WO2023049768A1

WO2023049768A1 - Fibp knockout in t cells amplifies antitumor activity by limiting cholesterol metabolism, suggesting a potentiator of adoptive cell therapy

Info

Publication number: WO2023049768A1
Application number: PCT/US2022/076809
Authority: WO
Inventors: Peng Jiang; Yu Zhang
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2021-09-21
Filing date: 2022-09-21
Publication date: 2023-03-30

Abstract

In one embodiment, the invention provides a T cell exhibiting reduced or diminished FIBP or TMEM222 expression. The T cell can comprise chromosomal DNA, wherein the chromosomal DNA lacks a genetic sequence encoding an intact FIBP, TMEM222 or both FIBP and TMEM222. In another embodiment, the invention provides an extrachromosomal nucleic acid comprising a genetic sequence which is substantially complementary to a genetic sequence encoding a FIBP or TMEM222. In another embodiment, the invention provides a method for making a T cell lacking functional FIBP and/or TMEM222 expression. In another embodiment, the invention provides a composition comprising a T cell lacking functional FIBP and/or TMEM222 expression and a carrier. In another embodiment, the invention provides a method and uses involving the inventive composition for adoptive T cell transfer, such as for therapy for treating a solid tumor.

Description

FIBP KNOCKOUT IN T CELLS AMPLIFIES ANTITUMOR ACTIVITY BY LIMITING CHOLESTEROL METABOLISM, SUGGESTING A POTENTIATOR OF ADOPTIVE CELL THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No.

63/246,743, filed September 21, 2021, which is incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 125,714 Byte XML file named "764442.xml," dated September 19, 2022.

STATEMENT REGARDING

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with Government support under project number ZIA BC 011889 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0004] Despite recent breakthroughs in cancer immunotherapy, T cell therapies achieve limited efficacy in solid tumors. Identifying regulators in T cell dysfunction remains challenging due to limitations of current screening platforms. For example, immune checkpoint blockades (ICB) on CTLA4 and PD1/PDL1 signaling can induce long-lasting responses in patients with a wide range of metastatic cancer types. Further, cell-based therapies include adoptive T cell transfer or chimeric antigen receptor (CAR) T cells, according to which, collected T cells are engineered to express CARs, typically CD 19, which is an antigen found on B cells. Despite promising research, however, most patients with most cancer types do not respond to ICB treatment, and CAR-T therapy seemingly is effective in hematological malignancies but not solid tumors. (See “CAR T Cells: Engineering Patients’ Immune Cells to Treat Their Cancers,” originally published by the National Cancer Institute (updated July 30, 2019) (available at Cancer . gov/about-cancer/treatment/research/car-T-cells, and which is incorporated herein in its entirety)).

[0005] Many factors in the solid tumor microenvironment can suppress anticancer T cells even after therapeutic intervention. For example, T cell dysfunction in tumors can result from mechanisms other than CTLA4 and PD1/PDL1 signaling, such as alternative checkpoints, immune-suppressive cells, or cytokine and metabolite release. In such cases, current ICB therapies will not be effective. T cells can also lose anticancer activities due to cell-intrinsic mechanisms that act independently of other immune-suppressive factors. For example, CAR-T cells may progressively upregulate FASL, TRAIL, and their cognate receptors, resulting in cell death. Such activation-induced T cell death is an essential mechanism in maintaining immune tolerance and homeostasis but can limit the long-lasting effect of T cell therapy.

[0006] Diverse genomics approaches have been developed to identify mechanisms and therapeutic targets to enhance anticancer T cells. For example, T cell transcriptomes from tumor samples of patients treated by ICB have been profiled to identify the molecular signatures of T cells associated with favorable or unfavorable clinical outcomes. However, molecular markers identified from genomics profiles reflect correlations, not regulatory casualties. To address this limitation, pooled CRISPR screens in human T cells have identified genes that regulate T cell proliferation upon T cell receptor (TCR) stimulation (Shifrut, et al. “Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function” [Internet, August 3, 2018]; available from: dx.doi. org/ 10.1101/384776, which is referenced throughout this specification and incorporated herein in its entirety). However, in vitro T cell proliferation in the context of a CRISPR screen is not a direct measurement of anticancer capability. In vivo genetic screens attempt to address these limitations by pooling genes to evaluate gene knockout effects on the T cell accumulation in murine tumors. Also, murine tumors can host only a limited number of T cells tagged with guide RNAs. Therefore, in a genome-wide screen, most genes receive a “zero” count and are dropped from further evaluations.

[0007] Accordingly, adoptive and CAR T cell therapies still have low efficacy in solid tumors due to many immunosuppressive factors in the tumor microenvironment. Developing new cancer immunotherapies will benefit from an increased understanding of T cell dysfunction mechanisms in tumors. Thus, there remains a need for technology to amplify and potentiate antitumor and cancer immunotherapy using T cells (such as adoptive and CAR T cell therapies), particularly solid tumors.

BRIEF SUMMARY OF THE INVENTION

[0008] In one embodiment, the invention provides a T cell exhibiting reduced or diminished FIBP or TMEM222 expression. In a preferred aspect, the inventive T cell comprises chromosomal DNA, wherein the chromosomal DNA lacks a genetic sequence encoding a functional FIBP or TMEM222, in other words a “knockout” T cell, lacking functional FIBP, TMEM222, or both genes.

[0009] In another embodiment, the invention provides an extrachromosomal nucleic acid comprising genetic sequence which is substantially complementary to a genetic sequence encoding FIBP or TMEM222.

[0010] In another embodiment, the invention provides a method for making a T cell lacking functional FIBP and/or TMEM222 expression. The method comprises (a) obtaining one or more source T cells, (b) expanding the population of source T cells and activating the expanded population of source T cells, and (c) genetically manipulating the activated T cells within the population to generate a resulting T cell lacking functional FIBP and/or TMEM222 expression.

[0011] In another embodiment, the invention provides a composition comprising the inventive T cell and a carrier, which can comprise a pharmaceutically acceptable carrier.

[0012] In another embodiment, the invention provides a method of adoptive T cell transfer, and uses of the inventive composition for protocols involving adoptive T cell transfer, comprising administering a pharmaceutical composition comprising the inventive T cell to a subject suffering from cancer and in need of therapy therefor, in an amount and at a location sufficient to treat the cancer within the subject. In a preferred embodiment, the cancer can comprise a solid tumor.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0013] Figs. 1 A through II concern identification the of genes associated with T cell resilience to immunosuppressive tumor microenvironments.

[0014] Fig. 1 A is a graphic presentation of a two-stage model with single-cell T cell transcriptomes as input. The Tres model first quantifies the degree to which the tumor microenvironment surrounding each T cell is immunosuppressive, based on signaling response signatures of TGFB1 and TRAIL. Then, Tres identifies gene signatures associated with T cells that are still proliferative despite an immunosuppressive environment.

[0015] Fig. IB is a plot presenting data concerning an example of negative correlation between immune suppression and T cell proliferation scores from a patient in a melanoma study. Each dot represents a CD8 T cell. The X-axis shows immune suppression scores computed as TGFB1 signaling activities. The Y-axis shows cell proliferation scores computed through the cell cycle and DNA replication gene sets.

[0016] Fig. 1C is a plot presenting data concerning correlations between immune suppression and T cell proliferation across single-cell cohorts. Each dot represents a patient in a single-cell RNASeq study, shown as cancer and database names (G: GEO GSE; A: ARRAYEXPRESS). The correlations between immune suppression and CD8 T cell proliferation were computed as for Fig. 2B. The thick line represents the median value. The bottom and top of the boxes represent the 25th and 75th percentiles (interquartile range). Whiskers encompass 1.5 times the interquartile range. The shape of each dot indicates the profiling platform.

[0017] Fig. ID is a plots presenting data concerning correlations between immune suppressions from different cytokines and T cell proliferation. Each dot represents a single-cell dataset. The shape of each dot indicates the profiling platform. The median correlation between immunosuppression from different cytokines and T cell proliferation was computed as for Fig. 1C. Distributions were shown with violin plots smoothed by the kernel density estimator. The two-sided Wilcoxon signed-rank test was utilized to compare group values and zero (** p < le'²; *** p < le'³).

[0018] Fig. IE are plots presenting data concerning the T cell resilience (Tres) model through the variable interaction test. The correlation between immune suppression (TGFB 1 activity) and CD8 T cell proliferation is plotted as Fig. IB, except individual cells were split into high (log2CPM > 2) and low (log2CPM < 2) groups according to the expression of IL7R or FIBP. P-values were from the two-sided interaction test using continuous values without any cutoffs, evaluating whether the correlation between immune suppression and T cell proliferation depends on a third gene.

[0019] Fig. IF is a plot presenting data concerning the relationship between T cell resilience scores from a patient and CRISPR screen phenotypes. Each dot represents a gene, with the X- axis showing Tres scores computed for a patient in a colorectal cancer study and the Y-axis showing the loss-of-function phenotypes in human primary T cells from a genome-wide CRISPR screen (Shifrut, et al.). Crosses highlight genes whose values are significant, accounting for both axes (false discovery rate < 0.05). Shaded regions include genes whose values on the two axes are consistent with each other.

[0020] Fig. 1G is a plot presenting data concerning CRISPR screen phenotypes of FIBP and TMEM222 from a previous study (Shifrut, et al.). Each dot represents a CRISPR guide RNA (gRNA) for two genes with significant scores as shown in Fig. IF. For each gRNA, Y-axis shows the log2 -fold-change (logFC) between fractions of gRNA-harboring cells between T cell receptor stimulation and control conditions. The dotted line presents the median logFC of a reference gene CBLB with the most significant phenotype in the study. The thick line represents the median value. The bottom and top of the boxes are the 25th and 75th percentiles (interquartile range). The whiskers encompass 1.5 times the interquartile range.

[0021] Fig. 1H is a bar chart presenting data demonstrating that FIBP and TMEM222 have significant T cell resilience scores and CRISPR screen scores across studies. For each patient in the single-cell RNASeq data collection reported herein, the consistency between T cell resilience gene scores and CRISPR screen phenotypes was evaluated in the same way as for Fig. IF. The stacked bar plot shows the count of patients with significant positive CRISPR screen phenotypes and negative T cell resilience scores related to immune suppressions from either TGFB1 or TRAIL (false discovery rate < 0.05).

[0022] Fig. II are plots presenting data demonstrating that high FIBP expression in tumors for lymphocyte expansion indicates low adoptive cell therapy efficacy. The overall survival of patients upon adoptive T cell transfer was shown for tumors with different levels of cytotoxic T lymphocytes (CTL, average expression of CD8A, CD8B, GZMA, GZMB, and PRFP) and FIBP expression. The interaction significance between FIBP and CTL on the survival outcome was evaluated by the two-sided student t-test without any cutoffs.

[0023] Figs. 2A through 2F present the results of experiments demonstrating that FIBP and TMEM222 knockouts in T cells enhanced the cancer killing efficacy.

[0024] Fig. 2A is a schematic presentation of the protocol for co-culture between CD8 T cells and cancer cells used in the experiments reported herein. For humans, CD8 T cells are derived from donors’ peripheral blood transduced with the NY-ESO-1 T cell receptor (TCR), targeting NY-ESO-1 antigen on A375 and Mel624 cell lines labeled with RFP. For mice, CD8 T cells were harvested from the Pmell TCR transgenic mice, and Pmell T cells target gplOO antigen on the B16-mhgpl00 cell line labeled with RFP. The co-culture readout is cancer cell growth measured by the INCUCYTE imaging system and cytokine release measured by ELISA.

[0025] Fig. 2B are Western blots showing CRISPR guide RNA (gRNA) knockout (KO) efficiency. For each protein with antibodies available, the protein levels in T cells from human donor 1 and Pmel transgenic mice were shown for control (C, AAVS1 for human and Rosa26 for mice) and three independent gRNAs (1, 2, 3).

[0026] Fig. 2C are photomicrographs presenting the results of image-based co-culture killing assay. A375 cells with the NY-ESO-1 antigen were labeled with the red fluorescent protein (RFP). Human T cells from donor #1 targeting NY-ESO-1 antigen were incubated together with the cancer cells at an effector-to-tumor (E:T) ratio of 2. The RFP intensity of cancer cells was measured using the INCUCYTE at 48 hours for both AAVS1 control and FIBP KO conditions with three independent gRNAs. The quantitative data relating to the images is presented in Fig. 2D.

[0027] Fig. 2D are plots presenting data demonstrating that /

and TMEM222 knockouts in human donor T cells enhanced the cancer-killing efficacy. The killing efficacy of T cells from donor 1 targeting the NY-ESO-1 antigen was evaluated with NY-ESO-1 positive A375 and Mel624 cell lines at an E:T ratio of two as described in Fig. 2C. The T cell efficacy, measured as the relative RFP intensity (Y-axis) from time zero, was compared across various gene knockouts (KO) at different time points (X-axis). Each data point represents the median value among three gRNAs with standard deviations as error bars; except for TMEM222, only two gRNAs demonstrated successful KO (Fig. 6A). The lower panels show FIBP and TMEM222 KOs in an additional human donor T cell.

[0028] Fig. 2E are plots presenting data demonstrating that FIBP and Tmem222 KOs in mouse Pmel T cells enhanced its killing efficacy on B16-mhGP100 cells with the gplOO antigen. The efficacy of T cell mediated cancer killing was shown for murine cells with two E:T ratios as discussed with reference to Fig. 2D.

[0029] Fig. 2F are plots presenting data demonstrating that FIBP and TMEM222 knockouts in T cells enhanced the release of T cell effector cytokines. The interferon-gamma (IFNG) and TNF-alpha (TNFA) cytokine concentrations were measured for different gene KOs (legend for Figs. 2D and 2E) by the ELISA assay. The comparison between gene KO and control conditions was performed with the two-sided Wilcoxon rank-sum test (*: p-value < 0.05).

[0030] Figs. 3 A through 3E present the results of experiments demonstrating that FIBP knockouts in T cells enhanced the in vivo efficacy of adoptive transfer therapy.

[0031] Fig. 3 A is a graphic presentation of adoptive T cell transfer (ACT) procedure. The B16-mhgpl00 cell line was implanted in C57BL/6 mice. Lymphodepletion through radiation was performed ten days after tumor injection, and T cells with different gene knockouts were injected the day after. Tumor size measurements were taken twice a week to evaluate the adoptive cell therapy efficacy. [0032] Fig. 3B is a plot presenting data concerning tumor growth curves in mice treated with T cells with FIBP and Rosa26 knockouts. The tumor area (length*width) was measured after adoptive cell transfer (ACT).

[0033] Fig. 3C is a plot presenting data concerning the average tumor size in mice treated with T cells with gene knockouts. The tumor area mean was shown when no mice had reached endpoints (long dimension > 20mm or death). Error bars indicate standard error. Comparisons between target groups and the Rosa26 control were through the two-sided rank-sum test (** p < 0.01).

[0034] Fig. 3D is a plot presenting data concerning tumor size at the last time point at which no mice had reached the endpoint. All tumor size distributions at day 19 were shown through violin plots smoothed by a kernel density estimator. The comparison between each target group and the Rosa26 control was through the two-sided rank-sum test (** p < 0.01).

[0035] Fig. 3E is plot presenting Kaplan Meier curves of event-free survival. For each group, the fraction of mice that did not reach the endpoint (long dimension > 20mm or death) was shown at different days after adoptive cell transfer (ACT). The survival comparison between the target group and Rosa26 control was made through the one-sided log-rank test (* p < 0.05), with line type legend as described for Fig. 3C.

[0036] Figs. 4A through 4H present the results of experiments demonstrating that FIBP knockout inhibits cholesterol metabolism to enhance T cell anticancer activity.

[0037] Fig. 4A is a plot presenting data concerning differential gene expression profiles of T cells upon FIBP knockout. Each dot represents one gene. The log2 fold change (log2FC) was computed by comparing RNASeq read counts between FIBP and control (Rosa26) knockout conditions with three independent guide RNAs for each target. The adjusted p-values were computed using the DESEQ2 software.

[0038] Fig. 4B is a chart presenting data concerning pathway enrichment analysis of FIBP knockout expression profiles. Ingenuity Pathway Analysis (IP A) analysis was performed to identify up and down-regulated pathways in differential expression profiles. [0039] Fig. 4C is a depiction presenting exemplary expression values in cholesterol metabolism pathway. The log2(FPKM+l) values (z-transformed to zero mean and standard deviation) are shown for example genes. The data only include genes with logFC absolute value larger than one in at least one condition.

[0040] Fig. 4D are bar charts presenting data concerning RT-qPCR validation of essential genes in cholesterol metabolism. For mouse and human T cells, normalized expression levels were shown for control (Rosa26 for mouse and AAVS1 for human) and FIBP knockout conditions. The mean and standard deviation were calculated through two independent guide RNAs.

[0041] Fig. 4E depicts Western blots of essential cholesterol metabolism regulators after FIBP knockout. Protein levels of SREBF2 (unactivated full-length and activated N-terminal NH2 fragments), LDLR, and Actin control were shown for Rosa26 control and FIBP knockouts in Pmel T cells.

[0042] Fig. 4F depicts Western blots of cholesterol metabolism regulators after FIBP overexpression. Protein levels were shown as in Fig. 4E for vector control and FIBP over-expression in Pmel T cells.

[0043] Fig. 4G is a bar chart presenting data concerning cholesterol concentration in FIBP versus control perturbed T cells via oxidation-based quantification. In the knockout (KO) group, FIBP and Rosa26 control knockouts have three independent gRNAs for each target. Mean and standard deviations were calculated through three gRNAs. In the overexpression group, plasmid vector is the control condition. Mean and standard deviations were calculated through three cell culture replicates. The comparison between control and FIBP conditions was done through the two-sided Wilcoxon rank-sum test (*: p < 0.05).

[0044] Fig. 4H are graphs presenting data concerning the efficacy of T cell mediated cancerkilling in a high cholesterol environment. The killing efficacy on B16 cells with gplOO antigen was shown for T cells with Rosa26 control and FIBP knockouts in regular and high-cholesterol (0.75 pg/ml) medium. Each knockout utilized a mixture of three gRNAs. The mean and standard error values were computed from 6 cell culture replicates. [0045] Figs. 5 A through 5H concern signature genes of T cell resilience to immunosuppressive tumor microenvironment and are related to the data presented in Figs. 1 A through II.

[0046] Fig. 5 A is a plot of the distribution of interaction test p-values for each gene score. This example is from a patient in a melanoma single-cell RNASeq study. Two-sided student t- test p-values were computed for interaction terms between Tres score (i.e., TGFB1 signaling) and every gene expression value.

[0047] Fig. 5B is a plot of data concerning the association between T cell resilience (Tres) and T cell sternness gene scores. Each dot presents a gene with the Tres score, computed for a patient from a melanoma study, on Y-axis and T sternness score on X-axis. The p-value was from the two-sided Wilcoxon rank-sum test, comparing the Tres scores between positive and negative T sternness markers.

[0048] Fig. 5C is a plot of the receiver operating characteristic (ROC) curve for the quality of Tres score. The ROC curve presents false-positive rates against true-positive rates of predicting T cell sternness markers through Tres scores at different thresholds. Tres scores were computed for immune suppression scores from either TGFB1 or TRAIL signaling activities. The diagonal line represents random expectation.

[0049] Fig. 5D is a graph presenting data concerning the quality of Tres scores in single-cell cohorts. Each dot presents a patient in a single-cell study. The area under the ROC curve (AUC) is the quality measure of Tres scores associated with TGFB1 signaling, shown in box-plots as for Fig. 1C.

[0050] Fig. 5E is a plot presenting data concerning the quality of Tres scores associated with immunosuppression through either TGFB1 or TRAIL signaling. The median AUC in each cohort was shown with violin-plots as for Fig. ID. P-values were computed through the two- sided Wilcoxon rank-sum test, comparing the group values and the random expectation 0.5 (*** p < le-3).

[0051] Fig 5F are plots presenting data concerning CD8 T cells with low FIBP or TMEM222 expression are resilient to the TGFB1 signaling inhibition, using data from a colorectal tumor. The correlation between TGFB1 activities and CD8 T cell proliferation is plotted as for Fig. IB, except the single cells were split into high (log2CPM > 1) and low (log2CPM < 1) groups by the expression of FIBP or TMEM222. The t-value and p-value were computed through the two- sided student t-test on interaction covariates of regression using continuous values without any cutoffs.

[0052] Fig. 5G is a plot of data concerning Tres scores of FIBP and TMEM222 across all patients. Each dot represents a patient included in the single-cell RNASeq data collection reported herein. Tres scores computed for TGFB1 and TRAIL signaling are shown through violin plots smoothed by a kernel density estimator. P values were computed using the two- sided Wilcoxon signed-rank test comparing the difference between group values and zero (** p < 0.01; *** p < 0.001).

[0053] Fig. 5H are plots of data demonstrating that FIBP and TMEM222 are up-regulated in CD8 T cells from COVID-19 patients with severe symptoms compared to mild controls. The gene expression value is from two single-cell RNASeq studies on peripheral blood samples. Each dot represents the average value across all CD8 T cells in an individual. The violin plots present expression distributions in different symptom groups, smoothed by a kernel density estimator. The p-value was computed by the two-sided Wilcoxon rank-sum test, comparing values between severe and mild groups (* p < 5e-2; *** p < le-3).

[0054] Figs. 6A through 6H present the results of experiments demonstrating that FIBP and TMEM222 knockouts in CD8 T cells enhanced the efficacy of T cell mediated cancer killing and are related to the data presented in Figs. 2A through 2F.

[0055] Fig. 6A are GEL images showing the T7El-treated (+) or non-treated (-) PCR products amplifying the Cas9 cutting sites in both human and mice cells with 3 independent gRNAs of TMEM222. Since western-blot antibody for TMEM222 is not available, T7E1 assay was performed as an alternative approach to validate CRISPR knockout efficiency. For humans, the gRNA #1 (SEQ ID NO:7) does not have sufficient knockout efficacy, thus is not included in further experiments. [0056] Fig. 6B are plots of data concerning the efficacy of T cell mediated cancer killing from human donor 1 on A375 or Mel624 cells. The T cell efficacy was measured as the relative RFP signal intensity (Y-axis) of cancer cells across different time points (X-axis) as for Fig. 2D. The culture conditions in comparison include three control gRNAs (AAVSI for human and Rosa26 for mouse), parental T cells, and cancer cells cultured without T cells.

[0057] Fig. 6C are plots of data concerning the efficacy of T cell mediated cancer killing from human donor 2 on A375 or Mel624 cells.

[0058] Fig. 6D are plots of data concerning the efficacy of T cell mediated cancer killing from Pmel TCR transgenic mice on B16-mhGP100 cells.

[0059] Fig. 6E are plots of representative CFSE signals of T cell proliferation assay in Pmel CD8 T cells. The proliferative ability of Pmel T cells was compared in FIBP knockout (KO) versus control cells (Rosa26 KO) after restimulation with anti-CD3/28 antibodies for 4 days. Representative CFSE signals from TCR stimulated and unstimulated T cells are shown.

[0060] Fig. 6F is a graph presenting data concerning a T cell proliferation assay in Pmel CD8 T cells. The proliferation index across 3 independent gRNAs for each gene was calculated using FLOWJO software.

[0061] Fig. 6G are graphs presenting data concerning early activation marker measured 12h after TCR re-stimulation in human cells. CD69 level was determined by flow cytometry in FIBP knockout T cells compared to control cells across 3 independent gRNAs for each targeted gene.

[0062] Fig 6H are graphs presenting data concerning early activation marker measured 12h after TCR re-stimulation in mouse cells. CD69 level was determined by flow cytometry in FIBP knockout T cells compared to control cells across 3 independent gRNAs for each targeted gene.

[0063] Figs. 7A through 7C present the results of experiments measuring in vivo efficacy of adoptive transfer therapy and are related to the data presented in Figs. 3 A through 3E.

[0064] Fig. 7A is a plot of data concerning tumor size in different groups randomized at day zero. All mice were randomized into different groups to achieve an even initial tumor size. The tumor area distribution was shown through violin plots smoothed with a kernel density estimator. [0065] Fig. 7B is a plot of data concerning the average tumor size in negative control groups. The tumor area mean was shown when no mice reached endpoints, with standard errors as error bars. The group comparison was made through the two-sided rank-sum test (* p < 0.05) for two comparisons: 1, between parental T cells and no-treatment control; 2, between T cells with Rosa26 knockout and parental T cells. Notably, T cells electroporated with Rosa26 CRISPR RNP even have higher antitumor efficacy than parental T cells. One possible explanation is that the dead cell removal after CRISPR RNP electroporation will enrich more potent T cells than parental T cells.

[0066] Fig. 7C is a plot of Kaplan Meier curves concerning mice survival at different time points. The fraction of mice that did not reach the endpoint was shown at different days after adoptive cell transfer (ACT). The survival comparison was made through the one-sided log-rank test (* p < 0.05) for two comparisons as for Fig. 7B.

[0067] Figs. 8A through 8J present the results of experiments demonstrating that F/FP knockout inhibits cholesterol metabolism to enhance T cell anticancer activity and are related to the data presented in Figs. 4A through 4H.

[0068] Fig. 8A is a chart presenting data concerning IPA Disease & Function analysis for deregulated genes in FIBP knockout cells.

[0069] Fig. 8B is a plot of data concerning the correlation between FIBP and ABCA1 among acute lymphoblastic leukemia samples from the MILE project.

[0070] Fig. 8C is a chart depicting the mean and standard error of correlations between FIBP and gene members in the cholesterol metabolism pathway. The comparison between the positive and negative groups is via the two-sided Wilcoxon rank-sum test.

[0071] Fig. 8D is a chart of data concerning differential expression values for SREBF2 ChlP- Seq targets and non-targets. The CISTROME database contains two public human ChlP-Seq profiles from B lymphocytes and HepG2 cells. The regulatory score on each target gene is computed by the RABIT framework. Target genes are those with regulatory scores larger than 0.5. None target genes are those with zero regulatory scores. The comparison between groups is through the two-sided Wilcoxon rank-sum test (***: p < 0.001). [0072] Fig. 8E is a bar chart depicting data concerning RT-qPCR validation of essential enzymes in cholesterol synthesis in mouse T cells. Normalized expression levels were shown for control (Rosa26) and FIBP knockout conditions. The mean and standard deviation were calculated through two independent guide RNAs.

[0073] Fig. 8F is a bar chart depicting data concerning RT-qPCR levels of cholesterol metabolic regulators in Pmel T cells with FIBP and eGFP control overexpression. The mean and standard deviation were calculated through three cell-culture replicates. The comparison was done using the two-sided Wilcoxon rank-sum test.

[0074] Fig 8G presents plots of data concerning cholesterol concentration in FIBP versus Rosa26 (3 independent gRNA for each gene) knockout T cells via Filipin III staining. The comparison between control (Rosa26) and FIBP knockout conditions was done using the two- sided Wilcoxon rank-sum test.

[0075] Fig. 8H presents plots of data concerning cholesterol concentration in Pmel T cells after FIBP overexpression. Cholesterol content as indicated by filipin III staining in FIBP overexpressing cells compared to control cells (eGFP'). Mean and standard deviation were calculated through three cell-culture replicates.

[0076] Fig. 81 presents plots of data concerning cholesterol concentration in FIBP versus Rosa26 (3 independent gRNA for each gene) knockout T cells cultured in Bib-conditioned medium via Filipin III staining. The comparison between control (Rosa26) and FIBP knockout conditions was performed using the two-sided Wilcoxon rank-sum test.

[0077] Fig. 8J presents plots of data concerning LDL uptake after lipoprotein deprivation.

CD8 T cells with FIBP knockout versus control cells (gRosa26 were treated with Dil-LDL after lipoprotein deprivation for 16h. The LDL uptake was determined using flow cytometry.

[0078] The data presented in Figures 9A through 9F demonstrate that Tres predicts clinical efficacies of ICIs and adoptive cell therapies.

[0079] Fig. 9A plots data demonstrating that Tres score correlations predict the efficacy of T cells in immunotherapies. [0080] Fig. 9B presents graphs concerning Tres prediction performance with respect to T cell clinical efficacy.

[0081] Fig. 9C graphically presents comparisons among T cell signatures in predicting clinical response.

[0082] Fig. 9D plots Tres score correlations in tumors for lymphocyte expansion predict ACT outcome.

