Genetically modified t-cells and methods of treating cancer

Abstract

The present disclosure relates to genetically modified T cells or chimeric antigen receptor (CAR) T cells, comprising genetic modifications that inhibit or inactivate the expression of prolyl 4 hydroxylase subunit alpha 1 (P4HA1) and / or programmed cell death 1 (PD1) in the T cells or CAR T cells. The present disclosure also relates to methods of treating cancer comprising administration of the modified T cells or CAR T cells as disclosed herein; or administration of P4HA1 inhibitors and / or PD1 / PD-1L inhibitors.

Description

GENETICALLY MODIFIED T-CELLS AND METHODS OF TREATING CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to the Singapore provisional application no. 10202403032T, filed on 30 September 2024, the contents of it being hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of cell biology. In particular, the present invention relates to genetically modified T cells and methods treating cancer.BACKGROUND OF INVENTION

[0003] Current cancer immunotherapies, including immune checkpoint blockades (ICBs) and adoptive cell therapy (ACT), such as chimeric antigen receptor T (CAR T) cell therapy, have delivered great promises for cancer therapy. While having shown clinical success in certain cancers, they are either not effective in treating most solid tumors or lack durable response resulting in fast tumor relapse.

[0004] Immune cell dysfunction within the solid tumor microenvironment undermines the control of cancer progression. Weak immune activation of T cells and exhaustion of T cells in the adverse solid tumor microenvironment limit the efficacy of immunotherapies in treating solid tumors. Therefore, there is an unmet need to provide methods or therapies to overcome the lack of response to immunotherapy in cancer, such as solid tumor cancer. Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the accompanying drawings and this background of the disclosure.SUMMARY

[0005] In one aspect, the present disclosure relates to a genetically modified T cell, comprising a genetic modification to P4HA1 gene that inhibits or inactivates the expression of of prolyl 4 hydroxylase subunit alpha 1 (P4HA1) in the T cell.

[0006] In another aspect, the present disclosure relates to a method of manufacturing a T cell as disclosed herein, wherein the method comprises modifying the expression of P4HA1 by genetic modification to the T cell that inhibits or inactivates the expression of P4HA1.

[0007] In another aspect, the present disclosure relates to a method of treating or alleviating cancer, comprising administering a therapeutically effective amount of the genetically modified T cell as disclosed herein to a subject in need thereof.

[0008] In another aspect, the present disclosure relates to a method of treating or alleviating cancer in a subject, comprising administering a therapeutically effective amount of a P4HA1 inhibitor to a subject in need thereof, wherein the cancer is resistant to treatment with an immune checkpoint inhibitor.

[0009] In yet another aspect, the present disclosure relates to a method of treating or alleviating cancer, comprising administering a combination comprising a therapeutically effective amount of a P4HA1 inhibitor and a therapeutically effective amount of a PD1 / PD-1L inhibitor to a subject in need thereof

[0010] In another aspect, the present disclosure relates to a method for determining endogenous T cell immunity in a subject receiving a cancer treatment, the method comprising measuring the expression level of P4HA1 in T cells using a sample obtained from the subject, wherein a decrease in the expression level of P4HA1 in T cells of the subject as compared to the expression level of P4HA1 in the T cells of the subject measured prior to receiving the cancer treatment is indicative that the subject has improved endogenous T cell immunity.

[0011] In another aspect, the present disclosure relates to a method for determining resistance to PD1 / PD-1L inhibitor treatment in a subject having cancer, the method comprising measuring the expression level of P4HA1 in T cells using a sample obtained from the subject, wherein a higher expression level of P4HA1 in the T cells of the subject as compared to a cancer patient responsive to PD1 / PD-1L inhibitor treatment is indicative that the subject is resistant to PD1 / PD-1L treatment.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0013] Figure 1 provides data showing transcriptome analysis identifying P4HA1 as a metabolic enzyme strongly induced in CD8 T cells upon activation, exhaustion, and hypoxia. Figure 1A provides a schematic diagram showing the process of CD8 CAR T cell activation expansion, followed by repeated cocultures with cancer cells. Naive CD8 cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and stimulated with anti- CD3 / CD28 and subsequently infected with CAR-virus. The anti-CD3 / CD28 and CAR virus were washed away on Day 5 and expanded in a medium with IL7 / 15 for another 7 days beforebeing subjected to repeated coculture under normoxia or hypoxia. CD8 CAR T cells were collected at indicated time points for RNA-seq. Figure IB provides a Venn Diagram and heatmap showing 37 genes commonly induced under different T cell conditions: early activation, repeated antigen stimulation, and hypoxia (fold change > 1.5, adjusted p < 0.05). Figure 1C provides a heatmap showing P4HA1, and other genes known in T cell regulation expressed in CD8 CAR T during cell T early activation, repeated normoxia, and hypoxic cocultures with or without treatment with a HIFla inhibitor, PX478. Figure ID shows the fold changes of the genes indicated in Figure 1C. Data are normalized to day 0. PX stands for PX478.

[0014] Figure 2 provides data showing P4HA1 expression in CD8 T cells obtained from blood sample of cancer patients. The figure shows FACS analysis of P4HA1+CD8“ T cells in the blood of treatment-naive patients with breast cancer of different stages. Figure 2 is an example showing that P4HA1 expression in T cell is correlated with cancer progression.

[0015] Figure 3 provides data validating the knockout of P4HA1 and PD1 in T cells using the Crispr knockout method. Figure 3A provides a schematic diagram showing the process of ex vivo CAR T cell generation, genetic modification, expansion and repeated tumor antigen stimulation in vitro. Peripheral blood mononuclear cells (PBMC) obtained from different healthy donors (n=5) were stimulated with anti-CD3 / CD28 followed by gene knockout and CAR-lentivirus transduction. Anti-CD3 / CD28 antibodies and CAR virus were washed away on Day 5, and EGFR CAR T cells were expanded in a medium with 1L7 / 15. Figures 3B and 3C provide representative images for the validation of P4HA1 knockout (P4HA1KO) and PD1 knockout (PD1KO) by Western blot analysis (Figure 3B) and flow cytometry analysis (Figure 3C), respectively. DKO stands for double knockout, wherein both P4HA1 and PD1 genes were knockout out.

[0016] Figure 4 provides data showing that P4HA1 knockout reprograms CAR CD8 T cells towards TCF1+progenitor sternness while ameliorating terminal exhaustion in EGRF CAR T cells. P4HA1 knockout CAR T cells and PD1 knockout CAR T cells are labelled as P4HA1KO and PD1KO on the graphs, respectively. Figure 4A shows the expansion kinetics of Epidermal Growth Factor Receptor (EGFR) CAR T cells post P4HA1 or PD 1 knockout. Figure 4B shows the flow cytometric analysis of memory (Tscmand Tcm) and effector (Tea) T cell percentage of CD8 CAR T cells on Day 7 of expansion. Cytogram on the left shows the gating strategy. The quantification results shown on the right panel are based on different donors (n=5 donors). Figure 4C shows the quantification of TCF1+CD8 CAR T cells on Day 7 (n=3 donors). Figure 4D shows the expansion kinetics of EGFR CAR T cells during repeated cocultures, i.e.,repeated tumor antigen stimulation. The EGFR CAR T cells were cocultured with DLD1 cells every 3 days at an Effector: Target ratio of 3: 1 (n=5 donors). Figure 4E provides flow cytometric analysis of IFNy and TNFa cytokine production in CD8 CAR T cells after the 5th coculture (n=5 donors). Figure 4F shows the cytolysis of DLD1 cancer cells by EGFR CAR T cells after 5th coculture from triplicated repeats. For CAR T cell expansion rate and flow cytometric analysis, data represent mean ± standard error of mean (s.e.m.) of n number of samples from each group (Figures 4A-4F). The p-values were determined using ANOVA followed by Tukey’s multiple comparisons test (Figures 4A-4F).

[0017] Figure 5 provides data showing that P4HA1 knockout reprograms CAR CD8 T cells towards TCF1+progenitor sternness while ameliorating terminal exhaustion in Trop2 CAR T cells. P4HA1 knockout CAR T cells and PD1 knockout CAR T cells are labelled as P4HA1KO and PD1KO on the graphs, respectively. Figure 5A shows the expansion kinetics of Trop2 CAR T cells with P4HA1 or PD1 knockout (n=2 donors). Figures 5B and 5C show flow cytometric analysis of memory (Tscmand Tcm) and effector (Teff) T cell populations (Figure 5B) and TCF1 expression (Figure 5C) in CD8 CART cells with P4HA1 or PD1 knockout, at Day 7 of expansion (n=5 repeats from 3 different donors). Figure 5D shows the expansion kinetics of Trop2 CAR T cells in repeated coculture with targeted MDA-MB-231 cells (n=3 repeats). Figure 5E shows flow cytometric analysis of fFNy and TNFa cytokines in CD8 Trop2 CAR T cells after 5th coculture (n=5 repeats from 3 different donors). Figure 5F shows cytolysis of MDA-MB-231 cancer cell by Trop2 CAR T cells after 5th coculture from triplicated repeats (n=5 repeats). For CAR T cell expansion rate and flow cytometric analysis, data represent mean ± standard error of mean (s.e.m.) of number of samples from each group. The p-values were determined using ANOVA followed by Tukey’s multiple comparisons test (Figure 5A-5F).

[0018] Figure 6 provides data to show thatP4HA1 accumulates in the mitochondria of CD8 T cells and disrupts tricarboxylic acid (TCA) cycle and mitochondria activity. Figure 6A shows western blot analysis of P4HA1 and other prolyl hydroxylase proteins in cytosolic and mitochondrial fractions at rest (rest) or upon anti-CD3 / CD28-stimulated CD8 CAR T cells (stimulation). ATP5A was used as a marker for mitochondria, and P-tubulin as a marker for cytosol. In this experiment, naive human CD8 cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors for CD8 CAR T cell generation. CAR T cells on Day 12 were re-stimulated with anti-CD3 / CD28 for indicated rounds and collected for western blot. CD8 CAR T cells on Day 12 without stimulation were collected as resting cells. Figure 6B shows the representative images of immunofluorescent staining of P4HA1 in CD8CAR T cells at different rounds of coculture. CD8, ATP5A (mitochondrial marker), and P4HA1. Colocalization of ATP5A and P4HA1 indicates that P4HA1 expression is increased at later rounds of coculture. Scale bar = 2 pm.

[0019] Figure 7 shows the effects of P4HA1 knockout on the fitness of mitochondria in CAR T cells. Figure 7A shows the representative images of mitochondria staining of CD8 CAR T cells at different rounds of cocultures. Scale bar = 2 pm. Figure 7B shows representative cytograms to demonstrate the gating strategy for MRhigh / MGhigh (healthy mitochondria) and MRlow / MGhigh (dysfunctional mitochondria) in CD8 CAR T cells at different rounds of coculture. Figure 7C provides flow cytometric quantification of healthy / dysfunctional mitochondria level in CD8 CAR T cells at different rounds of cocultures. CD8 T cells were genetically modified by P4HA1 knockout (n=3 donors). Figure 7D shows the cellular succinate level in rest or anti-CD3 / CD28 stimulated CD8 CAR T cells at cytosolic / mitochondrial fractions of T cells that were genetically modified by P4HA1 knockout (P4HA1K0; n=2 donors). Figure 7E shows a schematic diagram of P4HA1 -mediated succinate production in TCA cycle in CD8 T cells upon activation and exhaustion. Data are expressed as means ± standard error of mean (s.e.rn.) of n number of samples from each group. The p-values were determined using ANOVA followed by Tukey’s multiple comparisons test (Figure 7C and 7D).

[0020] Figure 8 provides data showing that ex vivo depletion of P4HA1 empowers CAR T in vivo efficacy and persistency in solid tumors. Figure 8A provides schematics of the experimental design showing ex vivo manipulation of P4HA1 in CAR T cells before injection into NSG mice bearing human tumor xenografts. Figure 8B shows flow cytometry assessment of P4HA1 expression in Trop2 CAR T cells in the blood collected on days indicated on the graph from naive NSG mice or NSG mice bearing MDA-MB-231 tumors. Trop2 CAR T cells before inoculation were nominated as day 0 (n=6 mice per group) Figure 8C shows the expression of P4HA1 by flow cytometry on CD8 HER2-CAR T cells in blood, spleen, and tumor of NSG mice bearing MDA-MB-361 tumors. CD8 HER2-CAR T cell before inoculation was included as day 0. Peripheral blood samples were collected on days indicated on the graph (n=6 per group), while spleen and tumor samples were collected on day 5 post-CAR T cells injection (n=5 per group). Figure 8D shows the flow cytometric analysis of Texcells: PD1+TIM3 TCFP (left) and Tpcxcells: PD1+TIM3-TCF1+(right) among CD8 HER2-CAR T cell in spleen and tumor of NSG mice bearing MDA-MB-361 tumors (n=5 per group). Samples were collected on day 35 post-CAR T injection. Figure 8E provides analysis of exhausted T cell population, PD1+Tim3+, expressing P4HA1” or P4HA1" within CD8 HER2-CAR T cellsinfiltrated into MDA-MB-361 tumor (n=5 per group). Figures 8F and 8G show the tumor progression (Figure 8F) and CAR T cell concentration in blood (Figure 8G) of DLD1 tumorbearing NSG mice treated with either vehicle or EGFR CAR T cell with negative control (NC), P4HA1 knockout (P4HA1K0-CAR T) or PD1 knockout (PD1K0-CAR T) Figure 8H shows the tumor volume of DLD1 colorectal tumor treated with vehicle (n=7) or EGFR 1 -CAR T cell over time with negative control (NC) (n=6), P4HA1K0 (n=6) or PD1KO (n=7). Each line represents an individual tumor. Data are expressed as means ± (standard error of mean) s.e.m. of n number of samples from each group. The p-values were determined using two-way ANOVA followed by Tukey’s multiple comparisons test.

[0021] Figure 9 provides data showing that ex vivo depletion of both P4HA1 and PD1 empowers CAR T in vivo efficacy and persistency in solid tumors. Figure 9A and 9B show tumor progression (Figure 9A) and CAR T cell concentration in blood and (Figure 9B) following treatment of Trop 2-CAR T cells to TNBC MDA-MB-231 -tumor bearing NSG mice. Figure 9C shows the survival rate of TNBC MDA-MB-231 in NSG mice treated with vehicle or control (NC-CAR T), P4HA1 knockout (P4HA1KO-CAR T), PD1 knockout (PD1KO-CAR T) and P4HA1-PD1 double knockout (DKO CAR T) Trop 2-CAR T cells. Figure 9D shows the time course of MDA-MB-231 tumor volume treated with vehicle (n=8) or Trop2-CAR T cell with negative control (NC) (n=6), P4HA1KO (n=6), PD1KO (n=4), or DKO (n=5). Each line represents an individual tumor. Data are expressed as means ± (standard error of mean) s.e.m. of n number of samples from each group. The p-values were determined using two-way ANOVA followed by Tukey’s multiple comparisons test (Figures 9A-9C).

[0022] Figure 10 provides data to show that P4HA1 inhibitor in vivo treatment remodels endogenous T cell immunity to suppress tumor recurrence and metastasis. The P4HA1 inhibitor used for this study is DPCA. Figure 10A provides the schematics of the 4T1 syngenic mouse model with neoadjuvant drug treatment followed by surgical removal of the tumors and monitoring of tumor recurrence. Figure 10B shows 4T1 tumor growth in mice treated with DPCA, anti-PD-1 antibody and a combination of DPCA and anti-PDl -antibody treatment before and post surgery as compared to the control group. The tumor was removed at day 14 post drug treatment, dotted line (n=10 for each group). Figure 10C shows individual tumor growth before and after surgery for each treatment group. The dotted line on the graphs in Figure 10B and 10C indicates the day the tumor resection was performed, which was on Day 14 post-tumor inoculation. The n-numbers and post-surgery relapse rates for each group are indicated in the graphs. Figure 10D provides representative images (left) and quantification of lung metastasis (right) using bioluminescence (n>5 for each group). Figure 10E shows therelapse-free survival rate of 4T1 bearing mice treated with DPCA, anti-PD-1 antibody or a combination of DPCA and anti-PDl -antibody as compared to the control group. (n>5 for each group). The p-values were determined by log-rank Mantel-Cox (survival) test. Figure 10F shows representative cytograms of TCF1 and TIM3 gating in PD1+CD44“ CD8 cells in 4T1 tumors. Figure 10G shows flow cytometric analysis of P4HA1+, Tpexcells (PD1+TIM3' TCF1“), and Texcells (PD1+T1M3+TCF1‘) CD8 cells in 4T1 tumors (n=7 per group). Figure 10H shows the flow cytometric analysis of overall CD8 T cell infiltration and IFNy+TNFa“ CD8 T cells in 4T1 tumors (n=7 per group). Figure 101 provides a schematic diagram showing the experimental design for CD8 T cell depletion followed by re-challenge with 4T1 cells. Figure 10J shows the tumor volume upon reinoculation of 4T1 cells (n>6 for each group). Figure 10K shows the flow analysis of CD8 Tscm(CD44’CCR7+Scal 1+), Tcm(CD44+CCR7+), and Teff (CD44+CCR7‘) in the blood of relapse-free mice after 4T1 re-challenge (n=4). Data are expressed as means ± standard error of mean (s.e.m.) of n number of samples from each group. The p-values were determined using ANOVA followed by Tukey’s multiple comparisons test (Figure 10E, 10G, and 10H).

