Methods and Compositions for Improved Immunotherapy

Abstract

Provided herein are nucleic acids, expression cassettes, modified lymphocytes, and compositions comprising the same, comprising sequences encoding the genes of Table 1. In some embodiments, the gene is LTBR. In certain embodiments, the cell is a T cell. In certain embodiments, the cell further comprises a CAR or an engineered TCR. Methods of treatment using the provided compositions are also described.

Description

[Technical field]

[0001] Statement regarding government support This invention was made with Government support under grants R00HG008171, DP2HG010099, and R01CA218668 awarded by the National Institutes of Health, and D18AP00053 awarded by the Defense Advanced Research Projects Agency. The Government has certain rights in this invention. [Background technology]

[0002] Cellular immunotherapy using engineered autologous patient T cells redirected against selected tumor antigens has demonstrated significant efficacy against hematologic cancers and has led to five U.S. Food and Drug Administration (FDA) approvals of chimeric antigen receptors (CARs) to date. 6 In contrast, CAR therapy for solid tumors has shown much lower efficacy overall due to suppression of T cell effector function in the tumor microenvironment. Even for hematological malignancies, with the exception of B acute lymphoblastic leukemia, most patients do not experience a sustained response, and resistance is primarily attributable to T cell dysfunction rather than antigen loss. 7 Considerable efforts have been made to identify genes and pathways involved in T cell dysfunction. 8、9 However, so far, comprehensive genome-wide screens for regulators of T cell function have been limited to loss-of-function screens. 2~4 .

[0003] Advances in CRISPR genome engineering have made it possible to easily knock out entire genes in a genome in a scalable and customizable manner. Its large size makes it difficult (if not impossible) to deliver Cas9 via lentivirus into primary T cells.10 ), an alternative approach has been developed, which uses the Cas9 protein 2 or mRNA 11 Transient delivery of Cas9 or constitutive Cas9 expression in engineered isogenic mouse lines 3 However, these approaches are not amenable to gain-of-function screening in human cells, which requires continuous expression of the transcriptional activator that drives target gene expression.

[0004] What is needed are improved compositions and methods for more effective immunotherapy. Summary of the Invention

[0005] In one aspect, provided herein is a modified lymphocyte comprising an exogenous nucleic acid encoding a gene in Table 1. In certain embodiments, the gene is LTBR. In other embodiments, the gene is LTBR, ​​ADA, IFNL2, IL12B CALML3 MRPL51, DBI GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

[0006] In one embodiment, the lymphocyte comprises an expression cassette comprising an expression control sequence and a nucleic acid encoding a gene of Table 1. In a specific embodiment, the gene is LTBR. In other embodiments, the gene is LTBR, ​​ADA, IFNL2, IL12B CALML3 MRPL51, DBI GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

[0007] In certain embodiments, the lymphocytes are updated with a nucleic acid encoding a chimeric antigen receptor (CAR). In certain embodiments, the CAR is Axicabtagene ciloleucel (Yescarta®), Brexucabtagene autoleucel (Tecartus™), Idecabtagene vicleucel (Abecma™), Lisocabtagene maraleucel (Breyanzi®), Tisagenlecleucel (Kyrmriah®), or one of those found in FIG. 19. In certain embodiments, the CAR is a chimeric autoantibody receptor (CAAR). In certain embodiments, the lymphocyte further comprises a nucleic acid encoding a T cell receptor (TCR). In certain embodiments, the TCR is selected from those found in FIG. 17. In certain embodiments, the lymphocyte is a T cell.

[0008] In another aspect, a vaccine composition is provided comprising a nucleic acid encoding a gene of Table 1 and a nucleic acid encoding a viral protein. In certain embodiments, the viral protein is a glycoprotein. In certain embodiments, the glycoprotein is a viral spike protein, optionally a coronavirus spike protein. In certain embodiments, the nucleic acid encoding a gene of Table 1 is an mRNA or the nucleic acid encoding the viral spike protein is an mRNA, or both. In certain embodiments, the gene is LTBR. In other embodiments, the gene is LTBR, ​​ADA, IFNL2, IL12B CALML3 MRPL51, DBI GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

[0009] In another aspect, an expression cassette is provided that comprises a nucleotide sequence encoding a chimeric antigen receptor and a nucleic acid encoding a gene in Table 1. In certain embodiments, the gene is LTBR. In other embodiments, the gene is LTBR, ​​ADA, IFNL2, IL12B CALML3 MRPL51, DBI GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

