response curve of T cell activation determined by flow cytometry measuring 41BB surface expression. (B) The fold change in P15 between CD4-overexpressing cells and wild-type cells from Figure A is plotted against TCR / pMHC affinity. F-test for non-zero slope. Each point represents the mean P15, and the error bars represent SD. ****=p=0.0001.

[0058] Figure 30 Combining CD8α knockout with CD4 overexpression results in an additive enhancement of T cell ligand discrimination. The fold change in p15 between CD4-overexpressing and wild-type cells, or between CD8 KO CD4-overexpressing and wild-type cells, is plotted against TCR / pMHC affinity. F-test for nonzero slopes. Each point represents the mean p15, with error bars representing the SD. **** = p = 0.0001; ****** = p < 0.0001. DETAILED DESCRIPTION

[0059] Modified T cells and factors

[0060] The present invention relates to modulating the function (eg, amount and / or activity) of factors associated with the correction of T cell receptor (TCR) kinetics. One or more of these factors may be modulated.

[0061] Therefore, the present invention also provides a modified T cell comprising a factor associated with the correction of T cell receptor (TCR) dynamics, wherein the function of the factor is regulated. The modified T cell may comprise one or more such regulatory factors.

[0062] The inventors discovered that modifying T cells to reduce the function of factors that promote progression through the KP mechanism enhances the T cell's discrimination of its cognate pMHC. Without wishing to be bound by theory, in unmodified T cells, the longer the pMHC is bound to the TCR, the greater the extent to which the TCR complex progresses through the KP mechanism, and therefore, the more likely the TCR is to signal through the classic TCR signaling cascade, thereby activating the T cell. In contrast, in the modified T cells of the present invention, the function of factors associated with the KP mechanism is modified, thereby reducing the rate of progression through the KP mechanism. This means that the pMHC must remain bound to the TCR of the T cell for a longer period of time before TCR stimulation. Therefore, the likelihood of off-target pMHC triggering TCR signaling is reduced, and antigen discrimination is enhanced.

[0063] The rate of progression of T cells by the KP mechanism can be reduced in many ways.For example, this can be achieved by reducing the function of the factor that promotes the multi-step progression to TCR activated state.This can also be achieved by increasing the function of the factor that resists (such as resisting, suppressing or reversing) this multi-step progression, and therefore making T cells recover to TCR resting state.The method for reducing or increasing the function of such factors is further described below.For example, if the co-receptor function is disturbed, it is preferentially destroyed by the activation of low-affinity peptides.

[0064] Thus, factors useful in the present invention may be factors that promote T cell progression via the KP mechanism. For example, the factor may help T cells progress from a TCR resting state to a TCR activated state (e.g., Figure 2 This factor may contribute to the phosphorylation of the cytoplasmic chains of the TCR-CD3 subunits (e.g., ITAMs on the cytoplasmic tail of the CD247ζ chain), ZAP70, and / or LAT (see Figure 1 ). The factor can be a kinase, a kinase recruiting factor (recruiter), a scaffold molecule or a co-stimulatory molecule. For example, the factor can be CD8α, CD8β, CD4, Lck, ZAP70 or LAT. The function of such a factor is generally reduced in the modified T cells of the present invention. The function of the factor can be reduced directly (e.g., by regulating the expression and / or activity of the factor) or indirectly (e.g., by regulating the binding partner of the factor so that the expression and / or activity of the factor is regulated, for example, overexpression of the binding partner causes the factor to be sequestered).

[0065] Factors useful in the present invention may be factors that counteract progression through the KP mechanism. For example, factors that can reset the TCR complex to a resting state after pMHC dissociates from the TCR are factors that counteract (or reverse) progression through the KP mechanism. The function of such factors is generally increased in the modified T cells of the present invention.

[0066] The modified T cells of the present invention are T lymphocytes. The T cells may be inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. The T lymphocytes may be CD4+ T lymphocytes. The T lymphocytes may be CD8+ cytotoxic T lymphocytes.

[0067] The modified T cells can be CD4+ T cells or CD8+ T cells.

[0068] The modified T cells may additionally comprise a TCR (also referred to herein as a target TCR) that is specific for the target antigen of interest. The modified T cells may be selected to express the target TCR prior to regulation of the regulated factor, or the target TCR may be transduced into the T cells before or after regulation of the regulated factor.

[0069] Preferably, the modified T cells are suitable for adoptive T cell transfer therapy (ACT). The modified T cells retain the suitability of the T cells for ACT.

[0070] The modified T cells exhibit enhanced discrimination for their target antigen relative to a reference T cell (eg, a corresponding T cell in which the function of the relevant factor is not modulated).

[0071] The modified T cell retains sensitivity to its target antigen compared to a reference T cell, for example 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.

[0072] Therefore, the present invention also provides a method for enhancing pMHC discrimination of T cells. The present invention also provides a method for enhancing pMHC discrimination of T cells while maintaining sensitivity to pMHC. Such methods include preparing modified T cells according to any of the methods described herein. For example, the method can include regulating (e.g., reducing) the function (e.g., amount and / or activity) of a factor associated with T cell receptor (TCR) kinetic correction. The factor can be CD4, CD8, Lck, LAT, and / or Zap70. For example, the factor can be CD8 and / or Lck. Methods for regulating factor function will be further explained below.

[0073] Also provided is a method for preparing a modified T cell population for adoptive cell therapy, for example, an in vitro method for preparing a modified T cell population for adoptive cell therapy. The method may include culturing the modified T cells of the present invention. The method may include preparing one or more modified T cells as provided herein and expanding the T cells. Also provided is a modified T cell population produced according to the methods herein.

[0074] The method of the present invention can also include the step of introducing the target TCR into the T cell. The introducing can be carried out by any method known in the art, for example, by transduction. The introducing can be performed before or after adjusting the factor related to the KP mechanism. The introducing can be carried out simultaneously with the adjustment of the factor, for example, by introducing TCR and a regulator (such as an inhibitor) in the same transduction step, optionally in the same vector, optionally on the same expression construct.

[0075] For example, in some cases, the method of the present invention may include: (i) regulating the amount and / or activity of the factor participating in the TCR KP mechanism in the T cell, and then (ii) the target TCR is transduced into the T cell. In other cases, the method of the present invention may include: (i) the target TCR is transduced into the T cell, and then (ii) regulating the amount and / or activity of the factor participating in the TCR KP mechanism in the T cell. In other cases, the method of the present invention may include: (i) regulating the amount and / or activity of the factor participating in the TCR KP mechanism in the T cell, and (ii) the target TCR is transduced into the T cell. In some cases, the method of the present invention may also include (iii) regulating the amount and / or activity of other factors participating in the TCR KP mechanism in the T cell. Step (iii) can be performed before, during or after any one of steps (i) and (ii).

[0076] T cell discrimination capacity can be assessed by, for example, (a) exposing a T cell or T cell population to its high-affinity target pMHC (i.e., its cognate pMHC) and determining the concentration of pMHC required to achieve signal transduction; (b) exposing a T cell or T cell population to one or more low-affinity pMHCs and determining the concentration of these pMHCs required for signal transduction; (c) comparing the concentration of high-affinity pMHC required to induce comparable signal transduction in cells expressing pMHC-binding receptors with the concentration of low-affinity pMHC, where a large difference in concentration indicates good discrimination. Weak or absent signal transduction induced by the low-affinity pMHC, while signal transduction is induced by the high-affinity target pMHC, also indicates good discrimination.

[0077] Signal transduction can be measured, for example, by induction of molecules downstream of the TCR signaling cascade, such as cytokines (e.g., IL-2) or surface molecules (e.g., CD69 or 4-1BB (CD137)), or target cell killing (e.g., LDH release).

[0078] Functional regulation

[0079] Modulating the function of a factor described herein includes modulating the amount and / or activity of the factor in T cells.

[0080] Reducing the function (e.g., amount and / or activity) of factors that promote progression through the KP mechanism (e.g., CD8α, CD8β, CD4, Lck, ZAP70, or LAT) can enhance T cell differentiation, as shown in Examples 1 and 4. Therefore, for embodiments of the present invention relating to factors that promote 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 CD8α, CD8β, CD4, Lck, ZAP70, and LAT can be reduced. The function (e.g., amount and / or activity) of one or more of Lck, ZAP70, and LAT can be reduced.

[0081] The reduction can be complete or partial. For example, the amount of a factor can be reduced by, for example, at least 10%, 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 100%. The activity of a factor can be reduced by, for example, at least 10%, 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 100%.

[0082] The degree of reduction, such as complete reduction (e.g., knockout) or partial reduction (e.g., knockdown), can be determined by those skilled in the art. For example, the skilled person will understand that it may not be desirable to completely inhibit the amount and / or activity of a factor (e.g., Lck, ZAP70, or LAT) that performs a key step in the TCR signaling pathway.

[0083] Thus, the present invention encompasses completely reducing the amount of factors (eg, CD4 or CD8) expressed on the surface of T cells.

[0084] The modified T cells may not express functional forms of the factors described herein.

[0085] For example, the modified T cells of the present invention may not express functional CD4. For example, the method of the present invention may include deleting the CD4 gene in the genome of a T cell (e.g., CD4+T cell or untyped CD4+CD8+T cell). The method of the present invention may include deleting the CD4 gene in the genome of an autologous T cell or an allogeneic T cell. Therefore, the modified T cells of the present invention may be CD4 knockout T cells, such as CD4+ typed CD4 knockout or CD4+CD8+ untyped CD4 knockout. The modified T cells of the present invention may be autologous T cells or allogeneic T cells.

[0086] CD4 stabilizes TCR:pMHC-II complex, and contributes to the progress of kinetic correction pathway to T cell activation state.When T cell recognition pMHC-II antigen (that is, T cell comprises the peptide presented by MHC II class molecule and has specific TCR), CD4 knocks out and enhances target antigen distinction.Therefore, the modified T cell of the present invention can be the T cell of identification pMHC-II, for example, through through engineering approaches to express the autologous or allogeneic T cell or T helper cell of the TCR identifying pMHC-II.In some embodiments, the modified T cell recognition pMHC-II of the present invention, and the amount of CD4 has reduced, optionally reduces completely.In some embodiments, the modified T cell recognition pMHC-II of the present invention, and CD4 has been knocked out.In some embodiments, the method of the present invention can include making the CD4 gene deletion in the genome of the T cell identifying pMHC-II.

[0087] The modified T cells of the present invention may not express functional CD8. For example, the method of the present invention may include deleting the CD8α and / or CD8β genes in the genome of T cells (e.g., CD8+ T cells or untyped CD4+CD8+ T cells). The method of the present invention may include deleting the CD8 genes in the genome of autologous or allogeneic T cells. Therefore, the modified T cells of the present invention may be CD8 knockout T cells. The modified T cells may be CD8+ typed CD8 knockout. The modified T cells may be CD4+CD8+ untyped CD8 knockout. The modified T cells of the present invention may be autologous T cells or allogeneic T cells.

[0088] CD8 stabilizes TCR:pMHC-I complexes and contributes to the progress of the kinetic correction pathway to the T cell activation state. When T cells recognize pMHC-I antigens (i.e., T cells include TCRs that are specific for peptides presented by MHC class I molecules), CD8α knocks out and enhances target antigen differentiation (as shown in Examples 1 and 7). Therefore, the modified T cells of the present invention can be T cells that recognize pMHC-I, such as autologous or allogeneic T cells or T helper cells that are engineered to express TCRs that recognize pMHC-I. In some embodiments, the modified T cells of the present invention recognize pMHC-I, and the amount of CD8 protein is reduced, optionally completely reduced. In some embodiments, the modified T cells of the present invention recognize pMHC-I, and the amount of CD8 alpha polypeptides has been reduced, optionally completely reduced. In some embodiments, the modified T cells of the present invention recognize pMHC-1, and CD8 protein has been knocked out, optionally wherein CD8 alpha polypeptides have been knocked out. In some embodiments, the methods of the present invention may include deleting the CD8α gene in the genome of T cells that recognize pMHC-I.

[0089] The present invention includes partially reducing the amount of a factor (eg, Lck, ZAP70, or LAT) expressed on the surface of T cells.

[0090] Means for reducing the amount and / or activity of a factor will be apparent to those skilled in the art. For example, the amount and / or activity of a factor can be reduced by impairing gene expression at the nucleic acid level and / or protein level, for example, by regulating the gene encoding the factor or its components, the expression of the gene, the expression of the mRNA transcript, the translation of the mRNA transcript, the protein or its activity. For example, the reduction in the amount and / or activity of a factor can be achieved by deleting (i.e., knocking out) a gene encoding the factor or its components in the genome, reducing the expression of the factor or its components, inhibiting the factor or its components, introducing one or more mutations that impair factor expression or function (e.g., binding activity and / or enzymatic activity) in a gene encoding the factor or its components, or promoting the degradation of the factor or its components.

[0091] For example, for example CD4 or CD8α and / or CD8β, means of knocking out 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.

[0092] Means for reducing factor activity include inhibiting the binding of the factor to its ligand or by inhibiting enzymatic activity. For example, CD4 or CD8 binding to MHC can be inhibited, the recruitment of Lck, ZAP70, or LAT can be inhibited, or the kinase activity of Lck, ZAP70, or LAT can be inhibited. Suitable modulators such as inhibitors will be readily apparent to those skilled in the art and include small molecules, proteins, peptides, antibodies and fragments thereof, scFv, VHH, and VNAR.