[0023] Figure 11 provides data to show that treatment with the P4HA1 inhibitor, DPCA, reduces mouse tumor relapse and improves metabolic fitness in tumor-infiltrated CD8 cells. Figures 11A and 11B show individual CT26 tumor growth before and after surgery in mice treated with DPCA, anti-PD-1 antibody and a combination of DPCA and anti-PDl -antibody treatment as compared to the control group. The dotted line on the graphs indicates the time point where tumor resection was performed, which was on Day 14. The sample size (n number) and post-surgery relapse rates for each group are indicated in the graphs. Figure 11C shows the relapse free survival rate of CT26 tumor-bearing mice with the treatments indicated on the graph (n>5 for each group). The p-values were determined by log-rank Mantel-Cox (survival) test. Figure 1 ID shows the flow cytometric analysis of mitochondrial fitness of 4T1 -infiltrated CD8 T cells by Red-CMXRos (MR) and Green-FM (MG) signals (n=6 for each group). Data are expressed as means ± standard error of mean (s.e.m.) of n number of samples from each group. The p-values were determined using ANOVA followed by Tukey’s multiple comparisons test. Figure HE shows the 4T1 tumor growth of individual tumors before and after surgery which were treated with combination of DCPA and anti-PDl antibody (aPDl) and a combination of anti-PDl antibody (otPDl) and HD AC inhibitor Endostat (ENT), as compared to the control group. Surgery was performed on Day 14 as indicated by the black dotted line. The sample size indicated by the n number and relapse rate for each group were indicated in the graph. Figure HF shows the relapse free survival rate of 4T1 tumor-bearingmice with the treatment as indicated on the graph (n>5 for each group). The p-values were determined by log-rank Mantel-Cox (survival) test.

[0024] Figure 12 provides data to show that P4HA1 inhibitor boosts and sustains systemic immune memory response through CD8 T progenitors expansion in tumor-drained lymph node (TDLN). The P4HA1 inhibitor used in this experiment is DPCA. Figure 12A shows flow analysis of CD8 TScm (CD44'CCR7+Scal l+) and Tcm(CD44+CCR7+) in lymph node (LN) of naive mice or tumor-draining lymph node (TDLN) of 4Tl-bearing mice collected on Day 14 post the treatments indicated on the graph (n=7 per group). Figure 12B shows the time course flow analysis of blood CD8 Tscm(CD44'CCR7+Scal l+) and Tcm(CD44+CCR7“) in mice with indicated treatments (n=7 per group). Figure 12C shows the time course flow analysis of blood P4HA1+CD8 T cells and TCF1+CD8 T cells in mice with treatment indicated on the graph (n=6 per group). Figure 12D shows a receiver operating characteristics (ROC) curve to demonstrate classification performance of blood P4HA1+, TCF1+, and PD1+CD8 T cells as biomarkers to predict relapse. Area under curve (AUC) values were indicated in the graph. Figure 12E provides schematics of the mouse experiment with neoadjuvant drug treatment followed by surgery, with or with FTY720 to lock lymphoid T cells. Figure 12F shows data of tumor volume of 4T1 before and after surgery. The dotted line indicates the tumor resection on Day 14. N number and relapse rate for each group were indicated in the graphs. Figure 12G shows the relapse-free survival rate of 4T1 bearing mice with the indicated treatment, n>6 for each group. The p-values were determined by log-rank Mantel-Cox (survival) test. Figure 12H shows the flow analysis of P4HA1 and TCF1 expression in TDLN on Day 62 post-treatment. The n-numbers are the same as in Figure 12F. Figure 121 shows the flow analysis of CD8 T cell percentage in TDLN and blood harvested on Day 62 post-treatment. The n-numbers are the same as in Figure 12F. Data are expressed as mean ± standard error of mean (s.e.m.) of n number of samples from each group The p-values were determined using ANOVA followed by Tukey’s multiple comparisons test (Figure 12A, 12B, and 12H).

[0025] Figure 13 provides data showing that inhibition of P4HA1 by DPCA treatment boosted and prolonged systemic anti-tumor immunity. Figures 13A and 13B show the flow cytometric analysis of Tscm(CD44'CCR7+Scal l+) and Tcmcell (CD44+CCR7+) percentages in CD8 T cells obtained from the blood (Figure 13A) and tumors (Figure 13B) of naive mice or 4Tl-tumor bearing mice on Day 14 post-drug treatment (n=7 per group). Figure 13C shows flow cytometric analysis of Tscm(CD44’CCR7+Scal 1+) and Tcm(CD44+CCR7+) cell percentages in splenic CD8 cells of naive mice or 4T1 tumor-bearing mice at Day 14 post drug treatment (n=7 per group). Figure 13D shows the time course flow cytometric analysis ofP4HA1 expression in blood CD8 cells in the mice in control group as in Figure 12C. Each graph represents one mouse.

[0026] Figure 14 provides data to show that P4HA1 expression in CD8 cells correlates with tumor response to immune checkpoint blockades (ICB). Figure 14A shows the time course of tumor growth of MC38 tumors in mice that are responsive and non -responsive to treatment with anti-PDl antibody (ctPDl) and the Z-score of tumor growth rate of MC38 tumors in mice treated with anti-PDl inhibitor at Day 14 post treatment. A cut-off at -0.5 was used to define a responder and a non-responder to the anti-PD l inhibitor treatment. Figure 14B shows the flow cytometric analysis of P4HA1 and TCF1 expression, Tpexcell and Texcell percentage of 4T1 or MC38-infiltrated CD8 T cells. MC38 tumors were stratified to responders and nonresponders as in Figure 14A, n> 7 for each group. Both 4T1 and MC38 tumor samples were harvested at Day 14 post treatment. Figure 14C shows a simple linear correlation analysis of P4HA1 and TCF1 expression levels in MC38-infiltrated CD8 cells with tumor growth rate at Day 14 post treatment (n=27). Squared R and p-values provided on the plot were calculated based on linear repression analysis. Figure 14D shows the normalized average expression level of P4HA1 in CD8 T cells from tumor samples of melanoma patients stratified as responders and non-responders to immune checkpoint blockade (ICB) therapy (n>9 for each group). Analysis was based on scRNA-seq profiling database published by Sade-Feldman, et. al. Figure 14E shows the normalized average expression level of P4HA1 in CD8 T cells from blood or tumor samples of triple negative breast cancer (TNBC) patients stratified as responders and non-responders (n>4 for each group). Analysis was based on scRNA-seq profiling database published by Zhang, et. al.DEFINITIONS

[0027] As used herein, the term “T cell” or “T lymphocyte” refers to an important type of white blood cell and play a central role in the adaptive immune response. T cells are differentiated from hematopoietic stem cells, which are stem cells in the bone marrow. T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell surface. There are two major types of T cells: the CD4+T cells (“helper T cells”) and the CD8+T cells (“cytotoxic T cells”, or “killer T cells”). The CD8“ T cells are able to directly kill virus-infected cells, as well as cancer cells, and utilise cytokines to recruit other types of cells when mounting an immune response. Unlike the CD8+killer T cells, the CD4+cells function by further activating memory B cells and cytotoxic T cells, which leads to a larger immune response.

[0028] As used herein, the term “naive T cells” or “Tn cells” refers to the immature T cells that have differentiated in the thymus. After the encounter with its cognate antigen within the periphery, a naive T cell will be matured. The differentiation and activation of T cells is dependent on signals transduced by three different receptors: TCRs (including the CD4 and CD8 receptors that respond to MHC-II displayed antigens and MHC-I displayed antigens, respectively), costimulatory receptors, and cytokine receptors. These signals drive naive T cells to differentiate into effector T cells or memory T cells.

[0029] As used herein, the term “effector T cells” or “Teff cells” refers to a subset of T lymphocytes that have a relatively short lifespan. Effector T cells actively respond to a stimulus and carry out the functions of an immune response. Effector T cells can be cytotoxic T cells (CD8+), helper T cells (CD4+), and regulatory T cells (Treg).

[0030] As used herein, the term “memory T cells” refers to a subset of T lymphocytes that are capable of mediating a faster and more potent immune response upon encounter with antigens they have prior exposure to. These cells are long-lived and can quickly expand to large numbers of effector T cells to protect against subsequent exposure to the same antigen. Memory T cells can be CD4+cells, or CD8+cells, depending on the type of antigen encountered. Memory T cells comprise several subtypes. In general, the memory T cells include T memory stem cell (Tscm) cells and central memory T (Tcm) cells, which have different specific phenotypes and functions. The terms “T memory stem cell”, “TSCm cell”, “stem cell-like memory T cell” and “T progenitor memory cell” are used interchangeably.

[0031] As used herein, the term “central memory T cells” or “Tcm” refers to one subtype of the memory T cells which express L-selectin, CD45RO and CCR7, and provide central immunosurveillance by patrolling the lymph nodes draining peripheral tissue sites in the body. Central memory T cells have several attributes in common with stem cells, the most important being the ability of self-renewal, mainly because of high level of phosphorylation on key transcription factor STAT5.

[0032] As used herein, the term “effector memory T cells” or “Tem” refers to another subtype of the memory T cells which express CD45RO but lack expression of CCR7 and L- selectin. Due to the lack the CCR7 lymph node-homing receptors, unlike central memory T cells, effector memory T cells are found in the peripheral circulation and tissues. Effector memory T cells are primarily active as tire CD8 variants, thus being mainly responsible for cytotoxic action against pathogens Temcells express higher levels of receptors responsible for migration to inflamed tissues and have a stronger immediate effector function than Tcmcells.

[0033] As used herein, the term “progenitor exhausted T cells” or “Tpex cells” refers to a specific type of CD8“ T cells that exhibit features of both exhaustion and stem-like properties. Tpex cells embody many functional properties of conventional memory CD8+T cells, such as the capacity to proliferate, differentiate, persist and also undergo self-renewal. They are characterized by the expression of TCF1 and PD-1, but not TIM-3; i.e., Tpex cells are PD1+TIM3'TCF1‘ CD8 T cells.

[0034] As used herein, the term “terminally exhausted T cells” or “Texcells” refers to a subset of dysfunctional T cells, characterized by a severely diminished ability to kill cells or to produce cytokines. Texcells are often found in chronic infections or tumors and represent the end stage of the T cell exhaustion process. Tcxcells are characterized by expression of PD-1 and TIM-3, but not TCF1 , i.e., they are PD1+TIM3+TCFT CD8 T cells.

[0035] As used herein, the term “exhaustion” refers to a dysfunctional and hypo-responsive cellular state commonly observed in response to persistent antigen exposure, for example, in chronic infection or cancer. In the context of the present disclosure, “T cell exhaustion” refers to such a state associated with T cells characterized by progressive loss of T cell effector functions and self-renewal capacity in a tumor microenvironment, thereby limiting the efficacy of immunotherapy. In other words, T cell exhaustion is a state of T cell dysfunction wherein the T cells lost their ability to effectively kill cells. Therefore, T cell exhaustion is often associated with poor tumor control in patients.

[0036] As used herein, the term “immunotherapy” refers to a method of treatment or prevention of disease by stimulation of the immune system to activate or suppress an immune response. The term “cellular immunotherapy”, or “adoptive cell therapy” refers to a type of immunotherapy in which patients’ own immune cells are given to the patients to help the body fight diseases such as cancer. The immune cells can be expanded ex vivo to improve in the total number of cells or are engineered to target specific tumor cell types. The term “adoptive T cell immunotherapy therapy” refers to an adoptive cell therapy that utilizes T cells.

[0037] As used herein, the term “immune checkpoint blockade” or “1CB” is an example of an immunotherapy approach which blocks checkpoint proteins on immune cell, preventing them from suppressing the immune response against cancer cell. Immune checkpoint blockade allows the immune system to better recognize and attack tumors or cancer cells. Some examples of immune checkpoint proteins include, but are not limited to PD-1, PD-L1, CTLA- 4 or LAG-3.

[0038] As used herein, the term “CAR” or “chimeric-antigen-receptor” refers to a recombinant receptor for antigens which redirect the specificity and function of T lymphocytesand / or other immune cells in a single molecule such that they are programmed to target tumor- associated antigens. Chimeric antigen receptors (CARs) usually consist of an extracellular domain that binds to a specific antigen on tumor cells, a transmembrane domain and intracellular domains that provide signals for T cell activation to attack tumor cells.

[0039] As used herein, the term “CAR-T cells” or “chimeric antibody receptor engineered T cell” refers to engineered T cells that express cancer specific artificial chimeric-antigen- receptor (CAR), which can be used in an adoptive T cell immunotherapy therapy. The T cells can be obtained from patient’s blood and are produced ex vivo. Large number of CAR-T cells are given to the patient by infusion to treat diseases such as cancer.

[0040] As used herein, the term “tumor microenvironment” or “TME” refers to the complex ecosystem surrounding a tumor, including cancer cells, stromal cells, immune cells, and the extracellular matrix. It is characterized by hypoxia, acidity, nutrient deficiency, and immunosuppression. It is an environment where cancer cells interact with other cells and structures, influencing tumor growth, metastasis, and response to therapy.

[0041] As used herein, the term “tumor-draining lymph nodes” or “TDLN” refers to lymph nodes that are closest to a primary tumor. Tumor-draining lymph nodes first to receive cancer cells and tumor-related antigens that have travelled through the lymphatic system. They play a crucial role in both anti-tumor immunity and cancer metastasis.

[0042] As used herein, the term “prolyl 4-hydroxylase alpha subunit 1” or “P4HA1” refers to the alpha catalytic subunit of an alpha-ketoglutarate (a-KG)-dependent metabolic enzyme, collagen prolyl 4 hydroxylase (C-P4H), which plays a role in catalysing collagen synthesis and succinate by-production. P4HA1 is encoded by the P4HA1 gene. P4HA1 requires assembly with the beta-subunit, P4HB to form the enzymatically active C-P4H. The term “P4H” broadly refers to multiple enzymes, including C-PH4 and other HIF propyl hydroxylases such as PHD1 / 2 / 3 Thus, P4HA1 being a subunit of C-PH4 is a distinctly different from P4H, which refers to a group of enzymes.

[0043] As used herein, the term “tricylic acid cycle” or “TCA cycle” refers to a central metabolic pathway in the cell. It comprises 8 enzymes within the mitochondrial matrix except the outlier succinate dehydrogenase, which is related to the respiratory chain on the inner mitochondrial membrane. The cycle is a gateway for aerobic metabolism for molecules that can convert to an acetyl group or dicarboxylic acid. Regulation of the TCA cycle occurs at 3 distinct points, including the following enzymes: citrate synthase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase.DETAILED DESCRIPTION

[0044] Effective and durable cancer immunotherapy relies on both intratumoral and systemic immune responses. For instance, tumor-draining lymph nodes (TDLN) have been known to be crucial for long-term immunotherapy outcomes Preclinical studies have shown that CD8+T progenitor memory cells generated from tumor-draining lymph nodes (TDLN) can serve as a central hub to pool progenitor exhausted T cells (Tpexcells) and memory T cells into tumor microenvironment for effective antitumor response to immunotherapy treatments. However, it is difficult to achieve effective intratumoral and systemic immune response for patients with solid tumor due to immune cell dysfunction within the solid tumor microenvironment (TME) which undermines the control of cancer progression.

[0045] CD8 T cell differentiation trajectory in cancer comprises an initial priming or activation phase of naive T cells in the tumor-draining lymph node (TDLN) followed by effector differentiation and exhaustion within the tumor. This process can be further influenced by hypoxia in solid tumors, which is known to facilitate CD8 T cell exhaustion and immune escape of the cancer cells. As described in the present disclosure, reduction in T cell sternness and increased in T cell exhaustion can be responsible for the ineffective immune response.

[0046] In light of the ineffective T cell response in solid tumor microenvironment, which undermines intratumoral immune response, the present disclosure identifies a T cell differentiation and exhaustion regulator, prolyl 4 hydroxylase subunit alpha 1 (P4HA1, see Figure 1). P4HA1 is the alpha subunit of an alpha-ketoglutarate (aKG)-dependent metabolic enzyme that catalyzes collagen synthesis and succinate by-production. Given P4HA1 function in regulating T cell differentiation and T cell exhaustion as described in the present disclosure, the present disclosure identifies P4HA1 as a therapeutic target in modulating both adaptive and endogenous T cell activity for treating solid tumor.