[0010] In another aspect, an expression cassette is provided that includes a nucleic acid encoding a T cell receptor and a nucleic acid encoding a gene of Table 1. In a particular embodiment, the gene is LTBR. In other embodiments, the gene is LTBR, ​​ADA, IFNL2, IL12B, CALML3 MRPL51, DBI GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

[0011] In another aspect, an expression cassette is provided that includes a nucleic acid encoding a viral protein and a nucleic acid encoding a gene of Table 1. In certain embodiments, the gene is LTBR. In other embodiments, the gene is LTBR, ​​ADA, IFNL2, IL12B CALML3 MRPL51, DBI GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

[0012] In another aspect, a method of producing an engineered lymphocyte is provided, comprising introducing into a cell an exogenous nucleic acid encoding a gene of Table 1. In certain embodiments, the lymphocyte comprises an expression cassette comprising an expression control sequence and a nucleic acid encoding a gene of Table 1. In certain embodiments, the gene is LTBR. In other embodiments, the gene is LTBR, ​​ADA, IFNL2, IL12B CALML3 MRPL51, DBI GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4. In certain embodiments, the lymphocyte further comprises a nucleic acid encoding a chimeric antigen receptor (CAR). In other embodiments, the lymphocyte further comprises an engineered T cell receptor (TCR). It further comprises a nucleic acid encoding the

[0013] In another aspect, a method of treating cancer in a subject in need thereof is provided. The method comprises administering to a subject in need thereof a composition described herein. In certain embodiments, the subject has a solid tumor. In certain embodiments, the subject has a lymphoma, optionally B-cell lymphoma, follicular lymphoma, or mantle cell lymphoma. In certain embodiments, the subject has a leukemia. In certain embodiments, the subject has multiple myeloma. In certain embodiments, the subject has a virus-driven cancer, optionally Burkitt's lymphoma, liver cancer, Kaposi's sarcoma, cervical cancer, head cancer, neck cancer, anal cancer, oral cancer, pharyngeal cancer, penile cancer, adult T-cell lymphoma, or Merkel cell carcinoma.

[0014] In another aspect, a method for treating a viral disease in a subject in need of such treatment is provided. The method comprises administering to a subject in need thereof a composition as described herein. In certain embodiments, the disease is HIV. In certain embodiments, the disease is HPV. In certain embodiments, the disease is an autoimmune disorder.

[0015] In another aspect, a method of treating an autoimmune disease in a subject in need thereof is provided, the method comprising administering to a subject in need thereof a composition described herein.

[0016] In another aspect, a method of increasing T cell effector function, including proliferation or cytokine production and / or secretion, the method comprising administering a composition described herein to the T cells. In a particular aspect, the T cells are obtained from a human before treating the T cells to overexpress the genes of Table 1, and the treated T cells are reintroduced into the human. In a particular embodiment, the gene is LTBR. In other embodiments, the gene is LTBR, ​​ADA, IFNL2, IL12B CALML3 MRPL51, DBI GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

[0017] In another aspect, a method of increasing a response to a vaccine composition is provided. The method comprises co-administering a nucleic acid encoding a gene of Table 1 with the vaccine. In a particular embodiment, the gene is LTBR. In other embodiments, the gene is LTBR, ​​ADA, IFNL2, IL12B CALML3 MRPL51, DBI GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

[0018] In certain embodiments of any of the methods described herein, expression of the genes in Table 1 is transient.