[0093] The amount and / or activity reduction of the factor can be achieved by a regulator such as an inhibitor. Regulators (such as inhibitors) can be stably or transiently expressed. Therefore, the modified T cells of the present invention can also include regulators (such as inhibitors) or one or more genes encoding the regulators (such as the inhibitors). Regulators can be inhibitors of gene expression of the factor (such as CRISPR / Cas related systems, shRNA) (see Example 4). Regulators can be inhibitors of the activity of the factor (such as small molecule inhibitors) (see Example 5).

[0094] Modulators can be small organic molecules, small inorganic molecules, nucleic acids (e.g., siRNA, guide RNA, antisense oligonucleotides), peptides, polypeptides, proteins, antibodies or antigen-binding fragments thereof, sugars, nucleic acid analogs or derivatives (e.g., analogs of NAADP), phospholipids (e.g., analogs or derivatives of phosphatidylinositol 3,5-bisphosphate), etc. Small molecules are typically molecules containing several carbon-carbon bonds. Small molecules typically weigh less than 5 kilodaltons, e.g., less than 3 kDa, less than 2 kDa, less than 1 kDa, or less than 500 Daltons.

[0095] Modulators can bind directly to a factor and alter its function.

[0096] An inhibitor can be, for example, a small molecule that specifically binds to a factor and acts as an antagonist of factor function. An inhibitor can be a nucleic acid agent that affects factor expression (transcription and / or translation).

[0097] For example, the inhibitor may be a small interfering RNA (siRNA). The design and production of siRNA is within the common knowledge of the skilled person and is also commercially available (eg www.thermofisher.com).

[0098] Inhibitors can be guide RNA (gRNA), for example, for use with CRISPR-type systems. gRNA can be designed to prevent the expression of functional factors, for example, by deleting a portion of a genomic locus (e.g., exon, start codon, promoter, or ORF). gRNA can be designed to delete the transcriptional repressor binding site on the factor gene promoter, thereby enhancing the expression of functional factors. The design and production of gRNA are within the common knowledge of technicians, and such gRNAs are commercially available (e.g., www.genscript.com).

[0099] The inhibitor may be an antisense oligonucleotide such as a morpholino. The design and production of morpholinos are within the common knowledge of the skilled person and are commercially available (eg www.gene-tools.com).

[0100] The inhibitor may be a small hairpin RNA (shRNA). The design and production of shRNA is within the common knowledge of the skilled person and is commercially available (eg, reference 8).

[0101] The present invention includes increasing the amount and / or activity of a first factor that competes for binding with a second factor involved in progressing the KP pathway to an active state. For example, the present invention includes expressing or overexpressing a factor that competes for binding with a factor involved in progressing the KP pathway to an active state. Thus, 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 CD8α, and the second factor is Lck.

[0102] CD4 stabilizes the TCR:pMHC-II complex; CD8 stabilizes the TCR:pMHC-I complex. The interaction of CD4 or CD8α with the intracellular molecule Lck promotes T cell signal transduction and activation (see Figure 1 , which shows CD4, but CD8 can be considered similar). If co-receptor function is disturbed, activation of T cells by low-affinity peptides is preferentially impaired. Thus, by increasing the amount of CD4 polypeptide in T cells that recognize peptide-MHC-I targets, CD4 competes with CD8α for Lck binding and effectively reduces the amount of Lck available in the cell. Similarly, by increasing the amount of CD8α polypeptide (or generally the CD8 complex) in T cells that recognize peptide-MHC-II targets, CD8α competes with CD4 for Lck binding and effectively reduces the amount of Lck available in the cell.

[0103] For example, the modified T cells of the present invention may comprise CD4. The modified T cells of the present invention may comprise a higher amount of CD4 polypeptides than corresponding unmodified T cells. For example, the methods of the present invention may include increasing the amount of CD4 polypeptides in T cells (e.g., CD8+ T cells, CD4+ T cells, untyped CD4+CD8+ T cells, or autologous T cells or allogeneic T cells). Therefore, the modified T cells of the present invention may be CD8+ T cells, CD4+ T cells, untyped CD4+CD8+ T cells, or autologous or allogeneic T cells, wherein the T cells comprise a higher amount of CD4 polypeptides than corresponding unmodified T cells.

[0104] When T cells recognize pMHC-I antigens (i.e., T cells contain TCRs specific for peptides presented by MHC class I molecules), CD4 overexpression enhances target antigen discrimination (see Examples 9 and 10). Similarly, when T cells recognize pMHC-II antigens (i.e., T cells contain TCRs specific for peptides presented by MHC class II molecules), CD8 overexpression will enhance target antigen discrimination.

[0105] Therefore, the modified T cells of the present invention can be T cells that recognize pMHC-I, for example, autologous or allogeneic T cells or cytotoxic T cells that are engineered to express a TCR that recognizes pMHC-I. In some embodiments, the modified T cells of the present invention recognize pMHC-I and contain a higher amount of CD4 polypeptide than corresponding unmodified T cells. For example, T cells may have been engineered to express or overexpress CD4. In some embodiments, the method of the present invention may include increasing the amount of CD4 polypeptide in T cells (optionally T cells that recognize pMHC-I). The method may include introducing a polynucleotide encoding CD4 into the T cells. For example, the method may include transducing the T cells with a polynucleotide encoding CD4, introducing the polynucleotide encoding CD4 into the T cells by electroporation, knock-in, transfection, or any other method known to the skilled person. The method may include introducing a polynucleotide encoding CD4 and a polynucleotide encoding a TCR that recognizes pMHC-I into the T cells. For example, the method may include transducing the T cells with a polynucleotide encoding CD4 and a polynucleotide encoding a TCR that recognizes pMHC-I. The transduction may be performed by the same vector or by separate vectors. The introduction may be performed by any other method known to the skilled person, such as electroporation, knock-in or transfection.

[0106] As another example, the modified T cells of the present invention may comprise CD8, optionally CD8α. The modified T cells of the present invention may comprise a higher amount of CD8 protein, optionally a higher amount of CD8α polypeptide, than corresponding unmodified T cells. For example, the method of the present invention may comprise increasing the amount of CD8 protein (optionally CD8α polypeptide) in T cells (e.g., CD8+T cells, CD4+T cells, untyped CD4+CD8+T cells, or autologous T cells or allogeneic T cells). Therefore, the modified T cells of the present invention may be CD8+T cells, CD4+T cells, untyped CD4+CD8+T cells, or autologous or allogeneic T cells, wherein the modified T cells comprise a higher amount of CD8 protein (optionally CD8α protein) than corresponding unmodified T cells.

[0107] Therefore, the modified T cells of the present invention can be T cells that recognize pMHC-II, such as autologous or allogeneic T cells or helper T cells that are engineered to express a TCR that recognizes pMHC-II. In some embodiments, the modified T cells of the present invention recognize pMHC-II and contain a higher amount of CD8 protein, optionally CD8α polypeptide, than corresponding unmodified T cells. For example, T cells may have been engineered to express or overexpress CD8, optionally CD8α. In some embodiments, the methods of the present invention may include increasing the amount of CD8 protein (optionally, CD8α polypeptide) in T cells (optionally, T cells that recognize pMHC II). For example, the method may include introducing a polynucleotide encoding CD8α (optionally, also encoding a polynucleotide encoding CD8β) into T cells. In another example, the method may include introducing a polynucleotide encoding CD8α (optionally also encoding a polynucleotide encoding CD8β) and a polynucleotide encoding a TCR that recognizes pMHC-II into T cells. The introduction can be performed by transduction, electroporation, knock-in, or any other method known to the skilled person. For example, the method may include transducing T cells with a polynucleotide encoding CD8α, optionally also transducing T cells with a polynucleotide encoding CD8β. In another example, the method may include transducing T cells with a polynucleotide encoding CD8α (optionally also with a polynucleotide encoding CD8β) and a polynucleotide encoding a TCR that recognizes pMHC-II. The transduction may be performed by the same vector or separate vectors.

[0108] The amount of the first factor can be increased, for example, by at least 10%, at least 20%, 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 can be expressed in cells of the present invention that do not express it under normal cellular conditions. The first factor can be expressed in the same cells that did not previously express it. The first factor can be overexpressed for example by at least 10%, at least 20%, 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 degree of increase can be determined by those skilled in the art.

[0109] As explained herein, increasing the function (e.g., amount and / or activity) of factors that resist (e.g., counter, inhibit, or reverse) progression through the KP mechanism can also enhance T cell differentiation. Thus, for embodiments of the present invention directed to factors that resist progression through the KP mechanism, the function (e.g., amount and / or activity) of the factor is increased. Methods for increasing the amount and / or activity of the factor will be apparent to those skilled in the art.

[0110] The expression of factor at nucleic acid or protein level can be measured by any means known to the technician (such as flow cytometry, Western blotting, qRT-PCR, genome sequencing, fluorescent labeling experiments, mass spectrometry, etc.). The expression of factor can be determined by any means known in the art (such as nucleic acid sequencing). Technicians can use any method common in this area to determine the polypeptide sequence encoded by the factor gene and determine whether the polypeptide will produce a functional factor. In addition, the technician will understand that frameshift mutations and / or mutations that cause premature termination codons (early termination codons) can lead to non-functional factors.

[0111] In some embodiments, the present invention includes regulating the function of one or more factors associated with the TCR KP pathway to increase the discrimination ability of T cells. In some embodiments, the present invention includes regulating the function of two or more factors associated with the TCR KP pathway to increase the discrimination ability of T cells. For example, two or more of CD8, CD4, Lck, ZAP70 and / or LAT can be regulated. In some embodiments, the present invention includes regulating the function of two, three, four, five or more factors associated with the TCR KP pathway.

[0112] For example, the modified T cells of the present invention may comprise a TCR that recognizes pMHC-I, wherein CD8α has been knocked out, and wherein the amount of CD4 polypeptide has been increased (see Example 10). Accordingly, the methods of the present invention may comprise (i) knocking out CD8α and (ii) increasing the amount of CD4 polypeptide in T cells (see Figure 27 In some embodiments, the T cells are pMHC-I restricted T cells (e.g., autologous or allogeneic T cells or cytotoxic T cells comprising a TCR that recognizes pMHC-I). In some embodiments, the method comprises the further step (iii) of expressing a pMHC-I TCR in the T cells. Steps (i), (ii), and optionally (iii) can be performed simultaneously or sequentially in any order. In some embodiments, step (i) is performed before step (ii).

[0113] For example, the method may include: (i) transducing T cells with sgRNA to knock out CD8α; (ii) introducing a polynucleotide encoding CD4 into T cells; and / or (iii) introducing a polynucleotide encoding a TCR that recognizes pMHC-I into T cells. The introduction can be performed by any other method known to the skilled person, such as transduction, electroporation, knock-in, or transfection.

[0114] For example, the method may include transducing T cells with: (i) an sgRNA for knocking out CD8α; (ii) a polynucleotide encoding CD4; and / or (iii) a polynucleotide encoding a TCR that recognizes pMHC-I. The sgRNA and polynucleotide (one or more) may be contained in the same vector or in separate vectors.

[0115] Similarly, the modified T cells of the present invention may include TCRs that recognize pMHC-II, wherein CD4 has been knocked out, and wherein the amount of CD8α polypeptides and / or CD8β polypeptides has increased. Accordingly, the method of the present invention may include (i) knocking out CD4 and (ii) increasing the amount of CD8α polypeptides and / or CD8β polypeptides in T cells. In some embodiments, the T cells are pMHC-II restricted T cells (e.g., autologous or allogeneic T cells or cytotoxic T cells comprising TCRs that recognize pMHC-II). In some embodiments, the method includes a further step (iii) of expressing pMHC-II TCR in T cells. Steps (i), (ii), and optionally (iii) may be performed sequentially simultaneously or in any order. In some embodiments, step (i) is performed before step (ii).

[0116] For example, the method may include: (i) transducing T cells with sgRNA to knock out CD4; (ii) introducing one or more polynucleotides encoding CD8α and / or CD8β into T cells; and / or (iii) introducing a polynucleotide encoding a TCR that recognizes pMHC-II into T cells. The introduction can be performed by any other method known to the skilled person, such as transduction, electroporation, knock-in, or transfection.

[0117] For example, the method may include transducing T cells with: (i) an sgRNA for knocking out CD4; (ii) one or more polynucleotides encoding CD8α and / or CD8β; and / or (iii) a polynucleotide encoding a TCR that recognizes pMHC-II. The sgRNA and polynucleotide(s) may be contained in the same vector or in separate vectors.

[0118] The regulation of the function of a factor (e.g., a decrease in amount and / or activity) can be measured relative to the function of the corresponding factor in a reference T cell. A reference T cell is typically a corresponding T cell in which the function of the relevant factor is not regulated. The reference T cell can be an unmodified T cell. The reference T cell can be a differentiated but immunologically immature T cell of the same class as the modified T cell. For example, in the case where the modified T cell can be a CD4+ T cell, the reference T cell will 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 can bind to the same antigen as the modified T cell, i.e., it can contain the same TCR as the modified T cell.