[0047] Therefore, in one aspect, the present disclosure provides a genetically modified T cell, comprising a genetic modification to P4HA1 gene that inhibits or inactivates expression of prolyl 4 hydroxylase subunit alpha 1 (P4HA1) in the T cell. In some examples, the T cells being modified as disclosed herein can be CD8+T cells, which are also known as cytotoxic T lymphocytes. The genetically modified T cell, comprising the genetic modification to P4HA1 gene that inhibits or inactivates expression of P4HA1 can further comprise a genetic modification to PD1 gene that inhibits or inactivates the expression of programmed cell death- 1 protein (PD1) in the T cell. Thus, the genetically modified T cell comprises genetic modifications that inhibit or inactivate the expression of both P4HA1 and PD1.

[0048] Expression of the P4HA1 and / or PD1 gene can be inhibited to reduce the expression of their protein product in the genetically modified T cell or, in some examples, inactivated, such that the gene does not express the protein product or express an active protein product. In some examples, a gene can be inactivated by a gene deletion. As used herein, "gene deletion" refers to removal of at least a portion of a DNA sequence from, or in proximity to, a gene. In some examples, the sequence subjected to gene deletion comprises an exonic sequence of a gene. In some examples, the sequence subjected to gene deletion comprises a promoter sequence of the gene. In some examples, the sequence subjected to gene deletion comprises a flanking sequence of a gene. In some examples, a portion of a gene sequence is removed from a gene. In some examples, the complete gene sequence is removed from a chromosome. In some examples, the T cell comprises a gene deletion as described in any of the examples herein. In some examples, the gene is inactivated by deletion of at least one nucleotide or nucleotide base pair in a gene sequence results in a non-functional gene product. In some examples, the gene is inactivated by a gene deletion, wherein deletion of at least one nucleotide to a gene sequence results in a gene product that no longer has the original gene product function or activity; or is a dysfunctional gene product. In some examples, the gene is inactivated by a gene addition or substitution, wherein addition or substitution of at least one nucleotide or nucleotide base pair into the gene sequence results in a non-functional gene product In some examples, the gene is inactivated by a gene inactivation, wherein incorporation or substitution of at least one nucleotide to the gene sequence results in a gene product that no longer has the original gene product function or activity; or is a dysfunctional gene product. In some examples, the gene is inactivated by an addition or substitution, wherein incorporation or substitution of at least one nucleotide into the gene sequence results in a dysfunctional gene product. Methods for modifying the expression of genes in general are known in the art. In some examples, the T cell as claimed herein comprises a gene deletion as described in any of the examples disclosed herein.

[0049] Methods and techniques for inhibiting or inactivating P4HAI gene and / or PD1 gene as described herein that can genetically modify the expression of P4HA1 protein and PD1 protein in a T cell can include, but are not limited to, small interfering RNA (siRNA), small hairpin RNA (shRNA; also referred to as a short hairpin RNA), clustered, regularly interspaced, short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), zine-finger nuclease (ZFN), homologous recombination, non-homologous endjoining, and meganuclease.

[0050] In some examples, the P4HA1 or PD1 gene can be inactivated by a small interfering RNA (siRNA) system As can be appreciated by a person skilled in the art, siRNA sequences to inactivate a target gene can be identified using considerations such as length of siRNA, e.g., 21-23 nucleotides, or fewer; avoidance of regions with 50-100 nucleotides of the start codon and termination codon, avoidance of intron regions, avoidance of stretches of four or more of the same nucleotide; avoidance of regions with GC content that is less than 30% or greater than 60%; avoidance of repeats and low sequence complexity region; avoidance of single nucleotide polymorphic sites, and avoidance of sequences that are complementary to sequences in other off-target genes.

[0051] In some examples, the siRNA system comprises a siRNA nucleotide sequence that is about 10 to 200 nucleotides in length, or about 10 to 100 nucleotides in length, or about 15 to 100 nucleotides in length, or about 10 to 60 nucleotides in length, or about 15 to 60 nucleotides in length, or about 10 to 50 nucleotides in length, or about 15 to 50 10 nucleotides in length, or about 10 to 30 nucleotides in length, or about 15 to 30 nucleotides in length. In some examples, the siRNA nucleotide sequence is about 10 to 25 nucleotides in length. In some examples, the siRNA nucleotide sequence is about 15 to 25 nucleotides in length. In some examples, the siRNA nucleotide sequence is at least about 10, at least about 15, at least about 20, or at least about 25 nucleotides in length. In some examples, the siRNA system comprises a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% complementary to a region of a target mRNA molecule. In some examples, the siRNA system comprises a nucleotide sequence that is at least at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% 20 complementary to a region of the target pro-mRNA molecule. In some examples, the siRNA system comprises a double stranded RNA molecule. In some examples, the siRNA system comprises a single stranded RNA molecule. In some examples, the T cell comprises a siRNA system as described in any of the examples herein. In some examples, the T cell comprises a pro-siRNA nucleotide sequence that is processed into an active siRNA molecule as described herein. In some examples, the T cell comprises a siRNA nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% complementary to a region of the target mRNA molecule. In some examples, the T cell comprises an expression vector encoding a siRNA molecule as described in any of the examples disclosed herein. In some examples, the T cell comprises an expression vector encoding a pro-siRNA molecule as described in any of the examples herein.

[0052] In some examples, the siRNA system comprises a delivery vector. In some examples, the T cell comprises a delivery vector. In some examples, the delivery vector comprises the pro-siRNA and / or siRNA molecule. The vector encodes for the siRNA molecule used to inhibit or inactivate P4HA1 gene and / or PD1 gene.

[0053] In some examples, the P4HA1 gene or PD1 gene can be inhibited or inactivated by a small hairpin RNA (shRNA; also referred to as a short hairpin RNA) system. In some examples, the shRNA system comprises a nucleotide sequence that is about 10 to 200 nucleotides in length, or about 10 to 100 nucleotides in length, or about 15 to 100 nucleotides in length, or about 10 to 60 nucleotides in length, or about 15 to 60 nucleotides in length, or about 10 to 50 nucleotides in length, or about 15 to 50 nucleotides in length, or about 10 to 30 nucleotides in length, or about 15 to 30 nucleotides in length. In some examples, the shRNA nucleotide sequence is about 10 to 25 nucleotides in length. In some examples, the shRNA nucleotide sequence is about 15 to 25 nucleotides in length. In some examples, the shRNA nucleotide sequence is at least about 10, at least about 15, at least about 20, or at least about 25 nucleotides in length. In some examples, the shRNA system comprises a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% complementary to a region of P4HA1 or PD1 mRNA molecule. In some examples, the shRNA system comprises a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% complementary to a region of a pro-mRNA molecule. In some examples, the shRNA system comprises a double stranded RNA molecule. In some examples, the shRNA system comprises a single stranded RNA molecule. In some examples, the T cell comprises a shRNA system as described in any of the examples herein. In some examples, the T cell comprises a pre-shRNA nucleotide sequence that is processed in an active shRNA nucleotide sequence as described in any of the examples herein. In some examples, the pro-shRNA molecule composed of DNA. In some examples, the pro-shRNA molecule is a DNA construct. In some examples, the T cell comprises a shRNA nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% complementary to a region of the T-cell inhibitory gene mRNA molecule. In some examples, the T cell comprises an expression vector encoding a shRNA molecule as described in any of the examples disclosed herein In some examples, the T cell comprises an expression vector encoding a pro-shRNA molecule as in any of the examples disclosed herein.

[0054] In some examples, the shRNA system comprises a delivery vector. Tn some examples, the host comprises a delivery vector. In some examples, the delivery vector comprises the pro-shRNA and / or shRNA molecule. In some examples, the delivery vector is avirus vector. In some examples, the delivery vector is a lentivirus. In some examples, the delivery vector is an adenovirus. In some examples, the vector comprises a promoter.

[0055] As disclosed herein, examples of siRNA sequences for inhibiting or inactivating P4HA1 expression can include, but are not limited to the sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. Also disclosed herein are examples of siRNA sequences for inhibiting or inactivating PD1 expression, which can include but are not limited to sequences of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. Exemplary sequences of siRNA for inhibiting or inactivation P4HA1 or PD1 expression are provided in Table 1 below.

[0056] Table 1 : Nucleotide sequences of siRNA for inhibiting or inactivation P4HA1 or PD1 expression.

[0057] In some examples, genetic modification to inhibit or inactivate the expression of P4HA1 gene orPDl gene is accomplished using CRISPR / CAS methodology. A CRISPR / Cas system includes a Cas protein and at least one to two ribonucleic acids (RNA) that hybridize to a target motif in the P4HA1 or PD1 gene and direct the Cas protein to the target motif. Any CRISPR / Cas system that is capable of altering a target polynucleotide sequence in a cell can be used. In some examples, the CRISPR Cas system is a CRISPR type I system, in some examples, the CRISPR / Cas system is a CRISPR type II system. In some examples, the CRISPR / Cas system is a CRISPR type V system.

[0058] The Cas protein used in the present disclosure can be a naturally occurring Cas protein or a functional derivative thereof. A "functional derivative" includes, but is not limited to fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof such as derivative Cas proteins. Suitable derivatives of a Cas polypeptide or a fragment thereofinclude but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof

[0059] There are three main types of Cas nucleases (type I, type II, and type III), and 10 subtypes including 5 type I, 3 type II, and 2 type III proteins. Type II Cas nucleases include Casl, Cas2, Csn2, and Cas9. These Cas nucleases are known to those skilled in the art. Nonlimiting examples of Cas nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, variants thereof, mutants thereof, and derivatives thereof.

[0060] Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to bacteria of the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Variants of any of the Cas9 nucleases provided herein can be optimized for efficient activity or enhanced stability in the host cell. Thus, engineered Cas9 nucleases are also contemplated. Cas 9 from Streptococcus pyogenes contains 2 endonuclease domains, including an RuvC-like domain that cleaves target DNA that is noncomplementary to crRNA, and an HNH nuclease domain that cleave target DNA complementary to crRNA. The double-stranded endonuclease activity of Cas9 also involves a short conserved sequence, (2-5 nucleotides), known as a protospacer-associated motif (PAM), which follows immediately 3"- of a target motif in the target sequence

[0061] As used herein, the term "Cas9 ribonucleoprotein" complex and the like refers to a complex between the Cas9 protein and a guide RNA (gRNA), the Cas9 protein and a crRNA, the Cas9 protein and a trans-activating crRNA (tracrRNA), or a combination thereof (e.g., a complex containing the Cas9 protein, a tracrRNA, and a crRNA guide RNA). It is understood that in any of the examples described herein, a Cas9 nuclease can be substituted with another RNA-mediated nuclease, e.g., an alternative Cas protein or a Cpfl nuclease.

[0062] In some examples, the Cas protein is introduced into T-cells in polypeptide form. Thus, in certain examples, the Cas proteins can be conjugated to or fused to a cell-penetrating polypeptide or cell-penetrating peptide that is well known in the art. In some cases, T cells may be genetically engineered to produce the Cas protein In some examples, the Cas9 nuclease and the gRNA can be introduced into the T cell as a ribonucleoprotein (RNP) complex. In someexamples, the T cells are cultured under conditions effective for expanding the population of genetically modified T cells.

[0063] Also disclosed herein is a population of T cells, in which the genome comprises genetic modifications to P4HA1 gene and / or PD1 gene or in which the T cell comprises heterologous polynucleotide that inhibits expression of P4HA1 gene and / or PD1 gene or P4HA1 and / or protein.

[0064] In some examples, the ribonucleoprotein (RNP) complex can be introduced into the T cells by electroporation. Methods, compositions, and devices for electroporating cells to introduce a RNP complex are available in the art. In some examples, the Cas9 protein can be in an active endonuclease form, such that when bound to target nucleic acid as part of a complex with a guide RNA (gRNA) or part of a complex with a DNA template, a double strand break is introduced into the target nucleic acid. In the methods disclosed herein, a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide can be introduced into the T cells. In some examples, the Cas9 protein is a nickase, such that when bound to target nucleic acid as part of a complex with a guide RNA (gRNA), a single strand break or nick is introduced into the target nucleic acid. A pair of Cas9 nickases, each bound to a structurally different guide RNA, can be targeted to two proximal sites of a target genomic region and thus introduce a pair of proximal single stranded breaks into the target genomic region.

[0065] In some examples, the Cas nuclease can be a high-fidelity or enhanced specificity Cas9 polypeptide variant with reduced off-target effects and robust on-target cleavage. Nonlimiting examples of Cas9 polypeptide variants with improved on-target specificity known in the art include the SpCas9 (K855A), SpCas9 (K810A / K1003A / R1060A) (also referred to as eSpCas9(1.0)), and SpCas9 (K848A / K1003A / R1060A) (also referred to as eSpCas9(l.l)) and the SpCas9 variants containing one, two, three, or four of the following mutations: N497A, R661 A, Q695A, and Q926A (e g., SpCas9-HFl contains all four mutations).

[0066] In some examples, the target motifs can be selected to minimize off-target effects of the CRISPR / Cas systems. For example, in some examples, the target motif is selected such that it contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some examples, the target motif is selected such that it contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. Those skilled in the art will appreciate a variety of techniques that can be used to select suitable target motifs for minimizing off-target effects (e g., bioinfor atics analyses).

[0067] As used throughout the present disclosure, a guide RNA (gRNA) sequence is a sequence that interacts with a site-specific or targeted nuclease and specifically binds to orhybridizes to a target nucleic acid within the genome of a cell, such that the gRNA and the targeted nuclease colocalize to the target nucleic acid in the genome of the cell. Each gRNA includes a DNA targeting sequence or protospacer sequence of about 10 to 50 nucleotides in length that specifically binds to or hybridizes to a target DNA sequence in the genome. For example, the targeting sequence may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some examples, the gRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence. In some examples, the gRNA does not comprise a tracrRNA sequence.

[0068] The gRNAs can be selected depending on the particular CRISPR / Cas system employed, and the sequence of the target polynucleotide, as will be appreciated by those skilled in the art. As indicated above, in some examples, the one to two ribonucleic acids (RNA) can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some examples, the one to two ribonucleic acids (RNA) hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell In some examples, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some examples, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent 30 to a deoxyribonucleic acid motif recognized by the Cas protein. In some examples, each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs. Guide RNAs (gRNAs) can also be designed using softwares that are readily available. The one or more gRNAs can be transfected into T cells in which Cas protein is present by transfection, according to methods known in the art

[0069] In some examples, the DNA targeting sequence can incorporate wobble or degenerate bases to bind multiple genetic elements. In some cases, the 19 nucleotides at the 3' or 5' end of the binding region are perfectly complementary to the target genetic element or elements. In some examples, the binding region can be altered to increase stability. For example, non-natural nucleotides, can be incorporated to increase RNA resistance to degradation. In some cases, the binding region can be altered or designed to avoid or reduce secondary structure formation in the binding region. In some cases, the binding region can be designed to optimize G-C content. In some examples, the G-C content is preferably between about 40% and about 60% (e.g., 40%, 45%, 50%, 55%, 60%).

[0070] In some examples, the sequence of the gRNA or a portion thereof is designed to complement (e.g., perfectly complement) or substantially complement (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% complement) the target region of the P4HA1 gene or PD1 gene. In some examples, the portion of the gRNA that complements and binds the targeting region in the polynucleotide is, or is about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more nucleotides in length. In some examples, the portion of the gRNA that complements and binds the targeting region in the polynucleotide is between about 19 and about 21 nucleotides in length. In some examples, the gRNA may incorporate wobble or degenerate bases to bind target regions. In some examples, the gRNA can be altered to increase stability For example, non-natural nucleotides, can be incorporated to increase RNA resistance to degradation. In some examples, the gRNA can be altered or designed to avoid or reduce secondary structure formation. In other cases, the gRNA can be designed to optimize G-C content. In some examples, G-C content is between about 40% and about 60% (e.g., 40%, 45%, 50%, 55%, 60%). In some examples, the binding region can contain modified nucleotides such as, without limitation, methylated or phosphorylated nucleotides.

[0071] In some examples, the gRNA can be optimized for expression by substituting, deleting, or adding one or more nucleotides. In some examples, a nucleotide sequence that provides inefficient transcription from an encoding template nucleic acid can be deleted or substituted. For example, in some cases, the gRNA is transcribed from a nucleic acid operably linked to an RNA polymerase III promoter. In such examples, gRNA sequences that result in inefficient transcription by RNA polymerase III can be deleted or substituted. For example, one or more consecutive uracils can be deleted or substituted from the gRNA sequence. In some examples, if the uracil is hydrogen bonded to a corresponding adenine, the gRNA sequence can be altered to exchange the adenine and uracil. This "A-U flip" can retain the overall structure and function of the gRNA molecule while improving expression by reducing the number of consecutive uracil nucleotides.