[0083] Fig. 9E graphically presents data demonstrating that Tres correlations in T cells for CAR-T manufacture predict a favorable response.

[0084] Fig. 9F graphically presents comparisons among T cell signatures in predicting survival outcome.

[0085] The data and other information presented in Figures 10A through 10H pertain to the control analyses of the median Tres signature in predicting immunotherapy responses.

[0086] Figure 10A graphically depicts correlations between the median Tres signature and T- cell bulk transcriptomic profiles lead to predictive value (responder vs. non-responder).

[0087] Figure 10B presents plots representing Tres score correlations with profiles from post-treatment tumors.

[0088] Figure 10C presents plots representing scores indicative of T-cell clinical efficacy.

[0089] Figure 10D presents data demonstrating a comparison among T-cell signatures in predicting clinical response.

[0090] Figure 10E presents data demonstrating a lack of associations between Tres score correlations and adoptive cell therapy efficacy with respect to tumors with T-cell infiltration lower than average.

[0091] Figure 10F presents data demonstrating performance of Tres on predicting ICI outcomes using bulk data.

[0092] Figure 10G presents data demonstrating Tres prediction performance with respect to different combinations of treatments and sample sites in a triple-negative breast cancer study.

[0093] Figure 10H presents data demonstrating the Tres prediction performance with respect to tumors when immunosuppressive signals are lower than average. [0094] Figures 11 A through 1 IB present data from in vivo flow analysis of T cells from mouse tumors, which demonstrates FIBP knockout potentiates T-cell efficacy through lowering cholesterol levels in T cells.

[0095] Figure 11 A presents data from in vivo flow analysis of cholesterol levels (Filipin III) in FIBP knockout (“KO”) T cells. Representative histograms are presented for gene KO T cells isolated from mouse tumors (left). Mean and s.d. are shown as error bars (n = 4 tumors per group) (right). KOs were compared using a one-sided Wilcoxon rank-sum test.

[0096] Figure 1 IB presents in vivo flow analysis of T-cell phenotype markers, such as T-cell sternness and exhaustion.

[0097] Figures 12A through 12D present data demonstrating that simvastatin does not lower cholesterol levels in T cells.

[0098] Figure 12A presents data concerning the cholesterol levels of T cells treated with simvastatin concurrently with anti-CD3/28 activation.

[0099] Figure 12B presents data concerning the cholesterol levels of T cells 72 hours after anti-CD3/28 activation.

[0100] Figure 12C presents data concerning the proliferation of T cells treated with simvastatin concurrently with anti-CD3/28 activation.

[0101] Figure 12D presents data concerning the proliferation of T cells 72 hours after anti- CD3/28 activation.

DETAILED DESCRIPTION OF THE INVENTION

[0102] In accordance with the present invention, TMEM222 and especially FIBP are identified as new target genes, the disrupted expression of which in T cells can enhance the efficacy of adoptive cell therapy and CAR-T therapies. Accordingly, in one embodiment, the invention provides a T cell exhibiting reduced or diminished FIBP and/or TMEM222 expression. In embodiment, the inventive T cell lacks functional expression of FIBP and/or TMEM222 achieved by elimination of (“knocking out”) all or a portion of the gene(s) encoding FIBP and/or TMEM222 from T cell chromosomal DNA to eliminate transcription of the genes. In this sense, the invention provides a T cell comprising chromosomal DNA, wherein the chromosomal DNA lacks an intact genetic sequence encoding FIBP, TMEM222 or both FIBP and TMEM222. Using another approach, the inventive T cell lacks functional expression of FIBP and/or TMEM222 through diminishing (“knocking down”) their transcription into or translation from mRNA such that insufficient quantity of FIBP and/or TMEM222 is produced by the inventive T cell to exert biological effect. Through either approach, the inventive T cell lacks functional FIBP and/or TMEM222 expression.

[0103] The FIBP protein (also called “FGF1 intracellular binding protein”) is an intracellular protein that binds selectively to acidic fibroblast growth factor (aFGF). Little is known about the molecular function of FIBP. It is postulated that FIBP may be involved in the mitogenic action of aFGF and to be an oncogene that induces chemotherapy resistance in colorectal cancer cells. The human genomic FIBP sequence is available at NCBI Reference Sequence: NG_047103.1 (ncbi.nlm.nih. gov/nuccore/1027250676, which is incorporated herein in its entirety) (the entire sequence therein is set forth below as SEQ ID NO:63) and is identified therein as nucleotides 5069-9800. Human FIBP mRNA variants are published as NCBI Reference Sequence: NM_198897.2 (ncbi.nlm.nih. gov/nuccore/ NM_198897.2, which is incorporated herein in its entirety) (the entire sequence therein is set forth below as SEQ ID NO:65) and NCBI Reference Sequence: NM_004214.5 (ncbi.nlm.nih. gov/nuccore/ NM_004214.5, which is incorporated herein in its entirety) (the entire sequence therein is set forth below as SEQ ID NO:66). The murine genomic FIBP sequence is set forth below as SEQ ID NO:68, which is extracted from >NC_000085.7:5510626-5515080 Mus musculus strain C57BL/6J chromosome 19, GRCm39 (ncbi.nlm.nih. gov/nuccore/NC_000085.7?report=fasta&from=5510626&to=5515080, which is incorporated herein in its entirety). Mouse FIBP mRNA variants are published as NCBI Reference Sequence: NM_001253832.1 (ncbi.nlm.nih. gov/nuccore/NM_001253832, which is incorporated herein in its entirety) (the entire sequence therein is set forth below as SEQ ID NO:69) and NCBI Reference Sequence: NM_021438.4 (ncbi.nlm.nih. gov/nuccore/NM_021438, which is incorporated herein in its entirety) (the entire sequence therein is set forth below as SEQ ID NO:70).

[0104] TMEM222 protein (also called “Transmembrane protein 222”) is a transmembrane protein. Little is known about the molecular function of TMEM222. The human genomic TMEM222 sequence is set forth below as SEQ ID NO:64, which is extracted from

NC_000001.11 :27322163-27336400 Homo sapiens chromosome 1, GRCh38.pl3 Primary Assembly, (ncbi. nlm.nih. gov/nuccore/NC_000001.1 l?report=fasta&from=27322163&to=27336400, which is incorporated herein in its entirety). The human TMEM222 mRNA sequence is published as NCBI Reference Sequence: NM_032125.3 (ncbi. nlm.nih. gov/nuccore/NM_032125.3, which is incorporated herein in its entirety)(the entire sequence therein is set forth below as SEQ ID NO:67). The mouse TMEM222 genomic sequence is set forth below as SEQ ID NO:71, which is extracted from >NC_000070.7:cl33005101-132993356 Mus musculus strain C57BL/6J chromosome 4, GRCm39, (ncbi. nlm.nih. gov/nuccore/NC_000070.7?report=fasta&from=l 32993356&to=l 3300510 l&strand=true, which is incorporated herein in its entirety). The mouse TMEM222 mRNA sequence is available at NCBI Reference Sequence: NM_025667.3 (ncbi. nlm.nih. gov/nuccore/NM_025667.3) (the entire sequence therein is set forth below as SEQ ID NO:72, and which is incorporated herein in its entirety).

[0105] In a preferred aspect, the inventive T cell comprises chromosomal DNA, wherein the chromosomal DNA lacks a genetic sequence encoding all or a portion of functional protein, wherein the functional protein comprises FIBP or TMEM222 (i.e., the inventive T cell lacks an intact genetic sequence encoding one or both proteins). In certain embodiments, the T cell can lack functional copies of the genes encoding both FIBP and TMEM222 proteins. In other words, the inventive T cell can be a “knockout” T cell, lacking functional FIBP, TMEM222, or both genes. Such knockout T cells can lack all or a portion of the genomic sequence encoding FIBP, TMEM222, or both genes. Alternatively, the inventive T cell can comprise genomic copies of FIBP, TMEM222, or both genes but be engineered to interfere with the expression of such genes, such as by mutating their regulatory regions (promoters, enhancers, and the like), by blocking or diminishing translation of mRNA transcripts (a “knockdown” approach) using RNA interference, or other suitable method. To assist in designing interfering RNAs, the cDNA sequences for the human and mouse FIBP and TMEM222 genes are known (see SEQ ID Nos 65, 66, 67, 69, 70, and 72 herein as well as other sources known to persons of ordinary skill).

[0106] In addition to comprising the genetic modification to suppress FIBP and/or TMEM222 transcription and/or translation, in some embodiments, the inventive T cell can be otherwise genetically modified. For example, the inventive T cell can comprise genes encoding one or more T cell receptor (TCR) or Chimeric Antigen Receptor (CAR). As non-limiting examples, in some embodiments, the inventive T cell can comprise genes encoding TCRs such as NY-ESO-1 (see Example 1 herein), an individualized tumor TCR, such as are the subject of ClinicalTrials.gov Identifier: NCT03891706 (see clinicaltrials. gov/ct2/show/ NCT03891706), MAGE-A3/A6, and the like. Suitable TCRs also are identified in Table 2 of Zhao et al., “Engineered TCR-T Cell Immunotherapy in Anticancer Precision Medicine: Pros and Cons” Front. Immunol., 12, article 658753 (30 March 2021) (available at doi. org/10.3389/fimmu.2021. 658753, and incorporated herein in its entirety). Also, as non-limiting examples, in some embodiments, the inventive T cell can comprise genes encoding CARs targeting antigens such as BCMA, Biotin, CD 123, CD171, CD 19, CD22, CD23, CD33, CLCB, EGFRvIII, FAP, FGFR4, FR, GD2, Glypican-3, HER2, IL13Ra2, Mesothelin, MUC1, NKG2D, PD1, PSMA, or ROR-1, etc. It will be observed that the inventive T cell thus can comprise such TCRs and/or CARs. [0107] As another non-limiting example, the inventive T cells also can be deficient in the transcription or translation of one or more genes in addition to FIBP and TMEM222. In this respect, in certain embodiments, the inventive T cell can lack functional genes encoding CBLB, PDCD1, or both CBLB and PDCD1 (i.e., knockout mutations implicating CBLB, PDCD1, or both CBLB and PDCD1) or genetic modifications knocking down the expression of CBLB, PDCD1, or both CBLB and PDCD1. Mutations for generating T cells lacking expression of CBLB and PDCD1 are known to persons of ordinary skill (see., e.g.., Shifrut et al., (for CBLB) and Lu et al., Nature Med., 26(5): 732-740 (2020), incorporated herein in its entirety), and any such (or other) approach can be used in the context of the present invention. Similarly, the inventive T cell can additionally lack functional genes, such as lacking intact genetic sequences thereof, encoding targets that can prevent T cell exhaustion/enhance CAR T activity.

[0108] Desirably, the inventive T cell comprises one or more CAR (and gene(s) encoding them) that targets one or more antigens present on a solid tumor. For example, for targeting EGFRvIII-positive cancers, the inventive T cell can comprise one or more genes encoding one or more CARs targeting Biotin (such as a CAR structure comprising CD3(^, CD28 and 41BB), and can comprise such CARs. Similarly, for targeting neuroblastoma, the inventive T cell can comprise one or more genes encoding one or more CARs targeting CD171 (such as a CAR structure comprising CD3(^ and 41BB or CD3(^, CD28 and 41BB), and can comprise such CARs. Similarly, for targeting glioma, the inventive T cell can comprise one or more genes encoding one or more CARs encoding a CAR targeting EGFRvIII (such as a CAR structure comprising CD3(^ and 41BB or CD3(^, CD28 and 41BB), and can comprise such CARs. Other CARs useful for targeting glioma include those targeting IL13Ra2 (for example, having CAR structures of CD3 CD3< and 41BB; CD3i and CD28; CD3< CD28 and 41BB; or CD3< CD28 and OX40-), and the inventive T cell, therefore, can comprise one or more genes encoding one or more CARs targeting IL13Ra2 and can comprise such CARs. Similarly, for targeting mesothelioma or lung cancers, the inventive T cell can comprise one or more genes encoding one or more CARs targeting FAP (such as a CAR structure comprising CD3(^ and CD28 or KIR2DS2 and DAP12-), and the inventive T cell can comprise such CARs. Similarly, for targeting ovarian cancer or breast cancer, the inventive T cell can comprise one or more genes encoding one or more CARs targeting FR (such as a CAR structure comprising CD3(^ and CD27), and the inventive T cell can comprise such CARs. Similarly, for targeting hepatocellular carcinoma, the inventive T cell can comprise one or more genes encoding one or more CARs targeting Glypican-3 (such as a CAR structure comprising CD3(^, CD28 and 41BB), and can comprise such CARs. Similarly, for targeting HER2 positive cancer or sarcoma, the inventive T cell can comprise one or more genes encoding one or more CARs targeting HER2 (such as a CAR structure comprising CD3(^ and CD28), and the inventive T cell can comprise such CARs. Breast cancer also can be targeted with an inventive T cell comprising one or more genes encoding one or more CARs targeting HER2 (and also comprising such CARs) and also (preferably in combination with HER2- targeting) one or more genes encoding one or more CARs MUC1 (and also comprising such CARs) (such as a CAR structure comprising CD3(^ and CD28), and glioblastoma also can be targeted with an inventive T cell comprising one or more genes encoding one or more CARs targeting HER2 (and also comprising such CARs) and also (preferably in combination with HER2 -targeting) one or more genes encoding one or more CARs targeting IL13Ra2 (and also comprising such CARs) (such as a CAR structure comprising CD3(^ and CD28). Similarly, for targeting mesothelioma, pancreatic cancer, or non-small cell lung cancer, the inventive T cell can comprise one or more genes encoding one or more CARs targeting Mesothelin (such as a CAR structure comprising CD3 CD3i and CD28; CD3^and 41BB; CD3i and ICOS; or KIR2DS2 and DAP12-), and the inventive T cell can comprise such CARs. Pancreatic cancer also can be targeted with an inventive T cell comprising one or more genes encoding one or more CARs targeting Mesothelin (and also comprising such CARs) and also (preferably in combination with Mesothelin-targeting) a gene encoding a CAR targeting CD 19 (and also comprising such CARs) (such as a CAR structure comprising CD3(^ and 41BB). Similarly, for targeting MUCl-positive solid tumors, the inventive T cell can comprise one or more genes encoding one or more CARs targeting MUC1 (such as a CAR structure comprising CD3(^ and 41BB), and the inventive T cell can comprise such CARs. Similarly, for targeting ovarian cancer or Ewing sarcoma, the inventive T cell can comprise one or more genes encoding one or more CARs targeting NKG2D (such as a CAR structure comprising CD3(^; CD3(^ and DAP10; CD3(^ and 41BB; or CD3(^ and CD28), and the inventive T cell can comprise such CARs. Similarly, for targeting prostate cancer, the inventive T cell can comprise the gene encoding a CAR targeting PSMA (such as a CAR structure comprising CD3(^ and CD28), and the inventive T cell can comprise such CARs. Similarly, for targeting PD-L1 positive cells, such as within solid tumors, the inventive T cell can comprise one or more genes encoding one or more CARs targeting PD1 and CD 19; or PD1 and Mesothelin, preferably in combination, (such as a CAR structure comprising CD3(^ and CD28; or CD3(^, CD28 and 41BB), and can comprise such CARs. These CARs are discussed in Table 2 of materials produced by CREATIVE BIOMART and available on the Internet (see creativebiomart. net/Targets-of-C AR-T-Cell-Therapy.htm, the entirety of which is incorporated herein, see especially Table 2).

[0109] Any suitable technique can be employed to generate the inventive T cell with reduced or diminished FIBP and/or TMEM222 expression. Accordingly, the invention provides a method for making a T cell lacking functional FIBP and/or TMEM222 expression, such as the inventive T cells. In general, the inventive method involves first obtaining one or more source T cells. In certain applications, the source T cells can be obtained from one or more subjects, for example by isolating splenocytes or peripheral blood lymphocytes (e.g., peripheral blood mononuclear cells (PBMCs)) from one or more subjects. Methods of obtaining primary T cells from human and animal subjects are known to those of ordinary skill in the art, and any suitable method (such as negative magnetic selection for CD8+ and/or CD4+ cells) can be used to obtain source T cells for use in generating the inventive T cell. Non-limiting examples of protocols for use in processes relating to the generation of the inventive T cells are discussed, for example, in Aksoy et al, “Human Primary T Cells: A Practical Guide” (Peer J. Preprints, June 19, 2018) (available at peerj. com/preprints/26993vl.pdf) (incorporated herein in its entirety). In other applications, preparations of T cells from commercial or institutional sources are available and can be employed as the source T cells for use in methods to generate and derive the inventive T cells with reduced or diminished FIBP and/or TMEM222 expression. For instance, in Example 1 herein, human CD8+ T cells transduced with a recombinant T cell receptor (TCR) specific for the NY-ESO-1 antigen (NY-ESO-1 : 157-165 epitope) were provided as a gift by Dr. Rigel Kishton from the Surgery Branch at National Cancer Institute (NCI), National Institute of Health (NIH).

[0110] The source T cells can be of any desired species, such as a mouse, rat, or primate (preferably a human), though typically the species from which the source T cells are representative is a mammalian species. It will be observed that the subject from which the source T cells are isolated can be a human subject, such as a patient in need of therapy (e.g., for which the source T cells obtained from the patient can serve as the source to generate the inventive T cells with reduced or diminished FIBP or TMEM222 expression, as discussed herein, are to be introduced into the same patient via adoptive T cell therapy). Alternatively, the source T cells can be isolated from one or several subjects irrespective of the end use to which the derived inventive T cells are employed. Where T cells are obtained from multiple subjects, they can be pooled into a population of source T cells reflecting the contribution from several donor subjects.

[OHl] The source T cells preferably comprise CD8+, CD4+, both CD8+ and CD4+, or a mixture of such cells. However, any T cells can serve as the source T cells for generating the inventive T cells with reduced or diminished FIBP and/or TMEM222 expression. Moreover, the source T cells can be otherwise genetically modified, for example to express recombinant T cell receptors (“TCRs”), such as those specific for predefined substrates (such as the NY-ESO-1 antigen (see Example 1 herein), an individualized tumor TCR, such as are the subject of ClinicalTrials.gov Identifier: NCT03891706 (see clinicaltrials. gov/ct2/show/ NCT03891706), MAGE-A3/A6, and the like. Suitable TCRs also are identified in Table 2 of Zhao et al., “Engineered TCR-T Cell Immunotherapy in Anticancer Precision Medicine: Pros and Cons” Front. Immunol., 12, article 658753 (30 March 2021) (available at doi. org/10.3389/fimmu.2021. 658753, and incorporated herein in its entirety)). In certain preferred embodiments, for example, the source T cells alternatively or additionally can lack functional genes encoding CBLB, PDCD1, or both CBLB and PDCD1 (i.e., knockout mutations implicating CBLB, PDCD1, or both CBLB and PDCD1) or genetic modifications knocking down the expression of CBLB, PDCD1, or both CBLB and PDCD1. As another example, in certain preferred embodiments, the source T cells alternatively or additionally can be engineered to express one or more chimeric antigen receptors (“CARs”), such as those targeting antigens such as BCMA, Biotin, CD123, CD171, CD19 (which is commonly targeted in CAR T cell therapy), CD22, CD23, CD33, CLCB, EGFRvIII, FAP, FGFR4, FR, GD2, Glypican-3, HER2, IL13Ra2, Mesothelin, MUC1, NKG2D, PD1, PSMA, or ROR-1, etc. It will be observed that the inventive T cell thus can comprise such TCRs and/or CARs. Desirably, the inventive T cell comprises one or more CAR (and gene(s) encoding them) that targets one or more antigens present on a solid tumor. As another example, in certain preferred embodiments, the source T cells alternatively or additionally can be engineered to lacks one or more intact genetic sequences encoding one or more targets that can prevent T cell exhaustion/enhance CAR T activity.

[0112] It will be observed that these qualities of the source T cells (e.g., CD4+, CD8+, possessing genetic modifications such as to express recombinant TCRs or CARs, knockout/knockdown of other genes, etc.) will be shared by the resulting inventive T cell with reduced or diminished FIBP and/or TMEM222 expression upon production thereof.

Accordingly, in certain embodiments, the inventive T cells provided by the present invention also can comprise such genetic modifications (i.e., the inventive T cell can be a TCR-expressing T cell or a CAR T cell, or otherwise genetically modified) in addition to having the genetic modification leading to the reduced expression (“knockout” or “knockdown”) of FIBP and/or TMEM222.

[0113] Upon isolation, the inventive method for making a T cell lacking functional FIBP and/or TMEM222 expression comprises expanding the population of source T cells and activating the expanded population of source T cells. In performance of these steps, the source T cells can be treated by suitable methods to expand their population and activate them for further use. For example, the source T cells can be stimulated with CD3 and CD28 and then expanded and activated in the presence of Interleukin-2, such as is described in Example 1 below for both human and mouse CD8+ T cells. Also, acceptable cell culture media for use in culturing and expanding populations of T cells are known to those of ordinary skill, and any such suitable media and culture conditions can be employed in connection with methods used to generate the inventive T cells with reduced or diminished FIBP and/or TMEM222 expression.

[0114] The inventive method for making a T cell lacking functional FIBP and/or TMEM222 expression also involves genetically manipulating the T cells within the population. The T cells can be genetically manipulated to generate the inventive T cells with reduced or diminished FIBP and/or TMEM222 expression (lacking functional FIBP and/or TMEM222 expression). Such genetic manipulation can occur before, during, or after the source T cells are obtained, or such as before, during, or after the population of T cells is expanded, such as before, during, or after the population of T cells is expanded. Thus, in one exemplary embodiment, the inventive method can involve first obtaining one or more source T cells, then expanding the population of source T cells and activating the expanded population of T cells, and then genetically manipulating the T cells within the expanded, activated population to generate a resulting T cell lacking functional FIBP and/or TMEM222 expression. However, the method is not limited to performing the process steps in this order. For example, the T cells can be genetically manipulated before, during, or after the initial population is expanded, or before, during, or after the activation process.

[0115] For generating embodiments of the inventive T cells with reduced or diminished FIBP and/or TMEM222 expression in which one or both of FIBP and/or TMEM222 are “knocked out,” techniques such as CRISPR, the use of Transcription Activator-like Effector Nucleases (TALENs) or Zinc Finger proteins can be employed for the process of genetic manipulation of the T cells. Such approaches also can be employed for embodiments in which the inventive cell lacks functional expression of either or both of CBLB and/or PDCD1 or as well in embodiments in which the inventive cell one or more intact genetic sequences encoding one or more targets that can prevent T cell exhaustion/enhance CAR T activity.

[0116] A preferred method to generate the inventive FIBP and/or TMEM222 knockout T cell involves CRISPR. CRISPR-Cas systems employ a tracrRNA which plays a role in the maturation of crRNA. The tracrRNA is partially complementary to and base pairs with a pre- crRNA forming an RNA duplex. This is cleaved by RNase III to form a crRNA/tracrRNA hybrid, which acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid. Typically, for CRISPR applications, the Cas9 nickase enzyme is co-transfected with a guide RNA (“gRNA”) to effectuate gene editing. However, enzymes other than Cas9 (such as, for example Casl2a) can be suitably employed in some embodiments.

[0117] CRISPR technology is well known to persons of ordinary skill in the art, and any suitable protocol can be employed in the context of the present invention. For instance, in Example 1 herein, tracrRNA and crRNAs (respectively containing the targeting sequences from Table 5 A) were suspended in a buffer and incubated at 95 °C for five minutes to generate gRNA, which was thereafter incubated with Cas9 protein to generate Cas9-ribonucleotide proteins (Cas9-RNP). Such Cas9-RNP then were mixed with activated CD8+ T cells, and the mixture subjected to electroporation to effect nucleofection of the Cas9-RNP into the T cells. A similar protocol, or some desired variant or other suitable protocol, can be employed in the context of the present invention to effect knockout of either or both of the FIBR and/or TMEM222 genes from T cells to generate the inventive cells.

[0118] As information concerning the genetic sequences for FIBP and TMEM222 is known (see, e.g., SEQ ID Nos: 63 and 64), suitable guide RNAs (gRNAs) for use in targeting CRISPR- Cas9 (or other suitable CRISPR system) to knock out all or a portion of the FIBR, TMFM222, or both genes can readily be designed by persons of ordinary skill in the art. Non-limiting examples of sequences for constructing CRISPR-Cas9 gRNAs for knocking out human FIBR are provided herein as SEQ ID Nos: 4, 5, and 6 (Table 5A). Non-limiting examples of sequences for constructing CRISPR-Cas9 gRNAs for knocking out human TMEM222 are provided herein as SEQ ID Nos: 7, 8, and 9 (Table 5A). Non-limiting examples of sequences for constructing CRISPR-Cas9 gRNAs for knocking out mouse FIBR are provided herein as SEQ ID Nos: 16, 17, and 18 (Table 5 A). Non-limiting examples of sequences for constructing CRISPR-Cas9 gRNAs for knocking out mouse TMEM222 are provided herein as SEQ ID Nos: 19, 20, and 21 (Table 5 A). Furthermore, for embodiments in which the inventive T cell lacks a functional gene encoding CBLB, non-limiting examples of sequences for constructing CRISPR-Cas9 are provided herein in Table 5A as SEQ ID Nos: 10-12 (for human) and 22-24 (for mouse). For CBLB and PDCD1, see the Shifrut et al. and Lu et al. publications referred to herein. The sequences set forth in Table 5A are recited as DNA sequences; however, for any of these sequences, when RNA is to be constructed, thymidine (“T”) is substituted with uracil (“U”), and such RNA sequences are included as if they were separately set forth herein.

[0119] As noted, other methods for gene editing can be employed as alternatives to CRISPR to generate embodiments of the inventive T cells in which one or both of FIBP and/or TMEM222 are “knocked out” (applicable as well for embodiments in which the inventive cell lacks functional expression of either or both of CBLB and/or PDCD1). Such methods include those employing transcription activator like effector nucleases (TALENs) and the use of Zinc finger proteins, for example. For each application, standard methodology known to those of ordinary skill can be employed to generate the genetically altered T cells of the present invention.

TALENs are customized artificial restriction nucleases that can be readily constructed to target a known genetic sequence, using methods known to persons of ordinary skill in the art. Similarly, Zinc finger domains can be engineered using methods known to persons of ordinary skill in the art to target specific desired DNA sequences, which this enables Zinc finger nucleases to target unique sequences within complex genomes to alter the chromosomal DNA of cells. Thus, knowledge of the sequences for FIBP and TMEM222 (for example, as set forth in SEQ ID Nos:63, 64, 68, and 71) and otherwise known in the art) can facilitate the design and construction of TALENs and Zinc finger nucleases, targeting the same genome loci that FZSP and TMEM222 guide RNAs bind (see Table 5A), suitable for generating the inventive T cells with reduced or diminished FIBP and/or TMEM222 expression (lacking functional FIBP and/or TMEM222 expression) in which one or both of FIBP and/or TMEM222 are “knocked out.” [0120] Also, while approaches for removing functional copies of the FIBP and/or TMEM222 genes from T cells (“knocking out” all or a portion of their coding sequences) are preferred, other methods for attenuating the expression of these genes can alternatively be employed in some embodiments. As an example, as noted above, the genetic regulatory elements controlling expression of the FIBP and/or TMEM222 genes can be altered or removed from the T cell genomes to attenuate transcription of FIBP and/or TMEM222 if one or both of their coding sequences is not “knocked out.” Alternatively, in performing the inventive method, the genetic manipulation can involve using RNA interference to block or reduce translation of FIBP and/or TMEM222 mRNA transcripts within the cells. Persons of ordinary skill in the art will be able to design suitable interfering RNA sequences for attenuating (“knocking down”) the expression of FIBP and/or TMEM222, as the sequences for these genes are known. Moreover, the sequences presented in Table 5A (specifically SEQ ID Nos:4-9 and 16-21) can serve as a basis for designing interfering RNAs, such as snRNAs, for such RNAs targeting FIBP and/or TMEM222, or for inclusion in larger vectors (e.g., lentiviral vectors) containing coding sequences. Such “knock-down” approaches also can be employed for embodiments in which the inventive cell lacks functional expression of either or both of CBLB and/or PDCD1.