[0119] As described herein, TCR limitation can be determined for enhancing the regulatory strategy that T cell distinguishes.For example, as described herein, in the case where T cell comprises the TCR identifying pMHC-I, distinguishing can be enhanced by reducing the amount and / or activity and / or increase the amount of CD4 relative to unmodified T cell CD8. In the case where T cell comprises the TCR identifying pMHC-II, distinguishing can be enhanced by reducing the amount and / or activity and / or increase the amount of CD8 relative to unmodified T cell CD4. Regardless of the limitation of TCR, distinguishing can be enhanced by reducing the amount and / or activity of the factor (such as Lck or LAT) that plays a role in CD4 or CD8 downstream.

[0120] Thus, in some embodiments, the T cells of the invention comprise a target TCR that specifically recognizes a pMHC-I antigen. In other words, the T cells of the invention may comprise a target pMHC-I restricted TCR.

[0121] The TCR that specifically recognizes a pMHC class I antigen can be c259. The c259 TCR is a well-studied receptor whose cognate peptide is the tumor-specific antigen NY-ESO-1 (SEQ ID NO: 13) (9, 10). The T cells of the present invention can comprise a TCR that specifically recognizes SEQ ID NO: 13, optionally wherein SEQ ID NO: 13 is presented via an MHC class I molecule or complex.

[0122] The TCR that specifically recognizes a pMHC-I antigen can be a3a. The a3a TCR is a well-studied receptor whose cognate peptide is a MAGE-A3-derived peptide (SEQ ID NO: 30), which is presented by HLA-A*01:01. The T cells of the present invention can comprise a TCR that specifically recognizes SEQ ID NO: 30, which is optionally presented by an MHC class I molecule or complex. Off-target binding of a3a to low-affinity peptides has been associated with fatal autoimmune toxicity seen in clinical trials (4). Therefore, by providing T cells with enhanced discrimination ability, improved T cells, such as a3a T cells, with reduced off-target binding and improved safety are provided herein.

[0123] The TCR that recognizes pMHC-I is also referred to as pMHC-I restricted TCR. Similarly, pMHC-II restricted TCR refers to the TCR that recognizes pMHC-II. In some embodiments, the T cell comprising pMHC-I restricted TCR or pMHC-II restricted TCR naturally expresses the TCR. In other embodiments, the T cell comprising pMHC-I restricted TCR or pMHC-II restricted TCR has been engineered to express the TCR. The engineering can be performed by any method known in the art, such as transduction (e.g., viral transduction), electroporation, knock-in, gene editing or transfection.

[0124] Also provided herein is a method for identifying T cells that have enhanced differentiation of their cognate peptides. For example, the method may include: (i) regulating the amount and / or activity of factors associated with TCR kinetic correction in T cells; (ii) identifying T cells with reduced sensitivity to one or more low-affinity (or "off-target") ligands relative to unregulated T cells. Step (ii) can be performed by any method known to technicians, such as screening for the one or more low-affinity ligands. For example, methods for identifying sensitivity to low-affinity ligands may include flow cytometry, ELISA, affinity determination, cytokine screening, surface 41BB determination, IFN-γ determination, TNF-α determination, IL 2 determination, killing determination. The present invention also provides T cells obtained or obtainable by the method.

[0125] T cell differentiation capacity can be assessed as described herein.

[0126] The T cell may comprise a target TCR, optionally wherein the target TCR recognizes pMHC-I. In other embodiments, the method may further comprise introducing a pMHC-I restricted target TCR into the T cell, wherein the introduction may be performed before or after the modulation step (i).

[0127] The T cell may comprise a target TCR that recognizes pMHC-I. In other embodiments, the method may further comprise introducing a pMHC-I restricted target TCR into the T cell, wherein the introduction may be performed before or after the adjustment step (i). In some embodiments of the method for identifying a T cell, the T cell may be a T cell population.

[0128] In some embodiments, the amount and / or activity of the regulatory factor is to reduce the amount and / or activity of the factor. For example, as described herein, the amount of the factor can be reduced by RNA interference or knockout. As described herein, the activity of the factor can be reduced by inhibition. As described herein, the factor to be reduced can be CD8, CD4, Lck, ZAP70 or LAT, optionally CD8, CD4 or Lck. When the target TCR is a pMHC-I restricted TCR, the factor to be reduced is preferably CD8 (optionally CD8α) or Lck.

[0129] In some embodiments, the amount and / or activity of the regulatory factor is the amount and / or activity of the factor that increases. The amount of the factor can be increased by any method known to technicians, for example, including transduction, transfection, electroporation, knock-in and / or genetic engineering (such as genetic modification or gene editing). For example, the amount of the factor can be increased by transducing the gene encoding the factor into T cells. As another example, the amount of the factor can be increased by entering T cells through electroporation. The amount of the factor can be increased by overexpression. As described herein, the factor to be increased can be CD8 or CD4. When the target TCR is a pMHC-I restricted TCR.

[0130] Nucleic acids, vectors and host cells

[0131] Also provided herein are nucleic acids encoding or constituting the modulators (e.g., inhibitors) described herein. The nucleic acids can be DNA sequences. The nucleic acids can be RNA sequences such as mRNA.

[0132] The inhibitor may be an shRNA. The shRNA may comprise 5 to 35, 10 to 30, or 15 to 25 consecutive nucleotides of a gene encoding a factor described herein (eg, CD4, CD8α, CD8β, ZAP70, LAT, or Lck).

[0133] For example, a nucleic acid inhibitor (e.g., shRNA) for inhibiting Lck can comprise a polynucleotide sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity to any one of SEQ ID NOs: 5-8, and / or a polynucleotide sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence complementarity to any one of SEQ ID NOs: 5-8. The inhibitor (e.g., shRNA) can comprise SEQ ID NO: 8 and SEQ ID NO: 10. In embodiments where the inhibitor is an shRNA, the shRNA can further comprise a loop having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity to SEQ ID NO: 9.

[0134] Nucleic acid inhibitors (e.g., shRNAs) 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: 21-24, and / or a polynucleotide sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence complementarity to any one of SEQ ID NOs: 21-24. In embodiments where the inhibitor is an shRNA, the shRNA may further comprise a loop having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity to SEQ ID NO: 9.

[0135] Nucleic acid inhibitors for inhibiting LAT (e.g., shRNA) can comprise a polynucleotide sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity to any one of SEQ ID NOs: 25-28, and / or a polynucleotide sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence complementarity to any one of SEQ ID NOs: 25-28. In embodiments where the inhibitor is an shRNA, the shRNA can additionally comprise a loop having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity to SEQ ID NO: 9.

[0136] The inhibitor may be based on a CRISPR / Cas system, such as 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 factors such as CD4, CD8α, and / or CD8β. 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 a gene encoding the factor. The CRISPR-Cas system may also additionally comprise one or more Cas proteins or encoding polynucleotides, such as Cas9 proteins or Cas9 encoding polynucleotides, optionally suitable for knocking out CD4, CD8α, and / or CD8β. The one or more sgRNAs for knocking out CD8α may comprise a sequence having a sequence identity of ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% to any one of SEQ ID NOs: 1-4.

[0137] Also provided herein is a vector comprising nucleic acid. The vector may be a viral vector. Conventional viral-based expression systems may include retroviruses, alpha-retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex virus (HSV) vectors for gene transfer. The vector may be a lentiviral vector. Non-viral transduction vectors include transposon-based systems, including PiggyBac and Sleeping Beauty systems. Methods for preparing and purifying such vectors are known in the art. In some embodiments, the vector is a dual vector that allows expression of both a regulator (e.g., shRNA) and a target TCR. In some embodiments, the vector is a lentiviral vector, optionally a dual lentiviral vector.

[0138] The vector may be a cloning vector or an expression vector. Suitable vectors may be any vector that can carry sufficient genetic information and allow the expression of the polypeptide of the present invention.

[0139] The vector is preferably an RNA vector. Suitable RNA vectors include those described in Schutsky, Keith, et al., Oncotarget 6.30 (2015): 28911 and Beatty, Gregory L., et al., Gastroenterology 155.1 (2018): 29-32.

[0140] The general methods for constructing vectors, transfection methods and culture methods are well known to those skilled in the art. In this regard, reference can be made to "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual published by Cold Spring Harbor Publishing.

[0141] The nucleic acid can be provided in the form of an expression construct (or "expression cassette") comprising a regulatory sequence operably linked to an insertion sequence, thereby allowing expression of the regulator and / or factor as defined herein in vivo. Thus, one or more expression cassettes encoding the one or more nucleic acids encoding the regulator and / or factor as defined herein are also provided. These expression cassettes are then typically provided in a vector (such as a plasmid or recombinant viral vector). Thus, a vector encoding one or more regulators and / or factors as defined herein is also provided herein. A vector encoding one or more regulators and / or factors as defined herein is further provided.

[0142] The vector may be a human artificial chromosome. Human artificial chromosomes are described in, for example, Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011) and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014).

[0143] The vector can be a non-viral delivery system, such as a DNA plasmid, naked nucleic acid (eg, naked RNA), and nucleic acid complexed with a delivery vehicle such as a liposome.

[0144] Nucleic acid as described herein, expression cassette or vector can be introduced into host cell, for example, by transduction or other means such as electroporation or transfection. Therefore, a host cell comprising one or more nucleic acid, expression cassette or vector of the present invention is also provided. Nucleic acid as described herein, expression cassette or vector can be introduced into host cell transiently or permanently, so that antibody is expressed by described one or more nucleic acid, expression cassette or vector. 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 bacterial cells). The specific example of cell includes mammalian HEK293 (such as HEK293F, HEK293T, HEK293S or HEK Expi293F), CHO, HeLa, NS0 and COS cells, or any other cell line used herein.

[0145] Typically, the host cell is a T cell of the present invention. The nucleic acid, expression cassette or vector described herein can be transiently or stably introduced into the host cell.

[0146] The modified T cells 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.

[0147] Also provided is a kit suitable for modifying T cells or T cell populations to produce modified T cells or modified T cell populations of the present invention. The kit comprises one or more nucleic acids or vectors described herein. The kit may comprise other reagents such as those discussed herein to improve transfection, transduction or transformation efficacy.

[0148] Pharmaceutical composition

[0149] Also provided is a composition comprising the modified T cells or modified T cell populations of the present invention. The modified T cells or modified T cell populations can 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 can consist of or consist essentially of the modified T cells or modified T cell populations of the present invention, i.e., no other cells are detectable in the composition.

[0150] The composition may be a pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include aqueous carriers, diluents, or excipients. Examples of suitable carriers include: all aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, and solutes to render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may contain suspending and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic agents, and absorbents, etc. It should be understood that the compositions of the present invention may also contain other supplementary physiologically active agents.

[0151] The carrier is generally pharmaceutically "acceptable" in the sense that it is compatible with the other ingredients in the composition and is harmless to the subject. Compositions include those suitable for parenteral administration (including subcutaneous, intramuscular, intravenous and intradermal administration). The composition can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical field. Such methods include preparing a carrier to bind to the isolated T cells. Typically, the composition is prepared by uniformly and tightly combining any active ingredient with a liquid carrier.

[0152] 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 that may contain antioxidants, buffers, bactericides, and solutes that render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents.

[0153] The compositions described herein can be prepared in a manner known in the art and are 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 1x10 6 About 1x10 12 The modified T cells of the present invention.

[0154] The present disclosure also contemplates the combination of the compositions described herein with other active agents and / or with other treatment regimens or modalities such as radiation therapy or surgery. When the compositions described herein are used in combination with known active agents, the combination can be administered sequentially (continuously or with intervals of no treatment) or simultaneously or as a mixture.

[0155] Suitable anticancer agents are known to those skilled in the art.

[0156] Combination therapy is also contemplated to include treatment with a composition of this invention followed by a known therapy, or treatment with a known agent followed by treatment with a composition of this invention, eg, as a maintenance therapy.

[0157] For example, in the treatment of cancer, it is contemplated that the compositions of the present invention may be administered in combination with alkylating agents (e.g., nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide cysplatin, or platinum-containing alkylating agents such as cisplatin, carboplatin, and oxaliplatin) and antimetabolites (e.g., purine or pyrimidine analogs or antifolates such as azathioprine and mercaptopurine), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin), and oxaliplatin. icin), valrubicin, mitoxantrone or anthracycline analogs), plant alkaloids (e.g., vinca alkaloids or taxanes, such as vincristine, vinblastine, vinorelbine, vindesine, paclitaxel or docetaxel), topoisomerase inhibitors (e.g., type I or type II topoisomerase inhibitors), podophyllotoxins (e.g., etoposide or teniposide), tyrosine kinase inhibitors (e.g., imatinib mesylate), mesylate), nilotinib, or dasatinib), adenosine receptor inhibitors (e.g., A2aR inhibitors, SCH58261, CPI-444, SYN115, ZM241385, FSPTP, or A2BR inhibitors such as PSB-1115), adenosine receptor agonists (e.g., CCPA, IB-MECA, and CI-IB-MECA), checkpoint inhibitors (including those of the PDL-1:PD-1 axis, nivolumab, pembrolizumab, monoclonal antibodies (pembrolizumab, atezolizumab, BMS-936559, MEDI4736, MPDL33280A or MSB0010718C), CTLA-4 pathway inhibitors (such as ipilimumab and tremelimumab), TIM-3 pathway inhibitors or known agonist monoclonal antibodies that promote T cell function (including anti-OX40 such as MEDI6469 and anti-4-BB such as PF-05082566).