[0072] In some examples, the gRNA can be optimized for stability. Stability can be enhanced by optimizing the stability of the gRNA: nuclease interaction, optimizing assembly of the gRNAmuclease complex, removing or altering RNA destabilizing sequence elements, or adding RNA stabilizing sequence elements. In some examples, the gRNA contains a 5' stemloop structure proximal to, or adjacent to, the region that interacts with the gRNA-mediated nuclease. Optimization of the 5’ stem-loop structure can provide enhanced stability or assemblyof the gRNA:nuclease complex. In some examples, the 5' stem-loop structure is optimized by increasing the length of the stem portion of the stem-loop structure

[0073] gRNAs can be modified by methods known in the art. In some examples, the modifications can include, but are not limited to, the addition of one or more of the following sequence elements: a 5' cap (e.g., a 7-methylguanylate cap); a 3' polyadenylated tail; a 20 riboswitch sequence; a stability control sequence; a hairpin; a subcellular localization sequence; a detection sequence or label; or a binding site for one or more proteins. Modifications can also include the introduction of non-natural nucleotides including, but not limited to, one or more of the following: fluorescent nucleotides and methylated nucleotides.

[0074] In some examples, expression cassettes and vectors for producing gRNAs can be introduced into a T cell The expression cassettes can contain a promoter (e g., a heterologous promoter) operably linked to a polynucleotide encoding a gRNA. The promoter can be inducible or constitutive. The promoter can be tissue specific. In some cases, the promoter is a U6, Hl, or spleen focus-forming virus (SFFV) long terminal repeat promoter. In some cases, the promoter is a weak mammalian promoter as compared to the human elongation factor 1 promoter (EFl A). In some cases, the weak mammalian promoter is a ubiquitin C promoter or a phosphoglycerate kinase 1 promoter (PGK). In some cases, the weak mammalian promoter is a TetOn promoter in the absence of an inducer. In some examples, when a TetOn promoter is utilized, the T cell is also contacted with a tetracycline transactivator. In some examples, the strength of the selected gRNA promoter is selected to express an amount of gRNA that is proportional to the amount of Cas9 or dCas9. The expression cassette can be in a vector, such as a plasmid, a viral vector, a lentiviral vector, etc. In some cases, the expression cassette is in the T cell. The gRNA expression cassette can be episomal or integrated in the T cell.

[0075] As disclosed herein, examples of gRNA sequences for targeting P4HA1 gene can include but are not limited to the sequences of SEQ IDNO: 1 or SEQ ID NO: 2. Also disclosed herein are examples of gRNA sequences for targeting PD1 gene, which can include but are not limited to sequences of SEQ ID NO: 3 or SEQ ID NO:4. The sequences of the P4PIA1 and PD-1 gRNA are provided in Table 1 below.

[0076] Table 2: Nucleotide sequences of P4HA1 and PD-1 gRNA

[0077] In another aspect, the genetically modified T cells as disclosed herein, which comprises a genetic modification to P4HA1 gene that inhibits or inactivates expression of P4HA1 or genetic modifications to P4HA1 and PD1 genes that inhibits or inactivates expression of P4HA1 and PD1, further comprises a genetic modification to express a chimeric antigen receptor (CAR) on the surface of the T cell. In such a case, the genetically modified T cell as disclosed herein expressing a CAR is referred to as a CAR T cell.

[0078] A CAR T cell can be obtained from a genetically modified T cell as disclosed herein by introducing a CAR transgene into the genetically modified T cell to produce a CAR-T cell Methods of designing a CAR transgene and methods of introducing a CAR transgene into T cells are known in the art. The CAR transgene comprises a target sequence that allows recognition and clearance of tumor cells by CAR-T cells. There are a variety of target sequences available for cellular immunotherapy, such as C-type lectin-like molecule-1 (CLL- 1), CD 19, CD20, B cell maturation antigen (BCMA), HER2, R0R1, EGFP1. Methods of designing a CAR transgene and methods of introducing a CAR transgene into T cells are known in the art. The CAR transgene comprises a target sequence that allows recognition and clearance of tumor cells by CAR-T cells. There are a variety of target sequences available for cellular immunotherapy, such as C-type lectin-like molecule- 1 (CLL-1), CD 19, CD20, B cell maturation antigen (BCMA), HER2, ROR1, EGFP1 or Trop 2. The present disclosure in Figure 9, for example, provides modified CAR-T cells targeting Trop2 for treatment of breast cancer. The present disclosure in Figure 8 also provides, for example, CAR-T cells targeting EGFR for treatment of colorectal cancer. Based on the disease to be treated and the genotype of the tumor specifically targeted, a person skilled in the art would be able to design a suitable CAR transgene for targeting the tumor to be treated.

[0079] Genetic modification for introduction of the CAR transgene into T cells or the genetically modified T cell as disclosed herein can be accomplished by transducing (or otherwise delivering) T cells with a recombinant DNA or RNA construct encoding the CAR, such as for example, a vector. A vector can be any agent capable of delivering or maintaining nucleic acid in a host cell, and includes viral vectors (e g. retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors), plasmids, naked nucleic acids, nucleic acids complexed with polypeptide or other molecules and nucleic acids immobilized onto solid phase particles. Preferably, a retroviral vector (either gamma-retroviral or lentiviral) isemployed for the introduction of the CAR transgene into the cell. Non- viral vectors may be used as well. As disclosed herein, the present disclosure provides a CAR T cell which is an anti-EGRF CAR-modified T cell. In another example, the CAR T cell is anti-HER2 CAR- modified T cell. In another example, the CAR T cell is anti-Trop2 CAR-modified T cells. The sequences of the single-chain variable fragment (scFv) of some examples of CAR are provided in Table 2. The scFv refers to the extracellular tumor-targeting component of the CAR transgene.

[0080] Table 3: Nucleotide sequences of the scFv of the CAR

[0081] In another aspect, the present disclosure provides a method of manufacturing a genetically modified T cell as disclosed herein. In one example, the method comprises modifying the expression of P4HA1 by genetic modification of the nucleotide sequence encoding P4HA1. In another example, the method further comprises modifying the expression of PD 1 by genetic modification of the nucleotide sequence encoding PD 1. In another example, the method comprises introducing into the T cell a ribonucleoprotein complex and a nucleic acid sequence encoding P4HA1 m / o^PDl gRNA targeting the genomic sequence of P4HA1 and / or PD1; or introducing a nucleic acid encoding an siRNA, an shRNA, a microRNA or an oligonucleotide targeting the P4PIA1 mRNA. In some examples, the ribonucleoprotein complex comprises a Cas9 protein and & P4HA 1 gRNA. In another example, the method further comprises comprising introducing into the T cells a nucleic acid encoding a chimeric antigen receptor. The introducing of nucleic acid or a ribonucleoprotein complex and gRNA can be accomplished by the methods and techniques as disclosed herein

[0082] The genetically modified T cells comprising genetic modification that inhibits or inactivates P4HA1 expression as disclosed herein are characterized by increased expansion rate, improved T cell sternness, increased population of T memory cell, increased antigenspecific cytotoxicity and / or reduced T cell exhaustion compared to unmodified T cells (see Figure 4). For example, the inhibition or inactivation of P4HA1 expression in the geneticallymodified T cells increases the sternness of the modified T cells and reduces terminal exhaustion of the modified T cells when subjected to repeated tumor antigen stimulation or the T cells (see Figure 4). Therefore, in one example, modifying the T cells by the inhibition or inactivation of P4HA1 expression increases the expansion rate of the T cells compared to unmodified T cells (Figure 4A). In another example, modifying T cells by the inhibition or inactivation of P4HA1 expression increases the percentage of T memory stem (TSCm) cell and T central memory (Tcm) cell populations in the expanded T cell population (Figure 4B). In another example, the inhibition or inactivation of P4HA1 expression in T cells increases TCF1 expression in the expanded T cell population (Figure 4C). In another example, the inhibition or inactivation of P4HA1 expression shows persistent expansion of the T cells upon repeated antigen stimulation as compared to control T cells (Figure 4D). In this example, CAR T cells that were obtained from the genetically modified T cells as disclosed herein showed enhanced T cell function, marked by enhanced cytotoxicity and improved cell lysis (Figure 4E and 4F; Figure 5E and 5F). The foregoing examples show that genetic modification that inhibits or inactivates of P4HA1 expression in T cells can increase T cell sternness and reduce terminal exhaustion in response to persistent antigen-induced stimulation. The foregoing examples shown in Example 3 (Figures 3 and 5) indicate that genetic modification to inhibit or inactivate the expression of P4HA1 in T cell is able to target and reprogram CD8 T cells towards TCF 1 progenitor expansion, which enhances the persistence of memory T cells and antitumor immunity of CD8 T cells. Accordingly, CAR T cells obtained from the genetically modified T cells showed increased cytotoxicity and effectiveness in killing cancer cells. In other words, in the examples as shown herein, inhibiting or inactivating P4HA1 expression in CD8 T cells drives T cell differentiation toward a TCF1+progenitor-like state, thereby increasing a renewable pool of anti -tumor T cells. The TCF1+population of T cells has the ability to self-renew, sustain proliferation and continuously generate cytotoxic effector T cells upon encounterment of tumor antigen. Thus, reducing the dysfunctional T cell exhaustion pool (Texcells) within the tumor microenvironment. In addition, T progenitor cells could be reinvigorated in tumor draining lymph node (TDLN), migrate to the tumor and generate more functional effector T cells to kill the cancer cells. Hence inhibiting P4HA1 in CD8 T cells also provides a pool of sustainable functional T cells. Collectively, the inhibition or inactivation of P4HA1 in T cells increases the durability of immune responses and reduces relapse risks of the cancer.

[0083] In another example, the genetically modified T cell comprising genetic modification to P4HA1 gene that inhibits or inactivates the expression of P4HA1 improves the fitness of Tcell, which affects the ability of T lymphocytes to effectively perform their immune function. As can be appreciated by a person skilled in the art, mitochondrial functions are crucial for CD8 T cell fitness. In one example, the present disclosure describes thatP4HAl is induced and accumulates in the mitochondria of CD8 T cells under T cell activation, differentiation and exhaustion (Figure 6A), wherein the accumulation of P4HA1 induces mitochondria unfitness through unconventional tricyclic acid (TCA) cycle perturbation. In another example, P4HA1 accumulation in the mitochondria catalyses the aberrant production of succinate that contributes to mitochondria damage leading to reprogramming of mitochondrial metabolic pathways in T cells, resulting in exhaustion and interference with T cell memory formation. Thus, the genetically modified T cells as disclosed herein are showed improve T cell fitness by reducing catalysis of alpha ketoglutarate (aKG) to succinate metabolism, thereby reducing production of succinate in mitochondria to prevent mitochondrial damage. Since metabolic fitness of CD8 T cells can enhance T cell persistence and antitumor activities, improving mitochondria function of T cells can enhance the efficacy of immunotherapy. Therefore, in one example, genetic modification of P4HA1 gene to inhibit or inactivate P4HA1 expression in the T cells can prevent P4HA1 aggregation in mitochondria induced by prolonged tumor antigen stimulation thereby preventing mitochondria damage in the genetically modified T cells. In another example, the genetically modified T cells as disclosed herein are characterized by reduced the percentage of dysfunctional mitochondria in the modified T cells as compared to unmodified T cells when subjected to repeated tumor antigen stimulation (Figures 7A and 7B).

[0084] Any of the methods disclosed herein can be used to genetically modify T cells from a human subject. The T cells modified in accordance with the methods as disclosed herein may be used to treat any diseases or conditions, including cancer. Thus, in another aspect, the present disclosure provides a method of treating or alleviating cancer, comprising administering a therapeutically effective amount of the genetically modified T cells or CAR T cells as disclosed herein to a subject in need thereof.

[0085] In some examples, the T cells are genetically modified to inhibit or inactivate the expression of P4HA1 and / or PD1 . In some examples, the T cells or CAR T cells are genetically modified to inhibit or inactivate the expression of P4HA1. In some examples, the T cell or CAR T cells are genetically modified to inhibit or inactivate the expression of both P4HA1 and PD1 . In some examples, the genetically modified T cells comprising genetic modification that inhibits / inactivates P4HA1 expression or inhibition or genetic modifications that inhibits / inactivates P4HA1 and PD1 expression, are further modified to express a CARtransgene to obtain a CAR T cell. The genetically modified CAR T cell can be, but are not limited to EGRF CAR T cell, HER2 CAR T cell, or Trop2 CAR T cell.

[0086] In some examples, the genetically modified T cells are further modified to express a CAR to obtain a CAR T cell. Both the genetically modified T cell and the genetically CAR T cell can be used for treating cancer. Thus, in some examples, there is provided a method of treating cancer comprising administering a therapeutically effective amount of the modified T cells or CAR T cells to a subject in need thereof. In some examples, there is provided a method of alleviating cancer, comprising administering a therapeutically effective amount of the modified T cells or CAR T cells to a subject in need thereof. As used herein, “alleviating cancer” refers to the managing and improving a subject’s condition when dealing with cancer. In one example, the cancer is a solid tumor cancer. In another example, the solid tumor cancer can include, but is not limited to: melanoma, lymphoma, cancer of the breast, colon, bladder, prostate, lung, kidney, pancreas, uterus, ovary, or testicle. As can be appreciated by a person skilled in the art, the ability of a CAR T cell to treat a specific cancer is dependent on the CAR that is expressed on the T cell and the tumor-associated antigen the CAR recognizes. However, in solid tumor microenvironment, the chronic antigen stimulation and the hostile environment limits T cell activity, driving T cells towards terminal exhaustion which is dysfunctional and irreversible In context of the modified CAR T cells as disclosed herein, wherein the CAR T cells are modified to inhibit or inactivate P4HA1 expression, the modified CAR T cells can persist, self-renew and resist terminal exhaustion under chronic antigen stimulation. The modified CAR T cells as disclosed herein can maintain progenitor like T cell pool with longterm proliferative capacity and the ability to continuously generate functional effectors. The inhibition or inactivation of P4HA1 can preserve T cell persistence and prevent T cell exhaustion, allowing the modified CAR T cells to kill tumor cells that express the relevant antigen more effectively.

[0087] In one example, the present disclosure describes the method of treating cancer using the genetically modified CAR T cells as disclosed herein. As shown in Figures 8B and 8H, treating tumor-bearing mice with a therapeutically effective dose of the genetically modified CAR T cell expressing EGFR can reduce the size of tumor in vivo more effectively than an unmodified T cell. In another example, treatment with genetically modified CAR T cells which have been modified to inhibit or inactivate P4HA1 expression enhances persistency of the CAR T cells in vivo. Thus, the number of CAR T cells remains higher in subjects treated with the genetically modified CAR T cells as compared to subjects treated with unmodified CAR T cells (Figure 8G). In another example, the increased persistence of genetically modified CART cells in vivo is associated with improved survival rate of the subject (Figure 9C). Thus, subjects that were treated with the genetically modified CAR T cells with inhibited or inactivated P4HA1 expression had improved survival rate compared to subjects treated with unmodified CAR T cells

[0088] In some examples, the present disclosure provides a method of treating cancer comprising administering of genetically modified T cells or CAR T cells, wherein the genetically modified T cells or CART T cells comprise genetic modifications that inhibit or inactivate the expression of both P4HA1 and PD1. The combined inhibition of P4HA1 and PD1 genetically modified T cells can render T cells or CAR T cells more effective in treating cancer than inhibition or inactivation of P4HA1 or PD1 alone. For example, the genetically modifying of T cells by inhibiting / inactivating expression ofboth P4HAl andPDl can increase T cell expansion upon repeated stimulation to tumor antigen compared to inhibiting P4HA1 or PD1 alone (Figure 4D). Thus, upon repeated antigen stimulation, inhibiting PD1 alone can lead to about 20-fold increase in T cell expansion and inhibiting P4HA1 alone can lead to about 25-fold increase in T cell expansion, respectively, inhibiting both PD1 and P4HA1 in combination increases T cell expansion to repeated tumor-antigen stimulation by about 50-fold (Figure 4D) In another example, genetic modifications which inhibit or inactivate both PD1 and P4HA1 in combination in T cells can further increase cytotoxicity of the T cell against cancer cells compared to PD1 or P4HA1 inhibition / inactivation alone (Figure 4F and Figure 5F). In another example, treating tumor-bearing mice with CAR T cells that were genetically modified to inhibit / inactivate both P4HA1 and PD1 expression resulted in 100% survival rate which was otherwise not observed in tumor-bearing mice treated with CAR T cells that were modified by inhibiting P4HA1 or PD1 alone (Figure 9C).