[0121] While, as noted, in some embodiments, the method begins with source T cells that are genetically modified (e.g., to express TCRs and/or CARs), which modifications typically will be retained in the inventive T cells generated by the method, in certain embodiments, the resulting T cells can be further genetically modified as desired, such as to express one or more TCRs and/or CARs, such as CARs targeting BCMA, Biotin, CD 123, CD171, CD 19, CD22, CD23, CD33, CLCB, EGFRvIII, FAP, FGFR4, FR, GD2, Glypican-3, HER2, IL13Ra2, Mesothelin, MUC1, NKG2D, PD1, PSMA, or ROR-1, etc., or otherwise genetically modified as may be desired depending on the end use of the resulting T cells (such as lacking functional expression of either or both of CBLB and/or PDCD1, or one or more targets that can prevent T cell exhaustion/enhance CAR T activity for example). Desirably, the inventive T cell comprises a CAR that targets an antigen present on a solid tumor.

[0122] Following the genetic manipulation to generate the T cells with reduced or diminished FIBP and/or TMEM222 expression, and any other genetic manipulation (e.g., to introduce genes encoding TCRs, CARs, or other desired transgenes or reduction of genetic expression), the resulting T cell is an inventive cell. The cell thereafter can be cultured and expanded to form a population comprising the inventive T cells. For example, following genetic manipulation, the resulting cells can be separated from the culture (e.g., through centrifugation), and resuspended in a suitable culture medium. Accordingly, the invention provides a population comprising one or more of the inventive T cells.

[0123] For culturing the inventive T cells, any desired culture conditions can be employed, using incubation parameters and culture media known to persons of ordinary skill in the art. In a preferred non-limiting embodiment, the T cells can be cultured completed X-VIVO medium, which is X-VIVO 15 SERUM-FREE HEMATOPOIETIC CELL MEDIUM (LONZA, BE02- 060Q) supplemented with 5% inactivated fetal bovine serum (GIBCO, 10082147), 50 pM 2- mercaptoethanol (GIBCO, 21985023), and 10 mM N-Acetyl L-cysteine (SIGMA, A7250-5G) at IE⁶ cells/mL. Desirably, the culture media can comprise Interleukin-2, as such reagent facilitates the stimulation and proliferation of T cells. The culture comprising the inventive T cells can be passaged to expand the population into any desired titer of T cells, such as at least 10³ cells/mL, or at least 10⁴ cells/mL, or at least 10⁵ cells/mL, or at least 10⁶ cells/mL, or at least 10⁷ cells/mL, or at least 10⁸ cells/mL(or at least “about” such values). A preferred titer for the culture is between IE⁶ cells/mL, up to 2E⁶ cells/mL (or between “about” such values). Within the culture, the inventive T cells can be adherent on the surface of a cell culture substrate (e.g., Petri dishes, or multiwell culture plates such as are known to persons of ordinary skill). Alternatively, the culture can be a suspension culture, in which at least some of the inventive T cells in culture are free-floating and not adherent to a substrate. Furthermore, if desired, the population comprising the inventive T cells can be frozen (e.g., at temperatures typical for maintaining frozen cells, such as at -20 °C) for future use, and the invention provides a composition comprising one or more of the inventive T cells, which is frozen.

[0124] Following culturing and expansion, the T cells can be separated from the culture medium and formulated into a composition. Accordingly, the invention provides a composition comprising the inventive T cells. The composition can comprise one component (e.g., a single solution comprising the inventive T cells, buffers, and other agents as described herein) or, optionally, can comprise separate components (e.g., a first component, a second component, a third component, etc.). Such components, when present as separate components, represent discrete volumes of constituents, each volume comprising discrete chemical constituents (e.g., buffer systems within the carrier of each respective component, active agents, adjuvants, etc.). The composition (or the first component, i.e., that comprising the inventive T cells) can comprise a suitable carrier which is physiologically compatible with the inventive T cells, which is taken to mean that the carrier is able to maintain the viability of the inventive T cells until the point at which the composition is to be used. In some embodiments, the carrier can be a suitable culture medium to support continued maintenance and expansion of the inventive T cells. In an embodiment, a component of the composition (e.g., first and/or second component) is frozen (e.g., lyophilized), and can be thawed and reconstituted prior to use. In an embodiment, the carrier of a component of the composition (e.g., first and/or second component) can be a pharmaceutically acceptable carrier; accordingly, the invention provides a pharmaceutical composition (or a first component thereof) comprising the inventive T cells and a pharmaceutically acceptable carrier.

[0125] The pharmaceutical composition (including any component, such as a first, second, third, etc. component thereof) of the present invention, whether comprising the inventive T cells or other agents, can be formulated for any desired mode of administration (e.g., as a solution, implantable structure, salve, etc.). A preferred formulation includes a liquid carrier, which facilitates administration to a patient via injection (such as intravenously, interperitoneally, intramuscularly, or by intratumoral injection, for example). A suitable carrier for injection can comprise sterile saline and can include excipients and adjuvants known to persons of ordinary skill, such as buffers, growth factors, preservatives, and the like, to facilitate storage and maintain the viability of the inventive T cells within the pharmaceutical composition.

[0126] The pharmaceutical composition of the present invention also can include active agents in addition to the inventive T cells, such as pharmaceutical agents (e.g., anticancer agents such as anti-PDl, anti-PDLl, or anti-CTLA4 therapeutic antibodies, offered here purely as nonlimiting examples). In certain preferred embodiments, the pharmaceutical composition of the present invention can include cytokines, such as Interleukin-2. Where present within the inventive composition, such active agents in addition to the inventive T cells can be present in the same component as the inventive T cells (i.e., the first component) or be present in a separate component (e.g., second component, third component, fourth component, etc.) within the composition, as desired.

[0127] Within the pharmaceutical composition of the present invention (or first component thereof), the inventive T cell or cells can be present at any desired concentration or titer such as at least 10³ cells/ml, or at least 10⁴ cells/ml, or at least 10⁵ cells/ml, or at least 10⁶ cells/ml, or at least 10⁷ cells/ml, or at least 10⁸ cells/ml (or at least “about” such values).

[0128] The inventive pharmaceutical composition can be manufactured according to standard methodology, which, in respect of the inventive T cells, involves suspending the inventive T cells in the desired carrier, under sterile conditions and desirably using Good Manufacturing Practices (GMP). Other agents for inclusion in the inventive composition, or any component thereof, can be formulated using methods known to those of skill in the art. Also, inventive composition can be packaged in a suitable manner to facilitate its use, such as in vials, ampoules, syringes, and the like.

[0129] For facilitating the construction of the inventive T cells, in an embodiment, the invention provides an extrachromosomal nucleic acid comprising genetic sequence which is substantially complementary to a genetic sequence encoding a functional protein, wherein the function protein comprises FIBP or TMEM222, with FIBP being preferred. The FIBP or TMEM222 can be of any desired species, such as a mouse, rat, or primate; preferably the FIBP or TMEM222 is a human ortholog.

[0130] By “ extrachromosomal” in this context is meant that the nucleic acid is not present within a chromosome but is, or is a component of, an oligonucleotide or vector (such as a plasmid or viral vector). Desirably, the extrachromosomal nucleic acid does not bind histones so as to not form chromatin. Preferably, the extrachromosomal nucleic acid is exactly complementary to a genetic sequence encoding FIBP or TMEM222. However, by “substantially complementary” in this context is meant that the inventive extrachromosomal nucleic acid is able to base-pair with FIBP or TMEM222 coding sequences (either chromosomal DNA or mRNA) such that specific intracellular binding occurs between the inventive extrachromosomal nucleic acid and either the genomic FIBP or TMEM222 coding sequences (e.g., when employed with gene editing technologies such as employing CRISPR, TALEN, and Zinc finger technology) or with FIBP or TMEM222 mRNA to facilitate RNA interference. [0131] The inventive extrachromosomal nucleic acid can be or comprise DNA, RNA, or other nucleic acid. Preferably, however, for facilitating generating the inventive T cells via CRISPR, the inventive extrachromosomal nucleic acid comprises RNA, such as being or comprising a crRNA or gRNA.

[0132] Exemplary sequences which the inventive extrachromosomal nucleic acid can comprise include those (a) selected from the group consisting of GATGAGTAGTGCCTGCCGGG (SEQ ID NO:4), CCGCTTTCCAGTGACCGACG (SEQ ID NO:5), GGTGCTGCAGAGCGACACCA (SEQ ID NO:6) (b) selected from the group consisting of TGAGGAGTACAAGCACCGCA (SEQ ID NO: 7), ACGGACATGAAGCAATATCA (SEQ ID NO: 8), GACTCACTGAGACAAAGTAG (SEQ ID NO: 9) (c) selected from the group consisting of CTTTAAACGAGTCTTC AAGG (SEQ ID NO: 16), ACCTGGCTAACCGGTCAGAG (SEQ ID NO: 17), and CTGGTGAGCACCTCTCGATG. (SEQ ID NO: 18), and (d) selected from the group consisting of CACTCGCATACACCTGTCCG (SEQ ID NO: 19), GACTCACCGAAACAAAATAG (SEQ ID NO:20), and GGAAGTAGAAACGCCGACGG (SEQ ID NO:21). It will be observed that, for any of these sequences, when the inventive extrachromosomal nucleic acid comprises RNA, one or more of the thymidine (“T”) nucleotides is substituted with uracil (“U”), and the invention includes such extrachromosomal nucleic acids comprising such sequences as if they were separately set forth herein.

[0133] The inventive extrachromosomal nucleic acid can be single-stranded or doublestranded, and it can form loop structures (e.g., a hairpin loop). In an embodiment, the inventive extrachromosomal nucleic acid is or comprises an oligonucleotide consisting of from about 20 to about 50 nucleotides (such as consisting of 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, 50, 51, 52, 53, 54, or 55 nucleotides). Preferably, for use in connection with CRISPR-Cas9 to generate the inventive T cell, the inventive extrachromosomal nucleic acid is an oligonucleotide that consists of 20 nucleotides. In other embodiments, the inventive extrachromosomal nucleic acid can comprise, and be incorporated within, larger genetic structures, such as plasmids or a viral genome (such as a viral genetic vector).

[0134] The inventive extrachromosomal nucleic acid can be formulated into a composition comprising the inventive extrachromosomal nucleic acid and a suitable carrier. Such a carrier can comprise reagents suitable for facilitating storage of the composition in lyophilized form (such as trehalose), and the composition can, therefore, exist in lyophilized form. The carrier also can comprise buffers, enzymes and other proteins, salts, and other constituents for stabilizing the composition or for facilitating reaction of the inventive extrachromosomal nucleic acid with other nucleic acids.

[0135] In a preferred embodiment, the composition includes an agent for facilitating gene modification, such as a Cas nickase, a TALEN, or a Zinc finger endonuclease, as discussed herein. Preferably, the composition comprising the inventive extrachromosomal nucleic acid comprises an enzyme for catalyzing CRISPR, a non-limiting example of which comprises the Cas9 nickase. Furthermore, in embodiments in which the inventive extrachromosomal nucleic acid comprises a crRNA, the composition preferably also comprises a tracrRNA.

[0136] In another embodiment, using the inventive T cells having reduced or diminished FIBP and/or TMEM222 expression (such as a “knockout” T cell lacking an intact FIBP and/or TMEM222 genomic coding sequence, or a T cell wherein the ZB and/or TMEM222 expression is attenuated due to alteration of genetic regulatory sequences or “knockdown” interference with translation of mRNA, as discussed herein), the invention provides a method of treating cancer by adoptive T cell transfer therapy, which can include CAR T cell therapy when the inventive cells express a CAR.

[0137] In accordance with the inventive method, a pharmaceutical composition comprising the inventive T cell is administered to a subject suffering from cancer and in need of therapy therefor. The subject can be a human or non-human patient (such as companion animal or “pet” (a cat or dog, for example), an animal of agricultural significance (such as cattle, horses, sheep, goats, pigs, and the like), or an animal employed as a subject in laboratory research (such as a mouse, rat, rhesus monkey, and the like). In this sense, the method has medical, veterinary, and research applications.

[0138] It will be observed that, inasmuch as the invention provides such methods for adoptive T cell transfer, in an embodiment, so too does the invention provide for the use of the inventive composition comprising T cells lacking functional FIBP and/or TMEM222 expression for preparing a medicament for adoptive T cell transfer comprising administering the composition to a subject suffering from cancer and in need of therapy therefor, in an amount and at a location sufficient to treat the cancer within the subject. Likewise, in an embodiment, the invention also provides the inventive composition comprising T cells lacking functional FIBP and/or TMEM222 expression for use in a method of adoptive T cell transfer comprising administering the composition to a subject suffering from cancer and in need of therapy therefor, in an amount and at a location sufficient to treat the cancer within the subject.

[0139] In accordance with the inventive method, use, or composition for use in adoptive T cell transfer, the cancer afflicting the subject can be any type of cancer, such as a blood-bom cancer, lymphoma, leukemia, and the like. Advantageously, the the inventive method, use, or composition for use in adoptive T cell transfer is employed to treat solid tumors, which, as noted above, has proven difficult with strategies employed heretofore involving T cell therapies such as adoptive T cell transfer and CAR T therapies. In this respect, the cancer can be or comprise any solid tumor, such as those within tissues such as brain or spinal cord, digestive tract (such as within the oral cavity, esophagus, stomach, small intestines, colon, or rectum), lung, heart, liver, pancreas, kidney, bladder, bone, skeletal smooth or cardiac muscle, breast, a reproductive structure (such as ovaries, fallopian tubes, uterus, cervix, vagina, testicles, prostate, seminiferous tubules, penis, etc.), meninges, interstitial tissue, gland (e.g., thyroid, parathyroid, adrenal, etc.), or other tissue of the subject.

[0140] In accordance with the inventive method, use, or composition for use in adoptive T cell transfer, the pharmaceutical composition is administered to the subject in an amount sufficient to treat the cancer within the subject. The amount administered will depend on the size of the subject and the mode of administration, and a suitable amount can be readily prescribed by a treating physician, veterinarian, or laboratory researcher, as appropriate. For instance, in Example 1 below, 10⁶ FIBP knockout CD8+ cells were administered to mice. However, for application in larger animals, correspondingly more of the inventive cells can be administered in connection with the inventive adoptive T cell transfer therapy method. For example, at least about 10⁶, such as at least about 10⁷, or at least about 10⁸ or, preferably, at least about 5 x 10¹⁰ or more of the inventive T cells can be administered to a human patient in accordance with the inventive method, use, or composition for use in adoptive T cell transfer. [0141] In accordance with the inventive method, use, or composition for use in adoptive T cell transfer, the composition is administered to the subject at a location sufficient to treat the cancer within the subject. The desired route of administration can be readily prescribed by a treating physician, veterinarian, or laboratory researcher, as appropriate. For instance, in Example 1 below, the FIBP knockout CD8+ cells were administered intravenously to mice. However, other routes of administration can be employed, such as intraperitoneally. Moreover, as the inventive method, use, or composition for use in adoptive T cell transfer has particularly beneficial application in the treatment of solid tumors, in practicing the inventive method, the composition can be administered by injection directly into the corpus of a desired tumor within the subject.

[0142] It will be observed that the outcome of the inventive method, use, or composition for use in adoptive T cell transfer can vary depending on many factors, including the stage of the cancer (tumor) within the treated subject, the age, medical history, and overall health of the subject, the impact of potentially other concomitant therapies employed to also treat the cancer/tumor in the subject, and other factors peculiar to a particular subject. While it is possible for the inventive method, use, or composition for use in adoptive T cell transfer to result in remission of the cancer or elimination of the tumor within the subject, a positive outcome also can be achieved through reduction in the rate of progression of the cancer or in the growth of the tumor, or reduction (if not elimination) of the cancer or tumor in the subject. Even in such situations, an improvement in the subject’s clinical condition associated with the cancer/tumor represents a successful application of the inventive method, use, or composition for use in adoptive T cell transfer.

[0143] Finally, it will be observed that the inventive method, use, or composition for use in adoptive T cell transfer can be employed alone, i.e., as monotherapy, to a human or animal having cancer, such as a tumor. Alternatively, the inventive method, use, or composition for use in adoptive T cell transfer can be used concomitantly or adjunctively with other anticancer therapies against the same cancer or tumor within the subject. Such therapies are known to those of ordinary skill in the art and include, but are not limited to, surgical resection of all or a portion of a tumor, treatment with radiation therapy, treatment with pharmaceutical agents (chemotherapy), other immunotherapies (such as therapeutic antibodies, e.g., anti-PDl, anti- PDL1, or anti-CTLA4, etc. therapeutic antibodies), and the like. Furthermore, Interleukin-2 preferably is administered to a subject undergoing treatment in accordance with the inventive method. The Interleukin-2 can be administered in accordance with any desired dosing protocol, a non-limiting example of which includes administration subcutaneous of Interleukin-2 125,000 lU/kg/day, maximum 9-10 doses over two weeks (see, e.g., Nguyen et al., “Phase II clinical trial of adoptive cell therapy for patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and low-dose interleukin-2,” Cancer Immunol. Immunother. 2019 May;68(5):773- 785, incorporated herein in its entirety).

EXAMPLES

[0144] The following experimental Example further illustrates the invention but, of course, should not be construed as in any way limiting its scope. In addition, data and experimental results concerning the invention have been published after the priority date; see Zhang et al., Nature Medicine 28, 1421-1431 (2022), which is incorporated herein in its entirety by reference.

EXAMPLE 1 (working Example)

[0145] In this Example, using single-cell transcriptomic data from tumors as input, a computational model (“Tres”) was developed to identify regulators of T cell resilience, defined as the ability of T cells to proliferate under immune-suppressive signals such as TGF-beta and TRAIL. The Tres model uses single-cell transcriptomes to search for gene markers of T cells that are resilient to immunosuppressive tumor microenvironments. Integrating 14 single-cell transcriptomic cohorts from seven tumor types, FIBP and TMEM222 were identified herein as top regulators of T cell resilience and as molecular markers of T cells that are resilient to the adverse immune microenvironment in many tumor types. Knocking out these genes, especially FIBP, in murine and human donor T cells significantly enhanced the efficacy of T cell mediated cancer killing and adoptive cell therapy. The data show that FIBP knockout in CD8 lymphocytes alleviates T cell dysfunction. Together, this T cell resilience model revealed FIBP as a candidate target to potentiate cancer immunotherapy, such as through adoptive cell therapy and CAR T therapy.

[0146] Furthermore, that data show that FIBP knockout in CD8 lymphocytes alleviates T cell dysfunction by limiting cholesterol metabolism, and high cholesterol level is known to inhibit T cell activity. This is consistent with previous studies indicating the importance of limiting cholesterol biosynthesis in tumors and the promise of combining immunotherapies with anti-cholesterol drugs.

[0147] It is worth noting that FIBP was not a top hit in previous CRISPR screens (see Shifrut, et al.), and its significance in T cell resilience was evident only in combination with the analysis presented in this Example, indicating the importance of data integration. The Shifrut, et al. CRISPR screen tested T cell proliferation upon TCR activation, rather than anticancer efficacy. The present study discussed in this Example shows that the Tres model can repurpose T cell proliferation screens to identify targets in T cell anticancer therapy. Thus, the Tres model has possible applications to future related CRISPR screens. The Tres model is generally applicable to single-cell datasets that capture a sufficient number of CD8 T cells in tumors. With the ever- increasing volume of single-cell data, the Tres model, thus, can assist in the systematic profiling of regulators and therapeutic targets in T cells to develop next-generation cell therapies.

Materials and Methods

T cell resilience score through variable interaction test

[0148] To evaluate whether T cells with high expression of a gene g are resistant to immune suppressive signaling, a variable interaction test in multivariate linear regression was used. The following variables for individual cells in a single-cell (sc) RNA-Seq dataset first were defined:

SuppressionTGFBi or Suppression iRAiL: Immune suppression level from TGFB1 or TRAIL signaling, predicted using the CYTOSIG platform based on the scRNA-Seq transcriptome. Proliferation: T cell proliferation score, computed through a linear regression approach. The output variable is the scRNA-Seq transcriptome. The explanatory variable is a binary vector with value 1 for all genes in the cell cycle and DNA replication pathways from the KEGG database; and value 0 for all other genes in KEGG. The proliferation score is computed as the t- value (coefficient / Stderr) of the explanatory variable, representing whether the current cell is proliferative.

[0149] For each gene g, the following regression was performed across all single CD8 T cells in a patient: d + a x SuppressionrGFBi (or TRAIL) + b x Expression^ + c x SuppressionrGFBi (or TRAIL) X Expressions ⁼ Proliferation

[0150] The T cell resilience score (“Zre ”) is defined as the t-value: c / StdErr(c). Expression “g” is the gene expression level of gene g. Suppression is as explained above. These numbers have no units, instead their t-values (i.e., c/StdErr) are used as a statistical significance level. To further understand the variable interaction test, the model can be rewritten as follows: [0151] d + b x Expressions + (a + e x Expressions ) ^x SuppressionTGFBi (or TRAIL) =

Proliferation. The association between the immune suppression and proliferation is (a + c x Expressions )• The coefficient a is typically negative, because a high immune suppression activity from TGFB1 or TRAIL signaling typically results in low proliferation level (Table 1). With a positive gene expression level, a positive coefficient c (T cell resilience score) will reduce the negative association between the immune suppression and T cell proliferation, while a negative coefficient will enhance the negative association.

Integrative analysis between T cell resilience score and CRISPR screening phenotypes [0152] To identify genes with significant scores from both the present T cell resilience (Tres) model and a genome-wide CRISPR screen from a previous study (Shifrut, etal. false discovery rates (FDR) were computed to test the statistical significance for each gene. A gene g could be selected as a significant hit in two situations: 1, The Tres score is positive and the CRISPR screen score is negative. In this situation, T cells with high expression of gene g tend to be resistant to immune suppressive signaling from TGFB1 and TRAIL. Also, knockout of gene g will undermine the T cell proliferation upon TCR activation.

2, The Tres score is negative and the CRISPR screen score is positive. In this situation, T cells with high expression of gene g tend to be sensitive to immune suppressive signaling from TGFB1 and TRAIL. Also, knockout of gene g will enhance the T cell proliferation upon TCR activation.

[0153] For situation 1, the FDR (thres_Tres, thres_c) (C: CRISPR)”) was calculated as Random Count (Tres > thres_Tres,Score_c < thres_c) I Gene Count (Tres > thres_Tres,Score_c < thres_c). “FDR’’ in this context refers to “false discovery rate.” The gene count derives directly from the data. The random count is equal to N x Probability (Tres > thres_Tres) x Probability (Score_c < thres_c N is the total number of genes. Both probabilities are computed from the score rank of each gene among all profiled genes. In this context, the “Probability” is a value between 0 and 1. In this context, “Tres” is the T cell resilience score (discussed above). In this context, “threshes” represents a threshold value at a particular Tres score. This value is not calculated; instead, an FDR value is estimated for each Tres score threshold. In other words, this procedure builds a function map from Tres score input value to FDR output value. In this context, “Scorec” refers to CRISPR screen score, measured in Shifrut, et al. Its units are expressed as a log-fold change. In this context, “thresc” means the threshold for each CRISPR score.

[0154] In summary, the FDR computations are as follows:

N * Prob (Tres > thres_Tres) * Prob (Score_c < thres_c) /Gene Count (Tres

> thres_Tres, Score_c < thres_c)

For situation 2, the FDR is computed as

N * Prob (Tres < thres_Tres) * Prob (Score_c > thres_c) /Gene Count (Tres

< thres_Tres, Score_c > thres_c) Human Specimen

[0155] Primary human CD8+ T cells transduced with a recombinant T cell receptor (TCR) specific for the NY-ESO-1 antigen (NY-ESO-1 : 157-165 epitope) were provided as a gift by Dr. Rigel Kishton from the Surgery Branch at National Cancer Institute (NCI), National Institute of Health (NIH).

Mice

[0156] All animal experiments were approved by the NCI Animal Ethics Committee of NIH and performed strictly according to the animal protocol. C57BL/6 mice were purchased from the Charles River Laboratories (NCI strains). Female mice at 6-8 weeks of age were used for tumor incubation and T cell transfer experiments. Pmel-1 T cell receptor (TCR) transgenic mice were provided as a gift by Dr. Chi-Ping Dai from Merlino Lab at NCI, NIH.

Cell lines and cultures

[0157] Human melanoma cell line A375 was recently purchased from American Type Culture Collection (ATCC). Mel624 and B16-mhgpl00 were kindly provided as a gift by Dr. Rigel J Kishton from the Surgery Branch at NCI, NIH.

[0158] A375, Mel624 and B16-mhgpl00 cells were routinely cultured in RPMI 1640 medium (GIBCO) supplemented with 10% Fetal Bovine Serum (FBS, GIBCO BRL) and 100 lU/mL penicillin/streptomycin (P/S). 293FT cells (THERMOFISHER, R70007) were cultured in complete medium, which is high glucose DMEM medium (GIBCO) supplemented with 10% Fetal Bovine Serum (FBS, GIBCO BRL), lOOIU/mL penicillin/streptomycin (P/S), 1 mM sodium pyruvate (Gibco), 0.1 mM MEM NEAA (GIBCO), and 0.5mg/mL geneticin (GIBCO). All cells were incubated in a humidified incubator at 37 °C with 5% CO2 supply.

Lentiviral production and cancer cells transduction for td-Tomato labeling

[0159] A375, Mel624 and B16-mgphl00 cells were labeled with td-Tomato via lentiviral transduction for cell growth assays through the INCUCYTE experiments. [0160] 293FT cells were seeded at the density of 1 million cells per well in a 6-well plate in complete culture medium. On the next day (Day2), cells were transfected with pLenti-V6.3 ULTRA-CHILI (ADDGENE, 106173) plasmid, together with the packaging plasmids psPAX2 (ADDGENE, 12260) and pMD2.G (ADDGENE, 12259) using the LIPOFECTAMINE 2000 transfection reagent (INVITROGEN, 11668030) according to the manufacturer’s protocol. On Day3, the supernatant was replaced with fresh transfection medium supplemented with the viral boost reagent (500X, ALSTEM, VB100) as per the manufacturer’s instructions. The virus was collected by spinning down the viral supernatant at 1000g, 4 °C for 15 mins to remove the cell debris at Day 4.

[0161] A375, Mel624 and B16-mgphl00 cells were mixed with lentivirus at the 1 : 1 dilution with culture medium; 10 pg/mL polybrene (SIGMA, TR-1003) was added to the mixture for 24 hours before refreshing the medium. Three days after infection, blasticidin (GIBCO, Al 113903) was added for selection and maintenance of cells with positive Ultra-Chili expression.

Activation and culture of human CD8 T cells

[0162] Human NY-ESO-1 CD8+ T cells were stimulated with plate-bound anti -human CD3 (TONBO, 40-0038-U100, clone UCHT1) at 10 pg/mL and anti-human CD28 (TONBO, 40- 0289-U100, clone CD28.2) at 2 pg/mL for 48 hours before expanded with recombinant human Interleukin-2 (BIOLEGEND, 589106) at 100 lU/mL. Cells were cultured in completed X-VIVO medium, which is X-VIVO 15 SERUM-FREE HEMATOPOIETIC CELL MEDIUM (LONZA, BE02-060Q) supplemented with 5% inactivated fetal bovine serum (GIBCO, 10082147), 50 pM 2-mercaptoethanol (GIBCO, 21985023), and 10 mM N-Acetyl L-cysteine (SIGMA, A7250-5G) at 1E6 cells/mL.

Isolation, activation, and culture of Pmel-1 mouse CD8 T cells

[0163] CD8+ T cells were isolated from the single-cell suspension of splenocytes of Pmel-1 TCR transgenic mice using the EASYSEP mouse CD8+ T cell isolation kit (STEMCELL, Cat# 19853) by magnetic negative selection. If cells were not used directly, the freshly isolated splenocytes were frozen in cry opreservation medium (GIBCO, Cat# 2176664). CD8+ T cells isolated from newly-thawed splenocytes using the same method were rested in medium for overnight before stimulation. Isolated CD8+ T cells were stimulated with plate-bound antimouse CD3 (TONBO, 40-0032-U100, clone 17A2) at 10 pg/mL and anti-mouse CD28 (TONBO, 40-0281-U100, clone 37.51) at 5 pg/mL for 48 hours before expanded with recombinant mouse Interleukin-2 (BIOLEGEND, 575406) at 100 lU/mL.