[0158] The present invention also provides a kit or an article of manufacture comprising the pharmaceutical composition described above.

[0159] The present invention also provides a kit for use in the above therapeutic applications, comprising: (a) a container containing the polypeptide, nucleic acid, vector or pharmaceutical composition of the present invention; and (b) a label or package insert with instructions for use.

[0160] Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container can be made of a variety of materials such as glass or plastic. The container contains a therapeutic composition effective for treating the condition and can have a sterile access port (e.g., an intravenous solution bag or a vial with a stopper pierced by a hypodermic needle). The label or package insert indicates that the therapeutic composition is used to treat the selected condition. In one embodiment, the label or package insert includes instructions for use and indicates that the therapeutic or preventive composition can be used to treat cancer or other conditions described herein.

[0161] The kit may also include another container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desired from a commercial and user perspective, which will be known to those skilled in the art, suitable examples of which include other buffers, diluents, filters, needles, and syringes.

[0162] Therapeutic uses

[0163] Also described herein are modified T cells, modified T cell populations, modulators (e.g., inhibitors described herein such as shRNA or sgRNA), polynucleotide(s), vector(s), expression cassette(s), or pharmaceutical compositions of the invention for use in enhancing T cell differentiation.

[0164] Also described herein are modified T cells, modified T cell populations, modulators (e.g., inhibitors described herein such as shRNA or sgRNA), polynucleotide(s), vector(s), expression cassette(s), or pharmaceutical compositions of the invention for use in methods of treating the human or animal body by therapy (e.g., as a drug).

[0165] For example, a method for treating cancer in a subject is also provided, comprising administering to the subject an effective amount of a modified T cell or a modified T cell population of the present invention. Therefore, the present invention also provides modified T cells, modified T cell populations, regulators (e.g., inhibitors as described herein such as shRNA or sgRNA), polynucleotides (one or more), expression cassettes (one or more), or pharmaceutical compositions of the present invention for use in methods for treating cancer. The present invention also provides modified T cells, modified T cell populations, regulators (e.g., inhibitors as described herein such as shRNA or sgRNA), polynucleotides (one or more), vectors (one or more), expression cassettes (one or more), or pharmaceutical compositions of the present invention for use in the preparation of a medicament for treating cancer. The present invention also provides modified T cells, modified T cell populations, regulators (e.g., inhibitors as described herein such as shRNA or sgRNA), polynucleotides (one or more), vectors (one or more), expression cassettes (one or more), or pharmaceutical compositions of the present invention for use in treating cancer.

[0166] The cancer can be any cancer, such as a solid cancer. The cancer can be a malignancy listed in Table 1. The cancer can be a hematological malignancy or a B-cell cancer.

[0167] Also provided is a method for treating or preventing an infection in a subject, comprising administering to the subject an effective amount of a modified T cell or a modified T cell population of the present invention. Therefore, the present invention also provides a modified T cell or a modified T cell population of the present invention, a modulator (e.g., an inhibitor as described herein such as shRNA or sgRNA), a polynucleotide (one or more), an expression cassette (one or more), or a pharmaceutical composition for use in a method for treating or preventing an infection.

[0168] Also provided is a method for 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 a modified T cell population of the present invention. Accordingly, the present invention also provides a modified T cell, a modified T cell population, a modulator (e.g., an inhibitor as described herein such as shRNA or sgRNA), a polynucleotide (one or more), a vector (one or more), an expression cassette (one or more), or a pharmaceutical composition of the present invention for use in a method for treating or preventing an inflammatory disease.

[0169] The present invention also provides the use of the modified T cells, modified T cell populations, modulators (e.g., inhibitors described herein such as shRNA or sgRNA), polynucleotides (one or more), vectors (one or more), expression cassettes (one or more) or pharmaceutical compositions of the present invention in the preparation of a medicament for treating or preventing infection.

[0170] The present invention also provides a modified T cell, a modified T cell population, a modulator (e.g., an inhibitor as described herein such as shRNA or sgRNA), a polynucleotide (one or more), a vector (one or more), an expression cassette (one or more), or a pharmaceutical composition of the present invention for use in treating or preventing an infection. The infection can be a chronic infection.

[0171] The present invention also provides the use of the modified T cells, modified T cell populations, modulators (e.g., inhibitors described herein such as shRNA or sgRNA), polynucleotides (one or more), vectors (one or more), expression cassettes (one or more) or pharmaceutical compositions of the present invention in the preparation of a medicament for treating or preventing an inflammatory disease.

[0172] The present invention also provides a modified T cell, a modified T cell population, a modulator (e.g., an inhibitor as described herein, such as shRNA or sgRNA), a polynucleotide (one or more), a vector (one or more), an expression cassette (one or more), or a pharmaceutical composition of the present invention for use in treating or preventing an inflammatory disease. The inflammatory disease may be an autoimmune disease.

[0173] Also provided is a method of performing adoptive cell therapy in a subject, the method comprising administering to the subject an effective amount of the modified T cells or modified T cell population of the present invention.

[0174] Therefore, the present invention also provides modified T cells, modified T cell populations, modulators (e.g., inhibitors described herein such as shRNA or sgRNA), polynucleotides (one or more), vectors (one or more), expression cassettes (one or more), or pharmaceutical compositions for use in adoptive cell therapy. The present invention also provides the use of modified T cells, modified T cell populations, modulators (e.g., inhibitors described herein such as shRNA or sgRNA), polynucleotides (one or more), vectors (one or more), expression cassettes (one or more), or pharmaceutical compositions of the present invention for the preparation of a medicament for adoptive cell therapy.

[0175] The present invention also provides the use of the modified T cells, modified T cell populations, modulators (e.g., inhibitors described herein such as shRNA or sgRNA), polynucleotides (one or more), vectors (one or more), expression cassettes (one or more) or pharmaceutical compositions of the present invention for adoptive cell therapy.

[0176] Therapeutic uses and methods may comprise administering a therapeutically effective amount of a modified T cell or a population of modified T cells.

[0177] Also provided is a method for formulating a composition for treating cancer, wherein the method comprises mixing the modified T cell or modified T cell population of the present invention with an acceptable carrier to prepare the composition.

[0178] The subject may have previously been treated for cancer, for example using adoptive cell therapy.

[0179] The treatment methods and uses may include determining whether the cancer expresses a target antigen specifically targeted by the modified T cells or modified T cell populations of the invention prior to treatment with the modified T cells or modified T cell populations of the invention.

[0180] The method may include selecting a modified T cell or a population of modified T cells based on the cancer's expression of a target antigen, such that the modified T cell or population of modified T cells is specific for the cancer. The method may include transfecting or transforming a T cell with a nucleic acid of the invention in response to information regarding the cancer's expression of the target antigen.

[0181] The methods of treatment and uses described herein may include inhibiting a disease state (e.g., cancer), for example, by preventing its progression and / or causing regression of the disease state, until a desired endpoint is reached. The methods of treatment and uses of the present invention may include achieving a partial response and a complete response to cancer. The methods of treatment and uses of the present invention may achieve remission of cancer.

[0182] The methods and uses described herein can slow the growth of cancer, stop the growth of cancer, and / or reverse the growth of cancer. The methods and uses of the invention can reduce the size of a 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 100%.

[0183] Typically, the methods of treatment and uses are directed to human subjects in need thereof. However, non-human animals, such as non-human mammals, are also contemplated. The non-human mammal can be a mouse, rabbit, sheep, pig, cow, cat, or dog.

[0184] The dosage of immune effector cells or immune effector cell populations can vary depending on the age and size of the subject, as well as the disease, condition, and route of administration. The modified T cells or modified T cell populations can be administered in an amount of about 1 x 10 6 to about 1x10 12 The modified T cells or modified T cell populations can be administered at a dose of about 1 x 10 5 cells / kg to about 1x10 11 cells / kg body weight.

[0185] Modified T cells or modified T cell populations can be administered in a single dose. Modified T cells or modified T cell populations can be administered in a multiple dose regimen. For example, a second dose or multiple subsequent doses can be administered after the initial dose. The second dose and subsequent doses can be spaced apart by a suitable time. For example, the dosage between doses can be administered approximately once a week, approximately every 2 weeks, approximately every 3 weeks, approximately every four weeks, or approximately every month.

[0186] The modified T cells or modified T cell populations can be administered intravenously.

[0187] The modified T cells or modified T cell populations can be administered together with one or more additional therapies (e.g., one or more additional therapeutic agents). The additional therapeutic agent can be an anti-tumor agent. The additional therapeutic agent can be another immune effector cell.

[0188] The combined administration of modified T cells or modified T cell populations with additional therapeutic agents can be achieved in a variety of different ways. All components can be administered together in a single composition. Each component can be administered separately as part of a combination therapy.

[0189] For example, the modified T cells or modified T cell populations of the present invention can be administered before, after, or simultaneously with an additional therapeutic agent. The additional therapy can be chemotherapy, radiotherapy, and / or surgery.

[0190] Prior to administering the modified T cells or modified T cell populations of the present invention, the subject can undergo lymphocyte clearance. Lymphocyte clearance can be achieved by administering fluradabine, cyclophosphamide, and / or bendamustine to the subject. Lymphocyte clearance can be performed for at least about one day, such as about 2 days or about 3 days.

[0191] The biological activity and / or therapeutic effect of the administered modified T cells or modified T cell populations can be measured by known methods. For example, the method may include imaging, such as magnetic resonance imaging.

[0192] Table 1 - Examples of antigen-associated malignancies that can be addressed by the pMHC binding receptors of the present invention.The pMHC binding receptors of the present invention can bind peptides from the antigen presented on any MHC molecule, including MHC-I or MHC-II and any allelic variants thereof.

[0193]

[0194]

[0195] Embodiments of the present invention

[0196] 1. A modified T cell, characterized in that the function of a factor associated with the correction of T cell receptor (TCR) dynamics is regulated.

[0197] 2. The modified T cell of embodiment 1, wherein the modified T cell is a CD4+ or CD8+ T cell.

[0198] 3. The modified T cell of embodiment 1 or 2, wherein the amount and / or activity of the factor is reduced.

[0199] 4. The modified T cell of any one of the preceding embodiments, wherein the factor is a factor that progresses the T cell toward a TCR activated state.

[0200] 5. The modified T cell of any one of the preceding embodiments, wherein the factor is a kinase, a kinase recruitment factor, a scaffold molecule or a co-stimulatory molecule.

[0201] 6. The modified T cell according to any one of the preceding embodiments, wherein the factor is CD8, CD4, Lck, ZAP70 or LAT.

[0202] 7. The modified T cell according to any one of the preceding embodiments, wherein the modified T cell further comprises an inhibitor for the amount and / or activity of the inhibitory factor.

[0203] 8. The modified T cell of embodiment 7, wherein the inhibitor is shRNA.

[0204] 9. The modified T cell according to any one of the preceding embodiments, wherein the amount and / or activity of the factor is partially reduced.

[0205] 10. The modified T cell of embodiment 9, wherein the factor is Lck, ZAP70 or LAT.

[0206] 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.

[0207] 12. The modified T cell of embodiment 11, wherein the factor is CD4.

[0208] 13. The modified T cell of embodiments 1 to 8 and 11 to 12, wherein the modified T cell does not express functional CD4, eg, the modified T cell is CD4 knocked out.

[0209] 14. The modified T cell of embodiment 11, wherein the factor is CD8.

[0210] 15. The modified T cell of embodiments 1 to 8, 11 and 14, wherein the modified T cell does not express functional CD8, eg, the modified T cell is CD8 knocked out.

[0211] 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 that progresses the T cell towards a TCR quiescent state.

[0212] 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 binding the same antigen.

[0213] 18. The modified T cell of any one of the preceding embodiments, wherein the modified T cell further comprises a TCR of interest.

[0214] 19. The modified T cell of any one of the preceding embodiments, wherein the function of an additional factor associated with correction of T cell receptor (TCR) kinetics is modulated, optionally wherein the additional factor is CD8, CD4, Lck, ZAP70 or LAT.

[0215] 20. An inhibitor of a factor that is associated with correction of TCR dynamics in a T cell and progresses the T cell toward a TCR activated state, optionally wherein the inhibitor is a shRNA.

[0216] 21. The inhibitor of embodiment 20, comprising: (a) any one of SEQ ID NOs: 5-8 and 21-28, and / or (b) a polynucleotide sequence complementary to SEQ ID NOs: 5-8 and 21-28.

[0217] 22. An sgRNA for knocking out a factor that is associated with TCR dynamics correction in T cells and progresses the T cells toward a TCR activated state, optionally wherein the sgRNA comprises one or more sequences of 5 to 35 consecutive nucleotides comprising a gene encoding the factor.

[0218] 23. The sgRNA of embodiment 22, for knocking out CD8α, wherein the sgRNA comprises SEQ ID NO: 1, 2, 3 or 4.