[0089] As disclosed herein, the present disclosure provides a pharmaceutical composition comprising the genetically modified T cells or the genetically modified CAR T cells as disclosed herein with a pharmaceutically acceptable carrier. In another aspect, the present disclosure provides a genetically modified T cell or a genetically modified CAR T cell obtained from the methods as disclosed herein for use in treating cancer. In another aspect, the present disclosure provides use of a modified T cell or a modified CAR T cell obtained from the methods as disclosed herein in the manufacture of a medicament for treating cancer. In one example, the cancer is a solid tumor cancer. In another example, the solid tumor cancer can include, but is not limited to: melanoma, lymphoma, cancer of the breast, colon, bladder, prostate, lung, kidney, pancreas, uterus, ovary, or testicle.

[0090] The present disclosure provides methods for improving the efficacy of CAR T cell immunotherapy in solid tumors. Although multiple factors can contribute to this limited CAR T cell efficacy in solid tumors, the lack of CAR T cell in vivo persistence due to insufficient stem-like T memory cell expansion and facilitated CAR-T cell exhaustion contributes to clinical failure of CAR-T cells immunotherapy. The present disclosure shows that that a substantial proportion of late-stage cancer patients have high levels of P4HA1+CD8 T cells in their blood (Figure 2), which indicates that autologous CAR T cell products prepared from these patients may have limited efficacy. Taken together, the present disclosure provides for P4HA1 targeting approaches to inhibit or inactivate P4HA1 expression during T cell or CAR T cell manufacturing for cancer immunotherapy.

[0091] As discussed herein, effective and durable cancer immunotherapy requires both intratumoral and systemic immunity. The present disclosure describes the induction of P4HAlin CD8 T cells, which is responsible for T cell dysfunction in response to repeated tumor antigen stimulation. For example, exhausted tumor infiltrating PD1+TIM3+CD8 T cells expressed higher P4HA1 but lower TCF1+CD8 T cells than that in non-exhausted TEVIS' CD8 T-cell The present disclosure also discloses that modifying T cells or CAR T cells to specifically inhibit or inactivate P4HA1 expression in T cells or CAR T cells reprograms the T cells or CAR T cells towards progenitor sternness, which supports persistence of T cell activity in vivo to increase the modified T cells’ antitumor activity as compared to unmodified T cells. Since inhibiting or inactivating P4HA1 in T cells can reinvigorate T cell sternness and reduce T cell exhaustion, the present disclosure also provides a method of treating cancer which can reduce the effect of P4HA1 induction in T cells in vivo. In some examples, the method to reduce the effect of P4HA1 induction in T cells in vivo can be accomplished by administration of a small molecule P4HA1 inhibitor.

[0092] Therefore, in another aspect, the present disclosure provides a method of treating or alleviating cancer in a subject, comprising administering a P4HA1 inhibitor. In another aspect, the present disclosure provides a method of treating or alleviating cancer in subj ect, comprising administering a P4HA1 inhibitor, wherein the subject or the cancer is resistant to treatment with an immune checkpoint blocker. In some examples, the cancer that is resistant to treatment with an immune checkpoint blocker can include but is not limited to microsatellite stable- colorectal cancer (MSS-CRC), pancreatic ductal adenocarcinoma (PDAC), ovarian cancer, prostate cancer, glioblastoma, hepatocellular carcinoma (HCC), triple negative breast cancer (TNBC). Cancers that are resistant to immune checkpoint blockade treatment are characterized by low response to treatment with the immune checkpoint blocker. In some examples, theimmune checkpoint blocker can be a PD1 inhibitor, a PD-1L inhibitor, a CTLA inhibitor or a LAG-3 inhibitor. In some examples, the immune checkpoint blocker is an antibody In some examples, the antibody can be an anti-PDl antibody, an anti-PD-lL antibody, an anti-CTLA- 4 antibody or an anti-LAG-3 antibody. In some examples, the cancer is resistant to treatment with an PD1 / PD1L inhibitor. In some examples, the subject is resistant to treatment with PD1 / PD-1L inhibitor. In some examples, the administration of a P4HA1 inhibitor to a cancer patient can inhibit the activity of P4HA1 in systemic and intratumoral T cells, which can improve the T cells activity and function to boost the treatment effect of an immune checkpoint inhibitor.

[0093] In one example, the effect of P4HA1 inhibition in tumor growth and post-surgery tumor recurrence was evaluated in a neoadjuvant setting. In other words, the P4HA1 inhibitor was administered to the subject before the surgery was performed to resect the tumor (see Figure 10). In such neoadjuvant setting, in vivo administration of a P4HA1 inhibitor prior to the surgical removal of tumor reduces tumor growth in a cancer that is resistant to treatment with an immune checkpoint blocker (Figures 10B and 10C). In another example, subjects treated with P4HA1 inhibitor show reduced cancer metastasis as compared to control subjects or subjects treated with an immune checkpoint blocker (Figure 10C). In another example, in vivo administration of a P4HA1 inhibitor reduces tumor relapse after surgical removal of the tumor. Thus, subjects treated with P4HA1 inhibitors have a tumor relapse rate of 58% compared to control subjects and subjects administered with an immune checkpoint blocker, which show a 100% tumor relapse rate (Figure 10E).

[0094] In another example, the combined administration P4HA1 inhibitor and an immune checkpoint blocker can reduce tumor relapse rate further to 17%, from 58% in subjects treated with P4HA1 inhibitor alone. This indicates a synergistic effect of combined treatment of P4HA1 and an immune checkpoint blocker on a cancer (Figures 10B-10D). Therefore, in another aspect, the present disclosure provides a method of treating or alleviating cancer, comprising administering a combination of a P4HA1 inhibitor and an immune checkpoint blocker. In one example, the cancer is resistant to treatment with an immune checkpoint blocker. In another example, the subject is resistant to treatment with an immune checkpoint blocker. Therefore, there is also disclosed a method of treating or alleviating cancer, comprising administering a combination of a P4HA1 inhibitor and PD1 / PD-1L inhibitor.

[0095] In some examples, inhibition of P4HA1 in T cells in vivo can be achieved by administration of a small molecule P4HA1 inhibitor. In another example, the small molecule inhibitor of P4HA 1 can be, but is not limited to 1 ,4-Hy droxy- 1 , 10-phenanthroline-3 -carboxylicacidEnarodustatDencichine2216, Fraxine, TP0463518, 1,4-DPCA ethyl ester, AKBA, SYP-5, JNJ, Ethyl 3,4-benzoate, Desidustat, Py-thiDC, AKB-6899, EL-102, IOX 2, MK-8617, Acriflavine chloride, Oltipraz,Hydroxycitric acid, GN44028, Molidustat, N-Oxyl, DMOG, PT-2385, Vadadustat, or Oroxylin-A.

[0096] In one example, the small molecule P4HA1 inhibitor can be, but is not limited to, l,4-Hydroxy-l,10-phenanthroline-3 -carboxylic acidEnarodustatRoxadustat

[0097] In one example, the small molecule inhibitor is l,4-Hydroxy-l,10-phenanthroline-3-carboxylic acid

[0098] In another example, the amount of the P4HA1 inhibitor can be between 10 mg / kg to 30 mg / kg. In other examples, the amount of the P4HA1 inhibitor can be between 15 mg / kg to 25 mg / kg, or between 18 mg / kg to 22 mg / kg. In another example, the amount of P4HA1 inhibitor can be about 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg or 30 mg / kg. In a preferred example, the amount of P4HA1 is 20 mg / kg. In some examples, the P4HA1 inhibitor can be formulated as a pharmaceutical composition In such examples, the P4HA1 inhibitor can be present in the pharmaceutical composition in an amount between 10 mg to 30 mg. In other examples, the P4HA1 inhibitor can be present in the pharmaceutical composition in an amount of about 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg. 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg or 30 mg.