[0019] In another aspect, a method is provided for identifying a gene that, when exogenously expressed in an engineered lymphocyte, alters a therapeutic function of the engineered lymphocyte. The method includes: (a) obtaining a lymphocyte population; (b) transducing the lymphocyte population with a plurality of viral vectors, each viral vector encoding a gene that can be linked to one or more barcodes; (c) stimulating the transduced lymphocytes to induce activation, proliferation, and / or effector function; (d) isolating the transduced lymphocytes from the lymphocyte population of (c); and (e) detecting the presence of a gene and / or linked barcode in the isolated lymphocytes, the detected gene being effective to alter a therapeutic function of the engineered lymphocyte expressing the gene. In certain embodiments, the gene is an open reading frame (ORF), or a non-coding RNA, optionally a microRNA (miRNA) or The nucleotide sequence encoding a long non-coding RNA (lncRNA, long ncRNA). In certain embodiments, the lymphocyte population comprises a cell population enriched for one or more of T cells, B cells, NK T cells, NK cells, or subpopulations thereof, and optionally, the cells are human. In certain embodiments, the plurality of viral vectors comprises a library of open reading frames (ORFs). In certain embodiments, the viral vector is a retroviral vector or a lentiviral vector. In certain embodiments, stimulating the transduced lymphocytes comprises culturing the lymphocytes with one or more of an antibody, a cytokine, an antigen, a superantigen, an antigen-presenting cell, a cancer cell, and a cancer cell line. In certain embodiments, stimulating the transduced lymphocytes comprises TCR stimulation, and optionally CD3 / CD28 stimulation. In certain embodiments, the method further comprises labeling the transduced lymphocytes with a cytoproliferative dye and isolating progeny cells. In certain embodiments, step (d) comprises identifying cells expressing one or more cell surface markers and / or one or more effector functions and / or one or more secreted cytokines. In certain embodiments, step (e) comprises obtaining genomic DNA from the isolated lymphocytes and PCR amplification of the gene and / or barcode sequence. In certain embodiments, step (e) further comprises single-cell transcriptome and / or proteome analysis. In certain embodiments, (e) comprises flow cytometry analysis, cell hashing, single-cell sequencing analysis, single-cell RNA sequencing (scRNA-seq), Perturb-seq, CROP-seq, CRISP-seq, ECCITE-seq, or Cellular Indexing of Transcriptome and Epitopes (CITE-seq).

[0020] In another aspect, a method is provided for analyzing the effect of overexpression of an ORF of interest on an individual cell, the method comprising: (a) introducing into the cell an expression cassette comprising a nucleic acid encoding the ORF of interest, and overexpressing the ORF; (b) providing in separate fractions a first set of nucleic acids from the individual cell and an oligonucleotide having a common barcode sequence, the oligonucleotides being releasably attached to beads, the first set of nucleic acids comprising endogenous transcriptome mRNA and the ORF mRNA; and (c) performing RT-PCR to generate a second set of nucleic acids derived from the first set of nucleic acids, the second set of nucleic acids in the fractions having an oligonucleotide sequence attached thereto comprising the common nucleic acid barcode sequence, and the RT-PCR identifying an ORF that is not a polyA sequence. (d) amplifying the second set of nucleic acids using PCR reagents that include a second primer that anneals specifically to a sequence on the ORF cDNA that is not a polyA sequence to generate a third set of nucleic acids; and (e) detecting and / or sequencing the barcode sequence, the transcriptome cDNA, and / or the ORF cDNA. In certain embodiments, step (e) further comprises single-cell transcriptome and / or proteome analysis. In certain embodiments, (e) comprises flow cytometry analysis, cell hashing, single-cell sequencing analysis, single-cell RNA sequencing (scRNA-seq), Perturb-seq, CROP-seq, CRISP-seq, ECCITE-seq, or Cellular Indexing of Transcriptome and Epitopes (CITE-seq).

[0021] In certain embodiments, the method includes obtaining a portion of the third set of nucleic acids and amplifying the ORF cDNA using a second set of PCR reagents that includes a third primer that specifically anneals to a sequence on the ORF cDNA that is not a polyA sequence to generate a fourth set of nucleic acids.

[0022] In certain embodiments, the method further comprises the step of: PCR a fourth primer using a third set of PCR reagents that includes a fourth primer that specifically anneals to a sequence on the ORF cDNA that is not a polyA sequence; and amplifying ORF cDNA in the third set of nucleic acids to generate a fifth set of nucleic acids, and step (e) comprises fragmenting the third set and the fifth set of nucleic acids, ligating adapters to the ends, and subjecting them to NGS.

[0023] Other aspects and advantages of the present invention will become readily apparent from the following detailed description of the invention. [Brief description of the drawings]