[0219] 24. A vector comprising the shRNA molecule of embodiment 20 or 21 or one or more sgRNAs of embodiment 22 or 23.

[0220] 25. The vector of embodiment 24, wherein the vector further comprises a polynucleotide encoding a TCR of interest.

[0221] 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.

[0222] 27. A method for preparing modified T cells, comprising regulating the function of a factor associated with TCR kinetic correction in T cells.

[0223] 28. The method of embodiment 27, wherein the T cells are CD4+ or CD8+ T cells.

[0224] 29. The method of embodiment 27 or embodiment 28, wherein modulating the function of the factor comprises decreasing the amount and / or activity of the factor.

[0225] 30. The method of embodiment 29, wherein the factor is a factor that progresses the T cell toward a TCR activated state, optionally wherein the factor is a kinase, a kinase recruitment factor, a scaffold molecule or a co-stimulatory molecule, such as CD8, CD4, Lck, ZAP70 or LAT.

[0226] 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 CD8.

[0227] 32. The method of any one of embodiments 29 to 31, wherein reducing the amount and / or activity of the factor comprises introducing one or more sgRNAs targeting a gene encoding the factor into T cells, and optionally further comprises introducing a Cas9 protein or a polynucleotide encoding a Cas9 protein into the T cells.

[0228] 33. The method of embodiment 29 or embodiment 30, wherein reducing the amount and / or activity of the factor comprises partially reducing the factor, optionally wherein the factor is Lck, ZAP70 or LAT.

[0229] 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.

[0230] 35. The method of embodiment 33, wherein the inhibitor is as defined in embodiment 20 or 21, or is a small molecule inhibitor.

[0231] 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.

[0232] 37. The method of embodiment 36, wherein the factor progresses the T cell toward a TCR quiescent state.

[0233] 38. The method of any one of embodiments 27 to 37, wherein the method further comprises transducing the target TCR into the T cell.

[0234] 39. The method of any one of embodiments 27 to 38, wherein the method enhances target antigen discrimination by the TCR of interest.

[0235] 40. A modified T cell obtainable or obtained by the method according to any one of embodiments 27 to 39.

[0236] 41. A method of enhancing target pMHC discrimination by a T cell, comprising preparing a modified T cell according to any one of embodiments 27 to 39.

[0237] 42. A method of preparing a modified T cell population for adoptive cell therapy, the method comprising culturing the modified T cells of any one of embodiments 1 to 19 and 40.

[0238] 43. A modified T cell population produced by the method of embodiment 42.

[0239] 44. A method of treating cancer, infection or inflammatory disease, comprising administering to a patient in need thereof a modified T cell according to any one of embodiments 1 to 19, a T cell population according to embodiment 43, an inhibitor according to embodiment 20 or 21, an sgRNA according to embodiment 22 or 23, a vector according to embodiment 24 or 25, or a pharmaceutical composition according to embodiment 26, optionally wherein the infection is a chronic infection, or the inflammatory disease is an autoimmune disease.

[0240] 45. The modified T cell according to any one of embodiments 1 to 19, the T cell population according to embodiment 43, the inhibitor according to embodiment 20 or 21, the sgRNA according to embodiment 22 or 23, the vector according to embodiment 24 or 25, or the pharmaceutical composition according to embodiment 26 for use as a medicament.

[0241] 46. ​​The modified T cell according to any one of embodiments 1 to 19, the T cell population according to embodiment 43, the inhibitor according to embodiment 20 or 21, the sgRNA according to embodiment 22 or 23, the vector according to embodiment 24 or 25, or the pharmaceutical composition according to embodiment 26, for use in a method for treating cancer, infection or inflammatory disease, optionally wherein the infection is a chronic infection, or the inflammatory disease is an autoimmune disease.

[0242] Other embodiments of the present invention

[0243] 1. A modified T cell, characterized in that the function of a factor associated with the correction of T cell receptor (TCR) dynamics is regulated.

[0244] 2. The modified T cell of embodiment 1, wherein the modified T cell is a CD4+ or CD8+ T cell.

[0245] 3. The modified T cell of embodiment 1 or 2, wherein the amount and / or activity of the factor is reduced.

[0246] 4. The modified T cell of any one of the preceding embodiments, wherein the factor is a factor that progresses the T cell toward a TCR activated state.

[0247] 5. The modified T cell of any one of the preceding embodiments, wherein the factor is a kinase, a kinase recruitment factor, a scaffold molecule or a co-stimulatory molecule.

[0248] 6. The modified T cell according to any one of the preceding embodiments, wherein the factor is CD8, CD4, Lck, ZAP70 or LAT.

[0249] 7. The modified T cell according to any one of the preceding embodiments, wherein the modified T cell further comprises an inhibitor for suppressing the amount and / or activity of the inhibitor, such as shRNA.

[0250] 8. The modified T cell of any one of the preceding embodiments, wherein the amount and / or activity of a factor is partially reduced, optionally wherein the factor is Lck, ZAP70 or LAT.

[0251] 9. The modified T cell of any one of embodiments 1 to 7, wherein the modified T cell does not express functional CD4 or functional CD8, e.g., the modified T cell is CD4 knockout or CD8 knockout.

[0252] 10. The modified T cell of any one of the preceding embodiments, wherein the modified T cell further comprises a TCR of interest.

[0253] 11. An inhibitor of a factor that is associated with TCR dynamics correction in T cells and causes T cells to progress toward a TCR activated state, optionally wherein the inhibitor is a shRNA comprising: (a) any one of SEQ ID NOs: 5-8 and 21-28, and / or (b) a polynucleotide sequence complementary to SEQ ID NOs: 5-8 and 21-28.

[0254] 12. An sgRNA for knocking out a factor that is associated with TCR dynamics correction in T cells and progresses the T cells toward a TCR activated state, optionally wherein the sgRNA comprises SEQ ID NO: 1, 2, 3 or 4.

[0255] 13. A method for preparing modified T cells, comprising modulating the function of a factor associated with the correction of TCR dynamics in T cells.

[0256] 14. The method of embodiment 13, wherein the T cells are CD4+ or CD8+ T cells.

[0257] 15. The method of embodiment 13 or embodiment 14, wherein modulating the function of the factor comprises decreasing the amount and / or activity of the factor.

[0258] 16. The method of any one of embodiments 13 to 15, wherein the factor is a factor that progresses a T cell toward a TCR activated state, optionally wherein the factor is a kinase, a kinase recruitment factor, a scaffold molecule or a co-stimulatory molecule.

[0259] 17. The method according to any one of embodiments 13 to 16, wherein the factor is CD8, CD4, Lck, ZAP70 or LAT.

[0260] 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 CD8.

[0261] 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 targeting a gene encoding the factor into T cells, and optionally further comprises introducing a Cas9 protein or a polynucleotide encoding a Cas9 protein into the T cells.

[0262] 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 Lck, ZAP70 or LAT.

[0263] 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.

[0264] 22. The method of any one of embodiments 13 to 21, wherein the method further comprises transducing the target TCR into the T cell.

[0265] 23. A modified T cell obtainable or obtained by a method according to any one of embodiments 13 to 22.

[0266] 24. The modified T cell according to any one of embodiments 1 to 10 and 23, the inhibitor according to embodiment 11, or the sgRNA according to embodiment 12 for use as a medicament.

[0267] 25. The modified T cell according to any one of embodiments 1 to 10 and 23, the inhibitor according to embodiment 11, or the sgRNA according to embodiment 12, for use in a method for treating cancer, infection or inflammatory disease, optionally wherein the infection is a chronic infection, or the inflammatory disease is an autoimmune disease.

[0268] other

[0269] It should be understood that the different applications of the modified T cells, methods or pharmaceutical compositions disclosed herein can be customized to the specific needs of the art. It should also be understood that the terms used herein are only used to describe specific embodiments of the present invention and are not intended to be limiting.

[0270] Furthermore, as used in this specification and the accompanying embodiments, the singular indefinite articles ("a", "an") and the definite article ("the") include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "modified T cell" includes two or more "modified T cells".

[0271] Furthermore, when "≥x" is mentioned herein, this means equal to or greater than x. When "≤x" is mentioned herein, this means less than or equal to x.

[0272] For the purposes of the present invention, in order to determine the percent identity of two sequences (e.g., two polynucleotides or two polypeptide sequences), the sequences are aligned (e.g., gaps can be introduced in the first sequence for optimal alignment with the second sequence) for optimal comparison. 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, the nucleotide or amino acid at that position is identical. The percent identity between the two sequences is a function of the number of identical positions shared by the two sequences (e.g., % identity = number of identical positions / total number of positions in the reference sequence × 100).

[0273] Typically, sequence comparisons are performed over the length of a reference sequence. For example, if a user wishes 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), one would align the sequence over the length of SEQ ID NO: 3 and determine how many positions in the test sequence are identical to positions in 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, gaps or missing positions would be considered non-identical positions.

[0274] The skilled artisan is aware of different computer programs that can be used to determine the identity between two sequences. For example, a mathematical algorithm can be used to perform sequence comparison and percent identity determination between two sequences. In one 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 a Blosum 62 matrix or a PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0275] Unless otherwise specified, the factors described herein are human homologs.

[0276] The TCRs described herein can be humanized.

[0277] References herein to "antigen" in the context of TCR binding will be understood to refer to the pMHC complex.

[0278] The "amount" of a factor mentioned herein will be understood to refer to the average amount of the factor in a cell population. For example, the amount of a protein or polypeptide in a T cell population (e.g., 10 6 ) is the average amount of protein or polypeptide in the

[0279] The "activity" of a factor mentioned herein will be understood to refer to the average activity level of the factor in a cell population. For example, the activity of a protein or polypeptide in a T cell population (e.g., 10 6 ) is the average level of activity of a protein or polypeptide in the

[0280] T cells modified according to the present invention exhibit enhanced discrimination of their target antigens relative to corresponding unmodified T cells. Corresponding unmodified T cells will be understood as T cells that contain the same target TCR as the modified T cells, but do not contain factors that have been regulated as described herein.

[0281] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0282] The following examples illustrate the invention.

[0283] Example 1 - Knockout of CD8α enhances ligand discrimination by T cells

[0284] In this example, T cells were modified to knock out CD8α, and the effect on ligand discrimination was investigated.

[0285] The CD8 co-receptor can be formed as a heterodimer consisting of α and β chains or a homodimer consisting of two α chains. CD8α binds to the invariant domain of MHC-I molecules, stabilizing the TCR-pMHC interaction. Furthermore, the intracellular tail of CD8α binds Lck via a zinc clasp and recruits it to the vicinity of the TCR, enhancing progression through a kinetic correction mechanism and improving signal transduction. Knockout of CD8α prevents the formation of CD8 co-receptors.

[0286] A CD8α negative T cell population was generated by Cas9 / sgRNA electroporation as described in Example 6. The CD8α sequences targeted by sgRNA are SEQ ID No: 1-4. The knockout efficiency was 95%. Subsequently, T cells were stained with anti-CD8α PE-conjugated antibodies, and the remaining CD8α positive cells were depleted by pull-down with magnetic anti-PE beads. A pure CD8α negative cell population ( Figure 3 ).

[0287] To quantify ligand discrimination by CD8α-negative T cells, T cells were transduced with c259 TCR and co-cultured with U87 cells pulsed with graded concentrations of 8 peptide sets as described in Example 6. pMHC potency (P15) was defined as the peptide concentration that elicited 15% of the maximal response achieved.

[0288] The amount of T cell activation was measured by measuring surface 41BB expression ( Figure 5 ), T cell cytotoxic activity (Nluc; Figure 6 ) and cytokine secretion (IL2; Figure 6 ) to indicate.

[0289] like Figure 5As shown, using surface 41BB expression as a measure of T cell activation, no significant differences in T cell potency were observed for the high-affinity 9V peptide. Potency was significantly reduced for intermediate and lower affinity peptides in CD8α-negative T cells. These results suggest that CD8α deletion in T cells transduced with the affinity-matured c259 TCR results in increased ligand discrimination without loss of on-target potency.

[0290] Similar results were observed when T cell cytotoxic activity and cytokine secretion were measured ( Figure 6 A). As ligand affinity decreases, the fold change in P15 between CD8α-negative cells and wild-type T cells increases significantly ( Figure 6 B).

[0291] Thus, CD8α-negative cells have higher ligand discrimination than wild-type T cells. This may be because activation to low-affinity peptides, which are more co-receptor-dependent, is preferentially impaired when co-receptor function (such as CD8) is perturbed.

[0292] Thus, modified T cells with reduced CD8α expression and / or activity exhibit enhanced ligand discrimination.

[0293] Example 2 - Knockout of CD43 has no effect on T cell activation

[0294] In this example, T cells were modified to knock out CD43, and the effect on ligand discrimination was investigated.

[0295] CD43 is an abundant and heavily glycosylated protein expressed on the surface of T cells. CD43 is known to participate in T cell antigen recognition by, for example, reducing T cell-APC interactions due to its negative charge or steric hindrance, or by reducing the effective on-rate of TCR / pMHC interactions due to its size and abundance on the cell surface. Furthermore, loss of CD43 has been reported to result in increased T cell proliferation, T cell adhesion, and T cell activation in mice.