[0099] In one example, the P4HA1 inhibitor can be administered daily, or once every 2 days, twice weekly or once weekly. In another example, wherein the P4HA1 is to be administered with PD1 as a combination therapy, the P4HA1 can be administered daily, while the PD1 inhibitor can be administered every other day.[000100] In one example the PD1 / PD-1L inhibitor can be a PD1 antibody or a PD-1L antibody. In another example, the PD1 antibody can be but not limited to: Nivolumab (ATC code: L01FF01), Pembrolizumab (ATC code: L01XC18), Cemiplimab (ATC code: L01FF06), Dostarlimab (ATC code: L01FF07), Retifanlimab (ATC code: L01FF10), Toripalimab (ATC code: L01FF13), or any other antigen binding protein that binds to PD1. In another example, the PD-1L antibody can be but are not limited to: Atezolizumab (ATC code: L01FF05), Avelumab (ATC code: L01FF04), Durvalumab (ATC code: L01FF03), and any other antigen binding protein that binds to PD1L.[000101] In another example, the dose of the PD1 / PD-1L inhibitor can be between 5 mg / kg to 15 mg / kg. In other examples, the dose of the PD1 / PD-1L inhibitor can be between 7 mg / kg to 13 mg / kg, 8 mg / kg to 12 mg / kg or between 9 mg / kg to 11 mg / kg. In some examples, the dose of the PD1 / PD-IL inhibitor can be 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 1 1 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg or 15 mg / kg. In another example, the dose of PD1 / PD-1L inhibitor can be 10 mg / kg. In some examples, the PD1 / PD1L inhibitor can be formulated as a pharmaceutical composition. In such examples, the PD1 / PD1L inhibitor can be present in the pharmaceutical composition in an amount between 5 mg to 15 mg. In other examples, the P4HA1 inhibitor can be present in the pharmaceutical composition in an amount of about 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg or 15 mg.[000102] In one example, PD1 / PD-IL inhibitor can be administered daily, or once every 2 days, twice weekly or once weekly.[000103] In a preferred example, the method of treating or alleviating cancer comprising administering a combination of a P4HA1 inhibitor and a PD1 / PD-1L inhibitor to a subject,wherein the P4HA1 inhibitorwherein the PD1 inhibitor is a PD1 blockade antibody; wherein the 1,4-DPCA is administered once daily and wherein the PD1 blockade antibody is administered once every two days. Tn some examples, the duration of treatment can be about 2 weeks to about[000104] In another example, the present disclosure provides a method of treating cancer involving administration of a P4HA1 inhibitor alone or administration of a combination of comprising a P4HA1 inhibitor and a PD1 / PD-1L inhibitor, wherein the treatment can reduce tumor growth in a subject having cancer. In one example, the cancer is resistant to treatment with an immune checkpoint blocker. In another example, the present disclosure provides a method of treating cancer involving administration of a P4HA1 inhibitor alone or administration of a combination of comprising a P4HA1 inhibitor and a PD1 / PD-1L inhibitor, wherein the treatment can prevent metastasis of cancer cells in a subject having cancer. In another aspect, the present disclosure provides a method of treating cancer involving administration of a P4HA1 inhibitor alone or administration of a combination of comprising a P4HA1 inhibitor and a PD1 / PD-1L inhibitor, wherein the treatment can delay and / or reduce tumor relapse in a subject having cancer in a neoadjuvant setting. In one example, administering the combination comprising a P4HA1 inhibitor and a PD1 / PD-1L inhibitor can delay and / or reduce tumor relapse compared to administering the P4HA1 inhibitor alone or administering the PD1 / PD-1L alone. In another example, the present disclosure provides a method of treating cancer involving administration of a P4HA1 inhibitor alone or administration of a combination of comprising a P4HA1 inhibitor and a PD1 / PD-1L inhibitor, wherein the treatment can activate endogenous T cell immunity in vivo in a subject having cancer. In one example, the cancer is a solid tumor cancer. In another example, the solid tumor cancer can be but not limited to: melanoma, lymphoma, cancer of the breast, colon, bladder, prostate, lung, kidney, pancreas, uterus, ovary, or testicle.[000105] As described in the present disclosure, the level of circulating P4HA1+CD8T cells coincides with tumor progression and is associated with resistance to immune checkpoint blockade therapy. Therefore, the aforementioned observation indicates that the level of P4HA1+CD8 T cells in blood and tumor can be used as a biomarker for immune monitoring of endogenous T cells immunity to predict a subject’s response to cancer treatment.[000106] Therefore, in another aspect, the present disclosure provides a method for determining endogenous T cell immunity of a subject receiving a cancer treatment, the method comprising measuring the expression level of P4HA1 in T cells using a sample obtained from the subject, wherein a decrease in the expression level of P4HA1 in T cells of the subject as compared to the expression level of P4HA1 in the T cells of the subject measured prior to receiving the cancer treatment is indicative that the subject has improved endogenous T cell immunity. In one example, the subject is receiving a cancer treatment. In another example, the cancer treatment can include, but are not limited to: chemotherapy, radiation therapy, immunotherapy, gene therapy, surgery and a combination thereof. In another example, the sample is a tumor sample, a blood sample, or a tumor-draining lymph node sample. Since the success of immunotherapy requires effective response of both intratumoral immunity and systemic immunity, using P4HA1 expression in CD8 T cells as a biomarker can predict a subject responsiveness to a cancer treatment and to monitor the immune system response to the cancer treatment.[000107] In another aspect, the present disclosure provides a method for determining resistance to immune checkpoint blockade (ICB) treatment in a subject having cancer, the method comprising measuring the expression level of P4HA1 in T cells using a sample obtained from the subject, wherein a higher expression level of P4HA1 in the T cells of the subject as compared to a cancer patient responsive to PD1 inhibitor treatment is indicative that the subject is resistant to PD1 treatment. In one example, the expression level of P4HA1 in the T cells of the subject is at least 1.5 folds higher than the cancer patient responsive to PD1 inhibitor treatment. In another example, the sample is a tumor sample, a blood sample, or a tumor-draining lymph node sample (Figure 14D and 14E). The present disclosure has shown that circulating P4HA1+CD8 T cell level is highly correlated with cancer progression in both mouse models and human cancers, indicating a use for P4HA1 as a peripheral biomarker for immune monitoring. Moreover, the number of P4HA1+CD8 T cells in the blood is also associated with immune checkpoint blockade (ICB) resistance. In one example, using analysis of public single-cell TNBC data, the present disclosure shows that blood P4HA1+CD8 T cells outperform TCF1+CD8 T cells in correlation with immune checkpoint blockade (ICB) response, pointing to a role of blood P4HA+CD8 T cells as a circulating biomarker to guide and monitor the effectiveness of immune checkpoint blockade ICB therapy.[000108] Current immunotherapy for cancer treatment, such as immune checkpoint inhibitors and adoptive cell therapy have faced obstacles in delivering long-term tumor control in solid tumors in part due to ineffective intratumoral and systemic immune response. As described inthe present disclosure, reduction in T cell sternness and increased in T cell exhaustion are partially responsible for the ineffective immune response. Considering the foregoing problem, the present disclosure has identified P4HA1 as a critical regulator of T cell differentiation and exhaustion, wherein inhibition of P4HA1 in T cells reprograms the T cells towards immune memory and systemic antitumor immunity. The present disclosure has provided non-limiting examples to demonstrate P4HA1 as a therapeutic target in modulating both adoptive and endogenous T-cell activity for treating solid tumors. Thus, inhibition of P4HA1 can increase in systemic immune memory response, leading to long-term eradication of tumors that are resistant to PD1 inhibitor treatment.[000109] AlthoughPDl andP4HAl show similar expression patterns in early T cell activation and late-stage exhaustion, PD1 inhibition does not enrich CD8 T cell progenitor memory formation in both in vitro and in vivo models. By contrast, P4HA1 inhibition can lead to both adoptive and endogenous CD8 T cell memory expansion, both in vitro and in vivo, generating a durable system immunity against cancer. Previous studies in clinical models have shown that system immunity generated from the T cell priming stage in TDLN is required for effective antitumor immunity and that CD8 T memory cells in TDLN are primary responders to PD1 / PD-L1 blockade.[000110] The present disclosure indicates that boosting systemic immune surveillance is critical to enabling long-term anticancer immunity and improving survival benefits. Recently, an example showing lymph node-targeted personalized cancer vaccination used alone or in combination with anti-PDl has shown promising clinical results in preventing post-surgery tumor recurrence. Given that P4HA1 inhibition only induces CD8 T progenitor memory cells in tumor-bearing mice but not in tumor-free mice, targeting P4HA1 in CD8 T cells can evoke cancer-directed memory immunity to prevent tumor recurrence.[000111] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such tenns and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations are considered to be within the scope of this invention.[000112] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof. [000113] As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically, + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.[000114] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.[000115] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.[000116] The invention has been described broadly and generically herein Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.[000117] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.[000118] It should further be appreciated that the exemplary examples are only examples, and are not intended to limit the scope, applicability, dimensions, or configuration of the disclosure in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing an exemplary embodiment of the disclosure, it being understood that various changes may be made in the function and arrangement of elements and method of fabrication described in an exemplary embodiment without departing from the scope of the disclosure as set forth in the appended claims.EXAMPLESExample 1 - P4HA1 is strongly induced in CDS T cells upon activation and exhaustion [000119] CAR T cell was used as a model to investigate the tumor antigen-stimulated CD8 T cell differentiation and exhaustion process in the tumor microenvironment (TME). Peripheral blood mononuclear cells (PBMCs) from normal healthy donors were activated with a single treatment of anti-CD3 / CD28 followed by lentiviral EGFR CAR transduction before further expanded for 5-10 days to mimic the priming and activation phase of T cells (Figure 1A). The expanded CAR T cells were further subject to repeated cocultures with EGFR-expressing DLD1 colorectal cancer cells in normoxia or hypoxia, mimicking the effector and exhaustion phase in the solid tumor microenvironment (Figure 1A). The dynamic transcriptomic changes in this process were profiled using RNA-seq analysis using CAR T cells that were collected from the early priming and activation stage and the late stage upon repeated cocultures undergoing exhaustion. In addition, HlFla inhibitor, PX478, was also included to allow characterizing HIF la-dependency in this process.[000120] Thirty-seven (37) genes whose expression was commonly induced in both early activation and late-stage exhaustion, as well as in hypoxia (fold change > 1.5, adjust P < 0.05) were identified. Twelve (12) actionable enzyme-encoding genes were identified among the 37 induced genes (Figure IB). Among them, P4HA1, encoding prolyl 4-hydroxylase subunit alpha 1, showed the highest progressive induction from activation to exhaustion and further in hypoxia (Figure IB and ID). P4HA1 is an alpha-ketoglutarate (a-KG)-dependent metabolic enzyme that catalyzes the collagen synthesis and succinate by-production, implicating a potential role of P4HA1 in CD8 T cell metabolic regulation. P4HA1 appears to be a distinct metabolism enzyme that is strongly induced in CD8 T cells upon activation and exhaustion, which was not seen in other reported metabolic regulators of T cells. Further comparisons with other established T cell regulators, including immune checkpoint molecules, transcription factors, and effector cytokines, still show P4HA1 as the most highly induced upon T cell activation and exhaustion (Figure 1C). Of note, unlike those inhibitory receptors, includingPDCD1 (PD1), HAVCR2 (T1M3), LAG3, or CTLA4, which have been previously shown to be induced by hypoxia but in a HIF la-independent manner, P4HA1 induction under hypoxia was also HIF la-independent as its expression was not affected by HIF la inhibitor PX478 treatment (Figure 1C). As opposed to P4HA1 induction, the immune effector cytokines IFNG and TNF were upregulated in T cell activation but downregulated upon exhaustion and hypoxia, but in a HIF la-dependent manner. T cell progenitor transcription factor TCF7 also showed a transient upregulation in the early cell activation stage but drastically downregulated upon exhaustion in repeated tumor antigen stimulation (Figure ID), consistent with T cell priming and exhaustion in TDLN and TME, respectively.Example 2 - P4HA1 expression in CD8 T cells obtained from the blood of cancer patients [000121] Blood analysis of patients with breast cancer also showed upregulation of P4HA1 ” CD8 T cells upon disease progression (Figure 2). The finding supports P4HA1+CD8 T cells as a circulating biomarker of cancer progression.Example 3 - P4HA1 knockout or chemical inhibition reprograms CDS T cells towards TCF1+progenitor sternness while ameliorating terminal exhaustion[000122] The functional role of P4HA1 in modulating CD8 T cell activity in early activation, differentiation, and tumor antigen-induced exhaustion were evaluated (Figure 3A). P4HA1 was depleted by CRISPR-Cas9 together with P4HA1 gRNA through electroporation in EGFR CAR T cells (hereafter P4HAPAO) (Figure 3B). PD1 knockout (77J / KO) was also included as a control (Figure 3C). / !<()-(’ AR T cells showed an enhanced proliferation compared to the negative control cells (Figure 4A). In contrast, / 7> / K0-C AR T cells only had a modest increase in cell expansion (Figure 4A). Flow cytometry analysis showed that C- / / / .4 / KO, but not / 7) / I<0, increased stem central memory (Tscm) and central memory (Tcm) cells but reduced CD8 T effector cells (Figure 4B), indicating that P4HA 1\<JJ has reprogrammed CD8 T cell towards stem-like T memory phenotype trait P4H A / KO, but not / 7 / KO, also resulted in progenitor memory transcription factor TCF1 induction in CD8 T cells (Figure 4C). These findings indicate that P4HA1 induction in CD8 T activation suppresses CD8 T cell progenitor memory maintenance. By contrast, PD1, albeit exhibiting a similar induction in CD8 T cell activation, does not seem to have this intrinsic capacity. In subsequent CAR T cell and cancer cell coculture experiments aimed to assess the tumor antigen-induced exhaustion and cytotoxicity, P4HA1KO-CAR T cells exhibited robust and persistent expansion compared to control CAR T cells that were quickly eliminated over repeated tumor antigen stimulation (Figure 4D). Although PD1KO only modestly increased CAR T expansion in the cocultures, its combination with P4HA1KO (DKO) led to a further increase in CAR T cell expansion thanP4HA1K0 alone (Figure 4D). Consistently, DKO-CAR T cells displayed further increase in polycytokine production and cytotoxicity against the cancer cells compared to P4HA1K0 and PD1K0 alone (Figure 4E and 4F). Similar results were also observed in the Trop2 CAR T model (Figures 5A-5F). These findings support a dual role of P4HA1 in regulating CD8 T cell progenitor differentiation and exhaustion, while PD1 does not seem to have an intrinsic activity to modulate T progenitor development in the monoculture model.Example 4 - P4HA1 accumulates in the mitochondria of CDS T cells, resulting in disruption of TCA cycle and mitochondria damage[000123] To investigate whether P4HA1 is present in mitochondria in CD8 T cells, cell fractionations of CAR T cells were obtained from the resting stage or stimulated with anti- CD3 / CD28 The Western blot result showed that P4HA1 was more enriched in mitochondria than the cytosol and was markedly induced in mitochondria upon repeated stimulation but not in the cytosol (Figure 6A). P4HA2 also showed a similar induction but to a much lesser extent, while P4HA3 was absent in mitochondria (Figure 6A). P4HB, though present in both cytosol and mitochondria, did not show induction upon stimulation (Figure 6A). On the other hand, HIF -prolyl hydroxylase (PHD) family proteins (PHD1, PHD2, PHD3) were found mainly in the cytosol (Figure 6A). Thus, among the a-KG-dependent prolyl hydroxylases, P4HA1 is the only one induced in mitochondria upon CD8 T cell activation. Further immune fluorescence staining also validated P4HA1 accumulation in mitochondria of CD8 T cells undergoing exhaustion following repeated cocultures with cancer cells (Figure 6B). Corresponding with the P4HA1 aggregation upon CD8 T cell exhaustion during cocultures, mitochondria exhibited morphological shrinkage reflecting extensive damage (Figure 6B). P4HAPKO treatment retained mitochondria in a normal elongated and tubular-like morphology in cocultures (Figure 7A), suggesting a role of P4HA1 in promoting mitochondria damage.[000124] Further, the MitoTracker assay was used to assess mitochondrial fitness. Red- CMXRos (MR) and Green-FM (MG) are probes that measure functional mitochondria with intact mitochondrial membrane potential and total mitochondria, respectively. It was found that, along with repeated cocultures, the percentages of CD8 CAR T cells with dysfunctional mitochondria (MRlow / MGhigh) gradually increased. In contrast, the healthy and active mitochondria (MRhigh / MGhigh) were reduced (Figures 7B and 7C). These changes were prevented by P4HA1KO treatment (Figure 7C).[000125] Aberrant succinate accumulation is also known to damage the mitochondria and induce CD8 T cell exhaustion and death. Preferential succinate induction in mitochondria over cytosol upon CD8 cell activation was detected (Figure 7D), which was abolished byP4HA1K0 treatment (Figure 7D), indicating that mitochondrial P4HA1 accumulation disrupts a-KG / succinate balance in the TCA cycle, leading to aberrant succinate accumulation and mitochondria damage (Figure 7E).Example 5 - Ex vivo genetic depletion of P4HA1 boosts CAR T in vivo efficacy and persistency in solid tumors[000126] Next, it was investigated whether targeting P4HA1 in CAR T cells can be translated into increased anticancer efficacy in vivo. To this end, several human CAR T cells and corresponding xenograft mouse models were used to monitor the effect of ex vivo manipulated CAR T cells on xenograft tumor growth and in vivo persistence (Figure 8A).[000127] First, whether P4HA1 induction in CAR T cells upon antigen stimulation also occurred in vivo and tumor microenvironment (TME), thus restricting the CAR T antitumor efficacy was determined. Following CAR T cell blood injection in tumor-bearing NSG mice, P4HA1 levels in both Trop2 CAR T and HER2 CAR T cells showed progressive induction in circulating blood (Figure SB and SC), as well in spleen and tumors (note, NSG mice have only small, residual lymph nodes thus inaccessible for analysis) (Figure 8C). CAR T cells injected in non-tumor-bearing NSG mice did not exhibit P4HA1 induction in the blood (Figure 8B). Analysis of CAR T cells in the tumor versus spleen revealed higher levels of terminal exhaustion population (Tex, PDl+TIM3+TCFr) but lower progenitor exhaustion CD8 T cells (Tpex, PD1+TIM3'TCF1+) in the tumors than spleen (Figure 8D), indicating increased CAR T exhaustion in the tumor. Moreover, tumor-infiltrating P4HA1- CAR T cells were much more exhausted than the P4HA1" CAR T cells (Figure 8E).[000128] P4HA1KO-CAR T cells when administered into tumor bearing mice achieved complete and durable tumor clearance, with great persistence in circulating blood (Figures 8F- 8H). PD1KO-CAR T cells failed to constrain tumor growth and did not display increased persistency in the blood (Figures 8F and 8H) In addition, the Trop2 CAR T-MDA-MB-231 model was also used to validate the above finding and to determine whether the double knockout of P4HA1 and PD1 (DKO) can further improve CAR T activity, as shown in in vitro experiments. The results show that the control and PD1KO-CAR T cells in this model only delivered partial and transient tumor control (Figures 9A and 9D). P4HA1KO CAR T induced an effective and persistent tumor remission, though 2 out of 6 relapsing tumors arose from Day 52 and Day 32, respectively (Figure 9A and 9D). DKO-CAR T cells achieved a durable complete response in all the tumors without tumor relapse beyond 90 days (Figure 9 A) Both P4HA1KO or DKO-CAR T cells displayed increased persistence in blood compared to controlor PD1K0 CAR T cells (Figure 5K) and improved the survival of mice up to 90 days (Figure 9C)[000129] Example 6 - P4HA1 inhibitor treatment in vivo activates endogenous T cell immunity to suppress tumor recurrence and metastasis[000130] It was next interrogated if the P4HA1 inhibitor DPCA would work in vivo to deliver therapeutic efficacy on endogenous T cells in syngeneic mouse tumor models. Immunotherapies in neoadjuvant settings have demonstrated clinical benefit by providing intratumoral and systemic immune responses. To investigate the effect of DPCA in tumor response and post-surgery tumor recurrence in a neoadjuvant setting, we treated the 4T1- bearing Balb / c mice with DPCA, aPDl or combination for two weeks before surgical resection of tumors (Figure 10A) 4T1 tumor is known to be highly resistant to aPDl treatment, while DPCA treatment, either given alone or with aPDl, substantially reduced tumor growth (Figure 6B). DCPA markedly delayed and reduced tumor relapse compared to control or aPDl treatment (58% vs 100% relapse rate). This was more evident in DPCA / aPDl combination treatment, which reduced the relapse rate to 17% (Figures 10B and 10C). DPCA or DPCA / aPDl also markedly suppressed lung metastasis (Figures 10D), leading to a substantial survival benefit (Figure 10E). The potent effect of DPCA or DPCA / aPDl neoadjuvant treatment on preventing post-surgery tumor recurrence was also seen in the mouse CT26 colon cancer model (Figures 11A-11C).[000131] Analysis of resected control 4T1 tumors revealed a higher prevalence of terminal exhausted CD8 cells (Tex, TCF1'PD1+TIM3T“ CD8 T cells) than TCF1+progenitor exhausted CD8 T cells (Tpex, TCF1+PD1+TIM3'), indicating an immune resistant state in 4T1 tumor (Figures 10F and 10G). DPCA treatment alone or combined with aPDl increased Tpexbut reduced Tcx(Figure 10G), indicating the in vivo reprogramming of CD8 T within tumor microenvironement (TME). aPDl did not induce such changes. DPCA or DPCA / aPD1 also enhanced the infiltration rate of CD8 cells and augmented effector cytokine expression in tumor-infiltrating CD8 cells (Figure 10H). Consistent with the in vitro finding in human CD8 T cells, the mitochondria fitness of 4T1 -infiltrated CD8 cells was improved by DPCA treatment (Figure 11C). These data indicate that P4HA1 inhibitor DPCA could induce endogenous CD8 T reprogramming, enabling immune response in tumors resistant to aPDl treatment.[000132] To exclude the concern that the decreased tumor recurrence may arise from smaller sizes of DPCA-treated tumors before the surgery, an additional control-aPDl treatment combined with HD AC inhibitor Endostat (ENT) was also included, which has been previously shown to reduce 4T1 tumor growth substantially. The result showed that ENT / aPDl treatmentreduced the 4T1 tumor growth similarly to DPCA / aPDl but did not prevent post-surgery relapses or improve survival (Figures HE and HD), thus excluding the above concern [000133] The data thus far shows that DPCA can establish long-term antitumor immunity to prevent tumor recurrence This effect is shown in CD8 T cells that were depleted by administration of anti-CD8 in 3 of the 7 DPCA / aPDl -treated mice that remained relapse-free, followed by 4T1 cell reinoculation (Figure 101). Relapse-free mice with CD8 T cell depletion quickly developed tumors upon 4T1 reinoculation (Figure 10J). 4T1 cell reinoculation in the other 4 relapse-free mice without CD8 T cell depletion failed to develop tumors (Figure 10J). This result shows that CD8 T cells are required to suppress tumor regrowth. Blood analysis of recurrence-free mice indicated the presence of a substantially high level of CD8 T memory populations (Tscmand Tcm) (up to 80%) compared to the Teff population (20%) (Figure 10K). Following the 4T1 re-challenge, an immune recall response was detected as TScm / TCm cells decreased with a corresponding increase in Terr simultaneously. After tumor clearance, T effector cells returned to baseline while memory T cells were comping up again (Figure 10K). These data indicate that DPAC / aPDl has established an immune memory capacity that prevented tumor recurrence.Example 7 - P4HA1 inhibitor generates systemic immune response through CD8 T progenitor expansion in tumor-draining lymphatic node (TDLN)[000134] It is next shown that DPCA or DPCA / aPDl can increase CD8 T memory cells in TDLN, blood, and tumor. Compared to lymph nodes (LN; from tumor-free naive mice), tumordraining lymph nodes (TDLN; from 4T1 bearing mice) showed substantially lower CD8 T memory cells, consistent with the tumor-antigen simulated T cell differentiation (Figure 12A). Upon 14 days of treatments, DPCA or DPCA / aPDl, butnot aPDl, significantly increased CD8 Tscm and Tcmin TDLN but not in LN (Figure 12A), indicating that DPCA-induced CD8 T memory enrichment is tumor-dependent. A similar trend was also observed in the blood and tumor but not in the spleen (Figure 13A), which is in indirect contact with tumors. This indicates that DPCA-induced CD8 T progenitor memory expansion in TDLNs. By contrast, aPDl treatment did not show such a capacity.[000135] Serial blood sampling and analyses across the entire course of mouse experiments indicated that CD8 T memory cells are sustained in DPCA or DPCA / aPDl -treated mice but gradually vanished in control or aPDl-treated mice (Figure 12B). Comparing all the tumor- recurrent and non-recurrent mice showed that the down regulation of CD8 T memory cells correlated with the development of tumor recurrence. Like CD8 T memory cells, TCF1+ CD8 T cells also showed downregulation alongside tumor recurrence but remained at higher levelsin DPCA / aPD 1 -treated mice free of tumor recurrence (Figure 12C). P4HA1+CD8 T cell elevation coincided with the tumor recurrence but was suppressed in DPCA / aPD 1 -treated mice (Figure 12C). PD1+CD8 T cells, however, did not correlate clearly with tumor burden or recurrence Analysis of post-surgery blood samples of recurrence and non-recurrence by receiver operating characteristic (ROC) curve indicated high sensitivity and specificity of P4HA1+CD8 T cells in detecting recurrence with the area under the curve (AUC) of 0.94, compared to TCF1+ CD8 T cells with an AUC of 0.841 (Figure 12D). In contrast, PD1+CD8 T cells showed poor utility for recurrence detection with an AUC of 0.650 (Figure 12D). A closer examination of the individual mice during the tumor recurrence shows that the upregulation of circulating P4HA CD8 T cells always occurred ahead of the unset of tumor recurrence (Figure 13E).