[0164] Cells were cultured in completed RPMI 1640 medium, which is RPMI 1640 Medium (GIBCO, 11875119) supplemented with 10% inactivated fetal bovine serum (GIBCO, 10082147), 20 mM HEPES (GIBO, 15630080), 1 mM sodium pyruvate (GIBCO, 11360070), 50 pM 2-mercaptoethanol (GIBCO, 21985023), 2 mM L-glutamine (GIBCO, 25030024), and 1% Penicillin-Streptomycin (P/S, GIBCO, 15140122) at IE⁶ cells/mL.

Cas9 Ribonucleotide Protein (RNP) preparation and nucleofection

[0165] Lyophilized tracrRNA (IDT, #: 1072533) and crRNA (predesigned and synthesized from IDT, the oligos targeting different genes are listed at Table 5A) were resuspended in duplex buffer (IDT, #: 11-01-03-01) to 100 pM stock concentrations. CrRNA and tracrRNA were combined at 1 : 1 volume ratio and incubated at 95 °C for 5 mins in sterile PCR tubes in the thermocycler and then cooled down the gRNA complexes at room temperature for 20-30 mins. Cas9 protein (BERKELEY MACROLAB, 40 pM) was added to the crRNA/tracrRNA complexes at 1 :2 v/v ratio and incubated at room temperature for 15 mins. Assembled Cas9- ribonucleotide proteins (Cas9-RNP) were aliquoted to the PCR tube at 5 pl per tube.

[0166] Pre-activated mice or human CD8+ T cells were spun down and resuspended in the LONZA P3 buffer at 1E6 cells/ 20 pL and added 60 pL cell resuspension to the PCR tube. The cells/ Cas9-RNP mixture were transferred to the 100 pL nucleofection cuvette (LONZA, V4XP- 3024) and electroporated using the pulse program CM137 for mice activated T cells and EH100 for human activated T cells. After nucleofection, immediately added 100 pL pre-warmed culture medium to the cuvettes and transferred the cells to a 6-well plate at the density of IE⁶ cells/mL. FIBP overexpression in mice primary CD8 T cells

[0167] For FIBP overexpression, the open reading frame (ORF) of murine FIBP gene with 3 Flag-tags at the N-terminal and fused eGFP at the C-terminal (SEQ ID NO:73) was cloned into the pLV-EFla-IRES vector (provided as a gift by Dr. Zuojia Chen from the Experimental Immunology Branch, NCI, NIH). Lentivirus was made according to a previously described method. To concentrate the virus particles, the virus was mixed with the precipitation solution (ALSTEM, VC 100) per the manufacturer’s protocols, refrigerated at 4 °C for 4 hours and spinned down at 1500g for 30 mins at 4 °C. The pellet was resuspended in cold PBS to make the 100X concentrated lentivirus.

[0168] Pmel mice primary CD8 T cells (2 millions/mL) were cultured with the concentrated lentivirus (50 ul/ mL) supplemented with 8 ug/ mL polybrene and centrifuged at 500g at 32 °C for 90 mins. The medium was refreshed 12 hours later, and cells were cultured in the complete culture medium. 72 hours later, cells were sorted for GFP-positive CD8 T cells by flow sorter.

Genome DNA extraction and gene editing efficiency test

[0169] The knockout efficiency of T cells for IMEM222 was determined by T7 endonuclease I (T7E1) assay at 72 hours after nucleofection. Genome DNA was extracted by incubating 1000 cells with the QUICKEXTRACT DNA extraction solution (LUCIGEN, QE09050) at 65 °C for 6 mins and subsequently vortexed and heated at 98 °C for another 2 mins. Genome DNA was then amplified using primers flanking the cutting region and purified PCR products were subjected to T7 endonuclease I (T7E1) assay for cleavage activity validations. The primers used for T7E1 assay were listed at Table 5B.

Flow cytometry and antibodies

[0170] For the evaluation studies of surface and intracellular markers on CD8+ T cells, the following antibodies were used: anti -mouse CD3 (BIOLEGEND, 100205), anti -mouse CD8 (BIOLEGEND, 100705), anti-human CD3 (BIOLEGEND, 300307), anti-human CD8 (BIOLEGEND, 344704), anti-mouse CD69 (BIOLEGEND, 104505), anti-human CD69 (BIOLEGEND, 301904). Flow cytometry was performed on either BD LSR FORTESSA SORP I instrument or FACS CANTOII analyzer and raw data was analyzed using FLOWJO software (Version 10.6.1).

CFSE staining

[0171] One vial of lyophilized CFSE dye (BIOLEGEND, 423801) was spun down and reconstituted in 36 pL of DMSO to make the 5 mM stock solution according to the manufacturer’s instructions. A 5 pL working solution of CFSE dye was prepared by 1 :1000 dilution of the 5 mM stock solution in PBS right before use. The cells were spun down at 90g for 10 mins and resuspend at 1E7 cells/mL in the CFSE working solution. The cells then were incubated at room temperature for 20 mins in dark and then the staining quenched with 5 times volume of completed culture medium. Thereafter, the cells were spun down and resuspend in regular culture medium prior to restimulation.

Cancer cell and T cell in vitro co-culture assay

[0172] Two settings were used: 1, A375, Mel624 tumor cell lines and human NY-ESO-1 T cells; 2, B16-mhgpl00 cells and mice Pmel T cells. Cancer cells were seeded at different densities in 100 pL of complete culture medium in 96-well plates (For A375 and Mel624 cells, 5000 cells per well; for B16-mhgpl00 cells, 2500 cells per well). On the next day, CD8 T cells were resuspended in complete medium and added in 50 pL per well on top of tumor cells at the effector to target ratio of 2: 1.

[0173] In the experiments involving cholesterol (SIGMA, C4951) treatment, T cells were pre-treated with cholesterol at the indicated concentration for 5 days and then washed before adding to the co-culture system. The plates were then subjected to the INCUCYTE real time cell imaging system and the number of RFP-labeling tumor cells were counted over time. Enzyme-Linked Immuno-Sorbent Assay (ELISA)

[0174] To detect the levels of cytokines released by T cells after co-culture with tumor cells, the tumor cells A375 and B16-mhgpl00 in 100 pL were seeded in complete culture medium in 96-well plates (for A375, seeded 10⁵ cells/well, and for B16-mhgpl00, seeded 5X 104 cells/well). On the next day, the matched human NY-ESO-1 or mice Pmel-1 edited CD8+ T cells were added on top of tumor cells at the indicated effector to target ratio. 6 hours later, the supernatant was collected, and the secretion level of TNF-alpha was detected using TNF-alpha DUOSET ELISA kit (R&D, DY410 and DY210). The amount of IFN-y was determined with supernatant collected 24 hours later using the IFN-y DUOSET Elisa kit (R&D, DY485 and DY285B) according to the manufacturer’s instructions.

Adoptive T cell Transfer

[0175] For adoptive T cell transfer therapy, B16-mhgpl00 cells that are responsive to Pmel CD8+ T cells in the C57BL/6 mouse setting were used. 5 x 10⁵ B16-mhgpl00 cells were injected subcutaneously into the right flank of the C57BL/6 mice, 8-9 days after tumor implantation, mice were sub-lethally irradiated with the dose of 600 cGy and randomly distributed into different treatment groups (15 mice for each group). On the next day, 10⁶ edited CD8+ T cells (7 days after Cas9-RNP nucleofection) were transferred intravenously into the mice and recombinant human Interleukin-2 was given (10⁵ IU/0.5 mL) by intraperitoneal injection twice daily for consecutive 3 days. PBS and primary T cells without editing were used as controls for the experiment. Tumor size was measured blinded twice a week after T cell transfer, and the tumor area was calculated as length x width. Mice were sacrificed when either diameter reached 2 cm.

Whole transcriptome sequencing

[0176] 2E⁶ T cells from each sample were harvested and the cell lysate was incubated with

500 pL TRIzol reagent at room temperature for 5 mins. 100 pL chloroform was added to each sample and the tube shaken vigorously by hand for 15 secs. The tubes then were incubated at room temperature for 5 mins and then centrifuged at 12000g for 15 mins at 4 °C. The upper phase was transferred to a new tube and an equal volume of 70% ethanol added. This then was vortexed, and the mixture was subjected to binding, washing and elution steps using the PURELINK RNA Mini Kit (THERMO, 12183020). RNA was then treated with DNAse I (THERMO, 89836) for genomic DNA removal according to the manufacturer’s instructions. The RIN values of all samples were above 8.0.

Quantitative PCR analysis

[0177] Total RNA was extracted from T cells using TRIZOL Reagent (THERMO, 15596026), and cDNA was synthesized through reverse transcription using the PRIMESCRIPT RT REAGENT KIT with gDNA eraser (TAKARA, RR047A). Quantitative PCR (qPCR) was performed using the TB GREEN PREMIX EX TAQ KIT (TAKARA, RR420A) and corresponding primers, Ct values were detected by STEPONE PLUS REAL-TIME PCR system, raw data were processed using SDS 1.9.1 software and the relative mRNA expression level was normalized to internal reference gene of ATCB. The sequences of primers are listed in Table 5B.

Western Blot

[0178] 5 E⁶ T cells from each sample were harvested and cell pellets were washed with cold

PBS for 3 times, then cells were lysed with RIPA buffer (THERMO, 89900) supplemented with protease and phosphatase inhibitor cocktail (THERMO, 78441). After protein quantification using BSA method, samples were separated on the NUPAGE 4-12% Bis-Tris Gel (INVITROGEN), transferred to nitrocellulose membrane (LIFE TECHNOLOGIES), blocked with 5% non-fat milk in TBST, and incubated with primary antibodies at 4 °C overnight.

Signals were detected using HRP-conjugated secondary antibodies and ECL western blotting detection reagent (GE HEALTHCARE). Primary and secondary antibodies were listed in Table 5C. Cellular cholesterol content measurement

[0179] Cellular free cholesterol content was measured using the cholesterol cell-based detection assay kit (CAYMAN, 10009779). Cells were fixed, washed, and stained with fllipin III before being analyzed using the BD FACSYMPHONY flow analyzer. For the oxidationbased quantification of cholesterol, total cholesterol was extracted using the CHOLESTEROL EXTRACTION KIT (SIGMA, MAK 175), and then analyzed using the AMPLEX RED CHOLESTEROL ASSAY KIT (INVITROGEN, A12216) per the manufacturer's instructions.

LDL uptake assay

[0180] For LDL uptake experiments in primary Pmel CD8 T cells with FIBP knockout or overexpression, cells were starved in low glucose RPMI 1640 (GIBCO, 21870076), complemented with low glutamine (1 mM), 0.3% BSA instead of standard FBS for 16 hours. Then, the diluted LDL-DYLIGHT 488 was added to each well at the dilution of 1 :200, cells were incubated at 37 °C for 4 hours in the dark. The staining was either measured by flow cytometry using the FACS CANTOII analyzer or captured by the fluorescence microscope.

Data and Materials Availability

[0181] The source code and test data of Tres model is publicly available at github. com/data2intelligence/Tres and is incorporated herein in its entirety. The processed RNA-Seq data is available at: hpc.nih. gov/~Jiang_Lab/Tres/DESeq.Fibp.gz and is incorporated herein in its entirety.

RESULTS

Quantification of the immunosuppressive context of individual CD8 T cells in tumors

[0182] The T cell resilience (Tres) model utilizes two stages to identify gene markers of CD8 T cells that are resilient to the immunosuppressive microenvironment in solid tumors (Fig. 1 A). The model first quantifies the immunosuppressive context of individual CD8 T cells in a singlecell study by analyzing cytokine signaling activities (Stage 1, Fig. 1 A). The basis for this calculation can be illustrated by a melanoma tumor example, where TGFB 1 signaling activities in CD8 T cells, computed by CELLSIG, positively correlate with lower T cell proliferation levels, which are inferred by the expression of cell cycle and DNA replication genes (Fig. IB). A consistent anti-correlation between TGFB1 signaling activity and T cell proliferation in multiple datasets was identified in this study (Fig. 1C).

[0183] Similarly, CELLSIG-predicted TRAIL signaling activities are a reliable indicator of low T cell proliferation (Fig. ID), which is consistent with the role of TRAIL signaling in promoting activation-induced T cell death. The activities of T cell effector cytokines IFNG and TNFA were also associated with T cell proliferation, although to a lesser degree than TGFB1 and TRAIL (Fig. ID). Therefore, the immune suppression score for each T cell was defined as the signaling activities predicted by CELLSIG for TGFB1 and TRAIL.

Identification of marker genes of T cell resilience to immunosuppressive tumor microenvironments

[0184] Having quantified the immunosuppressive context for each T cell, a variable interaction test was used to identify gene expression signatures of T cell resilience (Tres) to immune suppression (Stage 2, Fig. 1 A). The Tres model searched for genes whose status mitigates the negative correlation between immunosuppression scores (TGFB1 or TRAIL signaling, as described above) and T cell proliferation across many single cells. For example, in a previous example from a melanoma tumor, it was found that the negative correlation between TGFB 1 -related immune suppression and T cell proliferation is evident only for T cells with low IL7R or high FIBP expression levels (Fig. IE).

[0185] Relationships between immune suppression, T cell proliferation, and the expression status of a third gene can be evaluated through the student t-test on the interaction covariate in linear regression (Table 1, Methods). Therefore, the interaction test t-values were defined as the T cell resilience score for each gene. For each gene, a positive T cell resilience score indicates that T cells with positive expression for that gene maintain high proliferative values even under suppression from TGFB1 or TRAIL signaling. A negative T cell resilience score for a gene indicates that T cells with positive expression for that gene do not tend to proliferate under the influence of TGFB1 or TRAIL signaling. The interaction test p-values indicate the statistical significance of each gene’s score (Fig. 5 A). In a single-cell study, each tumor with a sufficient number of CD8 T cells sequenced has a T cell resilience signature, consisting of one resilience score for each gene. The source code and test data for the T cell resilience model is publicly available at github . com/data2intelligence/Tres. Additionally, a web interface (available at resilience. ccr.cancer . gov) enables users to make T-cell efficacy predictions and query all results generated from the Tres model.

[0186] In support of the validity and biological relevance of T cell resilience scores, it was found that T cell resilience signatures are significantly correlated with a T cell sternness signature from an independent study of T cell persistence in complete cancer regression (Fig. 5B through 5E).

Identification of candidate causal regulators of T cell anticancer activity

[0187] A limitation of the T cell resilience (Tres) model is that the computed scores reflect associations, which may arise from indirect effects rather than causality. Establishing the mechanism of regulatory causality is important for the development of therapeutic applications. To identify candidate causal regulators from our Tres model, a data integration approach was used.

[0188] A previous genome-wide CRISPR screen searched for genes whose knockout can enhance human T cell proliferation upon T cell receptor (TCR) stimulation (Shifrut, et a!.). Although the phenotypes explored in that study do not include tumor-killing effects, they reflect genetic causality on T cell proliferation activity. For the present study, it was hypothesized that the regulators of T cell proliferation upon TCR stimulation identified in the CRISPR screen could also serve as regulators of T cell resilience in tumors. A search was conducted for genes with significant T cell resilience scores and CRISPR screen scores, with a false discovery rate (FDR) lower than 0.05 for the combined score (Methods). For example, in a colorectal cancer tumor, FIBP and TMEM222 have a negative T cell resilience score (meaning low gene expression was associated with high T cell proliferation) and positive CRISPR screen scores (meaning a knockout could promote T cell proliferation) (Figs. IF and 5F). For both genes, the FDR for having a more extreme combined score by chance is less than 0.05. Notably, FIBP has a marginal phenotypic score and therefore was not a significant CRISPR screen hit (compared to the top hit CBLB in Fig. 1G) (Shifrut, et al.). Further, the Shifrut, et al. CRISPR screen study could not validate TMEM222 knockout effects on T cell proliferation upon TCR activation, and therefore did not list it as a significant hit. Thus, FIBP and TMEM222 were identified as candidate T cell resistance regulators only by joint analysis with the T cell resilience signature. [0189] The joint analysis described above was systematically performed across 14 single-cell

RNA-Seq datasets that span seven cancer types. Fifteen significant genes met the FDR threshold of 0.05 (Fig. 1H and Table 2). The top two genes are FIBP (ranking first) and TMEM222 (ranking second) (Figs. 1H Fig. 5G), indicating these genes are potential negative regulators of T cell proliferation in immunosuppressive tumor environments. It was found that high FIBP expression in tumors for lymphocyte expansions indicates a worse survival outcome upon adoptive T cell therapy (Fig. II). Consistent with an immunosuppressive role for FIBP in T cells, FIBP expression is higher in CD8 T cells from COVID-19 patients with severe symptoms than individuals with mild symptoms in two independent cohorts (Fig. 5H).

FIBP and TMEM222 knockouts in T cells significantly enhance the cancer killing efficacy of human and mouse T cells

[0190] The significant associations of FIBP and TMEM222 with T cell resiliency in solid tumors indicate that they are potential negative regulators of T cell proliferation in the anticancer immune response. Therefore, whether their genetic knockout (KO) could enhance the efficacy of T cells in an antigen-specific recognition system was explored. This question was examined in both human and mouse systems (Fig. 2A). For humans, NY-ESO-1+ melanoma cells, A375 and Mel624 were used as the target of NY-ESO-1 TCR+ T cells. For mice, the gplOO overexpressing cell line B16-mhgpl00 and the corresponding Pmel-1 TCR+ T cells were used. [0191] Genes were knocked out in primary CD8 T cells through the nucleofection of Cas9 ribonucleoprotein (Cas9-RNP), consisting of the Cas9 protein complex with CRISPR guide RNA (gRNA). The target genes were FIBP and IMEM222, with CBLB as the positive control and AAVS1 (humans) or Rosa26 (mice) as the negative controls. The positive control CBLB is a well- established negative regulator of T cell anticancer efficacy. The negative controls, human AAVS1 and mouse Rosa26, are widely used safe harbor loci for genetic editing experiments. Cas9-RNP electroporation can efficiently knock out genes in both human donor T cells and mouse primary T cells for each target’ s three independent gRNAs (Figs. 2B and 6A). Tumor cells were labeled with td-Tomato by lentivirus to facilitate real-time cell density tracking. The INCUC YTE imaging system (Methods) was used to evaluate the cancer-killing efficacy of T cells in co-cultures (Figs. 2A and 2C).

[0192] T cells with control knockouts (KO) have similar cancer-killing efficacy compared to parental T cells for two different human donors and Pmel-1 TCR transgenic mice (Figs. 6B through 6D). In contrast, T cells with FIBP and TMEM222 KOs kill cancer cells at a higher efficacy than control KOs in both humans (Figs. 2C and 2D) and mice (Fig. 2E). Moreover, both gene KOs enhance the release of T cell effector cytokines, including interferon-gamma (IFNG) and tumor necrosis factor-alpha (TNFA) in both human and mouse primary T cells (Fig. 2F). Overall, the effects of FIBP knockout are most significant, and FIBP KO has effects similar in amplitude to the positive control, CBLB KO (Figs. 2D through 2F). Consistent with these results, CD8 T cells had enhanced proliferative abilities (Figs. 6E and 6F) and higher levels of activation marker CD69 (Figs. 6G and 6H) after the FIBP knockout.

FIBP knockout in T cells enhances the in vivo efficacy of adoptive transfer therapy [0193] Whether knocking out FIBP or TMEM222 could enhance the in vivo efficacy of adoptive T cell transfer was next investigated (Fig. 3 A). 70 C57BL/6 mice were randomized into six different treatment groups: FIBP knockout (15 mice), Tmem222 knockout (15 mice), Rosa26 knockout (negative control, 15 mice), Cblb knockouts (positive control, 15 mice), wildtype T cells (no knockout, 5 mice), and no treatment controls (saline injection, 5 mice) (Fig. 7A). For each group except no treatment, Pmel T cells targeting the gplOO antigen expressed on inoculated tumor cells were injected. Controls indicated that the system was working properly. First, compared to no-treatment controls, the T cell adoptive transfer significantly repressed the growth of tumors with the gplOO antigen (Figs. 7B and 7C). Second, T cells electroporated with Rosa26 CRISPR RNP did not have lower antitumor efficacy than parental T cells (Figs. 7B and 7C), indicating the low toxicity from the nucleofection procedure.

[0194] Compared to the Rosa26 control knockout, both the FIBP and Tmem222 knockouts significantly reduced tumor size (Figs. 3B through 3D). The overall tumor size reduction effects of FIBP and Tmem222 knockouts are comparable to the positive control Cblb knockout before any mice reached endpoints (Figs. 3C and 3D). However, only FIBP and Cblb (the positive control) achieved a statistical significance on the survival effect (Fig. 3E, death or euthanasia endpoint in Methods). These results suggest FIBP knockout as a potential therapeutic approach to enhance adoptive T cell therapy in humans.

FIBP knockout limits cholesterol metabolism to enhance T cell antitumor efficacy

[0195] To determine the mechanism of FIBP’s inhibitory effects on T cells, RNA-Seq was performed in murine FIBP knockout Pmel T cells and control (Rosa26) knockout Pmel T cells (Fig. 4A). Ingenuity Pathway Analysis (IP A) revealed that all top enrichments upon FIBP knockout are related to the down-regulation of cholesterol metabolism (Fig. 4B) and enrichment of T cell activation genes (Fig. 8A). Compared to Rosa26 control knockouts, FIBP knockout down-regulated multiple essential enzymes in cholesterol biosynthesis, such as Hmgcs and Sqle, and the cell-surface receptor Ldlr, which modulates cholesterol intake (Fig. 4C). FIBP knockout also up-regulated the cholesterol efflux pump Abcal.

[0196] To further investigate associations between FIBP and cholesterol metabolism, the correlation between FIBP and cholesterol pathway genes was further analyzed across leukemia clinical samples from the MILE project, in which cancer cells have a hematopoietic lineage related to T cells (Kohlmann et al. “An international standardization programme towards the application of gene expression profiling in routine leukemia diagnostics: the Microarray Innovations in Leukemia study prephase.” Br. J. Haematol. 2008;142:802-7, incorporated herein in its entirety). FIBP expression is positively correlated with positive regulators of cholesterol metabolism and negatively correlated with repressors (Figs. 8B and 8C). Based on an analysis using public ChlP-Seq data, the target genes of SREBF2, the master transcription factor promoting cholesterol metabolism, are significantly down-regulated upon FIBP knockout (Fig. 8D and Table 4A). IPA analysis also revealed that SREBF2 was among the top enriched upstream transcriptional regulators in FIBP knockout T cells (Table 4B).

[0197] RT-qPCR was used in both Pmel and human donor T cells to validate the downregulation of several essential regulators (ABCA1, LDLR, and SREBF2) of cholesterol pathways (Fig. 4D) and key enzymes involved in cholesterol synthesis (Fig. 8E) upon knockout of FIBP. Decreases in LDLR protein and SREBF2 protein (both the unactivated full-length form and the NFL-cleaved activated form) also were confirmed by Western blotting in FIBP knockout cells (Fig. 4E). In contrast, FIBP overexpression in Pmel T cells through lentiviral transduction caused increases in the abundance of cholesterol metabolism regulators at both the mRNA and protein levels (Figs. 4F and 8F).

[0198] Consistent with these results, cholesterol levels in T cells dropped significantly upon FIBP knockout and increased significantly upon F77>/^J overexpression (Figs. 4G, 8G, and 8H). The cellular cholesterol level was also significantly lower inFffiP-KO T cells cultured with the tumor culture supernatant relative to control (Fig. 81). All of the analyses above indicate that FIBP knockout specifically down-regulates cholesterol metabolism in T cells.

FIBP knockout renders T cells resistant to the immunosuppressive effects of cholesterol [0199] Previous studies have revealed the effects of cholesterol on the anticancer activity of T cells through inhibiting effector T cell differentiation and inducing T cell exhaustion.

Consistent with these previous results, the co-culture assay performed in this Example shows that cholesterol treatment of T cells significantly inhibited cancer-killing efficacy (Fig. 4H, left panel, two-sided Wilcoxon rank-sum p-value < 0.05). However, FIBP knockout T cells retain their cancer-killing efficacy even after cholesterol treatment (Fig. 4H, right panel). Previous studies have also demonstrated that LDL uptake compromises the antitumor function of T cells. FIBP loss resulted in a marked reduction of low-density lipoprotein (LDL) uptake upon lipoprotein starvation (Fig. 8J). These results show that ZB knockout, through downregulation of cholesterol biosynthesis and transportation, renders CD8 T cells resistant to the immunosuppressive effects of high cholesterol levels, a common feature of the tumor microenvironment.

EXAMPLE 2 (Prophetic Example)

[0200] This Example concerns enhancement of CAR T cells killing efficacy on solid tumors, particularly cancer cells in muscle, through FIBP knockout.

[0201] Through mouse model studies, FGFR4 CAR T cells demonstrated anti-tumor efficacy in a subcutaneous implantation model but not in orthotopic implantation where sarcoma tumors cells are engrafted in the muscle. Therefore, an endpoint of the study discussed in this Example is to evaluate whether FIBP knockout in FGFR4-targeting CAR T cells can enhance its killing efficacy on solid tumors, particularly cancer cells in muscle.

[0202] The materials employed in this study include FGFR4 CAR construction plasmids and a RH30 sarcoma cell line with FGFR4 activation (described in Taylor etal., “Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models.” J. Clin. Invest. 119:3395-407 (2009), which is incorporated herein in its entirety). Human peripheral blood mononuclear cells (PBMCs) obtained from the NTH clinical center will serve as a source of CD4+ and CD8+ T lymphocytes.

[0203] The PBMC preparation will be spin-infected with CAR lentivirus, which will result in a mixture of CD4 and CD8 CAR T cells. These will be subjected to gene knockout of either FIBP or AAVS1 (control) using methods and reagents such as are discussed above in Example 1 (see Table 5 A for suitable gRNAs). This will result in CAR T cells with FIBP and AAVS1 (control) knockouts.

[0204] As a first in vitro experiment, the RH30-killing efficacy of CAR T cells with FIBP and A A CS 1 (control) knockouts will be compared using co-cultures between FGFR4-targeting CAR T cells and Rh30 and the INCUCYTE cell imager. The protocols will be similar to those described above in Example 1 (see, for example, experiments pertaining to Fig. 2A). A demonstration that CAR T cells with knockout is significantly more efficient than the control cells will be considered a promising result.

[0205] As a second in vitro experiment, the cytokine release difference between FIBP and AAVS1 (control) knockout CAR T cells will be compared using ELISA assays of interferongamma and TNF-alpha, both of which are T cell effector cytokines. The protocols will be similar to those described above in Example 1 (see, for example, experiments pertaining to Fig. 2A). A result indicating that the FIBP knockout enhances the release of these T cell effector cytokines relative to the control in CAR T cells will be considered a promising result.

[0206] If both above-mentioned in vivo assays yield promising results, in vivo studies will be conducted using mice. In a first in vivo experiment, the CAR T therapies (FIBP and AAVS1 (control) knockout CAR T cells) will be evaluated in NSG mice implanted with RH30 cells subcutaneously and orthotopically in the muscle. A second in vivo experiment will involve tail vein injection of CAR T cells with FIB or AAVS1 (control) knockouts and comparison of the tumor size and survival durations between treatment and control groups. The ideal outcome is that CAR T cells with FIBP knockout can repress cancer cells in the muscle. The suboptimal outcome is that CAR T cells with FIBP knockout kill subcutaneous cancer cells with higher efficacy than T cells with AAVS1 control knockout.

EXAMPLE 3 (Working Example)

[0207] This Example concerns the predictive potential of the Tres model as regards immunotherapy response using pre-treatment patient materials.

[0208] Pertinent data and other information are presented in Figures 9A through 9F and 10A through 10H. Materials and methods for the experiments underlying these data and other information are detailed in Zhang et al., Nature Medicine 28, 1421-1431 (2022), which is incorporated herein in its entirety by reference. For Figures 9A-9F and 10A-10H, reference to Caushi refers to Caushi et al., Nature 596, 126-132 (2021). Reference to Chen refers to Chen et al., Cancer Discov. 11, 2186-2199 (2021). Reference to Fraietta refers to Fraietta et el., Nat. Med. 24, 563-571 (2018). Reference to Lauss refers to Lauss et al., Nat. Commun. 8, 1738 (2017). Reference to SadeFeldman refers to Sade-Feldman el a\., Cell 175, 998-1013 (2018). Reference to Yost refers to Yost et al., Nat. Med. 25, 1251-1259 (2019). Reference to Zhang refers to Zhang et al., Cancer Cell 39(12) 1578-1593 (2021).