[0296] CD43 negative T cell populations were generated by Cas9 / sgRNA electroporation and CD43+ T cell depletion. sgRNA sequences are listed in SEQ ID NOs: 18-20. Subsequently, T cells were stained with anti-CD43 PE-conjugated antibodies, and a pure CD43 negative cell population was obtained ( Figure 14 ).

[0297] CD43 KO cells were transduced with the c259 TCR and expressed similar levels of the receptor as wild-type c259 cells.

[0298] Using a luciferase-based target cytotoxicity assay as a measure of T cell activation, we observed no differences in T cell activation against any of the peptides tested ( Figure 15 Similar results were observed when cytokine secretion and 41BB surface expression were measured ( Figure 16 Compared with wild-type primary human T cells, CD43-negative primary human T cells showed no difference in their ability to discriminate ( Figure 17 A).

[0299] In summary, genetic deletion of CD43 had no detectable effect on the sensitivity or discrimination of human T cell ligands in cellular antigen presentation assays. Thus, modified T cells with reduced CD43 expression and / or activity had no effect on ligand discrimination. CD43 is not involved in the kinetic correction mechanism of TCR activation.

[0300] Example 3 - Knockout of CD2 reduces TCR sensitivity but has no effect on ligand discrimination

[0301] In this example, T cells were modified to knock out CD2, and the effect on ligand discrimination was investigated.

[0302] The adhesion receptor CD2 is expressed on the surface of T cells and binds to CD58 on APCs. CD2-CD58 binding stabilizes tight contacts at the immunological synapse and has been shown to increase T cell sensitivity by lowering the antigen threshold for activation. Addition of CD58 has also been shown to enhance ligand discrimination by T cells in simplified assays using plate-immobilized pMHC. CD2 is not involved in the kinetic correction mechanism for TCR activation.

[0303] CD2 negative T cell populations were obtained by Cas9 / sgRNA electroporation and CD2+ T cell depletion as described in Example 6. The sgRNA sequences are listed in SEQ ID NOs: 14 to 17. Subsequently, T cells were stained with anti-CD2 PE-conjugated antibodies, and a pure CD2 negative cell population ( Figure 11 ).

[0304] CD2 KO cells were transduced with the c259 TCR and expressed similar levels of the receptor as wild-type c259 cells.

[0305] Using surface 41BB expression as a measure of T cell activation, small defects in sensitivity were observed among all peptides ( Figure 12 This loss of sensitivity was not amplified for lower affinity peptides and, therefore, had no impact on ligand discrimination. Similar results were observed when T cell cytotoxic activity and cytokine secretion were measured ( Figure 13 Compared with wild-type primary human T cells, CD2-negative primary human T cells showed no difference in their ability to discriminate ( Figure 17 B).

[0306] Example 4 - Inhibition of Lck Expression Enhances T Cell Ligand Discrimination

[0307] In this example, T cells were modified to suppress Lck expression, and the effect on ligand discrimination was investigated.

[0308] Lck is a protein kinase belonging to the Src family that catalyzes the phosphorylation of the ITAM domain in the cytoplasmic tail of the TCR-CD3 complex. This biochemical step initiates TCR signaling, leading to T cell activation following TCR / pMHC binding.

[0309] A dual lentiviral vector was constructed that constitutively expresses the c259 TCR (SEQ ID NO: 12) under the control of the EF1α promoter and also constitutively expresses the shRNA molecule under the control of the human U6 promoter.

[0310] The shRNA is a scramble shRNA molecule (SEQ ID NO: 29) or (B) an anti-Lck shRNA molecule, and the shRNA is constructed and transduced into human primary T cells. The target sequence of the shRNA (and therefore the sense portion of the shRNA) is given in SEQ ID NOs: 5-8. Each shRNA comprises a loop portion, as shown in SEQ ID NO: 9. The antisense sense portion of each shRNA is the reverse complement of the sense strand, for example, SEQ ID NO: 10 is the antisense portion to the sense portion given in SEQ ID NO: 5. Compared to T cells transduced with scramble (negative control) shRNA molecules, T cells transduced with the dual vector encoding the anti-Lck shRNA molecules had reduced intracellular Lck staining ( Figure 7 ).

[0311] To quantify the ligand discrimination of Lck knockdown T cells, T cells were co-cultured with U87 cells pulsed with eight peptide groups at graded concentrations as described in Example 6 ( Figure 8 ). pMHC potency (P15) was defined as the peptide concentration that elicited 15% of the maximal response achieved.

[0312] Using target cell killing as a measure of T cell activation, no significant difference in T cell potency against the high-affinity 9V peptide was observed between Lck knockdown and wild-type cells. Decreased potency against intermediate and lower affinity peptides ( Figure 8 and 9 A). Thus, these results demonstrate that Lck knockdown in T cells can lead to increased ligand discrimination without loss of on-target potency.

[0313] As ligand affinity decreased, the fold change in P15 between Lck knockdown cells and wild-type T cells increased significantly ( Figure 9 B).

[0314] Thus, Lck knockdown cells exhibited higher ligand discrimination than wild-type T cells, as Lck inhibition prolonged the kinetic correction time, leading to enhanced discrimination.

[0315] Thus, modified T cells with reduced Lck expression and / or activity exhibit enhanced ligand discrimination ability.

[0316] Example 5 - Chemical inhibition of Lck kinase enhances ligand discrimination by T cells

[0317] When Lck was inhibited by a chemical inhibitor (A-770041), enhanced ligand discrimination of Lck knockdown T cells was also observed. Figure 19 and 20 shown.

[0318] For the Lck chemical inhibition assay: A-770041 inhibitor was diluted in DMEM medium. T cells were treated with the appropriate dose of A-770041 for 1 hour and then co-cultured with peptide-pulsed U87 cells at 37°C, 5% CO2 for 4 hours. Subsequently, supernatants were collected for ELISA analysis, and cells were analyzed by flow cytometry to measure surface CD69 expression.

[0319] Cell co-culture assay: 50,000 U87 cells in 100 μl of DMEM were seeded in a 96-well flat-bottom plate and incubated overnight. Peptides were diluted to the appropriate concentration in DMEM, added to each well containing U87 cells, and incubated at 37°C, 10% CO2 for 60 minutes. The medium was discarded, and 50,000 T cells were added to 200 μl of RPMI medium in each well. The cells were incubated at 37°C, 5% CO2 for 24 hours. Supernatants were collected for cytotoxicity and ELISA analysis. 25 μl of 100 mM EDTA-PBS was added to each well containing cells, and the samples were incubated at 37°C, 5% CO2 for 5 minutes. Cells were detached by repeated pipetting of each well and transferred to a 96-well V-bottom plate. The cells were stained at 4°C for 20 minutes, washed with PBS, and analyzed by flow cytometry.

[0320] Example 6 - Materials and Methods

[0321] This example provides the materials and methods used in Examples 1 to 5.

[0322] c259 TCR and affinity peptide panel

[0323] The c259 TCR is a well-studied receptor whose cognate peptide is the tumor-specific antigen NY-ESO-1 (SLLMWITQC, SEQ ID NO: 13) (9,10). The c259 TCR recognizes the NY-ESO-1 peptide presented on the MHC-I molecule HLA-A2. Peptide libraries with varying affinities for this receptor have been developed ( Figure 4 ):9V>6V>3V>6T>4D>4A>5Y.

[0324] Human primary CD8+ T cell blasts were transduced with the c259 TCR and co-stimulated with U87 cells pulsed with a gradient of selected peptide pools. T cell activation was measured by different methods: (I) flow cytometric analysis of cell surface expression of activation markers (e.g., 41BB); (II) ELISA analysis of cytokine secretion such as IL-2; (III) luciferase-based assays to measure T cell cytotoxic activity.

[0325] This platform was used in Examples 1 to 5 to quantify antigen discrimination using multiple T cell responses with the goal of understanding how modulation of different T cell molecules enhances antigen discrimination.

[0326] Cell culture

[0327] U87 and HEK 293T cell lines were used. Primary human T cells were isolated from leukocyte cones.

[0328] Lentivirus production

[0329] HEK 293T cells were seeded in 6-well plates (Day 1) and incubated overnight to reach approximately 80% confluence. TM HP (Roche) co-transfected the cells in each well with 0.8 μg of the appropriate lentiviral transfer plasmid encoding the antigen receptor (1G4 TCR or c259 TCR) and the lentiviral packaging plasmid pRSV-Rev (0.25 μg), pMDLg / pRRE (0.53 μg) and pVSV-G (0.35 μg) (day 2). The medium was changed 18 hours after transfection (day 3). 24 hours after the medium was changed, the supernatant from one well was collected, filtered and used to transduce 1 million human T cells (day 4).

[0330] T cell blasts

[0331] T cells were isolated from leukocyte cones purchased from the NHS Blood Donor Centre at John Radcliffe Hospital (Oxford University Hospitals) (Day 3). RosetteSep was added at 150 μl / mL of sample. TM Human CD8+ or CD4+ T cell enrichment cocktail (STEMCELL Technologies) was incubated at room temperature for 20 minutes. The samples were diluted with an equal volume of PBS and washed with water at a ratio of 0.8:1 ( Sample) so that it The cells were layered onto Paque Plus (Cytiva) density gradient medium.

[0332] The samples were centrifuged at 1200 g for 30 minutes (brake off). Cells at the interface of medium and plasma (buffy coat) were separated and washed twice (centrifuged at 500 g for 5 minutes). Cells were resuspended in complete RPMI medium supplemented with IL-2 (50 U / mL) at a density of 1 million cells per ml. Human T-activator CD3 / CD28 (Thermofisher) (1 million beads per ml) and cells were incubated overnight.

[0333] Transduce 1 million cells with filtered lentiviral supernatant (Day 4). On Days 6 and 8, remove 1 mL of culture medium and replace with 1 mL of fresh medium. On Day 9, remove the culture medium using a magnetic stand. (6 days after isolation). Every other day, cells were resuspended in fresh medium at a density of 1 million cells per ml and used for co-culture experiments. 17 days after isolation, T cells were discarded. CRISPR / Cas9 knockout of T cell proteins

[0334] Cas9 ribonucleoprotein (RNP) was prepared by mixing 8.5 μg of Trucut Cas9 protein v2 (Thermofisher) with 150 pmol of sgRNA mixture (Truguide synthetic gRNA, Thermofisher) and Opti-MEM (Gibco) to a final volume of 5 μl. RNP was incubated at room temperature for 15 minutes.

[0335] One million freshly isolated T cells were washed three times with Opti-MEM (Gibco) and resuspended at a density of 20 million per ml. T cells were mixed with RNPs and transferred to a BTX Cuvette Plus electroporation cuvette (2 mm spacing, Harvard Bioscience). Cells were electroporated using a BTX ECM 830 square wave electroporation system (Harvard Bioscience) at 300 V and 2 ms. Immediately after electroporation, cells were transferred to a 4% PBS supplemented with IL-2 and 1% PBS. The cells were cultured in complete RPMI medium supplemented with human T activator CD3 / CD28 (Thermofisher).

[0336] Negative selection of T cell knockout cells

[0337] The knockout efficiency of different targets is 75-90%. T cells with residual target protein expression are exhausted by antibody staining and bead pull-down. T cells are resuspended in MACS buffer (PBS, 0.5% BSA, 2mMEDTA) at a density of 10 million cells per ml. The cells are stained with 5 μl of corresponding PE-labeled antibodies per million cells at 4 ° C for 15 minutes, washed with MACS and resuspended at a density of 100 million cells per ml. 1 μl Mojosort anti-PE nanobeads (Biolegend) are added to every million cells and incubated on ice for 15 minutes. The cells are washed with MACS and the beads are pulled down magnetically. The supernatant containing negatively selected cells is collected.

[0338] Cell co-culture assay

[0339] 50,000 U87 cells in 100 μl of DMEM were seeded in a 96-well flat-bottom plate and incubated overnight. The peptide was diluted to the appropriate concentration in DMEM, added to each well containing U87 cells, and incubated at 37°C, 10% CO2 for 60 minutes. The medium was discarded, and 50,000 T cells were added to 200 μl of RPMI medium in each well. The cells were incubated at 37°C, 5% CO2 for 24 hours. The supernatant was collected for cytotoxicity and ELISA analysis. 25 μl of 100 mM EDTA PBS was added to each well containing cells, and the samples were incubated at 37°C, 5% CO2 for 5 minutes. The cells were detached by repeatedly pipetting each well and transferred to a 96-well V-bottom plate. The cells were stained at 4°C for 20 minutes, washed with PBS, and analyzed by flow cytometry.

[0340] Flow cytometry

[0341] All samples were analyzed using a BD X-20 flow cytometer or a Cytoflex LX flow cytometer (Beckman Couter). Data were analyzed using FlowJo v10 (BD Biosciences) and GraphPad Prism (GraphPad Software).

[0342]

[0343] Table 2: Flow cytometry reagents ELISA

[0344] The levels of cytokines in diluted T cell supernatants were quantified using Invitrogen human IFN-γ, IL2, or TNF-α uncoated ELISA kits (Thermo Fisher Scientific) according to the manufacturer's protocol. The absorbance at 450 nm and 570 nm was measured using a SpectraMax M3 microplate reader (Molecular Devices).