[000136] To address whether tumor draining lymph node (TDLN) is responsible for DPCA- induced prevention of tumor recurrence, sphingosine 1 -phosphate receptor antagonist FTY720 was applied after the surgical removal of tumors (Figure 12E). Sphinosine-1 -phosphate receptor 1 is required for immune cells to leave the secondary lymphoid organs, and FTY720 blocks this process, thus retaining the T cells within the lymphoid organs. Administration of FTY720 largely rescued the effect of DPCA / aPD 1 on tumor recurrence (Figure 12F) and relapse-free survival (Figure 12G). Consistently, DPCA / aPD 1 -treated mice showed increased TCF CD8 T cells and decreased P4HA CD8 T cells in TDLN (Figure 12H), and adding FTY720 resulted in CD8 T cell depletion in the blood as expected (Figure 121). This finding confirmed that the tumor relapses are due to the lack of blood circulating immune memory cells arising from TDLN. These data support the conclusion that DPCA administration can target T cells in the TDLN, which induces and sustains the CD8 T progenitor expansion to enable durable systemic immunity for long-term tumor control.Example 8 - P4HA1 is a biomarker associated with anti-PDl resistance[000137] Given that P4HA1 contributes to CD8 T cell exhaustion and immune escape and that P4HA1 inhibitor treatment remains effective in aPDl -resistant tumors, it was asked if high P4HAU CD8 T cell is associated with aPDl resistance. Compared to the aPDl -resistant 4T1 tumor, the MC38 mouse tumor is sensitive to aPDl with a tumor growth inhibition range of 30-70%.[000138] aPDl treatment in Balb / c mice bearing MC38 resulted in marked tumor growth inhibition in over 50% of mice (Figure 14A). Tumor analysis showed that MC38 responding tumors expressed significantly lower P4HA1+CD8 T cells but higher TCF 1 CD8 T progenitor exhausted cells than MC38 non-responding and resistant 4T1 tumors (Figure 14B).Consistently, MC38 responding tumors express higher Tpexbut lower Texcells when compared with MC38 non-responding tumors and 4T1 tumors (Figure 14B). Further analysis of aPDl- treated MC38 tumors indicates that P4HA1"CD8 T cells were highly correlated with aPDl resistance, outperforming TCF1+CD8 T cells that correlated negatively with aPDl resistance (Figure 14C). Moreover, analysis of a public database of single-cell RNA-seq of melanoma data shows that P4HA1 expression in CD8 T cells is highly associated with aPDl resistance (Figure 14D. Single-cell analysis of aPDl-treated TNBC patients also indicates a significant correlation of higher P4HA1 in CD8T cells with otPDl resistance in blood, albeit less significant in tumors (Figure 14E). In contrast, TCF1 in CD8 T cells showed no significant correlation in both blood and tumor (Figure 14D). These findings support the potential utility ofP4HAl+CD8 T cells as a biomarker to evaluate aPD1 response.EXPERIMENTAL SECTION Cell lines[000139] For human cell lines, HEK-293T, MDA-MB-231, and MDA-MB-361 cells were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS). DLD-1 cells were obtained from ATCC and cultured in RPMI-1640 + 10% FBS. For mouse cell lines, 4T1 cells were from ATCC and cultured in RPMI-1640 + 10% FBS, CT26 and MC38 cells were from ATCC and cultured in DMEM + 10% FBS. Cells were tested for mycoplasma and confirmed negative before use in experiments.Human samples[000140] Human peripheral blood samples and FFPE were collected with informed consent for research. The study was approved by the Ethics Committee of Tan Tock Seng Hospital (Singapore) and the Ethics Committee of the Region of Southern Denmark (Denmark) Peripheral blood mononuclear cells (PBMC) isolated from healthy donors tested negative for HBV, HCV, HIV, and Syphlis were purchased from Humancells Biosciences (Cat No. PBMC- C100M).Human peripheral blood mononuclear cells (PBMCs)[000141] PBMC of non-cancer and cancer patients were collected by TTSH in EDTA-coated vacutainer tubes prior to surgery or treatment. PBMCs were isolated from peripheral blood by Ficoll gradient (GE Healthcare; Cat No. 17144002) and subjected to red blood cell lysis using RBC Lysis Buffer (Thermo Fisher Scientific; Cat No. 00-4333-57). PBMCs were frozen in a cry opreservation medium (FBS containing 10% DMSO) at -80C, followed by storage in liquidnitrogen. PBMCs were subjected to flow cytometry staining procedures immediately after thawing.Mice[000142] All experiments were conducted in compliance with animal protocols approved by the ASTAR-Biopolis Institutional Animal Care and Use Committee of Singapore. BALB / c and NSG mice were purchased from InVivos (Singapore). All mice were maintained at 21 °C ± 1, 55 to 70% humidity, and with a 12 h light / dark cycle from 7 am to 7 pm. The animal was euthanized when the tumor reached a maximum size of 1000 mm3CAR plasmid design and construction[000143] The plasmid pSLCAR-CD19-CD3z-CD28 was obtained from Addgene (USA). The sequence of human 4 IBB intracellular domain was inserted between CD28 and CD3z intracellular sequence in the plasmid to obtain third generation CART plasmid pSLCAR- CD19-CD3z-41bb-CD28, or pSLCAR-CD19-3G. The sequence of scfv targeting human EGFR, HER2 and Trop2 were obtained from work of Xia, et.al68, Zhao, et.al69 and Govindan, et.al70, respectively. The new CD19 scfv sequence in the pSLCAR-CD19-3G plasmid was replaced by the new scfv sequences based on the Bpil-directed golden gate cloning, as described previously, to generate pSLCAR-EGFR-3G, pSLCAR-HER2-3G and pSLCAR- Trop2-3G plasmids.CAR T cell generation and expansion[000144] Anonymous human healthy donor PBMCs were purchased from Humancell Biosciences (Cat No. PBMC -C100M). For generation of CAR T cells, bulk PBMCs were activated on day 0 using 10 pL T Cell Transact containing CD3 and CD28 (Miltenyi Biotech; Cat. No. 130-111-160) for each million PBMCs, cultured in ImmunoCultTM-XF T Cell Expansion Medium (Stemcell Technologies; Cat. No. 10981) supplemented with 10 ng / mL recombinant human IL2 for 5 days. Additionally, 2 pM DPCA or an equivalent volume of DMSO was added to the cell culture on day 0. On day 3, cells were transduced with 100 pL CAR lentivirus in the presence of 6 pg / mL polybrene. CD3 / CD28 agonist beads were removed on day 5 by washing the cells twice in lx PBS. Cells were further expanded in ImmunoCultTM- XF T Cell Expansion Medium containing 5 ng / mL recombinant human IL7 and 10 ng / mL recombinant IL15, maintaining a density of 0.25 million cells / mL from day 5 onwards. Subsequently, CAR T cells were sub-cultured under the same conditions every 3 days. The transduction efficiency of CAR T cells was evaluated based on GFP expression on day 7 Unless otherwise indicated, CAR T cells were used for in vitro cytotoxicity assays or inoculated into mice on day 12 post-activation.[000145] To characterize the expansion rate and differentiation profiles of CAR T cells, manual cell counts were conducted and recorded on day 5, 7, 12, 15 and subsequently every 5 days. FACS analysis was performed on day 5, 7 and 12 to assess CAR T cell subpopulations (Tscm, Tcm, Teff and Tem) and T cell markers (P4HA1 and TCF1)Generation of gene knockout CAR T cells[000146] Gene knockout CAR T cells were generated using the 4D-Nucleofector X kit S (Lonza, Switzerland), according to the manufacturer’s instructions. Briefly, the gRNA and SpCas9 nuclease (Integrated DNA Technologies IDT) were mixed at a molar ratio of 3: 1 for 20 min to form RNP complex under room temperature. During this time, CAR T cells were washed with I PBS for three times and resuspended in Nucleofector Solution. Then RNP complex was mixed with CAR T cells at 150 pmol gRNA per million cells per 20 pL solution and subjected to the 4D-Nucleofector X Unit (Lonza) for electroporation under the program EO-115. Post electroporation, cells were transferred to pre-warmed ImmunoCult™-XF medium for recovery and released to fresh complete medium for further analysis after 4 hrs. Sequences of gRNA were summarized in Supplementary Table 1 and synthesized by Integrated DNA Technologies IDT.Lentivirus production[000147] 1 x 106 HEK-293T cells were initially seeded in 10-cm plates in DMEM culture medium one day before transfection followed by replacement with OPTI-MEM medium the next morning. For lentivirus packaging, 2.25 pg pMDLg (Addgene plasmid #12251), 0.9 pg pRSV-Rev (Addgene plasmid #12253), 1.35 pg pMD2.G (Addgene plasmid #12259) and 5 pg pSLCAR plasmid were prepared with 24 pL Lipofectamine™ 2000 (ThermoFisher Scientific, CAT#11668019) in 1 mL OPTI-MEM medium for 20 min followed by addition to the HEK- 293T cells. A total of 10 mL fresh complete medium was used to replace OPTI-MEM medium 8 hr post transfection. The virus-containing supernatants were collected 24 and 48 hrs post transfection and concentrated by Amicon® Ultra Centrifugal Filters (Merck, UFSC050SL). The resulting concentrated lentivirus was aliquoted and stored frozen in -80 °C for further use.Coculture Systems[000148] Cancer cells were seeded in 24-well plates for 1 day to form monolayers. Culture media of cancer cells was then removed, and CAR T cells were subsequently added to the cancer cells 72 hours in normoxia (20% O2) or hypoxia condition (1% O2) at indicated effector to target (E:T) ratios. Cocultured CAR T cells were subsequently used for flow cytometry, or gene-expression analyses.Coculture cytotoxicity assay[000149] Coculture assays were performed using a luciferase-based killing assay with CAR T cells incubated with cancer cells stably expressing luciferase at indicated effector to target (E:T) ratios. After 48 hours of co-culture, lx luciferase substrate luciferin (Promega; Cat. No. Pl 043) was added to the cells and the chemiluminescent signals were detected by GlowMAX Explorer (Promega). The measurement was used to indicate cell viability against cancer cells without coculture. Percent cell cytotoxicity was calculated using the formula below:% cytotoxicity=100- (Luciferase measurement of cocultured samples / Luciferase measurement of noncocultured samples) * 100 %Flow cytometry analysis[000150] For in vitro human T cells analysis, cells were incubated in anti-human FcR antibody (Miltenyi Biotec; Cat. No. 130-059-901) followed by surface antibodies in FACS buffer (PBS with 0.5% BSA and 0. 1% sodium azide) for 30min. The fixation and permeabilization process was carried out with fixation buffer (BD Bioscinces) according to the manufacturer’ s protocols. Cells were stained with intracellular antibodies in washing buffer (BD Biosciences). The following antibodies were used for staining, anti-human APC CD8 (BioLegend, CAT#344722), anti-human BV421 CD197 (CCR7) (BioLegend, CAT# 353208), anti-human BV510 CD95 (BioLegend, CAT#305640), anti-human PE / Cy7 CD62L (BioLegend, CAT#304822), anti-human APC / Cy7 CD45RO (BioLegend, CAT#304228), anti-human PE TCF1 (BioLegend, CAT#655208), anti-human APC / Cy7 CD3 (BioLegend, CAT#344818), anti-human PE / Cy5 CD366 (BioLegend, CAT#345052), anti-human PE / Cy7 CD279 (BioLegend, CAT 329918), BV421 donkey anto-rabbit IgG (BioLegend, CAT#406410), antihuman P4HA1 (Invitrogen, CAT#PA5-106334), anti-human BV421 CD3 (BioLegend, CAT#317344), anti-human APC / Cy7 CD8 (BioLegend, CAT#344714), anti-human PE IL2 (BioLegend, CAT#500307), anti-human APC TNFot (BioLegend, CAT#502912), and antihuman PE / Cy7 fFNy (BioLegend, CAT#505826).[000151] For ex vivo analysis (mouse 4T1 or human MDA-MB-231, MDA-MB-361, CT26, MCA 8, and DLD-1), tumors, lymph nodes, and splenocytes were finely chopped using a scalpel and dissociated in either mouse tumor dissociation kit (Miltenyi Biotec; Cat. No. 130-096-730) or human tumor dissociation kit (Miltenyi Biotec; Cat. No. 130-095-929) according to manufacturer’s protocol. The samples were passed through a 0.45 gm filter to obtain singlecell suspensions and washed twice in lx PBS. Collected blood samples were washed twice in1x PBS. All samples were stored at -80°C in 10% DMSO containing FBS freeze buffer and thawed before flow staining procedures. For cytokine detection, cells were incubated with 10 nM PMA (Sigma- Aldrich) and 1 pM ionomycin (Sigma- Aldrich) in PBS at room temperature for 20min before staining. Dissociated cells were incubated with lx Live / Dead Fixable Dead Cell dye (Invitrogen) in ice-cold PBS for 20min to exclude dead cells from analysis All samples were stained with either anti-human or anti-mouse Fcr antibody (Miltenyi Biotec; Cat. No. 130-092-575) accordingly in ice-cold FACS buffer for 20 min. Cells were then processed for antibody staining as described above. The following antibodies were used for staining, antimouse PE / Cy5 CD3 (BioLegend, CAT# 100274), anti-mouse PE / Cy7 CD4 (BioLegend, CAT# 100422), anti-mouse APC / Cy7 CD8a (BioLegend, CAT#100714), anti-mouse APC CD197 (BioLegend, CAT#120108), anti-mouse / human BV785 CD44 (BioLegend, CAT#103059), anti-mouse BV711 Ly-6A / E (BioLegend, CAT#108131), anti-mouse PE / Cy7 CD3 (BioLegend, CAT#100220), anti-mouse BV650 CD4 (BioLegend, CAT#100546), anti-mouse FITC CD279 (BioLegend, CAT#135214), anti-mouse BUV661 CD366 (BD Biosciences, CAT# 753149), anti-mouse BV421 TCF7 / TCF1 (BD Biosciences, CAT#566692), and antimouse CoraLite594 P4HA1 (Proteintech, CAT# CL594-66101).[000152] All flow cytometry analysis was carried out using MACSQuant (Miltenyi Biotec), BD LSRFortessa (BD Bioscience), or NovoCyte Penteon (Agilent) flow cytometer. Data were analyzed using FlowJo software (BD Bioscience).In vivo tumor modeling[000153] For syngeneic mouse models, mouse tumor cells were inoculated in 6-8 weeks old female mice. 4Tl-luc cells were mixed with Matrigel at 1 : 1 and injected (5 x 104 cells in 50 pL of mix) into the fat pad of Balb / C mice to establish an orthotopic mouse TNBC model. CT26 and MC38 cells mixed with Matrigel at 1 : 1 were injected subcutaneously in Balb / C at 5* 105 cells in 50 pL of mix to establish a mouse colon cancer model.[000154] For the drug administration, 4T1, CT26 or MC38 tumors -bearing mice were randomized and treated when the tumor volume reached 70-100 mm3. 1,4-DPCA ethyl ester (DPCA) was given by intraperitoneal injection at the dose of 20 mg / kg, daily. Mouse PD1 blockade antibody (BioXcell, CAT# BE014) was given by intraperitoneal injection, 10 mg / kg, every other day. DPCA was dissolved in the formulation of 4% DMSO, 40% PEG300, 5% Tween-80 and 51% PBS. Mouse PD1 blockade antibody was diluted in PBS. The tumor size was measured twice a week. Tumor volume was calculated as V=L W *W / 2. For both 4T 1 and CT26 models, tumors were surgically removed at D14 post-drug treatment. Tumor volumesand lung metastasis were monitored after surgery. Lung metastasis was examined by bioluminescence measurement in IVIS Spectrum In Vivo Imaging System. Mouse blood was harvested from venous sinus every week and frozen down for further flow cytometry analysis. [000155] For the 4T1 rechallenge model, anti-CD4 / CD8 T cell depleting antibody (STEMCELL Technologies) was given by intraperitoneal injection 5 days before 4T1 cells reinoculation and 2 days after reinoculation. 4T1 cells were re-implanted subcutaneously at 2 x 105 cells. For the lymphoid T cell blockade experiment, FTY720 was given by intraperitoneal injection at 3 mg / kg daily after tumor surgery. Blockade of T cells in lymphoid organs was accessed by examining CD8 cell percentage in blood.[000156] For CAR T in vivo modeling, NSG mice were injected with 2* 106 MDA-MB-231 cells, 5x 106 cells MDA-MB-361 cells or 2x 106 DLD-1 cells in 50 pL of PBS to establish human breast or colon cancer models. Trop2, HER2 or EGFR1 -targeting CAR T cells were injected into relevant mouse models at 1.5x 106, 0.5x 106 and 2.5x 106 cells in 200 pL of PBS, respectively, through intravenous injection. Tumor volumes were measured twice per week. Blood was collected through retro-orbital sinus for FACS analysis once per week.Ex vivo tumor and tissue processing[000157] Syngeneic mouse tumors or human cell xenograft tumors were harvested at indicated time points. Tumors were either directly fixed in 10% formaldehyde or dissociated with a Mouse / Human Tumor Dissociation Kit (Miltenyi Biotec) following the manufacturer’s instructions and filtered with a 70-prn strainer to get single cells. The fixed tumor samples were processed for immunofluorescence and the dissociated cells were processed for flow cytometry analysis.[000158] For all the mouse models, mouse blood of 100 pL was collected through venous sinus once a week. Blood was either frozen down in FBS with 10% DMSO or incubated with 1 mL red blood cell (RBC) lysis buffer at room temperature for 10 minutes and washed with PBS. The blood cells after RBC lysis were immediately subjected to flow cytometric analysis. [000159] The spleens and lymph nodes at the lumbar side were harvested at indicated time points. The tissues were mechanically disrupted. The dissociated cell suspensions were filtered through a 70-pm constrainer to obtain single cells. Cells were frozen immediately in FBS with 10% DMSO for further flow cytometric analysisMitochondrial and Cytosol fraction[000160] Cytosol and mitochondria fraction was isolated with the Mitochondria / Cytosol Fraction kit from Abeam (USA) following manufacturer’s instructions. In brief, cells were harvested and washed with ice-cold PBS twice. Cell pellet was homogenized in CytosolExtraction Buffer (containing DTT and protease inhibitors) with a Dounce tissue grinder. Homogenate was centrifuged at 700 x g for 10 minutes to remove un-lysed cells and debris and subsequently centrifuged at 10000 x g for 30 minutes. The supernatant was collected as the cytosolic fraction. Pellets were washed once with Cytosol Extraction Buffer and lysed with Mitochondria Extraction Buffer (containing DTT and protease inhibitors) as mitochondria fraction.Seahorse analysis[000161] OCR was measured with the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, USA) following the manufacturer’s instructions. In brief, CD8 cells were harvested and re-suspended with a pre-warmed XF-RPMI medium containing 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. Cells were seeded into XF Cell Culture Microplate at 2 x 105 cells per well and incubated at 37 °C without CO2 supplement for 1 hours. During the incubation, Oligomycin, FCCP and Rot / AA were prepared in the XF RPMI medium and injected in the ports of pre-hydrated sensor cartridges for the final well concentration at 1.5 pM, 1 pM and 0.5 pM, respectively. The senor cartridges were loaded onto the XF Cell Culture Microplate with CD8 cells and subjected to Seahorse XFe Analyzers (Agilent Technologies, USA) for assay running after machine calibration.Cellular succinate measurement[000162] Succinate level in CD8 cells was measured with a Succinate Assay kit from Abeam (USA) following manufacturer’s instructions. In brief, CD8 cells were harvested and counted before washing with ice-cold PBS. The cell pellet was homogenized immediately with Succinate Assay Buffer and centrifuged at 12000 x g for 5 minutes to remove debris. The supernatant was filtered through 10 kDa spin column and 50 pL of supernatant was added into the reaction mix at 1:1 in a 96-well plate and mixed well with a gentle pipette up and down. The plate was incubated at 37°C for 30 minutes and absorbance at OD=450 was measured in GloMAX Explorer (Promega) The succinate concentration was calculated based on OD reading and succinate standard curve. The final results were normalized to CD8 cell numbers used in the assay to get nmol / 106 cells.Cellular a-ketoglutarate measurement[000163] CD8 cell a-ketoglutarate (KG) level was measured with an a-Ketoglutarate Assay kit from Sigma- Aldrich (USA) following manufacturer’s instructions. In brief, CD8 cells were harvested and counted before washing with ice-cold PBS. Cell pellet was homogenized immediately with a-KG Buffer and centrifuged at 12000 x g for 5 minutes to remove debris.The supernatant was filtered through 10 kDa spin column and 50 pL of supernatant was added into the reaction mix at 1 : 1 in a 96-well plate and mixed well with a gentle pipette up and down. The plate was incubated at 37 for 30 minutes, and absorbance at OD=570 was measured in GloMAX Explorer (Promega). The a-KG concentration was calculated based on OD reading and a-KG standard curve. The final results were normalized to CD8 cell numbers used in the assay to get nmol / 106 cells.Immunobloting[000164] All immunoblotting analyses were performed following SDS PAGE using standard methods. Antibodies used for immunoblotting were P4HA1 antibody (Thermo Fisher, CAT# PA5106334, dilution 1: 1000), P4HA2 (Thermo Fisher, CAT# PA5100166, dilution 1:1000), P4HA3 antibody (Thermo Fisher, CAT# PA5-89422, dilution 1 : 1000), P4HB antibody (Thermo Fisher, CAT# MA5-32542, dilution 1 :1000), PHD1 antibody (Proteintech, CAT# 12984-1-AP, dilution 1 :1000), PHD2 antibody (Proteintech, CAT# 19886-1-AP, dilution 1 :1000), PHD3 antibody (Proteintech, CAT# 18325-1-AP, dilution 1 :1000), ATP5A antibody (Abeam, CAT# AB14748, dilution 1 :1000).Immunofluorescence staining of FFPE slides[000165] Human TNBC FFPE slides were provided from the Department of Cancer and Inflammation Research, University of Southern Denmark (Denmark). The slides were deparaffinized and rehydrated, and antigens were retrieved using pH 9.0 Tris-EDTA buffer. Slides were blocked with 5% BSA in PBS for 20 min and incubated with rabbit anti-human P4HA1 antibody (Thermo Fisher, CAT# PA5106334, dilution 1: 100), rat anti-human CD8 antibody (Thermo Fisher, CAT# MA181692, dilution 1:50), mouse anti-human PD1 antibody (Abeam, CAT# ab52587, dilution 1 :100) and anti-human TIM3 APC conjugated antibody (Miltenyi, CAT# 130-120-700, 1 : 100) overnight under 4 °C. The slides were washed with 0. 1% Tween20 in PBS three times and then incubated with secondary antibodies at 1 : 100, including anti-rabbit Alex Fluor 546 (Invitrogen, CAT# A-11035), anti-rat Alex Fluor 488 (Invitrogen, CAT# A-l 1006) and anti -mouse Alex Fluor 633 (Invitrogen, CAT# A-21071). After washing with PBS, slides were stained with DAPI and mounted in FluorSave (Millipore, CAT# 345789). For each sample, quantifications were based on four images taken at random fields using a Leica Lightsheet microscope. Fluorescent signals were captured at the wavelength of 660-700 nm for TIM3-APC, 500-540 nm for Alex Fluor 488, 580-620 nm for Alex Fluor 546, and 640-670 nm for Alex Fluor 633. Images were analyzed using the software Leica Application Suite X.Immunofluorescence staining of human CD8 cells[000166] CD8 cells were seeded in an 8-well chambered coverslip (ibidi, German) at 2* 104 cells per well and incubated at 37 for 40 minutes. Cells were washed once with PBS and fixed in 4% paraformaldehyde for 15 minutes. Cells were washed twice with PBS, permeabilized with 0.2% Triton-X 100 in PBS for 20 minutes, and blocked in 0.1% Triton-X 100 and 3% BSA in PBS for 1 hour at room temperature. After washing with 0.1% Triton-X 100 in PBS, cells were incubated with anti-ATP5A antibody (Abeam, CAT# AB14748, dilution 1 :100), anti-CD8 antibody (Abeam, CAT# AB60067, dilution 1:100) and anti-P4HAl antibody (Thermo Fisher, CAT# PA5106334, dilution 1 : 100) diluted in 0.1% Triton-X 100 and 1% BSA in PBS at 4 overnight. Cells were washed with 0.1% Triton-X 100 in PBS once and incubated with anti-mouse Alex Fluor 488 (Invitrogen, CAT# A-32766, dilution 1 :100), anti-rat Alex Fluor 546 (Invitrogen, CAT# A-11081, dilution 1 : 100) and anti-rabbit Alex Fluor 647 (Invitrogen, CAT# A-3157311035, dilution 1 : 100) at room temperature for 1 hour. Cells were then washed with 0. 1% Triton-X 100 in PBS for twice and mounted with Vectashield medium with DAPI.RNA-seq[000167] Naive CD8 T cells were isolated from human healthy donor PBMCs using a human CD8+ T Cell Isolation Kit (Miltenyi) according to the manufacturer’s instructions. The CD8 cells were then stimulated, infected with CAR virus, expanded to generate CD8 CAR T cells, and subjected to coculture, following the same protocols as described above.[000168] Human CD8 T cells were harvested at indicated time points and lysed with Trizol (Invitrogen, USA). Total RNA was isolated and purified with the RNeasy Mini Kit (Qiagen, German) and reconstituted with Nuclease-free water. The RNA samples were sent toNovogene AIT (Singapore) for mRNA sequencing and directional mRNA library preparation.[000169] QC of sequencing data was performed in FastQC (vO.l 1.9) and MultiQC (vl .10). Paired fastq files were aligned to human genome hg38 with Spliced Transcripts Alignment to a Reference (STAR). BAM files were inspected for various alignment metrics using RSeQC and gene-level counts were quantified using Salmon. The raw counts data were imported in R (v4.0.4). The gene set enrichment analysis (GSEA) analysis and over representation analysis (ORA) of GO terms was performed using the packages edgeR (v4.0.1) and clusterProfiler (v4.10.1). The raw counts data were normalized and scaled to counts per 1 million (CPM) for gene fold change analysis and heatmap generation with packages edgeR (v4.0.1) and ggplot2 (v3.4.4). Raw RNA-seq data was uploaded in GEO with an accession number.Analysis of public scRNA-seq data[000170] The single-cell RNA sequencing data was obtained from the GEO database and analyzed in Seurat package (v.5.0.0). For the scRNA-seq data from the work of Zhang et.al. (GSE169246)33 and Sade-Feldman et al (GSE120575)5, CD8 cell data was extracted based on the metadata provided in the matrix file which was generated with two rounds of unsupervised clustering, as described previously. For the scRNA-seq data from the work of Steele et.al. (GSE155698)32, data was scaled and log normalized and PCA was run for dimension reduction. Unsupervised clustering of cells was done with UMAP algorithms and CD8 cells were identified based on Findclusters function. In the subsequent analysis, average expression levels of interest genes in CD8 cells were accessed by AverageExpression function in different tissues from different patients. Data was presented as the output values of AverageExpression function grouped by CD8 cells in each individual patient.Statistics analysis[000171] Statistics analyses were performed in GraphPad Prism (v9.0) or R programming (v4.0.4). For multiple comparisons, the P values were determined with one-way analysis of variance (ANOVA) or two-way ANOVA, as indicated in the figure legends For comparisons of two groups, the P values were determined with paired or unpaired two-tailed t-tests, as indicated in the figure legends. Survival analysis was based on a log-rank test. In GSEA analysis and Gene Ontology ORA analysis, p values were determined in edgeR or clusterProfiler, adjusted by the Benjamin-Hochberg model. Values of P < 0.05 were considered significant.Sequence Listing TableREFERENCES[000172] Sade-Feldman, M., Yizhak, K., Bjorgaard, S.L., Ray, IP., de Boer, C.G , Jenkins, R.W., Lieb, D.J., Chen, J.H., Frederick, D.T., Barzily-Rokni, M., et al. (2018). Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell 175, 998- 1013. e20. (Dataset reference number: GSE120575)[000173] Zhang, Y., Chen, H., Mo, H., Hu, X., Gao, R., Zhao, Y., Liu, B., Niu, L., Sun, X., Yu, X , et al. (2021). Single-cell analyses reveal key immune cell subsets associated with response to PD-L1 blockade in triple-negative breast cancer. Cancer Cell 39, 1578-1593. e8. (Dataset reference number: GSE169246)