[0209] The data presented in Figures 9A through 9F demonstrate that Tres predicts clinical efficacies of ICIs and adoptive cell therapies. In particular, Figure 9A reveals that Tres score correlations predict the efficacy of T cells in immunotherapies. Each data point represents a tumor, with sample counts around each box and cohort names under each panel. The y axis presents the correlation between the Tres signature and the T cell expression profile. P values were computed through the two-sided Wilcoxon rank-sum test, comparing responders and nonresponders. The thick line represents the median value. The bottom and top of the boxes are the 25th and 75th percentiles, respectively (interquartile range). Whiskers encompass 1.5 times the interquartile range.

[0210] Figure 9B presents graphs concerning Tres prediction performance on T cell clinical efficacy. The ROC curves present false-positive rates against true-positive rates of predicting responders versus non-responders based on signature correlations. The performance of Tres and other signatures was compared to that for random expectations, shown as diagonal lines.

[0211] Figure 9C graphically presents comparisons among T cell signatures in predicting clinical response. The ROC AUC is shown for T cell signatures in Table 6, with 0.5 as the random expectation. All box plots have the same format as in Figure 9A (n = 6 independent datasets per box).

[0212] Figure 9D plots Tres score correlations in tumors for lymphocyte expansion predictive of ACT outcome. This plot only included tumors with cytotoxic lymphocyte infiltration higher than average in a melanoma study. The y axis presents the fraction of patients with overall or progression-free survival (PFS) higher than each duration (x axis) for tumors whose transcriptomic profiles have positive or negative correlations with the Tres signature. P values were evaluated by the two-sided Wald test in the Cox proportional hazards regression without any cutoffs.

[0213] Figure 9E graphically presents data demonstrating that Tres correlations in T cells for CAR-T manufacture predict a favorable response. The B cell aplasia duration upon anti-CD19 CAR therapy was shown for patients whose pre-manufacture T cells have positive or negative correlations with the Tres signature as shown in Figure 9D.

[0214] Figure 9F graphically presents comparisons among T cell signatures in predicting survival outcome.

[0215] For datasets in Figure 9D and 9E (n = 3), the risk z scores were compared to box plots with the same format as in Figure 9 A for T cell signatures in Table 6, with zero as the random expectation.

[0216] The data and other information presented in Figures 10A through 10H pertain to the control analyses of the median Tres signature in predicting immunotherapy responses. As graphically represented in Figure 10A, correlations between the median Tres signature and T-cell bulk transcriptomic profiles lead to predictive value (responder vs. non-responder).

[0217] Figure 10B presents plots representing Tres score correlations with profiles from post-treatment tumors. Each point represents an ICI-treated tumor. The Y-axis presents the correlation between the Tres signature and T-cell expression profiles. P-values were calculated from the two-sided Wilcoxon rank-sum test comparing group values. The thick line represents the median value. The bottom and top of the boxes are the 25th and 75th percentiles (interquartile range). Whiskers encompass 1.5 times the interquartile range.

[0218] Figure 10C presents plots representing scores indicative of T-cell clinical efficacy. The ROC curve presents false-positive rates against true-positive rates of predicting whether T cells are from responders or non-responders. The performance of diverse signatures was compared. For Figures 10B and 10C, the top panel represents data concerning Sade-Feldman et al. (2018) and the bottom panel represents data concerning Caushi et al. (2021).

[0219] Figure 10D presents data demonstrating a comparison among T-cell signatures in predicting clinical response. The negative predictive value was shown for T-cell efficacy signatures in Table 6. All box-plots have the same format as Figure 10B (n = 6 independent datasets per box).

[0220] Figure 10E presents data demonstrating a lack of associations between Tres score correlations and adoptive cell therapy efficacy in tumors with T-cell infiltration lower than average. The survival of patients upon adoptive T cell transfer was shown for tumors with positive or negative Tres score correlations. P-values were calculated using the two-sided Wald test using continuous values.

[0221] Figure 10F presents data demonstrating performance of Tres on predicting ICI outcomes using bulk data. Each dot represents a pretreatment tumor transcriptomics cohort listed in Table 7. The first group presents the results using all samples. The other two groups present results using tumors with positive or negative CTL levels. The Y-axis presents Cox-PH risk z- scores as the association between overall survival and Tres signature correlations. Box plots have the same format as panel b. P-values were computed through the two-sided Wilcoxon signed- rank test.

[0222] Figure 10G presents data demonstrating Tres prediction performance on different combinations of treatments and sample sites in a triple-negative breast cancer study. The area under the ROC curve (AUC) and confidence intervals were shown with 0.5 as the random expectation.

[0223] Figure 10F presents data demonstrating the Tres prediction performance in tumors when immunosuppressive signals are lower than average. The data and box-plots are as shown as Figure 10F.

[0224] Taken together, the data presented in this Example demonstrate that the Tres model can predict immunotherapy response using pre-treatment patient materials, with superior performance than previous methods. EXAMPLE 4 (Working Example)

[0225] This Example concerns in vivo flow analysis of T cells from mouse tumors, which further demonstrates FIBP knockout potentiates T-cell efficacy through lowering cholesterol levels in T cells.

[0226] Materials and methods for the experiments underlying these data and other information are detailed in Zhang et al., Nature Medicine 28, 1421-1431 (2022) (see, e.g., Figures 6 and Extended Data 7), which is incorporated herein in its entirety by reference.

[0227] In particular, Figure 11 A herein presents data from in vivo flow analysis of cholesterol levels (Filipin III) in FIBP knockout (“KO”) T cells. Representative histograms are presented for gene KO T cells isolated from mouse tumors (left). Mean and s.d. are shown as error bars (n = 4 tumors per group) (right). For Figure 11 A, KOs were compared using a onesided Wilcoxon rank-sum test.

[0228] Figure 1 IB presents in vivo flow analysis of T-cell phenotype markers, such as T-cell sternness and exhaustion. Marker positive fractions of T cells with gene KOs were shown with mean values and standard deviations as error bars (tumor counts labeled under each group). Markers are as indicated in the left axis label of each respective panel. The growth curves are presented in Supplementary Fig. 3 of Zhang et al. (2022). Different groups were compared through the one-sided Wilcoxon rank-sum test. None of them achieved statistical significance, revealing that mechanisms such as T-cell sternness and exhaustion are not responsible for the potentiation of T-cell efficacy through lowering cholesterol levels in T-cells.

[0229] As noted above in Example 1, Figures 8G, 8H, and 81 present data demonstrating that demonstrates FIBP knockout potentiates T-cell efficacy through lowering cholesterol levels in T cells. The data presented in Figures 8G, 8H, and 81 were generated from in vitro experiments. This Example discusses data from in vivo flow analysis that reinforce this conclusion. Moreover, the data discussed herein, and as presented in Zhang et al. (2022), further rule out other mechanisms (such as T-cell sternness and exhaustion) as explanations for the potentiation of T- cell efficacy demonstrated by FIBP knockout. EXAMPLE 5 (Working Example)

[0230] This Example discusses an experiment demonstrating that statins, which are prescribed for lower cholesterol levels in patients, do not lower the cholesterol levels of T cells, specifically.

[0231] To conduct the experiment, CD8+ T cells isolated from C57BL/6 mice splenocytes were stained with CFSE dye. Briefly, one vial of lyophilized CFSE dye (BIOLEGEND, 423801) was spin down and reconstituted it in 36 pl of DMSO to make the 5 mM stock solution according to the manufacturer’s instructions. A 5 pl working solution of CFSE dye was prepared by 1 :1,000 dilution of the 5 mM stock solution in PBS immediately before use. After spinning down the isolated CD8+ T cells at 90g for 10 min and resuspending the cells at IxlO⁷ cells/ml in the CFSE working solution, the cells were incubated at room temperature for 20 min in the dark and then the staining quenched using five times the volume of complete culture medium.

Finally, the stained and quenched cells were spun down and resuspended in a regular culture medium before restimulation or simvastatin treatment.

[0232] For statin treatment, the stained, quenched, and resuspended CD8+ T cells were stimulated with anti-mouse CD3/28 and treated with simvastatin at several concentrations (0, 1, 5, 10 and 20 pM) simultaneously. The same CFSE staining and treatment of simvastatin were applied to CD8+ T cells after stimulation and during expansion with IL-2 for 3 days. At the end of treatment, cells were collected and washed before Filipin III and CFSE detection by flow cytometry.

[0233] The results of this experiment are presented in Figures 12A through 12D. In particular, Figure 12A presents data concerning the cholesterol levels of T cells treated with simvastatin concurrently with anti-CD3/28 activation, while Figure 12B presents data concerning the cholesterol levels of T cells 72 hours after anti-CD3/28 activation. The median Filipin III intensity was measured with flow cytometry at different simvastatin concentrations, with a representative plot on the left and the median intensity on the right.

[0234] Figure 12C presents data concerning the proliferation of T cells treated with simvastatin concurrently with anti-CD3/28 activation, while Figure 12D presents data concerning the proliferation of T cells 72 hours after anti-CD3/28 activation. For each condition, the T-cell expansion index was computed through CFSE staining and flow cytometry analysis. [0235] The results of these analyses reveal that simvastatin, a common drug for lowering cholesterol levels in patients, does not lower cholesterol levels in T cells. Thus, statins cannot replace FIBP knockout in T cells.

Table 1

Example of variable interaction test (related to Fig. IE)

[0236] The current examples are computed for a patient in a melanoma single-cell study. The interaction term is created by multiplying the immune suppression score (TGFB 1 activity) with the target gene expression. The t-value (coefficient / standard error) and two-sided p-value are computed using the student t-test in the ordinary least square regression. In the table, the top rows (A) concern the results for IL7R, while the bottom rows (B) concern the results for FIBP. Table 2 Identification of T cell activity regulators through the consistency between CRISPR screen phenotypes and Tres scores, related to Fig. 1H.

Table 2 (continued)

[0237] Genes with both significant T cell resilience (Tres) scores (column 1) and CRISPR screen phenotypes (column 2) are shown for different single-cell datasets (column 3) and patients (column 4). In the table, the top rows (A) concern results for Tres scores computed for the immune suppression through TGFB1, while the bottom rows (B) concern results for Tres scores computed for the immune suppression through TRAIL.

Table 3 Interaction effects between FIBP and cytotoxic T lymphocyte (CTL) levels on the overall survival outcome, related to Fig. II

[0238] The current table was generated using the data from a clinical trial of adoptive T cell therapy in melanoma. The CTL level is estimated through the average expression of CD8A, CD8B, GZMA, GZMB, and PRF1. 27 patients are included in the regression. The statistical significance was estimated through the two-sided Wald test in the Cox-PH regression.

Table 4A

FIBP knockout down-regulates SREBF2 transcription factor activity, related to Fig. 4B

[0239] Table 4 A presents data concerning ChlP-Seq target profiles from CISTROME database for SREBF2 from B lymphocyte and HepG2 cells. For each profile, the differential expression directions of SREBF2 target genes through linear regression and the two-sided student t-test was analyzed. The result indicates that SREBF2 ChlP-Seq target genes are significantly downregulated upon FIBP knockout. Table 4B

[0240] Table 4B presents data concerning upstream regulator analysis from the Ingenuity Pathway Analysis (IP A). IPA analyzed diverse regulatory molecules with known target genes and predicted the inhibited or activated status in the FIBP knockout differential expression profile.

Table 5A - CRISPR gRNA sequences.

[0241] The sequences set forth in Table 5A are recited as DNA sequences; however, for any of these sequences, when RNA is to be constructed, thymidine (“T”) is substituted with uracil (“U”), and such RNA sequences are included as if they were separately set forth herein. Table 5B - Primer sequences.

Table 5B - Primer sequences (continued)

Table 5C - Antibodies for Western blots

Table 6 - Gene signatures associated with T-cell efficacy

[0242] The column “Type” presents whether each signature is a positive (+) or negative (-) indicator. For each negative indicator, the sign of signature scores on each sample will be reversed in the comparison.

Table 7 - The prediction performance of Tres signature on ICI overall survival outcomes using bulk RNA-seq data from whole tumors

[0243] The Cox-PH risk z-scores present whether the correlations between Tres signature and pretreatment bulk transcriptomics profiles can predict the overall survival durations. The column “All” presents the results using all samples. The right two columns present results using tumors with positive or negative cytotoxic T lymphocyte levels (CTL), estimated by the median of GZMA/B, CD8A/B, PRF1. N/A: The Cox-PH regression may fail when patient sample counts are insufficient.

[0244] References listed in the “Cohort” column are as follows:

• Braun et al., Interplay of somatic alterations and immune infiltration modulates response to PD-1 blockade in advanced clear cell renal cell carcinoma. Nat. Med. 26, (2020).

• Gide et al., Distinct Immune Cell Populations Define Response to Anti-PD-1 Monotherapy and Anti-PD-l/Anti-CTLA-4 Combined Therapy. Cancer Cell 35, (2019).

• Hugo et al., Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. Cell 165, (2016). • Liu et al., Integrative molecular and clinical modeling of clinical outcomes to PD1 blockade in patients with metastatic melanoma. Nat. Med. 25, (2019).

• Mariathasan et al., TGFP attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells. Nature 554, (2018).

• Riaz et al., Tumor and Microenvironment Evolution during Immunotherapy with Nivolumab. Cell 171, (2017).

• Van Allen et al., Genomic correlates of response to CTLA-4 blockade in metastatic melanoma. Science 350, (2015).

• Zhao et al., Immune and genomic correlates of response to anti-PD-1 immunotherapy in glioblastoma. Nat. Med. 25, 462-469 (2019).

SEQ ID NO: 63

Genomic Sequence of Human FIBP, Reproduced from Homo sapiens FGF1 intracellular binding protein (FIBP), RefSeqGene on chromosome 11 : NCBI Reference Sequence: NG 047103.1.

1 ggattacagg ccggagtcac tgcctggtgc cagcaatctc ctaaaacgtc tgggcctgat

61 gggagttgtg cccattttct cccaggggct tatgggagtt gtagtccaga cttgctgggg

121 actctccact gccccccctc tccccccgcc caaccccagc tcctcagaac cctgagtcca

181 aacgtctgcg gctctggggt attcagccct cacctttctg actcctctcc tgatcttcta

241 caggagactg acaaactgga agatgagaaa tctgggctgc agcgagagat tgaggagctg

301 cagaagcaga aggagcgcct agagctggtg ctggaagccc accgacccat ctgcaaaatc

361 ccggaaggag ccaaggaggg ggacacaggc agtaccagtg gcaccagcag cccaccagcc

421 ccctgccgcc ctgtaccttg tatctccctt tccccagggc ctgtgcttga acctgaggca

481 ctgcacaccc ccacactcat gaccacaccc tccctaactc ctttcacccc cagcctggtc

541 ttcacctacc ccagcactcc tgagccttgt gcctcagctc atcgcaagag tagcagcagc

601 agcggagacc catcctctga cccccttggc tctccaaccc tcctcgcttt gtgaggcgcc

661 tgagccctac tccctgcaga tgccacccta gccaatgtct cctccccttc ccccaccggt

721 ccagctggcc tggacagtat cccacatcca actccagcaa cttcttctcc atccctctaa

781 tgagactgac catattgtgc ttcacagtag agccagcttg gggccaccaa agctgcccac

841 tgtttctctt gagctggcct ctctagcaca atttgcacta aatcagagac aaaatatttc

901 ccatttgtgc cagaggaatc ctggcagccc agagactttg tagatcctta gaggtcctct

961 ggagccctaa ccccttccag atcactgcca cactctccat caccctcttc ctgtgatcca

1021 cccaacccta tctcctgaca gaaggtgcca ctttacccac ctagaacact aactcaccag

1081 ccccactgcc agcagcagca ggtgattgga ccaggccatt ctgccgcccc ctcctgaacc

1141 gcacagctca ggaggcgccc ttggcttctg tgatgagctg atctgcggat ctcagctttg

1201 agaagccttc agctccaggg aatccaagcc tccacagcga gggcagctgc tatttatttt 1261 cctaaagaga gtatttttat acaaacctac caaaatggaa taaaaggctt gaagctgtgg

1321 cctgagtgcc tcactggacc cagaggccaa tgggagagta tttggagccc taggtcccag

1381 ccttagctct acagactcac tgcatgacct tggacaaatt ctttgatatt tttggacttt

1441 gtcttatctg acaagtgggg ctacatccgc tcggcctcat ctccgggact gaggggagac

1501 agtcctctgg ggtaaaagcg aagaggctta ccgcccctct ccccgtgggg gcccacagtg

1561 ctgctgcgtg tgccacggag gggcgctgtt atgccccctt ccagcctcag aggaggcggc

1621 cgagtggttc taggccccca gcacctgctt cctgccccat tgtcttggac aggaaggagc

1681 cgggaaggga gctggagcca cttcaggggc tggattaagg ccggtcccgg cgcccgatgg

1741 gggaagtctg gtttaccact gccccgcatc ccaccccagg ctcgaagtaa tgggggaatc

1801 attagttctg cagtccttct ggatgggaat tgtaccctgt atcagcaccc caccctcgcc

1861 ccaaccccgg cgagtaccaa acagacgctg cccaggccgt gggctccgct tccgtccggg

1921 tttattaccc ccaatccaac ccccagcaag tccctccgct ccagccttca gtccccctgc

1981 tgccctgcca gcaagagcga ggggcccttc ttggtgtcag gatttcttag tccatgccag

2041 cggcccctcc tgcgcagcca ggacggcgtc caggtcccga ggtccacagc ggtcccgggg

2101 ctcggggagc aatccgggcg ggcgtcggcg tggaggaagc agtgccttca atcatcgggg

2161 gaacaggcca ggggcaactg atccggactg cccacgctgc cagtgctgga gcttccatcg

2221 cctaggtcgc ggggcccgca cgggggcaag gcaagctcgg gtgctggccc ggctcctgat

2281 cccccggcgc cgctggggcc gccgcggggg ccccattctt cgcccagcgc caggcagagc

2341 tccttaagcg ctaggttctc ccgcagcagc tcctcctggc ggccctccag ctcggccagc

2401 ttctgccaac agccgcccag gtcctcgcgc acggcccggg atgcttgggt cccgaagagc

2461 tgccactggc gtgcggcgcg ccgcccgcgc tggcgctccg agtccaggaa gcagcagagg

2521 tcgcgcagct cacggttctc tgcctgcaga cgccggttga gctgcttgag ctcgcggatc

2581 tcgcccaggt ggccctgcag ctgccgattc acctcctgca tgaggcggcc gcgctgcacc

2641 agtgccgcca ggcgcgccgc ctcctcccgc cgcaggcgcc gcactagctc ttccttgcct

2701 agcgccgcca tctcctcgtc cgtcagctcc tccaggccgc ctgcctcggc ctccatggct

2761 ggtcaggcct ctggagcatc gcccgcccgc cgccgcggcc caggagcccc acgtcgggcc

2821 cggcgccact caggctcggg ctccggctcc gcacgcggca ggcagtggcc gaggcagcgt

2881 aggctgcagc agctacccgg gctgggcacg cggcggggcg cgcgccgggg cggggccgca

2941 tgtcgttatg gaaggacagc caatccggtg aaacgtcccc ggaaaggggc ggggcctgag

3001 ctcaggggcc gcccccctcg ttccccaccc actccgggtg ttactgatcc tcgatgtcaa

3061 ggaacccgcg cgtcatcgcc ctccacatcg gtctcaaccc gaaactagga gcttcgaacc

3121 tccaagtacc ggatcgcccc aagcttgagc ctaggcctct cggctcttct gagaaacaag

3181 attcaggcgt ccgcccctga ctcctgccca tcccccacag aagtcagatg gaccgctctc

3241 cagtccctct ttacctttga tcaaattcgg aagtctcggc ccactaagga tcgctcctgg

3301 ttcctcggaa ccctgagtcc aaacgtccta gctcaccaat agactaccct ccctcccagg

3361 gagcgtttcc tgcctggggg gaggaacata caggattcgt ttgttcaata aacgtttttt

3421 atgtgcaagg cccatgcaga aataaattta accaaaatac cgattccatc ctcaaggagg

3481 tcgcagtctg tccgggaagt caagctgtgt taagcaagaa ataaatccgg gaggagtatg

3541 gtctgagggt ccttaagagg gttcccaaaa gagatgctat ccaagaggct ctggtagatc

3601 cataggactt tgccaagtaa agagctattt cagggagaga gaataacata gtgaaagaca

3661 ctggtgcccg aaagagccta gctgctttag gaacaatgag gatagcttgg tttttgaaaa

3721 gcagatgtgt cgagaaaatg ctaagagaga gagagcgagg cttcaaaggt aggtaaaagc 3781 cagattctta agaacttcca gtaccaggtt ggagcatttc caaaggccac taaggatgtt

3841 aagcaagaga gtaacatttg taaggatgtt aagcaaggga gtaacatttt aaaatatgcc

3901 tttaactggc caagatggtg aaaccccgtt tctactaaaa atacaaaaat tagccgggcg

3961 tggtgggagg agcctgtaat ctcagctact cgggaggctg aggcagagaa ctgcttgaac

4021 ccgggaggcg gaggttgcag tgagccgaga tcgcgccact gcactccagc ctgggcgaca

4081 gagcgagacg ctgtctcgaa aaaaaaaatg tatttaaaaa gattgggctg atctgtgtgg

4141 gccggaccct gcggggaggg gacataaaca gggagattag taacggtgtc aaagagcaag

4201 agcaaggaga gctaagcaga aagacggctg gggcagcatc agtgggggtg gagaggacaa

4261 acagatacta gaaatattcg ggaggcaaat cagccgaact gaatgtgggg agagaggagt

4321 ctggaagaac gcccaggttt ctcgtatgag aactggtgga tgttagggct gtcactgaga

4381 tagggacgca ggaaagaaac aaatggggag aggcagaggt ctgtctagaa tatgttgagc

4441 ttttgagggg cttacgggaa gagtgtggat agacttggcc gataggcggc ccctccggat

4501 gtggacttcc aggcagagct cggaggggca gaattagaga cgtcagtagc tcgtggggct

4561 gggggtggga ttaaaaccaa ggtgggagca ggtgccccaa acccaggaga ggtgctcatg

4621 cgcaggatgg acaaaccagt ctccagcaag agcccgagga agtcgggtca taccccattt

4681 ttgcggagat ttaggataga gccctgtgaa acccctaagt ttaggataca ggtcaaactt

4741 aggaggtccc gaagagggca gagaaggtgg gagaaaagcc aggaggaagt gaggggagaa

4801 gttacgggga ccgaggagac gaagaggagg cactcccttc gtgctcctgc agacggccgg

4861 ggagggagac aggacgcagg agcccgggac tccccctccc ggcgggagga agcggtgctc

4921 ctccgcgcag gcctccccct ctcttccctc gccgctcccg ccccgtcccc tccccgactg

4981 gctgtgtccc ggaagtggtc ataagctggg tcccgatgcc gcgagcggaa tctcggcgct

5041 cccggaagtg gcctgaaggc ggcgcgccag tcccgagcag tgctcgctcc tgctcggggc

5101 gctgcggccc cgggcgtcgc catgaccagt gagctggaca tcttcgtggg gaacacgacc

5161 cttatcgacg aggacgtgta tcgcctctgg ctcgatggtt actcgggtcc gtgaggggcg

5221 tgtggggaag ccagtcggcg gagcggttgt gttgaatggg agagggggcg gggctaagac

5281 ttgggcatcc tttagggaag tgggcgagcg cctgggaata agaggtgtta taggggtctg

5341 ggatggaaac ccttaagggc ctctggcgtc ggcggggcgg ggcctgatgc tagcgtccgc

5401 tttccagtga ccgacgcggt ggccctgcgg gtgcgctcgg gaatcctgga gcagactggc

5461 gccacggcag cggtgctgca gagcgacacc atggaccatt accgcacctt ccacatgctc

5521 gagcggctgc tgcatgcgcc gcccaagcta ctgcaccagc tcatcttcca gattccgccc

5581 tcccggcagg cactactcat cgagaggtgc tcaccctctg ggatggtgga gacatcaacc

5641 agtctgactg tatacccatt ttgccggctg aaacaggccc agggagggat gggatttacc

5701 cagccatcag aaagttgaga gtggaagggg tcagacctag gccctgctag gccccgccag

5761 actccctttc ctgtctttta tggagggtca atggtgttct ctgactccac aaggtactat

5821 gcctttgatg aggcctttgt tcgggaggtg ctgggcaaga agctgtccaa aggcaccaag

5881 aaagacctgg atgacatcag caccaaaaca ggcatcaccc tcaagagctg ccggagacag

5941 gtatgcactg catccacacg gacacagcat cccatctaca cttcactccc tccctttggc

6001 cactggccca gttccagcct ctgattgtgg cagcagcttc atacctagtc ttctcctccc

6061 ttcccctttc ccctcctttt tttttttttt tttcgagatg gagtcttgct ctgtggccca

6121 agctggagtg cagtggcgcg acctcagctc actgcaacct ccgcctcctg ggttcaggta

6181 attcttgtgc ctcagcctct gcagtagctg agattacagg cgcccgctcc cacacccagc 6241 taatttttgt attgttagta gagacagggt ttcaccatgt tggccaggct tgtcttgaac 6301 tcctggcctc aagtgatcat ccgcctcggc ctcccaaagt gctgacatta caggcgtgag