[0345] Cytotoxicity assay

[0346] Coelenterazine (CTZ) 2 mM stock solution was prepared in methanol, aliquoted and stored at -80° C. Supernatant from the co-culture assay was mixed with 10 μM CTZ in PBS at a 1:1 ratio and luminescence was read using a SpectraMax M3 microplate reader (Molecular Devices).

[0347] Measurement of c259 TCR / pMHC binding affinity and surface plasmon resonance

[0348] The 3D binding affinity of the 8-peptide panel to the c259 TCR was determined by SPR using an equilibrium binding assay at 37°C. The average equilibrium binding affinities are summarized in Figure 4 As shown in .

[0349] All SPR experiments were performed at the SPR facility of the Sir William Dunn School of Pathology, Oxford, UK, following the method published by 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 the running buffer. The CAP chip was saturated with streptavidin and biotinylated pMHC was immobilized to the desired level. A gradient of TCR concentrations was flowed through at 37°C. CD58 was immobilized on a reference flow cell at a level that matched the pMHC level on the remaining flow cell. The signal from the reference flow cell was subtracted (single reference) and the average signal from the most recent buffer injection was subtracted (double reference). Steady-state binding affinities were calculated by fitting the double-referenced equilibrium RU values ​​using a single-site specific binding model on GraphPad Prism. Bmax was constrained to the Bmax inferred from the empirical standard curve, relating maximal antibody binding to maximal TCR binding.

[0350] Example 7 - Knockout of CD8α eliminates the cross-reactivity of a3a to titin

[0351] The a3a TCR is a well-studied receptor whose cognate peptide is a MAGE-A3-derived peptide (EVDPIGHLY, SEQ ID NO: 30). a3a recognizes MAGE-A3 presented on the MHC-I molecule HLA-A1. During clinical trials, when T cells engineered with the a3a TCR were administered to patients, they produced fatal autoimmune toxicity against cardiac tissue (3). A subsequent study determined that the a3a TCR recognizes a peptide (ESDPIVAQY, SEQ ID NO: 31) derived from the muscle protein titin, which was the most likely cause of the fatal autoimmune reaction (4). Several studies have determined that the a3a TCR has lower affinity for off-target titin peptides than for its target MAGE-A3 peptide (4, 11).

[0352] Example 1 demonstrates that CD8α knockout can increase ligand discrimination of T cells engineered with the c259 TCR. This example shows that CD8α knockout can eliminate the cross-reactivity of T cells engineered with the a3a TCR with the lower affinity titin peptide while maintaining activation of their higher affinity target, the MAGE-A3 peptide.

[0353] result

[0354] The CD8 co-receptor is a heterodimer formed by an α chain and a β chain. CD8α binds to the invariant domain of the MHC-I molecule (12), which stabilizes the TCR-pMHC interaction. In addition, the intracellular tail of CD8α binds Lck via a zinc clasp and recruits it to the vicinity of the TCR, which improves signal transduction (13). Without wishing to be bound by theory, it is speculated that if co-receptor function is disturbed, activation by low-affinity peptides will be preferentially disrupted, which have a stronger co-receptor dependency (14). CD8α is required for the expression of CD8β on the cell surface (15). It is therefore predicted that knocking out CD8α by CRISPR / Cas9 will prevent the formation of CD8 co-receptors and produce T cells with enhanced ligand discrimination. To test this prediction, a population of CD8α-negative T cells ( Figure 21 ).

[0355] Human primary CD8+ T cell naive cells were transduced with a3a TCR and co-stimulated with T2 cells pulsed with graded concentrations of MAGE-A3 or titin peptide. T cell activation was measured by different methods: (I) flow cytometric analysis of cell surface expression of activation markers such as 41BB; (II) ELISA analysis of cytokine secretion such as IL-2; and (III) measurement of T cell cytotoxic activity using a luciferase-based assay.

[0356] A protocol for electroporating human primary T cells with Cas9:sgRNA ribonucleoprotein (RNP) and transducing with a3aTCR was optimized. A knockout efficiency of 95% was achieved. Subsequently, T cells were stained with anti-CD8α PE-conjugated antibodies, and the remaining CD8α-positive cells were depleted by pull-down with magnetic anti-PE beads. A pure CD8α-negative cell population was obtained ( Figure 21 ).

[0357] T cells were co-cultured with T2 cells pulsed with graded concentrations of MAGE-A3 or titin peptides. Using surface 41BB expression as a measure of T cell activation, no significant difference in T cell potency was observed against the high-affinity MAGE-A3 peptide. No activation against titin was detected in CD8αKO T cells ( Figure 22 ).

[0358] When measuring T cell cytotoxic activity ( Figure 23 ) and cytokine secretion ( Figures 24-26 ), similar results were observed.

[0359] Thus, this example demonstrates that knockout of CD8α abolishes activation of a3a TCR-engineered T cells against the cross-reactive, lower affinity peptide titin, while maintaining a potent response to the higher affinity MAGE-A3 peptide.

[0360] Example 8 - Materials and Methods

[0361] This example provides the materials and methods used in Example 7.

[0362] Cell culture

[0363] T2 cells were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS, 50 μg / mL streptomycin, and 50 units / mL penicillin at 37° C. and 5% CO 2 .

[0364] Primary human T cells were isolated from leukocytes and cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS, 50 μg / mL streptomycin, 50 units / mL penicillin, and 50 U / mL IL-2 at 37° C. and 5% CO. Every other day, cells were resuspended in fresh culture medium at a density of 1 million cells per ml.

[0365] Lentivirus production

[0366] HEK 293T cells were seeded in 6-well plates (Day 1) and incubated overnight to reach approximately 80% confluence. TM HP (Roche) co-transfected the cells in each well with 0.8 μg of the appropriate lentiviral transfer plasmid encoding the antigen receptor (a3a TCR) and the lentiviral packaging plasmids pRSV-Rev (0.25 μg), pMDLg / pRRE (0.53 μg) and pVSV-g (0.35 μg) (day 2). The medium was changed 18 hours after transfection (day 3). 24 hours after the medium change, the supernatant from one well was collected, filtered and used to transduce 1 million human T cells (day 4).

[0367] T cell blasts

[0368] T cells were isolated from leukocyte cones purchased from the NHS Blood Donor Centre at John Radcliffe Hospital (Oxford University Hospitals) (Day 3). RosetteSep was added at 150 μl / mL of sample. TM Human CD8+ or CD4+ T cell enrichment cocktail (STEMCELL Technologies) was incubated at room temperature for 20 minutes. The samples were diluted with an equal volume of PBS and washed with water at a ratio of 0.8:1 ( Sample) so that it The cells were layered onto Paque Plus (Cytiva) density gradient medium.

[0369] The samples were centrifuged at 1200 g for 30 minutes (brake off). Cells at the interface of medium and plasma (buffy coat) were separated and washed twice (centrifuged at 500 g for 5 minutes). Cells were resuspended in complete RPMI medium supplemented with IL-2 (50 U / mL) at a density of 1 million cells per ml. Human T-activator CD3 / CD28 (Thermofisher) (1 million beads per ml) and cells were incubated overnight.

[0370] Transduce 1 million cells with filtered lentiviral supernatant (Day 4). On Days 6 and 8, remove 1 mL of culture medium and replace with 1 mL of fresh medium. On Day 9, remove the culture medium using a magnetic stand. (6 days after isolation). Every other day, cells were resuspended in fresh medium at a density of 1 million cells per ml and used for co-culture experiments. 17 days after isolation, T cells were discarded. CRISPR / Cas9 knockout of T cell proteins

[0371] Cas9 ribonucleoprotein (RNP) was prepared by mixing 8.5 μg of Trucut Cas9 protein v2 (Thermofisher) with 150 pmol of sgRNA mixture (Truguide synthetic gRNA, Thermofisher) and Opti-MEM (Gibco) to a final volume of 5 μl. RNP was incubated at room temperature for 15 minutes.

[0372] One million freshly isolated T cells were washed three times with Opti-MEM (Gibco) and resuspended at a density of 20 million per ml. T cells were mixed with RNPs and transferred to a BTX Cuvette Plus electroporation cuvette (2 mm spacing, Harvard Bioscience). Cells were electroporated using a BTX ECM 830 square wave electroporation system (Harvard Bioscience) at 300 V and 2 ms. Immediately after electroporation, cells were transferred to a 4% PBS supplemented with IL-2 and 1% PBS. The cells were cultured in complete RPMI medium supplemented with human T activator CD3 / CD28 (Thermofisher).

[0373] Negative selection of T cell knockout cells

[0374] The knockout efficiency of different targets is 75-90%. T cells with residual target protein expression are exhausted by antibody staining and bead pull-down. T cells are resuspended in MACS buffer (PBS, 0.5% BSA, 2mMEDTA) at a density of 10 million cells per ml. The cells are stained with 5 μl of corresponding PE-labeled antibodies per million cells at 4 ° C for 15 minutes, washed with MACS and resuspended at a density of 100 million cells per ml. 1 μl Mojosort anti-PE nanobeads (Biolegend) are added to every million cells and incubated on ice for 15 minutes. The cells are washed with MACS and the beads are pulled down magnetically. The supernatant containing negatively selected cells is collected.

[0375] Cell co-culture assay

[0376] 100,000 T2 cells in 100 μl of DMEM were seeded in a 96-well flat-bottom plate. The peptide was diluted to the appropriate concentration in RPMI, added to each well containing T2 cells, and incubated at 37°C, 5% CO2 for 120 minutes. The T2 cells were centrifuged at 500g for 5 minutes. The supernatant was discarded, and 50,000 T cells were added to 200 μl of RPMI medium in each well. The cells were incubated at 37°C, 5% CO2 for 20 hours. The supernatant was collected for cytotoxicity and ELISA analysis. The cells were detached by repeatedly pipetting each well and transferred to a 96-well V-bottom plate. The cells were stained at 4°C for 20 minutes, washed with PBS, and analyzed by flow cytometry.

[0377] Flow cytometry

[0378] All samples were analyzed using a BD X-20 flow cytometer or a Cytoflex LX flow cytometer (Beckman Couter). Data were analyzed using FlowJo v10 (BD Biosciences) and GraphPad Prism (GraphPad Software).

[0379] name illustrate source Anti-human 41BB (mouse monoclonal antibody) Clone 4B4-1, 1:200 dilution Biolegend Anti-human CD45 (mouse monoclonal antibody) Clone HI30, 1:200 dilution Biolegend Anti-human CD8α (mouse monoclonal antibody) Clone HIT8a, 1:200 dilution Biolegend

[0380] Table 3: Flow cytometry reagents ELISA

[0381] The levels of cytokines in diluted T cell supernatants were quantified using Invitrogen human IFN-γ, IL2, or TNF-α uncoated ELISA kits (Thermo Fisher Scientific) according to the manufacturer's protocol. The absorbance at 450 nm and 570 nm was measured using a SpectraMax M3 microplate reader (Molecular Devices).

[0382] Cytotoxicity assay

[0383] Coelenterazine (CTZ) 2 mM stock solution was prepared in methanol, aliquoted and stored at -80° C. Supernatant from the co-culture assay was mixed with 10 μM CTZ in PBS at a 1:1 ratio and luminescence was read using a SpectraMax M3 microplate reader (Molecular Devices).

[0384] Example 9 - Overexpression of CD4 enhances ligand discrimination by T cells engineered with MHC-I restricted TCRs

[0385] Typically, cytotoxic CD8-positive T cells recognize peptides presented by MHC-I molecules, while helper CD4-positive T cells recognize peptides presented by MHC-II molecules. In contrast, in adoptive T cell transfer therapy, both CD8 cytotoxic cells and CD4 helper cells are engineered with the same TCR (which recognizes peptides presented by either MHC-I or MHC-II molecules).

[0386] The CD8 co-receptor is a heterodimer formed by an α chain and a β chain, while the CD4 co-receptor is formed by a single chain. CD8α binds to the invariant domain of MHC-I molecules (12), while CD4 binds to the invariant domain of MHC-II molecules. In addition, the intracellular tails of CD8α and CD4 bind to Lck through zinc clasps and recruit it to the vicinity of the TCR, which improves signal transduction (13).

[0387] Without wishing to be bound by theory, it is speculated that if co-receptor function is disturbed, activation by low-affinity peptides is preferentially disrupted. It is therefore predicted that by overexpressing CD4 in T cells that recognize peptide-MHC-I targets (e.g., c259 TCR and a3a TCR), CD4 will compete with CD8 for Lck, thereby improving ligand discrimination. In other words, CD4 overexpression is expected to increase discrimination of any T cell expressing a TCR that recognizes a peptide-MHC-I target.

[0388] To test this prediction, a population of cytotoxic CD4-overexpressing T cells was generated ( Figure 27 ).

[0389] A dual lentiviral vector encoding c259 TCR and CD4 was constructed. Flow cytometry confirmed that human primary CD8+ T cells transduced with the dual vector expressed both c259 TCR protein and CD4 protein ( Figure 28 ).