Claims

CLAIMSWhat is claimed are:

1. A genetically modified T cell, comprising a genetic modification to P4HA1 gene that inhibits or inactivates expression of prolyl 4 hydroxylase subunit alpha 1 (P4HA1) in the T cell.

2. The genetically modified T cell of claim 1, further comprising a genetic modification to PD1 gene that inhibits or inactivates the expression of programmed cell death- 1 protein (PD1) in the T cell.

3. The genetically modified T cell of any one of claims 1-2, further comprising a genetic modification to express a chimeric antigen receptor (CAR) on the surface of the T cell.4 The genetically modified T cell of claim 3, wherein the genetically modified T cell is a CAR T cell.

5. A method of manufacturing a T cell as defined in any one of claims 1 to 4, wherein the method comprises modifying the expression of P4HA1 by genetic modification to the T cell that inhibits or inactivates the expression of P4HA1.

6. The method of claim 5 comprising: introducing into the T cell a ribonucleoprotein complex and a nucleic acid sequence encoding a P4HA1 gRNA targeting the genomic sequence of P4PIA1; or introducing a nucleic acid encoding an siRNA, an shRNA, a microRNA or an oligonucleotide targeting the P4PIA1 mRNA; wherein the ribonucleoprotein complex comprises a Cas9 protein and a P4HA1 gRNA.

7. The method of claim 5 or 6, wherein the method further comprises modifying the expression of PD1 by genetic modification to the T cells that inhibits or inactivates the expression of PD1.

8. The method of claim 7 comprising: introducing into the T cell a ribonucleoprotein complex and a nucleic acid sequence encoding a PD1 gRNA targeting the genomic sequence of PDP, or introducing into the T cell a nucleic acid encoding an siRNA, an shRNA, a microRNA or an oligonucleotide targeting the PD1 mRNA; wherein the ribonucleoprotein complex comprises a Cas9 protein and a P4HA1 gRNA.

9. The method of any one of claims 5-8, further comprising introducing into the T cells a nucleic acid encoding a chimeric antigen receptor.

10. A method of treating or alleviating cancer, comprising administering a therapeutically effective amount of the genetically modified T cell of any one of claims 1-4 to a subject in need thereof.

11. A method of treating or alleviating cancer in a subject, comprising administering a therapeutically effective amount of a P4HA1 inhibitor to a subject in need thereof, wherein the cancer is resistant to treatment with an immune checkpoint inhibitor.

12. The method of claim 11, wherein the immune checkpoint inhibitor is a PD1 / PD-1L inhibitor.

13. A method of treating or alleviating cancer, comprising administering a combination comprising a therapeutically effective amount of a P4HA1 inhibitor and a therapeutically effective amount of a PD1 / PD-1L inhibitor to a subject in need thereof.

14. The method of claim 13, wherein administering the combination delays and / or reduces tumor relapse compared to administering the P4HA1 inhibitor or PD1 / PD-1L inhibitor alone.

15. The method of any one of claim 11-14, wherein the P4HA1 inhibitor is selected from the group consisting of l,4-Hydroxy-l,10-phenanthroline-3-carboxylic acid (1,4-DPCA;Fraxine, TP0463518, 1,4-DPCA ethyl ester, AKBA, SYP-5, JNJ, Ethyl 3,4-benzoate, Desidustat, Py-thiDC, AKB-6899, EL- 102, IOX 2, MK-8617, Acriflavine chloride, Oltipraz, Hydroxycitric acid, GN44028, Molidustat, N-Oxyl, DMOG, PT-2385, Vadadustat, and Oroxylin-A.

16. The method of any one of claims 12-14, wherein the PD1 / PD-1L inhibitor is a PD1 antibody.

17. The method of any one of claims 11-16, wherein the cancer is selected from any one of the following: leukemia, myeloma, sarcoma, melanoma, lymphoma, or cancer of the breast, colon, bladder, prostate, lung, kidney, pancreas, uterus, ovary, or testicle.

18. The method of any one of claims, 10-17, wherein the cancer is a solid tumor cancer.

19. The method or use of claim 18, wherein the solid tumor cancer is selected from any one of the following: melanoma, lymphoma, cancer of the breast, colon, bladder, prostate, lung, kidney, pancreas, uterus, ovary, or testicle.

20. A method for determining endogenous T cell immunity in a subject receiving a cancer treatment, the method comprising measuring the expression level of P4HA1 in T cells using a sample obtained from the subject, wherein a decrease in the expression level of P4HA1 in T cells of the subject as compared to the expression level of P4HA1 in the T cells of the subject measured prior to receiving the cancer treatment is indicative that the subject has improved endogenous T cell immunity.

21. A method for determining resistance to PD1 / PD-1L inhibitor treatment in a subject having cancer, the method comprising measuring the expression level of P4HA1 in T cells using a sample obtained from the subject, wherein a higher expression level of P4HA1 in the T cells of the subject as compared to a cancer patient responsive to PD1 / PD-1L inhibitor treatment is indicative that the subject is resistant to PD1 / PD-1L treatment.

22. The method of claim 21, wherein the expression level of P4HA1 in the T cells of the subject is at least 1.5 folds higher than the cancer patient responsive to PD1 inhibitor treatment.

23. The method of any one of claims 20-22, wherein the sample is a tumor sample, a blood sample or a tumor-draining lymph node sample.

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