6361 ccaccgtgcc tggctccctg ttgtgccttc caagctatcc tgtactgtat ggccagagtg

6421 acctttcaga atgacctgaa attttatcag acccctgata aactttcatt ggctcccact

6481 gcctcctggg tagcagctgc actccttggc tttgacctat ggactgtcct gttatctcat

6541 ccctgcagac ctctctaggc ttacatttct ccagccccag gctttccttt actcatgttg

6601 cttctacctg gagggtactg ttcttggcaa tatctacccc ttgaaatcca agacaagagg

6661 tgagatgcca ccttctccac aagccttttt ggtacgccag gaagaagtaa cccttctggg

6721 tctttgatgc ctttcttttg tatctcctgt gagctttctg tttatagctt agcatttgtt

6781 ttttattttt tagtttctag ggcttagcca ggaaagtctc ttccaactca aacttgtgaa

6841 gatctggaga gcatggtgct aaaatggggt tggatttaga gataagattg agtaaacatt

6901 tatccaaggc aagatacagc aggagttgct aagcatttga tttgataaca agtactaata

6961 actaatagtt ggttgaagga ataacaaatg ctaaatcagc atctgctgac tggataggtg

7021 gatggggaag ggagagatgt tcaataagtg gtttattggg tgagcgacac agtgtacact

7081 ctgaccagtc ctctcttcta ccctaactgc cctgcagttt gacaacttta aacgggtctt

7141 caaggtggta gaggaaatgc ggggctccct ggtggacaat attcagcaac acttcctcct

7201 ctctgaccgg ttggccaggt gaggagetag ccacctcccc atcccgcaca ctacttcctg

7261 ggcccggagg ctcgtccacc ttccctaccc caaaccttct gacctcctga gtatgctggg

7321 ggaatgatta tcccactttt ggtctgtact tttttttttt tttttttttt aatgagaegg

7381 agtttcactc ttgttgccca ggctggaatg caatggtgca atcttggctc actgcaacct

7441 ctgcctcctg gttcaagcaa ttctgcctca gcctcccgag tagctgggat tacaggcttg

7501 agccaccgca cccagcttgg tctgtacttt ggaccagact gacctggatt caaagcacag

7561 tccagcgatt tccagctggg ctgttgagag gtaccatatg taaagcccat ggcacagccc

7621 tggcacttag gaagtgetea gtagatagta actgttaccc ttatggtctc ccactgtatc

7681 cactagtaaa ettgagetgg aaccttctct tcgcacagat ggggaagggt catagagaag

7741 tgaettgaga cacgtcacac agtgacttga ggcagagaga ggccagaact cggacaccca

7801 gctgaggttg caggtgggag ctgcttccta agaaggaatt tcaagcccta agaggaccct

7861 cctctcgtct gtgcagggac tatgcagcca tegtettett tgctaacaac egetttgaga

7921 cagggaagaa aaaactgcag tatetgaget teggtgaett tgccttctgc getgagetea

7981 tgatccaaaa ctggaccctt ggageegteg gtgaggcccc cactgaccca ggtgcctgga

8041 cgcctcccca tcctcaggac ctgattcccc accagtagtt tctcccacag gccctcagtt

8101 cctggcatcc gctcctgttc atggccccca cccaggctcc ccctgcccat agaatgtttt

8161 ccccacacac accttgtctg cagacaaagg gcccattgcc tctctgtggc ctggggcatg

8221 tcactccatc tctctgacac tccatttcct cttttgtccc caggggggaa ataaggtggt

8281 gggtgaggaa aggettagge tatcacagga ggggttttta tcacccaccc ccaccccctt

8341 catggcctca cagactcaca gatggatgac atggacatgg acttagacaa ggaatttctc

8401 caggacttga aggagctcaa ggtgctagtg gctgacaagg accttctgga cctgcacaag

8461 aggtgacctt gtaggggccc atggggcggg geetgeagta ggggcccagg tggetatagg

8521 cgtgaccctg tctctctgca gcctggtgtg cactgctctc cggggaaagc tgggcgtctt

8581 ctctgagatg gaagccaact teaaggtetg tggtcccatc cagcctctga cccttggcat

8641 cttcagcctc cagttcgcca cccctaccgg gcagctctgc ccctgccagg gattggeegg

8701 caggtggcac tgctgctccc tctggtggcc getgaeggga gcagctcata aacaatgggg

8761 acttggttga tggagggagg tggatggatc tggggagggg cctgctcctc ctcccccagc 8821 ttctgtgctc agctgccaac ttctggggca gaaagcagag ctccaaggat ccctgcccca 8881 cagaacctgt cccgggggct ggtgaacgtg gccgccaagc tgacccacaa taaagatgtc 8941 agagacctgt ttgtggacct cgtggagaag gtgagctgtc gtgctctctg acacccaacc 9001 tggtctgtcc ctggcctata caagtgctaa gtattctctt gggcccgcag tttgtggaac 9061 cctgccgctc cgaccactgg ccactcagcg acgtgcggtt cttcctgaat cagtattcag 9121 cgtctgtcca ctccctcgat ggcttccggt gagcagccct gtcctcctgt ccagcttggc 9181 tttgccccag cctgccacct gccaggacct cgcctcctgc agactcaagg ttccccattc 9241 tgctaccctg ggggtgggac caaagtctct ggaccaaagc tctggctctc cctcttagaa 9301 gctgtgtgat ttggggcagg tttcttaacc tctccatgcc tcagtttcct catgtacaaa 9361 ctagggttca tcatgagaag aattgtaagg attttctaag ggcaaaatga atgaaggtgt 9421 agaaagcacg cagggcaggg cctccatcac ggtgagtgtt gtgaaaactc agctgtcact 9481 gttcatgttc ttacagacac caggccctct gggaccgcta catgggcacc ctccgcggct 9541 gcctcctgcg cctgtatcat gactgaggtg cctcccaacg ctccgcccac gctgacaata 9601 aagttgctct gagtttggag actggtcctc gctccgggga gcaagtgggg ggcgtgcaga 9661 tgtgcctgtg tctgtctctg agcacctggt gtccgtgtac aaggatggat gtgtacagtg 9721 gctccttggg aactgagaca tatctcaggg aatggtgtct gtgctcagcc catccaccag 9781 aagagtctgc tcacaagcca gaggtcttgt cttggtttat tcaggctgta ttgagattgg 9841 gaggatgggc aaaaacctgg gggtggggct ggcaaggagg cagttggcct aacaggacag 9901 agctgagggg gccaggtggg ttcagggagg gcaggagact cggggcttca tatccggttt 9961 ctgcacacgg gcagtgagcg ggaacttggt gatgccacag gtattgctcc ctcggtgcag 10021 ccggaaatag ccctaggagg ttaggggaca taggggcggg aaggtgggaa gaggcctgtt 10081 ctgccttgcc ccctccccca atagatcaca ctcaccttct ctccccattg ggccccccag 10141 gagttcttca ggatccagta tggggtgggg tgtggaggct gaggctgaga ctgcgatgag 10201 actgtctctg cccatatccc ctcctctgac ttgacgctgc caaaacccac cagcaggaca 10261 gagtggtcca caagctgggg gtcacaggtg gtgggtgtgg ccttgatcac acctttccgg 10321 tatagctgca gggggtgggg gccgaggggc tgagtgtggg gccaagtctc ctgtatgccc 10381 cttccccatc agctccccca tctcacctga aggggcttca tgttgatggt cacggtgatg 10441 gggccataag tggccaggta ctgcgcaatt cctggggaca ggaacatcat cagacccttg 10501 gcatgtgggt gtgtgggtct ctcgctcctt cgctcccccc accccctctt gtcttctagc 10561 ccagtagccc agcgtgtctg cctctgcccc ggtgtctgtc cctgccatcc gcccgtgtcc 10621 ctgccctgcc cgcactgtgc tcgttgttct gcagcatgat gaagtcctgg atccaggcca 10681 ccttctggta cttcttgggg tggcacctgt gggctctgac tttgccctgg aacgggtagt 10741 ccttttcact ggccaggccg cctgcagata agcaagacaa gggaggaggt tccgctcctg 10801 cccaccctgt ccccctgcag atgtccagag tggcggggca gtgcactcac tgttgttgag 10861 gacagttatg aacgcgtccc agacgaagcc accgtggcag ccatccccac agcggccaca 10921 gtccagcagt tctggtgcaa gaagggacag tgtgggtgac caggccccta ggcctccctc 10981 ccctgtcaca cgcgcaccct ctcccaaccc taccctgcac ggagacatcc acaaaatccc 11041 agaaactgat gcgccacagg gtctctatgt tgcctgccgc tgccatggcc cagcagcagt 11101 tgcagttttt ctggagggcg agagggtggg gcggggctag gtgttcgggc cgtgcccctc 11161 tccctcccac cacttagctg aattagagcc agctgggtag cggcagatac ctggtccttg 11221 atgggtgaga tggcgctggc caccttccgc cagtcacagc tgaaaggtac tgactcctct 11281 ggctcttcag accttatttc tctgcccatg ctggggaccc ctccagctgc cctccgatag 11341 ccatagagct ggccaaactc ctcctctggg gaacagacag ggccactgag gtctccattt

11401 tgaccccttc tccctgttct gcctctccca ctccagctgt tggtcccata attccctgag

11461 gggcaaaaag gcaaggatag gccgggcgtg gtggctcaca cctataatcc cagcactttg

11521 ggaggccaag gtgggtggat cacttgaggt caggagtccg agaccccatc tccacaaaaa

11581 gtagaaaaat tagctgggcg tggttgtgcg ggcttgtagc cccagctact tagaaggctg

11641 aggggtcggg ggaattgctt gagtctagga ggtggaggct gagtgagctg tgattgcact

11701 attgtactgc agcctggaca acagagcaag accctgtctc aaaaaggcaa gggtgccaag

11761 cctagagagg ctgggctccc tgctgggtgc ttccctgcag

SEQ ID NO: 64

Genomic sequence of Human TMEM222 extracted from NCBI Reference Sequence:

NC 000001 .1 1 :27322 l 63-27336400

.S///9/C//.S chromosome 1, GRCh38.pl3 Primary

Assembly agagccggggccagtcggagcggggcgcgcgccgcatggcggaagcggaagggagttctctgctcttgtt gccgccgccgccacccccgcccaggatggcggaagtggaggcgccgacggcggccgagacggacatgaag caatatcaaggctccggcggcgtcgccatggatgtggaacggagtcgcttcccctactgcgtggtgtgga cgcccatcccggtgctcacgtgagtctcttccggcatccgcctggggacgaggagggcccagggagagcc ggagtcgggccgggctagcctccggccccaacgcgcagcaagccttttcctcgggtctggcccgcacgcc tcgcccagtcaccagaaccccgccatccatgtagtgccagtttgtgcacacggctgggacacgctccctg ctctcaatacacctggtgtagccagggagagagttgtaggccagcaggaaagggcaagtggctgggagac tcgagctgtgagtgtgtagaagagggagccccttatcggatctgaggagacatttttattaattcactgg tttattctctcattgagcaaatgcttactggtcgcttaccatattccagacatactgctgggtctgggag caagagtgggagtttggttgggaccacacgagatcctgggatgtcaacattggaagggcccccagagatc tgagagtccaaccctttgtctcacgtgccatctcagttgcaaactgggtttagatcccaggttgtctgcc tcccagaccaaagttctttctgtaacatcagcctttgtcaggggagaagcaggtgctctggtactacttg gcgccttttgggaggagctctcatatggggagaggtggtttgccctcctgcttcaaccttgacagctgat aaacgtaacccctcctcaccgtgctggatagtttgccaagctcttaaacacccattttatcagaattaaa catccattttctaaggtaggttatcttctcagatggtgaagctgagctgcttgtagcattaagtgtcttg atcaggttcgcaccccaataagcaaaggcatgaagagctcagtcttctgatctaacttgctctttgcact acatcttatccactctcacctgatgggatagcttgttccgctatcaataacagccagtggtgatgtggag aagggatcatttggggccttaggtgttgctgattctaaaaagatcgtagtttttgaattatgttaaaata cttgaattattttgtgttttcaagtacttatgcatataagagcatccaaatctgttctttaaaaataaaa tattgggacaggcgcggtggctcatgcctgtaatcccagcactctgggaggcagtggagggtggatcggt aggtagttcgagaccaacctggccatcgtggcgaaacccgtctctactaaaaatacaaaaattagctggg catagtggcacgtgcctgtaatcccagctactctggaagctgaggcaggagaatcgcctcaacccgggaa gtggagtttgcagtgagccgagattgcaccactgcacttcagcatgggcaacacagtgaaactccgtctc aaaataaatagtgaaataaaatcttaggaaaatgtgcacattaattcattcagtgttcttctaacacatg tgctaagtgacccctgtgttcccagatagttctaggtgatgcgaatatacagtgatgaataaaggagata tggtctttagacttttgagagtttaagtcatagtggaaggagatagacagtaaacaagtaagctaataaa agagcacctgatagtgatgagtgctgtgaagaaaataaaatattagtgtgaaaataacaggggccgggcg tggtggctcaggcctgtaatcccagcactttgggaggcccaggcgggcgaatcacctgaggtcaggagtt caagacaagcctgaccaacatggagaaaccccatctctactaaaaatacaaaattagccggggtggtggc acatgcctgtaatcccagctactcaggaggccgaggcaggagaatcacttgaacccgggaggtggaggtt gcggtgagccaagatcgtgcttttgcactccagcctggacaacaagagcaaaactccgtttcaaaaaaaa agaaaataactgggagggatactcaggaaatgtattggttttcttaaaacctgtcttgtgatcctaaact ctctttagcctttcttattctcaaaaacaccatctgtggcgtggtcctctgcggagaatggtagcagcgt gaggtggaggcaccacaatcccttgtcacctgggccaaaagcagctcatagcccatttctattcagatct gtgtacaggtgttcataagaccaagatgaataagacactgtgttcctgtcaaagggttcatgttcccttc tgtgttagtccattcttgcactaatacttgagactggataatttctaaagaaaagaggtttaattgaccc atgcttctgcaggctatacaggaagcatagtggcttctgcttctggggagacctcaggaagcttccagtt atggtgtaaggcaaagggggagggaggcatctcacatggcgggattaggagcaagagggagtgagggggg agatgctacacacttttaaacaaccagatcttgggaaaactcactatcacgagaacagcaccaaagggat ggtgctgaaccatttatgagaaaaccagcctcatgatcccaccaccagcccccacctccaatatggggac tacagtttgacaggagatttggcggggacagatccaaaccatgtcaccttcaaaggactcacctccagca ggagggagagaaaggtcaattagccatcgtagtattactgtatagtgatgattgcagagctaggcataga tgtggcgcaagtaactgtcacctgtctttggaggagagatcagaggaggaaatggaacccaaagatctgt ctaccccctcagagatgttggtaattctgcatgaaaaaccagtttagtgctcgcctcagcagcacatata ctaaaattggaacgatacagagaagattagcatggcccctgcgcaaggatgacatgcagattcgtgaagc gttccatatttttgtctccctggacttcgagcaggagatggccactaccacatcctcctccctggagaag agctacaagctgccggatggccaggtcatcaccatcagcaacaagcggttccagtgtccggaggcgctgt tccagccttccttcctgggtatggaatcttgcggcatccacgagaccacgttcaactccatcatgaagtg tgacgtagacatccgcaaagacctgtacaccaacatagggctatccagaggcaccaccatgtacccgggc atcaccgacaggatgcagaaggagattaacgccctggcatccagcaccatgaagatcaagctcattgtgc ccccagagtgcaagtactctgtgtggatcagcggctccatcctggcctcactgtccaccttccagcagat gtggattagcaagcaggagtatgacgagtcaggcccctgtatcgtccaccgcaaatgcttctaaatggac tgcgagccgatgcgtagcatttgctgcatgggttaattcagaagtataaattggccctggcaaatgcata tacctcatgctagcctcacgaaaatggaataagccttcgaaaagaaattgtcgttgaagcttgtatctaa tatcagcactggattgtagaacttgttgctgattttgaccttgtattcaagttaactgttccccttggta tttgtttaataccctgtacatatctttgctttcaacccttagtacatgtggcttggtcactgcgtggcaa ggtaagaatgtgcttgtggaagacaagtgcgacttggtgagtctgcatggccagcagtctccgatctttg cagggtattaatatgtcatggctgagtgttctgggatttctctagaggctgacaagggctcctgaaccag ttgtttctgtcctgctggtctgtcagggttggaaaggccaagccataggacccagtttcctttcttagct gatgttttcctgccagaacactgtgggctgtgacttgctttgagttggaagcagtttgcatttacacctg taaatgtattcatccttttaatttatgtaaggtttttttttgtatgcaattctcgattctttaaggagat gagaacagattttggttttctactgttatgtgagaacattaggccccagcggcatgtcattgtgtaagga aaaataaaagtgctgctgtaaccaaaaaaaagggaaaaaaaagaaactagtttaatagctgctgctacta ataaaaacagctaagattagatgcttactctgtcctaaacattgtatgttcactcactcaatcctaatgt caacctatgaaagtagatactgttgtcaccattttatagatgaggaagtaaggcttagaagccagtgtgg tgctcttttgctgagttagcctaccttcccagattgagctccaggatgagtgagtcatttattcatttat tgtatctttataaagcacctattatgtactaggtcctgaatagaaggtagtccacaaagcggctctggag ctgccttcctggagcttccaggctgacaaaccactttgagggtttcttctctggggcttccagcctttac tctgatttccatccactgctgccagcacagctgtggcgtgtggaatcctggttggacccagacttcctcc caagcagatgaaaggattagggggcagagatgggggtggagggcagtcctgagatcaggatgagctactt tcccataatcctgaatgtccatcctgtggcctgtggatcttctgcaacttattctatgttagtgcagtca gtcagatagtgcttatgtattgaacacctcgagatgacaacattgagcagctgccaagcggaaggcagag tcctaggcactgtgtaaatggataagtctatccatttaatcctcacaatagctcctcattaaggtaattt ttactatccacagagagatgggaattagtaacttgcccagaacaaaggcaagcctcaacccagatgatat ctgagcatgacttcactgttggcgtttgtttgttgtttttaatttttatttatttatttatttagagaca gagtcttgctctgtcacccaggctggaatgcagtggcgtgatctcagctcactgcatcctccgcctccgg ggttcaagtgattctcgtgcctcagcctctcgagtagctgggattacaggcacatgccaccatgcccatc taatttttgtatttttagtatagacagggtttcgccatgtttcccaggctggtctcgaactcctgacctc aagtcatccaccggcctctgcctcccaaagtgctgggattacaggcgtgagccactgagcccagccttga ctcactgcagcctcaacttcctgggcccagccttggctcactacagcctcaacttccttggatcaagtga tcctccagctaatttttagatttttttagatacagggtctcactgtgttgcccaggctggtctcaaattc ctgggctcaagcaatccaactgccttggccttccaaagtgctatgattacaggcgtgagccacagcaccc agccccagactctactcttaaatgcaggatgttcattcattatttagtcaacaaatatttagtgagttgc agctatatacaggcactgccgtagtaaacaagaaaaaatcctgtctctcttggagcttatgttctagtgg agagacagcatataaataaccaagataattttggatggtagtagatactatgagggaagtaaagcattgt agtgggatctgggattgggtggctgtgctggaagacaccgttcaggcaagacctccgaaggtgacagtga ggctgaggcctaatcagtgaggagtcagctgtgccatgctctctgggaagagtgttccagacggaggaga gggcagatacagagaaagcatggggtgggaggatttggcgtgtttgaggccacaagcaggcagtgtagct agagaggggactgagatgaagttcaagggataattagcaccacagtgttggaagaagttcgaagtgtatt ataagtgtagtaggaagccattaataggtgttgaacaggggaatgatggaatctgagttcaattctcaaa agaccactctggccctaccattgtctcaagcgctcaagctgtagacctggaagacccccatcctgtcagt cgtaaactctccagtcttcatttgaaagatcttttgaatccatgtgtttctcaccaccctgcccctgatc ctctagtcaggacctccatcatctcttgccagtggtacagcagtagcccctgagctggcctctccccctt tcgttcttccaggcacatgtaccactttcttgctctgcagtgttgttttctgcctgttgatgttgtgtcc tttcacaggtcaaggttaaaagtcacctcctctgtgaagtctttactggctccaccccagccccctgtaa ctgcttttttaaactgcgtctctggaggcttggcctgcacccctagccaggtgctttatgctcgtgccca ggcagcattgaggccaggactcggccactggccctgtgagttctccttaaactggatcagtctctccagc acccaggtcctgtagggccatggttctcagaacacagacaggtctggggttcctatgtgtgacatattgg ggtgcatctgagatttattgttgttttttaatgcccatggtttataaaaaaggaacttttatttcctggc atacaccatgcttctgttccaatttctacccttgcacattctgtttctttgttatttcctcagggaagcc tttcctgaactcccccacccatactggtgtcatgaccgcccacctttttttttttttttaactttcttat tgaggtataacttatgtaccaaacggtgtgcgaatctcaagtatacagcaggaagaatttttgcatatca cacactcatgtaaccgccacccagacgggggctagaaagcaatgctaagacccagaggacccccttgtgc cctcccagtcagttctaccacagattccaccccgctgtggacctctagccatagatcagtcttgcctttt cttggacttcacgtaaacagagtgctatgttacgtactctgtgtaagatttccttcattcacccttatgt ctgtgaaagcctgtgttgtgtgtagcagaagtgacactgtttttcattgttatggactatttcaatgtat aactataccacacttattcatcgtactgttgttgaacatttggattctttctagttgtttttatcaattt tattgaattatagtttacatataataaaatgtacacattttaagagtacagttcaatgagttttggcaaa tgttatttcccaggggtgagggcaaagaaggttccaaaagtgaaactgctgaggcatttagtatagtgcc aaactgttttccagaatggtagaaacaggccgggtacggtggctcacgcctgtaattccagcactttggg aggccgaggcaggcagatgacttgaggtcaggagttcgagaccagcctggccaacgtgatgaaacctcat gtctactaaaaatacaaaaattagccaggcatggtggtgcactcctgtagtcccagctactcgggaggct gaggcaggagaattgcttaaacccaggaggcggaggttgcagtgagctgagattgtgccaccgcacttca gccagggcaagagagcgagactccatctcaaaaacaaaatggtaggccgggtgcagtggcttatgcctgt aatcccagcactttgggaggccaaggcacgtggatcacgaggtcaagagatcgagaccatcctggccaac atggtgaaaccccatctctactaaaaatacaaaaattacccaggcgtggtggtgcacacctgtagtccca gctactcaggaggctgaggcaggagaattgcttgaactcgggaggcggaggttgcagtgagccaatattg tgccactgcactccagcctgggcgacagagcgagactccatttgaaaaaaaaaaaaaggtagaaacagtt tatccctcccaccagcagtgaatgagagctctggttgtcccttgccctcacctgcattggtgttgctgtc gtttgagttacagccatcctggcgggatgctgaggtatcccattgtgctgtcggtttgcagttacccttg tctgtgcttccccagccttctggaccaccctaatggccacctgtcaatgaaacgtaatattcacatgctt ctgtactgtatgaagggtccccagcgtccgtctaactggctgaaagatcccctaagctctgtcccttccc cgtccttttcctcacaggtggtttttccccatcatcggccacatgggcatctgcacatccacaggagtca ttcgggacttcgcgggcccctactttgtctcagtgagtccccattctgcccacccggggggttccaaggt ttaagtggaaggctgtcctgtcttgcctgcaggcaggcttcgtctccccagacccaggagaacgtagata acccgcagtcctggccctggccctctgcccctcaccagtgtttgacccctttcccccttctcttgcctcc aggacaggccgggagggcagtgtggccagaaggattcttaagtaactgacccagccctttgcccccaccc ctggggtaccgagacatgggtagggattagaggcaagagtggagagtcagaccatccaggaaccacatct ctggaccttcagaaggtgggagtcaaggtggggggacacgggaggctgattcggttccctgggtgtggcc atggagaagagggggtggggctttagaatgaagcagctgtgttctcaggtctgttccctggggctgtgac caagactggtccagtgggctagcagcagaggagcatgcatcatcctgtctccaggtgtcagaggctggac tgaaggggtgtggtggcaccaacctgtttatattaatagttgtcatgatcatcttaacacgacaaaggtc tgtcactttgtacttaaaaatactttcacatccattactttatcatatatttttatggtgattaatcatt gaccaggtctgtcagcagggtctccctgatctgaacccagagtctgtctctgtgtaagtcctgtacccca ggaataatgtgatgagtctaggacttcactgcagtgtctccctcacaggaagtccctctgggcccagcgc ccccgccctgctatgaatgcagtcagactgctgggcgaggagatggtctccatgtgggccaccactgttg agtcaggaggccagaccatgatggaaacctcctgctggtcctcagagatcagagaagactgaccagagaa cttggagaaaatgaggcccagagagggcaagagaattcctgggtgccacaccaagggacagtggcagctg gagccccagatctttggtctctgatgcagtgctctgtgcacttgtgccacactgccttcttcctgcagaa cagctgctagctctttagatgccatgccagccccagggcagggcccaggccccacttctggcccccccac ccctgacataccctcttctaagtcactgcacttctgccacctacccaggtttgtcatctggcccaaatgt aagttcctcactgacctctgcttttgtccctcaacaggaggacaacatggcctttggaaagcctgccaag taagtgatgaacacccatgtgactggctctagaggcaggtcgctcctgccggggcgggccaggccttctg gggcacaggaggggccagcaccctgagaatagagtatttggggtcgggggagaggtcagccagggtccac cagagcatggcaaccccatacagagtgtgtaccgcaccagcccctggctagggcctttacctgtagacca tctgggccttgccacagctcttcaggtttgtattttcaactccatggcaagggtgaggaaagggaaggga cttggtcaaggtcacacaggaagtggcagagctgggacccacacccagatctgtctccctctagactcac tctcctgccctttgggaacaaatgaggcatggaaggtagaagagaggcattgtttggagctctgctggaa agttctggttggagagaataaaaaccgttcaaccttctgggagctattgctggtttggtttgggacattt ggtcttcatctttgcagtctcgggtgcccacctcagctgtgggcctggtgagagtgcctcagtcatcagt gtcctcaggtgacctgttgcccaaggctgcactgggaggagagactgggccgaggaggagttggtgtccc acacagctgagatggcctggagcagggcttcctgctgccctctctggcttcctccggcaggcagcagtgt agtccaggagtctctgggccaccaggtgttcgctgccagactgctcttcaaggacagttttaagggcatc attttccaagcagtagcccctaagcggccccagtccaggccatggtctctagactcctccaccaagccat tcccctacacaacagccagggggcgccctgacctcccagctctccttggcctgagacccaccgggcactc tggtgcttggaacagcaattctcacccaccttgaggtttatgggctttagcaccatcagcttccctgcca ctcaccctggcaagctgcctgggagactaggggagagtgcttgctgctgggtaaactccccgcgtgatgt ggcctcacctgcatctccagccttagctgccagcattccatcaccgtgtttctctttctgcatcctccag gagggctcagtcacttcagttatgggacatgctgcacagttttatgcctgtcacttagcttaagctgttc cctcagcctggaatgcccacctcttctttctatgcctgcctaaccctcttccttcatactggacccaggt gtcacctccaggaagccttctcacaccccatcttagtccgttctggctgccataacaaaatctcatcaat tgggtatcttagaaacaacagaaatgtatttctcacagttccgaagactggacagtcctgggtgcgggtg ctggtagagtcagtgtctggtgagggcctgaggtgcctttccactgtgtccccacgtggtggaggggtga ggggtctccctcagggctcttttataaggacacggatcccattcatgagagctaatcaccccatggccta atcacctcccaaaggccccacctcctcataccatcaccttgagggttaagatttcaacatatgaacttgg ggacacagactttcagagcatagcacccccaatttcattccatatccccccaggatcccccatggcacca gccacctcaccctgtgtcacagttgactgccacataacacttgccccagatctggcttactgtacatctc agcacccagctcaggcccgggcacagggcaggcctcagaggacgtgcgtagagctgagggcacaaaggag ccaagcaagtgtccagagcccttctctccccccaggtactggaagttggaccctgctcaggtctatgcta gcgggcccaacgcatgggacacggctgtgcacgacgcctctgaggagtacaagcaccgcatggtaggtgg gccagggcggcaccggcactccccaggtggggaccaggggggaggctcccctgcagcccatcctgaccag cccttctggtgcctccagcacaatctctgctgtgacaactgccactcgcacgtggcattggccctgaatc tgatgcgctacaacaacagcaccaactggaatatggtgacgctctgcttcttctgcctgctctacgggaa gtacgtcaggtgagctgccctcctgcctgcccacccacacactgcccagaggctgctctcccaaggatcc tagaaagaccattcctagcgtccttcagtcagccaggacagttggaacagatgccagttggagccgtggt ggttctagagtgacgagctgagggcctgatggagagggaggtggctgtgctgaagtccagctccacctgg gccccaaatgcagctctggctcttgcctgctgtgagaccatgggcaactgccttggtctctcttagccac agctgtctctggtgaaatgcaggcaatgattccggctcctcagggaggcttctacggatccctgtgctcc ggtaaagtgagcagatagatcctctcataatgactgccccacaggtcccttcctaggcagagagattgag tgagtcgcccatggtcgccacagcatggacacagcagagcccagactcgtgagccagcataggttagtgc tgatctggccgcctctccaagctttcctcttagccacgttcatcattaagaggttctgaaaagaagactt ttagggatatttttaagtactgcatggcttttagaagtcccctgagaataactggtcacacagaggttga acccagacccctggctcccagctgcttggctccagccacaacactgctgacccttgccagccccagagct cccagtagtccccattatgaagtccataggcagagggcacacgtggcttccatacccaaactgcaattcc tggtcctcttgctggagaccctagaggtaggtggctatagatggagccagtgaggcctagaaatggggag catgtttcctgttcccaaccagcgaggtgtcagtaaatcaccaggagcgcggagcagcccctgcccggga ccatagctggtgatcccccatagtgggcaaccatgcccagcctgcttccttgattggccaagtttggggt gagggggtggttgggttttccttagggatggcagccctattggggtctgtgggtgctcagggctgcgggc cgcatgcctgctcaccaggccctgcccacctatgctcgctcctttctctctgtcagcgttggggccttcg tgaagacctggctgcccttcatccttctcctgggcatcatcctcaccgtcagcctggtctttaacctccg gtgatggctgctcggtggccccacacccaccagggtcccgaggaaacagccgccatcccttttggttcca gatttttttctcctcaccccaaaaggcagggttgggcctgctgttgtggaccgggggtcggggctggcag gatggaaggactgaggaccagcatgaagtgggggtttgttgtctccctgcctctcagaagcaccctgtcc cctcctccccaggcctgtgactccggccctggaagcccctttgttcttctgttgaaaggctttggcttcc cgctgtagagctgctcccgccaccacctgctggggtcctgcctcagcccagtgcccagtatggggagagg aggacatttgggctcacctgtcaaggtggccctgggaccagagctggtcccagcatggggtgcaccgggt acacttaacgtgtctctataagccaagttgcttcaggaccttcaccactggcctctagaatggtccagag gggctggctgggtccctttgtcagactcctgccggcagctgccctgggggacatgtgtgcccatctggca tcctccagcccgtgcagtccgctcttcactgttccacggcctcccagtgcctcccagcattggacccatc tccccctgcagtttgaggccagagaggtgagtggacctgacaagtgccagagtaaccgtgtagacagagc agtgtagacagcactcagccccagccccaggtgtggacctcatgctggtgatggctcccctgggtggcct gccagcacagccagtgccatcagggagctgaaggggctgtcccccacctaactccagctcccccttcacg ttgtcaccaaggccctgtgccgcccgcctcgcccccctgctctgtggattcctttgggaagggctccctg ggcaggacaataaagagttttgactcca