[0390] To quantify ligand discrimination of CD4-overexpressing T cells, the transduced T cells were co-cultured with U87 cells pulsed with a gradient of 8 peptides. pMHC potency (P15) was defined as the peptide concentration that elicited 15% of the maximum response achieved. Using surface 41BB expression as a measure of T cell activation, no difference in T cell potency was observed for the high-affinity 9V peptide. Potency decreased for intermediate and lower affinity peptides ( Figure 29 ). Thus, these results indicate that CD4 overexpression in T cells transduced with the c259 TCR can lead to increased ligand discrimination without loss of on-target potency. As ligand affinity decreased, the fold change in P15 between CD4-overexpressing cells and wild-type T cells increased significantly ( Figure 29 B) Thus, CD4-overexpressing cells have higher ligand discrimination than wild-type T cells.

[0391] Example 10 - Combining CD8α knockout with CD4 overexpression results in ligand discrimination of T cells Accumulation Enhancement

[0392] CD8α knockout human primary T cell populations were generated as described in Example 1. CD8α knockout T cells were transduced with a dual c259-CD4 lentiviral construct. The c259 TCR recognized peptides presented via pMHC-I.

[0393] To quantify ligand discrimination by CD8 KO T cells overexpressing CD4, the transduced T cells were co-cultured with U87 cells pulsed with a gradient of 8 peptides. pMHC potency (P15) was defined as the peptide concentration that elicited 15% of the maximal response achieved. As ligand affinity decreased, the fold change in P15 between CD8α-negative CD4-overexpressing cells and wild-type T cells increased significantly ( Figure 30 ). Thus, CD8α-negative cells have higher ligand discrimination than wild-type T cells. Moreover, the observed increase in discrimination is higher than that observed for either CD8 KO cells or CD4 overexpressing cells alone, suggesting an additive effect ( Figure 30 ).

[0394] Thus, Example 10 demonstrates that CD8+CD4+c259 T cells have enhanced ligand discrimination compared to wild-type CD8+c259 T cells ( Figure 29 Furthermore, the study showed that the increase in discrimination provided by CD4 was additive to the previously observed increase in discrimination provided by CD8α knockout. Thus, CD8αKO CD4-overexpressing T cells exhibited the highest increase in discrimination ( Figure 30 ).

[0395] Example 11 - Materials and Methods

[0396] This example provides the materials and methods used in Examples 9 and 10.

[0397] Cell culture

[0398] U87 and HEK 293T cell lines were cultured in DMEM D6429 medium (Sigma-Aldrich) supplemented with 10% FBS, 50 μg / mL streptomycin, and 50 units / mL penicillin at 37° C. and 10% CO 2 .

[0399] Primary human T cells were isolated from leukocytes and cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS, 50 μg / mL streptomycin, 50 units / mL penicillin, and 50 U / mL IL-2 at 37° C. and 5% CO. Every other day, cells were resuspended in fresh culture medium at a density of 1 million cells per ml.

[0400] Lentivirus production

[0401] HEK 293T cells were seeded in 6-well plates (Day 1) and incubated overnight to reach approximately 80% confluence. TM HP (Roche) co-transfected the cells in each well with 0.8 μg of the appropriate lentiviral transfer plasmid encoding the antigen receptor (1G4 TCR or c259 TCR) and the lentiviral packaging plasmid pRSV-Rev (0.25 μg), pMDLg / pRRE (0.53 μg) and pVSV-g (0.35 μg) (day 2). The culture medium was changed 18 hours after transfection (day 3). 24 hours after the culture medium was changed, the supernatant from one well was collected, filtered and used to transduce 1 million human T cells (day 4).

[0402] T cell blasts

[0403] T cells were isolated from leukocyte cones purchased from the NHS Blood Donor Centre at John Radcliffe Hospital (Oxford University Hospitals) (Day 3). RosetteSep was added at 150 μl / mL of sample. TM Human CD8+ or CD4+ T cell enrichment cocktail (STEMCELL Technologies) was incubated at room temperature for 20 minutes. The samples were diluted with an equal volume of PBS and washed with water at a ratio of 0.8:1 ( Sample) in The cells were layered onto Paque Plus (Cytiva) density gradient medium.

[0404] The samples were centrifuged at 1200 g for 30 minutes (brake off). Cells at the interface of medium and plasma (buffy coat) were separated and washed twice (centrifuged at 500 g for 5 minutes). Cells were resuspended in complete RPMI medium supplemented with IL-2 (50 U / mL) at a density of 1 million cells per ml. Human T-activator CD3 / CD28 (Thermofisher) (1 million beads per ml) and cells were incubated overnight.

[0405] Transduce 1 million cells with filtered lentiviral supernatant (Day 4). On Days 6 and 8, remove 1 mL of culture medium and replace with 1 mL of fresh medium. On Day 9, remove the culture medium using a magnetic stand. (6 days after isolation). Every other day, cells were resuspended in fresh medium at a density of 1 million cells per ml and used for co-culture experiments. 17 days after isolation, T cells were discarded. CRISPR / Cas9 knockout of T cell proteins

[0406] Cas9 ribonucleoprotein (RNP) was prepared by mixing 8.5 μg of Trucut Cas9 protein v2 (Thermofisher) with 150 pmol of sgRNA mixture (Truguide synthetic gRNA, Thermofisher) and Opti-MEM (Gibco) to a final volume of 5 μl. RNP was incubated at room temperature for 15 minutes.

[0407] One million freshly isolated T cells were washed three times with Opti-MEM (Gibco) and resuspended at a density of 20 million per ml. T cells were mixed with RNPs and transferred to a BTX Cuvette Plus electroporation cuvette (2 mm spacing, Harvard Bioscience). Cells were electroporated using a BTX ECM 830 square wave electroporation system (Harvard Bioscience) at 300 V and 2 ms. Immediately after electroporation, cells were transferred to a 4% PBS supplemented with IL-2 and 1% PBS. The cells were cultured in complete RPMI medium supplemented with human T activator CD3 / CD28 (Thermofisher).

[0408] Negative selection of T cell knockout cells

[0409] The knockout efficiency of different targets is 75-90%. T cells with residual target protein expression are exhausted by antibody staining and bead pull-down. T cells are resuspended in MACS buffer (PBS, 0.5% BSA, 2mMEDTA) at a density of 10 million cells per ml. The cells are stained with 5 μl of corresponding PE-labeled antibodies per million cells at 4 ° C for 15 minutes, washed with MACS and resuspended at a density of 100 million cells per ml. 1 μl Mojosort anti-PE nanobeads (Biolegend) are added to every million cells and incubated on ice for 15 minutes. The cells are washed with MACS and the beads are pulled down magnetically. The supernatant containing negatively selected cells is collected.

[0410] Cell co-culture assay

[0411] 50,000 U87 cells in 100 μl of DMEM were seeded in a 96-well flat-bottom plate and incubated overnight. Peptides were diluted to the appropriate concentration in RPMI, added to each well containing U87 cells, and incubated at 37°C, 5% CO2 for 60 minutes. The medium was discarded, and 50,000 T cells were added to 200 μl of RPMI medium in each well. The cells were incubated at 37°C, 5% CO2 for 24 hours. The supernatant was collected for cytotoxicity and ELISA analysis. 25 μl of 100 mM EDTA PBS was added to each well containing cells, and the samples were incubated at 37°C, 5% CO2 for 5 minutes. The cells were detached by repeatedly pipetting each well and transferred to a 96-well V-bottom plate. The cells were stained at 4°C for 20 minutes, washed with PBS, and analyzed by flow cytometry.

[0412] Flow cytometry

[0413] All samples were analyzed using a BD X-20 flow cytometer or a Cytoflex LX flow cytometer (Beckman Couter). Data were analyzed using FlowJo v10 (BD Biosciences) and GraphPad Prism (GraphPad Software). Flow cytometry reagents are listed in Table 4.

[0414]

[0415] Table 4: Flow cytometry reagents ELISA

[0416] The levels of cytokines in diluted T cell supernatants were quantified using Invitrogen human IFN-γ, IL2, or TNF-α uncoated ELISA kits (Thermo Fisher Scientific) according to the manufacturer's protocol. The absorbance at 450 nm and 570 nm was measured using a SpectraMax M3 microplate reader (Molecular Devices).

[0417] Cytotoxicity assay

[0418] Coelenterazine (CTZ) 2 mM stock solution was prepared in methanol, aliquoted and stored at -80° C. Supernatant from the co-culture assay was mixed with 10 μM CTZ in PBS at a 1:1 ratio and luminescence was read using a SpectraMax M3 microplate reader (Molecular Devices).

[0419] surface plasmon resonance

[0420] All SPR experiments were performed at the Dunn School SPR facility according to the method published by Pettmann et al. (2). Briefly, c259 TCR / pMHC steady-state binding affinity was measured on a Bioacore T200 (GE Healthcare) with a CAP chip using HBS-EP as the running buffer. The CAP chip was saturated with streptavidin and biotinylated pMHC was immobilized to the desired level. A gradient of TCR concentrations was flowed over at 37°C. CD58 was immobilized on a reference flow cell at a level that matched the pMHC level on the remaining flow cell. The signal from the reference flow cell was subtracted (single reference) and the average signal from the most recent buffer injection was subtracted (double reference). Steady-state binding affinity was calculated by fitting to the double-referenced equilibrium RU values ​​using a single-site specific binding model on GraphPad Prism. Bmax was constrained to the Bmax extrapolated from the empirical standard curve, relating maximal antibody binding to maximal TCR binding.

[0421] References

[0422] 1 Wooldridge et al., 2012, J Biol Chem., 287(2): 1168-77.

[0423] 2 Pettmann, et al., 2021, eLife 10:e67092.

[0424] 3 Linette et al., 2013, Blood, 122(6): 863-71.

[0425] 4 Cameron et al.,2013,Science translational medicine,5(197):197ra103.

[0426] 5 McKeithan,1995,Proc Natl Acad Sci USA,92(11):5042-6.

[0427] 6 https: / / www.cellsignal.co.uk / pathways / t-cell-receptor-signaling.

[0428] 7 Lever et al.,2014,Nat Rev Immunol,14(9):619-29.

[0429] 8 Moore et al.,2010,Methods Mol Biol.,629:141-158.

[0430] 9 Bethune et al.,2018,Proc Natl Acad Sci USA,115(45):E10702-E10711

[0431] 10 Linette,G.P.et al.,2013,Blood,122(6),863-871.

[0432] 11 Zhao,X.et al.,2022,Science,376(6589).

[0433] 12 Gangadharan,D.and Cheroutre,H.(2004),Current Opinion inImmunology,16(3),264-270

[0434] 13 Horkova,V.,et al.,2020.Cell Reports,30(5),pp.1504-1514.e7.

[0435] 14 Laugel et al.,2007,Journal Of Biological Chemistry,282(33),23799-23810.

[0436] 15 Norment and Littman, 1988, The EMBO Journal, 7(11), 3433-3439.

[0437] Sequence Listing

[0438]

[0439]

[0440]

[0441]

Claims

1. A modified T cell comprising a target TCR, characterized in that The function of factors associated with the correction of T cell receptor (TCR) kinetics is modulated relative to unmodified T cells containing the target TCR.

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

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

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 target TCR.

5. The modified T cell of any one of the preceding claims, wherein the factor is a factor that progresses the T cell toward a 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 Lck.

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 shRNA, optionally wherein the factor is Lck.

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 CD8α.

12. The modified T cell of claim 12, wherein the modified T cell does not express a functional CD8α polypeptide, eg, the modified T cell is CD8α knocked 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 target TCR, optionally wherein the factor is a factor that progresses the T cell toward a TCR quiescent 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 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 that binds the same antigen.

17. The modified T cell of any of the preceding claims, wherein the function of an additional factor associated with correction of T cell receptor (TCR) kinetics is modulated, optionally wherein the additional factor is CD4, CD8 or Lck.

18. The modified T cell of any one of the preceding claims, wherein the amount of CD4 polypeptide is increased and the modified T cell is CD8α knocked out.

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

20. An inhibitor of a factor that is associated with correction of TCR dynamics in a T cell and progresses the T cell toward a TCR activated state, optionally wherein the inhibitor is a shRNA.

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

22. An sgRNA for knocking out a factor that is associated with TCR dynamics correction in T cells and causes the T cells to progress toward a TCR activated state, optionally wherein the sgRNA comprises one or more sequences of 5 to 35 consecutive nucleotides comprising a gene encoding the factor. The sgRNA of claim 22 , for knocking out CD8α, 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, and optionally further 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 for preparing a modified T cell comprising a target TCR, comprising modulating the function of a factor associated with the correction of TCR kinetics in the T cell.

28. The method of claim 27, wherein the target TCR 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 Lck.

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

32. The method of claim 31, wherein the factor is a factor that progresses the T cell toward a 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 decreasing the factor, optionally wherein the factor is Lck 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 CD8α.

37. The method of claim 36, wherein the method comprises knocking out CD8α.

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 targeting a gene encoding the factor into the T cell, and 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 further comprises transducing the target TCR 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 toward a TCR quiescent 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 CD4 polypeptide in the T cells.

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

45. The method of any one of claims 27 to 33 and 36 to 44, wherein the method comprises knocking out CD8α and increasing the amount of CD4 polypeptide in the T cells.

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

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

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

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

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

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

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

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

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

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

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