[0024] [Figure 1A] Genome-scale overexpression screen to identify genes that promote proliferation of primary human T cells. Pooled ORF screen overview. CD4+ and CD8+ T cells were isolated separately from peripheral blood from three healthy donors. The barcoded genome-scale ORF library was then introduced into CD3 / CD28 stimulated T cells, followed by selection of transduced cells. After 14 days of culture, T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and restimulated to induce proliferation. ORFs enriched in the CFSE-low population were identified by comparing the number of specific ORF barcodes before and after cell sorting. These genes included LTBR, ​​VSTM1, CD59, IL12B, 1L23A, MAPK3, ADA, and DBI. [Figure 1B]A genome-scale overexpression screen to identify genes that promote proliferation of primary human T cells is shown. Robust rank enumeration of genes in both CFSE low CD4+ and CFSE low CD8+ T cells is shown based on consistent enrichment of individual barcodes in each gene. [Figure 2A] We show that overexpression of the top-ranked ORFs increases proliferation, activation, and cytokine secretion in CD4+ and CD8+ T cells. CD4+ and CD8+ T cells from a donor not involved in screening were isolated separately and then transduced with lentiviruses encoding the top-ranked ORFs together with a selection marker. After transduction and selection, T cells were restimulated before measuring proliferation, expression of activation markers, and cytokine secretion. [Figure 2B] Figure 1 shows that overexpression of the top ranked ORFs increases proliferation, activation, and cytokine secretion of CD4+ and CD8+ T cells. Proliferation of T cells transduced with the top ranked genes as relative proliferation defined as the ratio of stimulated cells to the corresponding unstimulated control, normalized to tNGFR. A minimum of two donors were tested in biological triplicates for each overexpressed gene. Boxes indicate 25th-75th percentiles with a line of the mean and whiskers extending to the maximum and minimum values. DUPD1 is also known as DUSP29. [Figure 2C] Figure 1 shows that overexpression of top-ranked ORFs increases proliferation, activation, and cytokine secretion in CD4+ and CD8+ T cells. Mean relative proliferation of ORF-transduced T cells normalized to tNGFR in CD4+ and CD8+ T cells. Significant genes in both or either T cell subsets are marked (two-tailed Student's t-test P<0.05 and false discovery rate<0.1). [Figure 2D]Figure 1 shows that overexpression of the top-ranked ORFs increases proliferation, activation, and cytokine secretion of CD4+ and CD8+ T cells. Representative expression of CD25 or CD154 after restimulation. Numbers on the histograms correspond to the percentage of gated cells (CD8+CD154+) or mean fluorescence intensity (MFI). Dashed lines indicate the gate used to enumerate the MFI of CD154+ cells (CD8+) or control (tNGFR) cells. [Figure 2E] Overexpression of top ranked ORFs increases proliferation, activation, and cytokine secretion of CD4+ and CD8+ T cells. IL-2 and IFNγ secretion after restimulation normalized to tNGFR. Only genes that significantly increase T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. A minimum of two donors were tested in triplicate per gene. Boxes show 25th-75th percentiles with mean lines and whiskers extending to maximum and minimum values. [Figure 2F] Figure 1 shows that overexpression of the top-ranked ORFs increases proliferation, activation, and cytokine secretion in CD4+ and CD8+ T cells. Cross-sectional relationship between different T cell activation phenotypes significantly (P<0.05) improved by a given ORF in either CD8+ or CD4+ T cells. [Figure 3A] We show that single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. OverCITE-seq captures overexpressed (ORF) constructs, transcriptomes, TCR clonotypes, cell surface proteins, and therapeutic hashtags in single cells. [Figure 3B] Figure 1. Single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. ORF assignment ratios in resting and CD3 / CD28 stimulated T cells. [Figure 3C]Figure 1 shows that single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. Antibody-derived tag sequencing (ADT, right) yields similar NGFR expression in tNGFR-transduced T cells as flow cytometry with tNGFR-transduced T cells (left). Non-transduced cells (left) or cells assigned with a non-tNGFR ORF (right) are shown in grey. [Figure 3D] Figure 1 shows that single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. Uniform manifold approximation and projection (UMAP) representation of single-cell transcriptomes after unsupervised clustering of OverCITE-seq-captured ORF singlets. The top left insert identifies stimulated and resting T cells given the treatment hashtag. For each cluster, a subset of the top 20 differentially expressed genes is shown. HIST1H1B is also known as H1-5 and HIST1H3C is also known as H3C3. [Figure 3E] Figure 1. Single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. ORF prevalence in two representative clusters. Normalized residuals are from chi-squared tests. ORFs of interest are indicated. [Figure 4A] Figure 1 shows that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a 2-fold or greater change in expression and adjusted P<0.05. [Figure 4B] Figure 1 shows that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. Significantly enriched