SEQ ID NO: 65

Sequence of Homo sapiens FGF1 intracellular binding protein (FIBP), transcript variant 1, mRNA, Reproduced from NCBI Reference Sequence: NM_198897.2

1 agtcccgagc agtgctcgct cctgctcggg gcgctgcggc cccgggcgtc gccatgacca

61 gtgagctgga catcttcgtg gggaacacga cccttatcga cgaggacgtg tatcgcctct

121 ggctcgatgg ttactcggtg accgacgcgg tggccctgcg ggtgcgctcg ggaatcctgg

181 agcagactgg cgccacggca gcggtgctgc agagcgacac catggaccat taccgcacct

241 tccacatgct cgagcggctg ctgcatgcgc cgcccaagct actgcaccag ctcatcttcc

301 agattccgcc ctcccggcag gcactactca tcgagaggta ctatgccttt gatgaggcct

361 ttgttcggga ggtgctgggc aagaagctgt ccaaaggcac caagaaagac ctggatgaca

421 tcagcaccaa aacaggcatc accctcaaga gctgccggag acagtttgac aactttaaac

481 gggtcttcaa ggtggtagag gaaatgcggg gctccctggt ggacaatatt cagcaacact

541 tcctcctctc tgaccggttg gccagggact atgcagccat cgtcttcttt gctaacaacc

601 gctttgagac agggaagaaa aaactgcagt atctgagctt cggtgacttt gccttctgcg

661 ctgagctcat gatccaaaac tggacccttg gagccgtcgg tgaggccccc actgacccag

721 actcacagat ggatgacatg gacatggact tagacaagga atttctccag gacttgaagg

781 agctcaaggt gctagtggct gacaaggacc ttctggacct gcacaagagc ctggtgtgca

841 ctgctctccg gggaaagctg ggcgtcttct ctgagatgga agccaacttc aagaacctgt

901 cccgggggct ggtgaacgtg gccgccaagc tgacccacaa taaagatgtc agagacctgt

961 ttgtggacct cgtggagaag tttgtggaac cctgccgctc cgaccactgg ccactcagcg

1021 acgtgcggtt cttcctgaat cagtattcag cgtctgtcca ctccctcgat ggcttccgac

1081 accaggccct ctgggaccgc tacatgggca ccctccgcgg ctgcctcctg cgcctgtatc

1141 atgactgagg tgcctcccaa cgctccgccc acgctgacaa taaagttgct ctgagtttgg

1201 agactggtcc tcgctccggg gagcaagtgg ggggcgtgca gatgtgcctg tgtctgtctc

1261 tgagcacctg gtgtccgtgt acaaggatgg atgtgtacag tggctccttg ggaactgaga

1321 catatctcag ggaatggtgt ctgtgctcag cccatccacc agaagagtct gctcacaagc

1381 ca SEQ ID NO: 66

Sequence of Homo sapiens FGF1 intracellular binding protein (FIBP), transcript variant 2, mRNA, Reproduced from NCBI Reference Sequence: NM_004214.5

1 agtcccgagc agtgctcgct cctgctcggg gcgctgcggc cccgggcgtc gccatgacca

61 gtgagctgga catcttcgtg gggaacacga cccttatcga cgaggacgtg tatcgcctct

121 ggctcgatgg ttactcggtg accgacgcgg tggccctgcg ggtgcgctcg ggaatcctgg

181 agcagactgg cgccacggca gcggtgctgc agagcgacac catggaccat taccgcacct

241 tccacatgct cgagcggctg ctgcatgcgc cgcccaagct actgcaccag ctcatcttcc

301 agattccgcc ctcccggcag gcactactca tcgagaggta ctatgccttt gatgaggcct

361 ttgttcggga ggtgctgggc aagaagctgt ccaaaggcac caagaaagac ctggatgaca

421 tcagcaccaa aacaggcatc accctcaaga gctgccggag acagtttgac aactttaaac

481 gggtcttcaa ggtggtagag gaaatgcggg gctccctggt ggacaatatt cagcaacact

541 tcctcctctc tgaccggttg gccagggact atgcagccat cgtcttcttt gctaacaacc

601 gctttgagac agggaagaaa aaactgcagt atctgagctt cggtgacttt gccttctgcg

661 ctgagctcat gatccaaaac tggacccttg gagccgtcga ctcacagatg gatgacatgg

721 acatggactt agacaaggaa tttctccagg acttgaagga gctcaaggtg ctagtggctg

781 acaaggacct tctggacctg cacaagagcc tggtgtgcac tgctctccgg ggaaagctgg

841 gcgtcttctc tgagatggaa gccaacttca agaacctgtc ccgggggctg gtgaacgtgg

901 ccgccaagct gacccacaat aaagatgtca gagacctgtt tgtggacctc gtggagaagt

961 ttgtggaacc ctgccgctcc gaccactggc cactcagcga cgtgcggttc ttcctgaatc

1021 agtattcagc gtctgtccac tccctcgatg gcttccgaca ccaggccctc tgggaccgct

1081 acatgggcac cctccgcggc tgcctcctgc gcctgtatca tgactgaggt gcctcccaac

1141 gctccgccca cgctgacaat aaagttgctc tgagtttgga gactggtcct cgctccgggg

1201 agcaagtggg gggcgtgcag atgtgcctgt gtctgtctct gagcacctgg tgtccgtgta

1261 caaggatgga tgtgtacagt ggctccttgg gaactgagac atatctcagg gaatggtgtc

1321 tgtgctcagc ccatccacca gaagagtctg ctcacaagcc a

SEQ ID NO: 67

Sequence of Homo sapiens transmembrane protein 222 (TMEM222), transcript variant 1, mRNA, Reproduced from NCBI Reference Sequence: NM_032125.3

1 agagccgggg ccagtcggag cggggcgcgc gccgcatggc ggaagcggaa gggagttctc

61 tgctcttgtt gccgccgccg ccacccccgc ccaggatggc ggaagtggag gcgccgacgg

121 cggccgagac ggacatgaag caatatcaag gctccggcgg cgtcgccatg gatgtggaac

181 ggagtcgctt cccctactgc gtggtgtgga cgcccatccc ggtgctcacg tggtttttcc

241 ccatcatcgg ccacatgggc atctgcacat ccacaggagt cattcgggac ttcgcgggcc

301 cctactttgt ctcagaggac aacatggcct ttggaaagcc tgccaagtac tggaagttgg

361 accctgctca ggtctatgct agcgggccca acgcatggga cacggctgtg cacgacgcct

421 ctgaggagta caagcaccgc atgcacaatc tctgctgtga caactgccac tcgcacgtgg

481 cattggccct gaatctgatg cgctacaaca acagcaccaa ctggaatatg gtgacgctct

541 gcttcttctg cctgctctac gggaagtacg tcagcgttgg ggccttcgtg aagacctggc

601 tgcccttcat ccttctcctg ggcatcatcc tcaccgtcag cctggtcttt aacctccggt

661 gatggctgct cggtggcccc acacccacca gggtcccgag gaaacagccg ccatcccttt

721 tggttccaga tttttttctc ctcaccccaa aaggcagggt tgggcctgct gttgtggacc

781 gggggtcggg gctggcagga tggaaggact gaggaccagc atgaagtggg ggtttgttgt

841 ctccctgcct ctcagaagca ccctgtcccc tcctccccag gcctgtgact ccggccctgg

901 aagccccttt gttcttctgt tgaaaggctt tggcttcccg ctgtagagct gctcccgcca

961 ccacctgctg gggtcctgcc tcagcccagt gcccagtatg gggagaggag gacatttggg

1021 ctcacctgtc aaggtggccc tgggaccaga gctggtccca gcatggggtg caccgggtac

1081 acttaacgtg tctctataag ccaagttgct tcaggacctt caccactggc ctctagaatg

1141 gtccagaggg gctggctggg tccctttgtc agactcctgc cggcagctgc cctgggggac

1201 atgtgtgccc atctggcatc ctccagcccg tgcagtccgc tcttcactgt tccacggcct

1261 cccagtgcct cccagcattg gacccatctc cccctgcagt ttgaggccag agaggtgagt

1321 ggacctgaca agtgccagag taaccgtgta gacagagcag tgtagacagc actcagcccc

1381 agccccaggt gtggacctca tgctggtgat ggctcccctg ggtggcctgc cagcacagcc

1441 agtgccatca gggagctgaa ggggctgtcc cccacctaac tccagctccc ccttcacgtt

1501 gtcaccaagg ccctgtgccg cccgcctcgc ccccctgctc tgtggattcc tttgggaagg

1561 gctccctggg caggacaata aagagttttg actcca

SEQ ID NO: 68

Genomic Sequence of Mus musculus FIBP, extracted from NCBI Reference Sequence:

>NC_000085.7:5510626-5515080 Mus musculus strain C57BL/6J chromosome 19, GRCm39 aaaatctccgaactcccggaagtcgctctgcggctactcgcccggccggggcccgtacaagctccagctt cgggcgaacccggcccgggcatcgtcatgaccagcgaactagacattttcgtggggaacacgacccttat agatgaagacgtgtatcgcctctggctggatggttactcaggtctgtgagccgccctcccgggaagctgt tgttggggatcgggaagagggggcgtggctaggatttaggcttggtttagggaagtggacagactgcggg aagtcggggaagtgtaaggattggaggtggaggtggaacccctggagggcctctagggtcttatggtagc gcccactcctcagtgaacgatgcagtggctctgcgagtacgctccggaatcttggagcagacgggagcca ccacaggagtgctgcagagcgacaccatggaccactaccgcacctttcacatgcttgagcgtctgctgca cgcgccgccgaagctgctgcaccagctcatcttccagattcctccctcccgacagacactcctcatcgag aggtgctcaccaggggtgggggacgggggtgggtactggcagggatgtcagtcaatctgacttataccca ttttgtagactgaagctgagtctggggggagggtattgggatatacacaggggtagatggggtgaactca ggaaattagaaaattgagaacaagagaaaccaattccaaactcccttctccttccctaatggatcaccaa taatgacctgtgcctccacaaggtactacacctttgatgaggcctttgttcgggaggtcttgggcaagaa gctgtccaagggtaccaagaaagacctggatgacatcagcaccaaaacaggaattactctcaagagctgc cggaggcaggtgggttctacacataacaccactacctgtctagcccacctctggatccttcttactactc tagccccacctctagcccgtggtctagcccatgattgtggaaccagttccttcttcatataaaccctctc ctccctccacctcccctctggccaggcggaccaggcttaggctcagtctgaccgtggctttcctttgctt atagattttcattggctcccactgtcctcgaccttcagactcaggttgggtcactatagacctctctgga cttaacatttcttgagccccaagtttgtttttgttcatggtactcctgcctagaagatgctgttcttggc cacatctatcccttgaaatcgacaagggagctagatcctaccttatccatgagccgttttggtagcattc atgccttgtctttaagggagaagtaccagggtgtaaactgggcgcagatggctatgggtatgggtctggg ctcttgctacaacctttataggccttgagtgcctttcctttgtgtctagaatgagttcgtgtgtgtatgc tgcttggttcactccaagttactcaggagaatccgttctaattcaggctcttgaagagccagagattgac ggtcgtgatgctaaattgaacttaaggacaggaataagtaagcattcttttaaggaaaaatgccaaaggc tggcaaacattgtcaaatgatggtagttattaatacctagtcatgccaggggtgctggcgcatgctttta atcccagcactcaggaggcagaggcaggtggatttctgagttcgaggccagcctggtctacagagtgagt tccaggacagccagggctacacagagaaaccctgtctcgaaacaaacaacaaaaacctagtcatttgttg aagtgttagtgctaaattagtacctgcccaccagataggaggacaggagggagagatcccatagcagctc gctcatgaacgtctcctgaccagtgttctgtaattgccggacagtttgacaactttaaacgagtcttcaa ggtggtggaagaaatgcggggctccctggtggacaacatccagcagcacttcctcctctctgaccggtta gccaggtgagggtcgccgcctcctctctgaccggttagccaggtgagggctgccacctcctctctgaccg gttagccaggtgagggccgccgcctcccttcctactcgaggtttcctgggcccttagcctgtacgccttc cctctcctaaccctttcccctccgctcttctggaggaatgattattctgttagtgacctgtaaactgcat caaattgccaagcgtaaggcatttgtaaagcattgaagaagtgcccagtgatgcagtgtggccttccgct gtactccttaggaactcctcgtagaggtgggaggttggtaaggaaatggtccgtggttggtcccttggtg agttagagcagaaacacaccagagctaaaatgttgaaccaagactgcaggtgggagctacctgctaagag ggatttaaaacaataaagagtttcttcaagcatggttctgtgtaagcttactcatcagacactgctgtgt gaagcagaaaaagggatttctggtcctaagagagccactgtccctgtctgcagggattacgcagccatcg tcttttttgccaacaaccgctttgaaacaggaaagaaaaagctgcagtacctgagctttggggactttgc cttctgtgcagagcttatgatccagaactggacccttggagccgtcggtgaggcccccactgacccaggt gcctggactccctacccccttggggactggttccctgctgtcagtttgtcctagcgtatagatagcttcc acagcgccctcagccccagaccacctctacggatagtcccctggctccacgcacacctgcctgcaggcca agggttcaccactgcgtggcctggggcatgacattcctctttccatctctgagactccagtgaggtagtg gtgaagagggttcacctcccaagatggtctttcatgcacccacaacgtctttcatggccttcgcagactc tcaggtggacgacatggatgtggacttagataaggagtttctccaagacttgaaggagctcaaggttctc gtggctgacaaggacctcctggacctgcataagaggtgactgtggggtccgtgagaccagggataaaata ctcttagtcggctagagacataactgcatcttcctacagcctggtgtgcactgccctccggggaaagctg ggtgtcttctctgagatggaaaccaacttcaaggtccgttgccctgtgcagacccttccccctaaagtgt caagagcctcctgtgcgctacccctgccaggcagctctgctcattcctgaaggtgaccagcaggtggcac tgctgctctccctgtctgcccttgagggggtcccaagctcataacaaaagggacttgattgtctgaggga ggtggatgaacttggggaggggcctggccctccttccgcctcagtgcgcagcctccgcatctggagcaga agacagaactcccgagatccttgccccacagaatctgtctcgggggctggtgaacgtggctgccaagctg acccacaataaggatgtcagagacctatttgtggacctcgtggagaaggtgagcagtcctgtccccaccc cctggtccccagcctgtcctgtccccacccctggtccccagcctgtcctgtgtctaagcagtcctagtgg tgccacctgttcccttggacctacagtttgtggaaccctgccgctctgaccactggccactgagtgatgt gcggctcttcctcagccagtattcagcgtcagtccactccctggatggcttccggtgagaggtgccaggc ctgctggctaacttccttgccctccaggagactcaggcttctctgttgaactgccctgtgggtggaagct ccgcctctccttgttgtgaccttggccaagttcttaacccttctgtgccttagctctcttaaatgtaaac tgagactaaagctagaagactcataaggtttatggaaactaaatagtcctcagatcggccctcagcactg ggtggagagcagggagaaggaagccgggcagaatggctcaagtttgtattcccagcacatcggaagctgg gacatgaggattgccgctgttttccaggccagcttgagccacactgtgagaccctgtctcaaaaacaaac tatccaaaaaaaaaaaaaaaaaggaggggctgtggtgaggaagaaagtgtagaaaacactgagcggtggt gtggcaggcatggcggtgagtggacatcagctctcaccgttatcttcttcattgcaggcaccaggcactc tgggaccgctacatgggcaccctccgtggctgccttctgcgcctctatcatgattaaagcccttttcctt cctccctccgacataccctgagaataaagttgccataagtttgga

SEQ ID NO: 69

Sequence of Mus musculus fibroblast growth factor (acidic) intracellular binding protein (FIBP), transcript variant 1, mRNA, Reproduced from NCBI Reference Sequence: NM_001253832.1

1 gaactcccgg aagtcgctct gcggctactc gcccggccgg ggcccgtaca agctccagct

61 tcgggcgaac ccggcccggg catcgtcatg accagcgaac tagacatttt cgtggggaac

121 acgaccctta tagatgaaga cgtgtatcgc ctctggctgg atggttactc agtgaacgat

181 gcagtggctc tgcgagtacg ctccggaatc ttggagcaga cgggagccac cacaggagtg

241 ctgcagagcg acaccatgga ccactaccgc acctttcaca tgcttgagcg tctgctgcac

301 gcgccgccga agctgctgca ccagctcatc ttccagattc ctccctcccg acagacactc

361 ctcatcgaga ggtactacac ctttgatgag gcctttgttc gggaggtctt gggcaagaag

421 ctgtccaagg gtaccaagaa agacctggat gacatcagca ccaaaacagg aattactctc

481 aagagctgcc ggaggcagtt tgacaacttt aaacgagtct tcaaggtggt ggaagaaatg

541 cggggctccc tggtggacaa catccagcag cacttcctcc tctctgaccg gttagccagg

601 gattacgcag ccatcgtctt ttttgccaac aaccgctttg aaacaggaaa gaaaaagctg

661 cagtacctga gctttgggga ctttgccttc tgtgcagagc ttatgatcca gaactggacc

721 cttggagccg tcggtgaggc ccccactgac ccagactctc aggtggacga catggatgtg

781 gacttagata aggagtttct ccaagacttg aaggagctca aggttctcgt ggctgacaag

841 gacctcctgg acctgcataa gagcctggtg tgcactgccc tccggggaaa gctgggtgtc

901 ttctctgaga tggaaaccaa cttcaagaat ctgtctcggg ggctggtgaa cgtggctgcc

961 aagctgaccc acaataagga tgtcagagac ctatttgtgg acctcgtgga gaagtttgtg

1021 gaaccctgcc gctctgacca ctggccactg agtgatgtgc ggctcttcct cagccagtat

1081 tcagcgtcag tccactccct ggatggcttc cggcaccagg cactctggga ccgctacatg

1141 ggcaccctcc gtggctgcct tctgcgcctc tatcatgatt aaagcccttt tccttcctcc

1201 ctccgacata ccctgagaat aaagttgcca taagtttgga

SEQ ID NO: 70

Sequence of Mus musculus fibroblast growth factor (acidic) intracellular binding protein (FIBP), transcript variant 2, mRNA, Reproduced from NCBI Reference Sequence: NM_021438.4

1 gaactcccgg aagtcgctct gcggctactc gcccggccgg ggcccgtaca agctccagct

61 tcgggcgaac ccggcccggg catcgtcatg accagcgaac tagacatttt cgtggggaac

121 acgaccctta tagatgaaga cgtgtatcgc ctctggctgg atggttactc agtgaacgat

181 gcagtggctc tgcgagtacg ctccggaatc ttggagcaga cgggagccac cacaggagtg

241 ctgcagagcg acaccatgga ccactaccgc acctttcaca tgcttgagcg tctgctgcac

301 gcgccgccga agctgctgca ccagctcatc ttccagattc ctccctcccg acagacactc

361 ctcatcgaga ggtactacac ctttgatgag gcctttgttc gggaggtctt gggcaagaag

421 ctgtccaagg gtaccaagaa agacctggat gacatcagca ccaaaacagg aattactctc

481 aagagctgcc ggaggcagtt tgacaacttt aaacgagtct tcaaggtggt ggaagaaatg

541 cggggctccc tggtggacaa catccagcag cacttcctcc tctctgaccg gttagccagg

601 gattacgcag ccatcgtctt ttttgccaac aaccgctttg aaacaggaaa gaaaaagctg

661 cagtacctga gctttgggga ctttgccttc tgtgcagagc ttatgatcca gaactggacc

721 cttggagccg tcgactctca ggtggacgac atggatgtgg acttagataa ggagtttctc

781 caagacttga aggagctcaa ggttctcgtg gctgacaagg acctcctgga cctgcataag

841 agcctggtgt gcactgccct ccggggaaag ctgggtgtct tctctgagat ggaaaccaac

901 ttcaagaatc tgtctcgggg gctggtgaac gtggctgcca agctgaccca caataaggat

961 gtcagagacc tatttgtgga cctcgtggag aagtttgtgg aaccctgccg ctctgaccac

1021 tggccactga gtgatgtgcg gctcttcctc agccagtatt cagcgtcagt ccactccctg

1081 gatggcttcc ggcaccaggc actctgggac cgctacatgg gcaccctccg tggctgcctt

1141 ctgcgcctct atcatgatta aagccctttt ccttcctccc tccgacatac cctgagaata

1201 aagttgccat aagtttgga

SEQ ID N0:71

Genomic Sequence oiMus musculus TMEM222, extracted from NCBI Reference Sequence: >NC_000070.7:cl33005101-132993356 Mus musculus strain C57BL/6J chromosome 4,