GO biological processes in LTBR-overexpressing T cells (p<0.05). [Figure 4C]Figure 2 shows that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. Cell viability of LTBR or tNGFR lentiviral transduced CD8+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). [Figure 4D] Figure 2 shows that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway.PD-1 expression on resting LTBR or tNGFRT cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. [Figure 4E] Figure 2 shows that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants normalized to wild-type LTBR (n=6 replicates across two experiments). [Figure 4F] Figure 1. LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). [Figure 4G] Figure 1 shows that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated time periods. [Figure 4H] Figure 1 shows that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. Quantification of phosphorylated IκBα in resting or CD3 / CD28 stimulated (15 min) LTBR or tNGFR cells. [Figure 4I] Figure 1 shows that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. Quantification of mature NF-κB2 in resting or CD3 / CD28 stimulated (15 min) LTBR or tNGFR cells. [Figure 4J]Figure 1 shows that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. IFNγ secretion by stimulated LTBR or tNGFR cells following CRISPR knockout of the indicated genes (n=18, 3 sgRNAs in 2 donors in 3 biological replicates). IFNγ amounts are normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow comparison of the relative effects of gene knockout on T cell activation. [Figure 4K] Figure 4 shows that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. Expression levels of core LTBR genes (n=274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to non-targeted control in LTBR cells). Boxes indicate 25-75 percentiles with median lines and whiskers extend 1.5-fold between quartiles. Independent two-tailed t-test P values ​​(Figure 4C, Figure 4G-Figure 4K): not significant (NS) P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars, sem, n=3 biological replicates unless otherwise stated. [Figure 5A] Top ranked genes show improved antigen-specific T cell responses and tumor killing. Co-delivery of anti-CD19 CAR and ORF to T cells from healthy donors. Schematic of tricistronic vector and CAR T cell experiments. [Figure 5B] Top ranked genes show improved antigen-specific T cell responses and tumor killing. Co-delivery of anti-CD19 CAR and ORF to T cells from healthy donors. IFNγ secretion after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n=3 biological replicates, representing 2 donors). [Figure 5C] Top ranked genes show improved antigen-specific T cell responses and tumor killing. Co-delivery of anti-CD19 CAR and ORF to T cells from healthy donors. IL-2 secretion after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n=3 biological replicates, representing 2 donors). [Figure 5D] Top ranked genes show improved antigen-specific T cell responses and tumor killing. Co-delivery of anti-CD19 CAR and ORF to T cells from healthy donors. Nalm6 GFP+ cell proliferation (normalized total GFP per well) after co-incubation with T cells co-expressing 19-28z CAR and LTBR or tNGFR (negative control) at the effector to target ratios shown. [Figure 5E] Showing that the top ranked genes improve antigen-specific T cell responses and tumor killing. Co-delivery of anti-CD19 CAR and ORF to T cells from healthy donors. Quantification of Nalm6 GFP+ clearance for T cells co-expressing 19-28z or 18-BBz CAR and top ranked genes (n=3 biological replicates, representing 2 donors) normalized to tNGFR after an effector-to-target ratio of 0.25 and 48 hours of co-incubation. [Figure 5F] The top ranked genes show improved antigen-specific T cell responses and tumor killing. Co-delivery of anti-CD19 CAR and ORF to T cells from healthy donors. 19-BBz CAR T cells co-expressing LTBR or tNGFR were co-incubated with Nalm6 cells at a 1:1 ratio every 3 days for up to 3 rounds of stimulation (n=3 biological replicates). 7 days after repeated antigen stimulation, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. [Figure 5G] Top ranked genes show improved antigen-specific T cell responses and tumor killing. Co-delivery of anti-CD19 CAR and ORF to total PBMC from a patient with diffuse large B cell lymphoma. Transduced T cells were incubated alone or co-incubated with CD19+Nalm6 or CD19-Jurkat cell lines at a 1:1 ratio (n=3 biological replicates, representing 2 patients). IFNγ and IL2 secretion was measured after overnight incubation. For Nalm6 conditions, the numbers above the indicated column pairs are the fold increase in cytokine secretion by LTBR cells over tNGFR (negative control) cells. [Figure 5H] Top ranked genes show improved antigen-specific T cell responses and tumor killing. Delivery of ORFs to Vy9V52 T cells. IFNγ and IL-2 secretion after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2, pretreated with zoledronate to promote phosphoantigen accumulation (n=3 biological replicates). Data are mean ± sem, where applicable. [Figure 6A] Figure 2 shows the design of human ORF library screening in primary T cells. Barcoded vector design for ORF overexpression. [Figure 6B] Figure 1 shows the design of the human ORF library screening in primary T cells. Distribution of the number of barcodes per ORF in the library. [Figure 6C] Figure 6 