GRCm39 cccggaagtgatgacacaggggcgagcggcggcgcggtcggccttcccgagcgggacgcgcgcgggatgg cggaagcggaagggagttctccgcttctgttacagccgccgccgccccctcctcggatggcggaagtaga aacgccgacgggggccgagacggacatgaagcagtaccacgggtccggtggcgttgtcatggacgtggag cggagccgcttcccctactgcgtggtgtggacacccatcccggtgctcacgtgagtgtcttccgatgtca gcgcgaagtcgccgagggcccgagcatcctccggggtgccatgctagcttttttttccccagacttgcag caacccgtactccgggccaggctagcccccagtcccctagagctctcgtcacctatgaggtgcgcggtcc ccttggatgtggggcatagttccggccctggttcccccaggtctaactggagaggaggagaagtgtagat cagcagcaaagatctggtcattagcacgcccttaagctgcaggcgtacagaaaagacccttaatcattgt ctgaagaggcatcgtcattcattccccgttggtttttgtttttgttttttcactcattgagcaaacgagc gcgtacttgtcgtctgttatgcacaagacgtttcgcctttagaaacagaactgagtgtttgatcgaagcc acactggttcgtggaatgtcagcattgggaggtccgtgctggcgtttttccccctacatcatttgagtta caaagcagtctcagcgacctaggtatctgcctcccagtggaagaatgcagcttacaaccctcctacctta atcctaacagtctggacagtttacaagctttattttctgtagtagaaatccccttcccccatggggaagc tgagttcctttgtaacatcgtcttggtccgttgactatcccagcaagaagatattcagaagccagttagt tcactgactatatataactcatctttcagctgtggcatcctacctctcacctgaggaggtactcttttct ttccttctatcaaaatcagctaccaggcacggggatccatacttttaaccatagcacttggaggcagagg caagcagatgtctgtgagttggagggcagcctgaattatatggtaggagttccaggccagccaggcatgt atagtgagccccaatctcaaacaaacaaacaaacagagccaagttgtgtggagaaggaatatttttagtc tcaaatgtttctgatcttcaaatcattttaatctttcgattttgtatttatttattttattggttttgac tgagacatgagcttgatatgtagccctggctggtctggtatttatttcgtagcccaacctgactttgaac ccctgttccccctgccttctgtgttctgggactacaggtgtatgccatttcatctatcaaatgaccctaa tttttaaagtatgtaaagctacttgggccagggtggatggctcagtcagtaaaagtgctttctgtgaata acctgagtttggatccttgttaaccatgtaaaaaaaccttcatgcagcagtgcattgcctgaagtcccag agctgaggaagtggaggtgagagcatctctgaggcttgctggctacctactcctgtcaagtcaatgagag atactgtctcaaaatgtagagggcaggggtggtccacacctctattcccagttctcagcaggcaaagcca gaatgggggtctgtgacggtgttgaggtctgtgacggtgttgaggccattgtaagctgcagaggaagggc caacctagggttccagagttagacactggatccaattcattaaaatttcctcttaacacgtttgctgact gactccttgtggtctgtagtagcaggtgtaaagcctagctgtccggtgtgccgcggcagggtgtgccgct tgtttccaggtgttgatgggagccgtagagggtaaataagtaactgacggacaggagaactccagatgtg gaggagagctgtgaagagaatgggctgtcactgtgaaagcggctgggtactcaggtggccgtgtggtttg tgtggaatccaatcatgttggccatgttggctttttcctcatttcttcttaaagattaccttgtgtgtgt aggagcgctgtggccggcagaggacatcattcaggagtcagttgtcccctgctgtgtgtgagtcaagttc aggtcttcagacttggcaacaggtgcctttatatactgaacaatttgcagcccctcattttcaggaatat tgtttctctgttgagaaatggaagcaacaagaggtaaagagacacggttccccatttctaggctagaagt agctggtgtctcagttctcttcagatctgtgtccacaagaccaacatgagtaagactgttcctgacagag gactcgccccttagcagaagggacagggcaaattaacttttgtaatgccatagttatgtgatgttgctaa aggtgtgtgcagatgtaggggcagaggaggtaactctcatgtctatgacagggagatcagaggtggaaac tgagtccatgacctatccacctcttagtgatagctactgctgctaataataaaaacagctaaacatttct tgcacactgtcatgcagtccttacatcaacctgtctcaatatctgaaagcagatattgaggcatgaaact ccagcatgagcaactaatacattcactaaagctttacttttaggtcagaagagacagcagttagcctagc ttgggagcttgtctttggggagctgacaagctgcatcgagaggttagtgttgtctctccaaccttcatcc ccagttccatttgccatcagcagccgagatgtagacagtactgctcatggcggagggaggagtctggatc aggaagagctgactgggttctgaggagccagaggagttgtttcccacagttcttggtgtccgtcctatgt tcactgtgggtcctcagctgcttaccctctgccagtcaggtagcgactgttcaccaagcacctcagcatc actgagcaccacgcatgtgagcagtgttgttctacatagactgtgagcagtagcccctccgcaaagtact tgttgctctgcagaaaggtggaaatgaaaaaactgctctagcctgctcagacttcatctgaaacaagcac aacccgactgtactctctttttttttttctgttggttttgagacagggtctctctatatagtcctggctg ctccggaactcactatgtagaccaggctggccttgaactcagagagatccacctccctctgctgacttct catatagagcaacctaatgtgtgttggggacaatgtgactttccagcattccagttaaatgggcgtgcac agtgcttgcctcacaaacatgaggatctgagttcaagtccccagaactcagcagaggcgggcatgggagt ggtcctgtcatcccaggagactccccaaaagttcataggcagctacctggagtagacagcaatgagatcc tgtcacaaggcagaaggcaaagcagcacccgaggttgtctgtctttatactccacacacttcagggcccg gggcgcatgcacacacacacacacacacacacacacacacacacacacacaccggcgggatgtaaggatc aggttagagagaaccgtgtatccgtacctcggctctcagttctctgggtggagagattcagcaccctctc ttggctcttttgaaatcagcagttaattgtcagttacagttaccttactatgatgtagaatattgaaacg tattccttctacccagctacacccctctactcttaaatatggtggatcatttgtccctcgttctgctgac agatacatagggagtttggaactatgtgccgagacaagcatcatcttagaagctatggcagtaaagggga cacagtctgtgttctcatggagtgtaaggatttagtgaggagaaacgagcagttgatgggctttagtgca tggatatgatgaagagaataaagcagggtgatgggtctggagttccatcaatggctgcttgtgtgtgaag ctgggattgtgatgggggaggtggggagagcttgcagggatctgactcttaaagctcttaaagtccagga acaagctggagagctggttcagtggttaaaagcccacactgctcttgcagaggacctagattcggttctc agcacagcatcctctggttacaactccgaactcctgctgcagcggatcagatgccttcttctggcctctg caggcgcctgtactcatgccccacactgcacacacagatacacaagtatttacataagtaaagattaatc ttgaaaaaaagaaaatctcttcctttagagacaagacaaacctatggctattgaactggggctggcaaaa tggctcagcagagaattacctatgattctctagtcagttggctattgaactgcggctgggactggagcag tagcctccacactggcctcctcctgccttcttgaccactgcccctcactctttgggctgtgtaccaagca ccttacttacctcctgagccatcttaccagccctacatatcctgccctgctgttaaacttcatgcttgaa gggttacttccatattttttttaaagattttatttattttatgtatatgagtacacactttagttgtaca gatggttgtgagccttcatgtgattgttaggaattgaatttaggacctctgcttgctctagccaacccca cttgccaaagatttacttattagaataagttcactgtagctgtcttcaaacgcaccagaagagagtatca gatctcattatgggtggttgtgagccaccatgtggttgctgggatttgaactcagtacctttggaagagc agtcggtgctcttacccactgaaccatctcaccagcccggttacttccatattataagatacaaaaatct taagcatgcagctcagggaagcttttatggttttctttctttttcaacttacggggctggagaaacagtt cagcagcagacctgcactgttaagaggacccagcacccgtgtcaggtggctgacagccccagtggggtcc agtgcttgtgagtgtccatacatgtccacaggcagacacataaaataacatgtgaaatttccttcatttt attattaatgtgtgtgtgtgtatacaggagtgagtgtgtcacagtacacatgtggaggtcagaggacagc tttatggagtcgttttccaggagtcatgggtcaccaggcatggtggcaggcaccctcaccactgagccat ttgccagccccacttcaatagccataggtttgtcttggcttctggaacataaacagtactatattttata ctcttgggtaaggctttcttccaggcacacactcttgagactctgtgtcatatagcagcagtgtgctttt ccttgttacatatgactgctctctctctctctctctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt gtgtgtgagagagagagagagagagagagagagagagagagagagagagagagagagagagagcagtgag gattgtttttaaagaatgttattttgtgcctatgggtgttttgtctgcatgtatatatgcacactgaatg catgcctggtgcccacagaggtcagaatagggtgtcacatcctgtggaattggagtgacgtagctttgag ctaccatgtgaatactgggaactgaacccaggtcttctgcaagaacagcaagtactcttaatggctaagc catctctccagtcccattgttttgggggtttgggtttttttgtttttttgtttttgagacaagatctcac tgtgtagacaaggcaagtgctgggatcacaggcatataccaaccgtgtccagctcagtcgggttttgttg tctttttttaaaatacactttgagataatttacgtataacaaaaaagtatactttttttttcattttatg tgtagggtgttttgcttgcatatatgtctgtgcaatacacctgcctggtgcctacagaggccagatggtt gtgagctgccatgtgtggggttggggattgaatccaggtcccctagaagagcagtgagtggtcttaacca ctgacccatctctccagccccaatcgaagtacacttttttttttttttttggttttttgagacagggttt ctctgtgtagccctggctgtcctggaactcactctgtagaccaggctggccttgaactctgaaattcgcc tgcctctgcctcccaagtgttgggattcaaagcacactttttaagagcacagttcaatcaggatttttgc cttttgcggttcaaagccaggtccttgtatatgctaagcaggcactgaggggaaaatgtatgtgtttgta catcctcagaatagagctgcggaggcatagcttacagagcctgacattcttccgtcagagagctggtttc tcttcccagtagagatccggctgctctgcacccggtgctgcacgtggtgttggtgtcctttcagtggtag ccccggggacctccctgctgtgactcactgtttgacatgcatttctggatggcagacctgagctctgtcc tttgctgctgtcttctcctgcaggtggtttttccccatcattggccacatgggcatctgcacatctgcag gggtcattcgtgacttcgctggcccctattttgtttcggtgagtccccagtttgccctgtcctggagaga ctccaggctcacatagaaggctggcctgggcccaggatgtcacacataactctcacccgggccctagcct tctgcccctctccagtgtttgacccgtttccttctcttgcctccaggacaggccaggagggcagtgtggc cagaggcatcttaagtaactggttcagcctctgtgcccatccctgggcaccaagacatgggtagggtaga tcaagagtggagtcagaccatcccggaaccacatctttggaccttcagaaggtgggagccattgttgggg gacaggggaggctgacatagttcccatggtggggatcttagaatgaagcactgtgttctcaaatccgctt cgtggggatggacggacactaatccaggggactctttctacatgagtgcttgcatttgcctgtttcaggt ggctggagctgaagaggtgacaacatgacctctttatgttagtaattgttagtaccatcctaacaggaca agacctattgctatacacttaatacattgactagctaaacataacaggccatgcctctaatcccaggatt gagaatctgaggcaggaggagtgccaagagttcatggctagcctgagctgtgtagtgagttccaggtcaa tgtgtacagagctagaccctgcttcaacaaaacaaaagaaagctgggcagaatagctcacacctttagtg ccagcactcaggaagcagagacaatgagttcagggccagcctgatctccatagacagttccaggacagcc agggctatactgagagaccctgtctcaatatagagagagataatatttctggggtgtgtgttgcctaaag agtctaggactccccacagtctcctcctcatgagaaagttctgtggtccagcaacctctcactgtggtgc tgagaggtcagagcgttgatcttgcacatgaaggagacctggccatggtgaaagcccagagtctgccctg agggccaggagagggccagagaagcgctgtgttgtggctctgccatccccttctaagtcactgctcttca gccactgatccggggatgttagcagactggatgtagtttctctactgacttctgctcttgtatctcaaca ggaagacaacatggccttcggaaagcctgccaagtaagtgaatgcctttgtgcctagtcagggccaagct cctcctgctcagagctacaggccaggccctgtgaagcacagcaggagccagcaccctgagtgccctgtac caaacacctagagtgggtccctgtctaggcctcatcttgaagcccattggatcttgggcaaggtgacaca gttagaaagcaccagggctggtcttacagccagtctgtctccctgtgtagccgggagtgctgcctcccct tgggacaccccagaaatatcatctcctatacatacgagacagggcctctcagtatcgccctggctgtcct ggtcagtctatggagaccagcctggccttggggaagtagaccatttaaggaataggaggctgcttggtgc gccgttgcatctttctgtttgaaaggcaggaaagcaactcagaccctccaaccatgtaagggtgacaatt caggtctgcatctcctgccagtcttgtccctgtcaccttgttttctcctgaaccctccaggattgctcag ccacttatttctgggacacacagtacagtttatgctcctcaccttgttcaagccgttcccacagccgcca atgcgacctaaccctcttccattagcccaggctcaggtgtttcctgttctcatccactcaagttgccatg acaaatgccattcagtgggtggctcatcaacaacaaaagtgtcacatctttggagactaggagggccaag atgaggtgctggtacatgacatggcttgtggggccaacatgtgaagaaaggaatgagggtctcttaggcc tccccacccacaaagggagttcctcaggggttaagatgtcagcagaatagggggacagacatgcagacca ccacactctggtcttgcctggttgttgtctgctgatgtatctcttcctgggaagatgtgaatagatggtt caccccaggtcggacacgggttgtgcccaaatcaagcttttagacctccctgaatttacagttacaactt agagaaccctggcaagggtcttgagacttgcttcgggagtttcctgagccctgtgactttctacatccta agtctcaggagctccgctcttccctccagaagagaatgttgggaagaagtagccacacaactagatctgg acctggtttcccctgtcgcccacagccttgggcatcccacacagagaacattcataaagttgaaagctgc aaaggggctgagccagtgcccagactatcccccctttcccttcaggttctggaaattggaccccggacag gtgtatgcgagtgggcccaatgcatgggacacggctgtgcacgatgcctccgaagagtataagcaccgaa tggtaggtgggggctgggctcagggggtctgtgtgccccaccccaccccaccctaccctgacccagctgc tcttgtgtctccagcacaatctctgctgtgacaactgccactcccacgtggctttagccctgaacctgat gcgttacaacaacagcaccaactggaacatggtgacgctctgctgcttctgcctcatttatgggaagtat gtcaggtgagtggtcttcagcctgcaggcctgctctggccactctcccaaggatcttggaaaggctgtca ctggagtccctcggtcactagggacagttggaacagaaactgaggtcttagagcaactgagttcctggtg gagagcaaggtaccagagtcctgcctccctcacgtgggttccctgctggcttgtcactgcttactgccag gttctgtttcgctgctctgggctgcagctggccctgggaaaggactgcagtggttctagcttctagggaa gctgagtctacaggtgctcggaggagcgtggatcctcttggatagtgttacccacctccataggctcctg cccctgcctgctttgtcagaggcaggcttctggacagttgggctcacagtccagggagactttggaagcc acagcctcaggaccagcccacactaagaactcaggttgggtgaattccctgtatcccacagcagacccca gacttagaaaccatctcacatgctggatgcggtggggcacacttaatctcagcacttggaggatttctga gttgaaggcgcctgtctccaaaacaaaacagaaaacatcttattacacttgagctggtcccccactctaa cttcatgttttgggttttgttttgttttgtttttttaaaatatttacctttattttatatgcattggtgt tttgcctgcatatgtgtctatgtgagaatgtcagatcttgaaattagagttgtatgcttcccatgtggat ggatttgaacccctgtccactggaagagtagtcattgccccaactgctgagccatctctccagcccttca ccaactttgttcttaattatacttatcaagaccttctggaagggaaactcgggacttctatgcccctaaa gctttagagacccccagaaaaggacagaacacaggcctgaggcagagctggggttgaggtcaggccctga tcagctcctatcctatgagtagccatatctgtgagctgaccttcattaccaccccagtggatcccacgct caactggaactttgtggtcaggagtgcttgtagatgagtgaggcccagcctgttggcttctttaatggcc tccatagtattcatcaggccaggccccatcccttgactggccaagttgaaggataagttccaggaggccc taggaacctaaattctgtcagcgttggggaaggaggagttctgggccataaccctgctcacttgaccctg tgttcttctcccctgtcagtgttggagcctttgtgaagacctggctgccctttgtccttctcctcggcat tatcctgaccgtcagtctcgtcttcaatctgcggtgatagctttcagatggtctaagcctcaaggaggct cctgacactcaccccttttggtcccaattcttttattccccacccctcaggcagtgttgcctcctgggtt gaagtagggtcaggacttgggacagaagtgccgatactgagcacctgatgaggtcacttagagcctcctt gtcctttctccctgggcctgagacctcacccttgtggcattcccagtgccatcctgggaaggccttcctt cccttagagcatttcctgccaccatcactgggctactcttcccatgcccagggcacagtgaggacaagaa cccacgggcaaagcagtgttcagagctggatttgaagctgtttgctggctggtggttgtgtcccaaagta gggcatgtgttatgagaagtgctgcacgccaagagacttggaaccttctctgctggcctcgaatggaccg ggagggctggctatgttctttctgccacaccagcacagtgtgtgcctcatcaccgctaatgtgggccctg gctagcaagggctgagggcacttacgtgcagagtgggtcctctgtaaattaggtaacgtgaatcccactt gtttccagcccaggaggcctcgtgctggggatagaaccctttaggtccaaaatgtgaataacttgtcaga gcagctggtacccctgatcggctgggtctgcttccggttgctacaatgggccctatgcagcctccccctg gtctgcaggctcctttataaggggctccctggcaggaaataaagttttaagtctaa

SEQ ID NO: 72

Sequence oiMus musculus transmembrane protein 222 (TMEM222), transcript variant 1, mRNA, Reproduced from NCBI Reference Sequence: NM_025667.3

1 cccggaagtg atgacacagg ggcgagcggc ggcgcggtcg gccttcccga gcgggacgcg

61 cgcgggatgg cggaagcgga agggagttct ccgcttctgt tacagccgcc gccgccccct

121 cctcggatgg cggaagtaga aacgccgacg ggggccgaga cggacatgaa gcagtaccac

181 gggtccggtg gcgttgtcat ggacgtggag cggagccgct tcccctactg cgtggtgtgg

241 acacccatcc cggtgctcac gtggtttttc cccatcattg gccacatggg catctgcaca

301 tctgcagggg tcattcgtga cttcgctggc ccctattttg tttcggaaga caacatggcc

361 ttcggaaagc ctgccaagtt ctggaaattg gaccccggac aggtgtatgc gagtgggccc

421 aatgcatggg acacggctgt gcacgatgcc tccgaagagt ataagcaccg aatgcacaat

481 ctctgctgtg acaactgcca ctcccacgtg gctttagccc tgaacctgat gcgttacaac

541 aacagcacca actggaacat ggtgacgctc tgctgcttct gcctcattta tgggaagtat

601 gtcagtgttg gagcctttgt gaagacctgg ctgccctttg tccttctcct cggcattatc

661 ctgaccgtca gtctcgtctt caatctgcgg tgatagcttt cagatggtct aagcctcaag

721 gaggctcctg acactcaccc cttttggtcc caattctttt attccccacc cctcaggcag

781 tgttgcctcc tgggttgaag tagggtcagg acttgggaca gaagtgccga tactgagcac

841 ctgatgaggt cacttagagc ctccttgtcc tttctccctg ggcctgagac ctcacccttg

901 tggcattccc agtgccatcc tgggaaggcc ttccttccct tagagcattt cctgccacca

961 tcactgggct actcttccca tgcccagggc acagtgagga caagaaccca cgggcaaagc

1021 agtgttcaga gctggatttg aagctgtttg ctggctggtg gttgtgtccc aaagtagggc

1081 atgtgttatg agaagtgctg cacgccaaga gacttggaac cttctctgct ggcctcgaat

1141 ggaccgggag ggctggctat gttctttctg ccacaccagc acagtgtgtg cctcatcacc

1201 gctaatgtgg gccctggcta gcaagggctg agggcactta cgtgcagagt gggtcctctg

1261 taaattaggt aacgtgaatc ccacttgttt ccagcccagg aggcctcgtg ctggggatag

1321 aaccctttag gtccaaaatg tgaataactt gtcagagcag ctggtacccc tgatcggctg

1381 ggtctgcttc cggttgctac aatgggccct atgcagcctc cccctggtct gcaggctcct

1441 ttataagggg ctccctggca ggaaataaag ttttaagtct aaaaaaaaaa aaaaaaa SEQ ID NO: 73

Open reading frame (ORF) of murine FIBP gene with 3 Flag-tags at the N-terminal and fused eGFP at the C-terminal gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttg tgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttaggg ttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcatt agttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgt caataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagt acatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatg ggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtt tgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaac aactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactg cttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggatccact agtccagtgtggtggaattctgcagatatccagcacagtggcggccgctcgagatggattacaaggatcatgatggcgattacaaggatcat gatatcgattacaaggatgacgacgataagatgaccagcgaactagacattttcgtggggaacacgacccttatagatgaagacgtgtatcg cctctggctggatggttactcagtgaacgatgcagtggctctgcgagtacgctccggaatcttggagcagacgggagccaccacaggagt gctgcagagcgacaccatggaccactaccgcacctttcacatgcttgagcgtctgctgcacgcgccgccgaagctgctgcaccagctcatc ttccagattcctccctcccgacagacactcctcatcgagaggtactacacctttgatgaggcctttgttcgggaggtcttgggcaagaagctgt ccaagggtaccaagaaagacctggatgacatcagcaccaaaacaggaattactctcaagagctgccggaggcagtttgacaactttaaac gagtcttcaaggtggtggaagaaatgcggggctccctggtggacaacatccagcagcacttcctcctctctgaccggttagccagggatta cgcagccatcgtcttttttgccaacaaccgctttgaaacaggaaagaaaaagctgcagtacctgagctttggggactttgccttctgtgcaga gcttatgatccagaactggacccttggagccgtcggtgaggcccccactgacccagactctcaggtggacgacatggatgtggacttagat aaggagtttctccaagacttgaaggagctcaaggttctcgtggctgacaaggacctcctggacctgcataagagcctggtgtgcactgccct ccggggaaagctgggtgtcttctctgagatggaaaccaacttcaagaatctgtctcgggggctggtgaacgtggctgccaagctgacccac aataaggatgtcagagacctatttgtggacctcgtggagaagtttgtggaaccctgccgctctgaccactggccactgagtgatgtgcggct cttcctcagccagtattcagcgtcagtccactccctggatggcttccggcaccaggcactctgggaccgctacatgggcaccctccgtggct gccttctgcgcctctatcatgattctagaggaagcggagctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctgg acctatgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcg tgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc accctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgc ccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacac cctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagcc acaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcag ctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccct gagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacggcatggacgagctgt acaagtaagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttg accctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtgg ggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcgga aagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtga ccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatc gggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgc cctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggt ctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaat gtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtg gaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccat cccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctg agctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaa gagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatga ctgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgt ccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttg tcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatc catcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagc acgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggct caaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttct ggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcg aatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcg ggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttggg cttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgca gcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaat gtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaat tccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgc ccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttc cgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacag aatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttc cataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggc gtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcg ctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgacc gctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagc agagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctg ctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagc agattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaaggg attttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttgg tctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataa ctacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaa ccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagta agtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctcc ggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaag ttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtact caaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaact ttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtg cacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagg gcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgt atttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc [0245] Within this sequence (SEQ ID NO:73), the following features are present: Nucleotides 895-909: Start of MCS

Nucleotides 1,066-1,081 : Start of mFibp

Nucleotides 2,139-2,051 : End of mFibp

Nucleotides 769-789: CMV promoter (sequencing primer)

Nucleotides 994-1065: 3Flag

Nucleotides 2,158-2,940: P2A+eGFP

Nucleotides 985-990 and 2,052-2,057: Xhol+Xbal restriction consensus recognition sites

[0246] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. Reference to information published on the Internet by way of URLs employ extra spaces in the text of the URLs to comply with regulations barring the use of live URLs in this document. It will be understood that the referenced, and incorporated, documents can be obtained by typing such URLs without extra spaces, and adding prefixes such as “www,” “http, “https,” and the like, with appropriate punctuation, into a suitable web browser software application.

[0247] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0248] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

97 CLAIM(S):

1. A T cell comprising chromosomal DNA, wherein the chromosomal DNA lacks an intact genetic sequence encoding FIBP, TMEM222 or both FIBP and TMEM222.

2. The T cell of claim 1, wherein the chromosomal DNA lacks an intact genetic sequence encoding FIBP.

3. The T cell of claim 1 or 2, wherein the chromosomal DNA lacks an intact genetic sequence encoding CBLB, PDCD1, or both CBLB and PDCD1.

4. The T cell of any one of claims 1-3, which is human.

5. The T cell of any one of claims 1-4, which is CD4+ or CD8+.

6. The T cell of any one of claims 1-5, which comprises a recombinant T cell receptor (TCR).

7. The T cell of any one of claims 1-6, which comprises a Chimeric Antigen Receptor (CAR).

8. The T cell of claim 7, wherein the CAR targets an antigen present on a solid tumor.

9. The T cell of claim 7, wherein the CAR targets BCMA, Biotin, CD123, CD171, CD 19, CD22, CD23, CD33, CLCB, EGFRvIII, FAP, FGFR4, FR, GD2, Glypican-3, HER2, IL13Ra2, Mesothelin, MUC1, NKG2D, PD1, PSMA, or ROR-1.

10. An extrachromosomal nucleic acid comprising genetic sequence which is substantially complementary to a genetic sequence encoding a protein comprising FIBP or TMEM222.

11. The nucleic acid of claim 10, wherein the protein comprises FIBP. 98

12. The nucleic acid of claim 10, which comprises RNA.

13. The nucleic acid of claim 10, wherein the genetic sequence encoding the protein comprising FIBP or TMEM222 is human.

14. The nucleic acid of claim 10, which comprises a sequence selected from the group of sequences consisting of: GATGAGTAGTGCCTGCCGGG (SEQ ID NO:4), CCGCTTTCCAGTGACCGACG (SEQ ID NO: 5), and GGTGCTGCAGAGCGACACCA (SEQ ID NO: 6), optionally for any of each, wherein thymidine nucleotides are replaced with uracil.

15. The nucleic acid of claim 10, which comprises a sequence selected from the group of sequences consisting of: TGAGGAGTACAAGCACCGCA (SEQ ID NO:7), ACGGACATGAAGCAATATCA (SEQ ID NO: 8), and GACTCACTGAGACAAAGTAG (SEQ ID NO: 9), optionally for any of each, wherein thymidine nucleotides are replaced with uracil.

16. The nucleic acid of claim 10, which comprises a sequence selected from the group of sequences consisting of: CTTTAAACGAGTCTTCAAGG (SEQ ID NO: 16), ACCTGGCTAACCGGTCAGAG (SEQ ID NO: 17), and CTGGTGAGCACCTCTCGATG. (SEQ ID NO: 18), optionally for any of each, wherein thymidine nucleotides are replaced with uracil.

17. The nucleic acid of claim 10, which comprises a sequence selected from the group of sequences consisting of: CACTCGCATACACCTGTCCG (SEQ ID NO: 19), GACTCACCGAAACAAAATAG (SEQ ID NO:20), and GGAAGTAGAAACGCCGACGG (SEQ ID NO:21), optionally for any of each, wherein thymidine nucleotides are replaced with uracil.

18. The nucleic acid of any one of claims 10-17, which consists of 20 nucleotides.

19. The nucleic acid of any one of claims 10-17, which comprises crRNA or gRNA. 99

20. The nucleic acid of any one of claims 10-17, which comprises an oligonucleotide consisting of from about 20 to about 50 nucleotides.

21. The nucleic acid of any one of claims 10-17, which comprises a plasmid.

22. The nucleic acid of any one of claims 10-17, which comprises a viral genome.

23. A composition comprising the nucleic acid of any one of claims 10-19, comprising a tracrRNA.

24. A composition comprising the nucleic acid of any one of claims 10-19 or 23, comprising an enzyme for catalyzing CRISPR.

25. The composition of claim 24, wherein the enzyme for catalyzing CRISPR comprises Cas9.

26. The composition of any one of claims 23-25, comprising a T cell.

27. The composition of claim 26, wherein the T cell is human.

28. The composition of claim 26 or 27, wherein the T cell CD4+ or CD8+.

29. The composition of any one of claims 26-28, wherein the T cell comprises a recombinant TCR, a CAR, or both a recombinant TCR and a CAR.

30. The composition of claim 29, wherein the CAR targets BCMA, Biotin, CD 123, CD171, CD19, CD22, CD23, CD33, CLCB, EGFRvIII, FAP, FGFR4, FR, GD2, Glypican-3, HER2, IL13Ra2, Mesothelin, MUC1, NKG2D, PD1, PSMA, or ROR-1.

31. A method for making a T cell lacking functional FIBP and/or TMEM222 expression comprising (a) obtaining a population comprising one or more source T cells, (b) expanding the population of T cells (c) activating the expanded population of T cells, and (d) genetically manipulating the T cells within the population to generate a resulting T cell lacking 100 functional FIBP and/or TMEM222 expression, wherein (d) can occur before, during, or after one or both of (b) and (c).

32. The method of claim 31, wherein the one or more source T cells is CD4+, CD8+, CD4+ and CD8+, or comprises a mixture of such cells.

33. The method of claim 31 or 32, wherein the one or more source T cells is genetically modified.

34. The method of any one of claims 31-33, wherein the one or more source T cells expresses a recombinant TCR, a CAR, or both a recombinant TCR and a CAR.

35. The method of any one of claims 31-34, wherein, before, during, or after (b), (c), and/or (d), the T cell is genetically modified to express a recombinant TCR, a CAR, or both a recombinant TCR and a CAR.

36. The method of claim 34 or 35, wherein the CAR targets BCMA, Biotin, CD123, CD171, CD19, CD22, CD23, CD33, CLCB, EGFRvIII, FAP, FGFR4, FR, GD2, Glypican-3, HER2, IL13Ra2, Mesothelin, MUC1, NKG2D, PD1, PSMA, or ROR-1.

37. The method of any one of claims 31-36, wherein the one or more source T cells lacks an intact genetic sequence encoding CBLB, PDCD1, or both CBLB and PDCD1.

38. The method of any one of claims 31-36, wherein, before, during, or after (b), (c), and/or (d), the T cell is genetically modified to lack functional expression of CBLB, PDCD1, or both CBLB and PDCD1.

39. The method of any one of claims 31-38, wherein (b) and (c) comprises stimulation with CD3 and CD28 and then activation in the presence of Interleukin-2.

40. The method of any one of claims 31-39, wherein (d) comprises the use of CRISPR, Transcription Activator-like Effector Nucleases (TALENs) or Zinc Finger proteins 101 targeting FIBP and/or TMEM222 and results in the complete or partial elimination of the FIBP and/or TMEM222 from the genome of the T cell.

41. A T cell lacking functional FIBP and/or TMEM222 expression produced by the method of any one of claims 31-40.

42. The method of any one of claims 31-40, wherein the resulting T cell lacking functional FIBP and/or TMEM222 expression is cultured and expanded to form a population comprising said T cells.

43. A composition comprising a first component comprising the T cell of any one of claims 1-9 or 41 and a carrier and optionally a second component comprising a carrier.

44. The composition of claim 43, wherein the first and/or second component comprises Interleukin-2.

45. The composition of claim 43 or 44, wherein the first and/or second component is frozen.

46. The composition of claim 43 or 44, wherein the carrier is a pharmaceutically acceptable carrier.

47. The composition of any one of claims 43-46, wherein the first and/or second component is a liquid formulated for injection.

48. The composition of any one of claims 43-47, wherein the first and/or second component comprises an active agent, which comprises an anti-PDl, anti-PDLl, or anti-CTLA4 therapeutic antibody.

49. The composition of any one of claims 43-48, wherein the T cells are present at a concentration sufficient to deliver about 5E¹⁰ cells per patient. 102

50. A method of adoptive T cell transfer therapy comprising administering the composition of any one of claims 43-49 comprising T cells lacking functional FIBP and/or TMEM222 expression to a subject suffering from cancer and in need of therapy therefor, in an amount and at a location sufficient to treat the cancer within the subject.

51. Use of a composition of any one of claims 43-49 comprising T cells lacking functional FIBP and/or TMEM222 expression for preparing a medicament for adoptive T cell transfer comprising administering the composition to a subject suffering from cancer and in need of therapy therefor, in an amount and at a location sufficient to treat the cancer within the subject.

52. The composition of any one of claims 43-49 comprising T cells lacking functional FIBP and/or TMEM222 expression for use in a method of adoptive T cell transfer comprising administering the composition to a subject suffering from cancer and in need of therapy therefor, in an amount and at a location sufficient to treat the cancer within the subject.

53. The method of claim 50, the use of claim 51, or the composition for use of claim 52, wherein the T cells lack functional FIBP expression.

54. The method, use, or composition for use of any one of claims 50-53, wherein the T cells lack functional CBLB and/or PDCD1 expression.

55. The method, use, or composition for use of any one of claims 50-54, wherein the subject is a human patient.

56. The method, use, or composition for use of any one of claims 50-55, wherein the cancer comprises a solid tumor.

57. The method, use, or composition for use of claim 56, wherein the cancer is a tumor within the brain or spinal cord, digestive tract (such as within the oral cavity, esophagus, stomach, small intestines, colon, or rectum), lung, heart, liver, pancreas, kidney, bladder, bone, 103 skeletal muscle, breast, a reproductive structure (such as ovaries, fallopian tubes, uterus, cervix, vagina, testicles, prostate, seminiferous tubules, penis, etc.), meninges, interstitial tissue, gland, or other tissue of the subject.

58. The method, use, or composition for use of any one of claims 50-57, wherein between about 10⁶ and IO¹⁰ or about 5E¹⁰ of the T cells lacking functional FIBP and/or TMEM222 expression are administered to the patient.

59. The method, use, or composition for use of any one of claims 50-58, wherein the T cells lacking functional FIBP and/or TMEM222 expression also comprise a recombinant TCR, a CAR, or both a recombinant TCR and a CAR.

60. The method, use, or composition for use of claim 59, wherein the CAR targets BCMA, Biotin, CD 123, CD171, CD 19, CD22, CD23, CD33, CLCB, EGFRvIII, FAP, FGFR4, FR, GD2, Glypican-3, HER2, IL13Ra2, Mesothelin, MUC1, NKG2D, PD1, PSMA, or ROR-. l

61. The method, use, or composition for use of any one of claims 50-60, wherein the composition is administered by intravenous or intratumoral injection.

62. The method, use, or composition for use of any one of claims 50-61, wherein Interleukin-2 is administered to the patient.

63. The method, use, or composition for use of any one of claims 50-62, wherein the adoptive T-cell transfer is employed in conjunction with surgical resection of the cancer, the administration of radiation or chemotherapy to the subject, immunotherapy, or a combination thereof.

64. The method, use, or composition for use of claim 63, wherein the chemotherapy or immunotherapy comprises the administration of an anti-PDl, anti-PDLl, or anti-CTLA4 therapeutic antibody to the subject.