shows the design of human ORF library screening in primary T cells. Vector design to quantify the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS-Elongation factor-1α short promoter, CMV-Cytomegalovirus promoter, PGK-Phosphoglycerate kinase-1 promoter. (Figure 6D) Percentage of positive cells and (Figure 6E) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n=3 biological replicates). Error bars are SEM. [Figure 6D] Figure 1 shows the design of human ORF library screening in primary T cells. Vector design to quantify the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS-Elongation factor-1 alpha short promoter, CMV-Cytomegalovirus promoter, PGK-Phosphoglycerate kinase-1 promoter. Percentage of positive cells for rat CD2 (rCD2) expressed from EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n=3 biological replicates). Error bars are SEM. [Figure 6E] Figure 1 shows the design of human ORF library screening in primary T cells. Vector design to quantify the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS-Elongation factor-1 alpha short promoter, CMV-Cytomegalovirus promoter, PGK-Phosphoglycerate kinase-1 promoter. Mean fluorescence intensity (MFI) of rat CD2 (rCD2) expressed from EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n=3 biological replicates). Error bars are SEM. [Figure 6F] Figure 6 shows the design of the human ORF library screening in primary T cells. Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels Figure 6D and Figure 6E are shown. [Figure 6G] Figure 2 shows the design of the human ORF library screening in primary T cells. CD3 / CD28 antibody titration. T cells were labeled with CFSE, stimulated and incubated for 4 days. Proliferating T cells were gated at least twice to include expanded cells (third CFSE peak). [Figure 6H] Figure 2 shows the design of human ORF library screening in primary T cells. Expansion of T cells from three healthy donors transduced with the ORF library. [Figure 6I] Figure 2 shows the design of the human ORF library screen in primary T cells. Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. [Figure 6J] Figure 2 shows the design of human ORF library screening in primary T cells. Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Each sample from three donors was computationally pooled together with equal number of reads and then counted how many barcodes or ORFs were present with a minimum of one read. [Figure 6K]Figure 1 shows the design of the human ORF library screen in primary T cells. Gene enrichment in both CFSE low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. [Figure 6L] Figure 6K shows the design of the human ORF library screening in primary T cells. GO biological processes for significantly enriched genes in Figure 6K. [Figure 6M] Figure 2 shows the design of the human ORF library screen in primary T cells. Overlap of differentially expressed and significantly enriched genes between CD3 / CD28 stimulated and naive T cells41. [Figure 7A] 1 shows overexpression of selected ORFs in donors irrelevant to screening. Histogram of expression of selected ORFs in T cells after puromycin selection. [Figure 7B] Overexpression of selected ORFs in donors irrelevant to screening is shown. Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. [Figure 7C] Figure 1 shows overexpression of selected ORFs in donors irrelevant to the screen. Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. [Figure 7D] Overexpression of selected ORFs in donors irrelevant to screening. Proliferation of restimulated CD8+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Means and SEM are shown. [Figure 7E]Overexpression of selected ORFs in donors irrelevant to screening. Proliferation of restimulated CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Means and SEM are shown. [Figure 7F] Overexpression of selected ORFs in donors irrelevant to screening is shown. Proliferation of T cells transduced with ORFs (see FIG. 2C) that significantly improved T cell proliferation as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions are shown (n=3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). [Figure 7G] Overexpression of selected ORFs in donors irrelevant to screening is shown. Proliferation of T cells transduced with ORFs (see Figure 2C) that significantly improved T cell proliferation as measured by dilution of CellTrace Yellow. Quantification of proliferation index is shown (n=3 biological replicates). P-values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). [Figure 7H] Figure 1 shows overexpression of selected ORFs in donors irrelevant to screening. Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of 4 donors tested) are shown (n=3 biological replicates, CD8 left bar, CD4 right bar). [Figure 7I]Overexpression of selected ORFs in donors irrelevant to screening is shown. Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 h. Gating was based on isotype and fluorescence minus one controls. Representative gating quantification is shown (n=6 biological replicates from 2 donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right). Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Dunnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 7J] Overexpression of selected ORFs in donors irrelevant to screening is shown. Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Quantification of cells (for stimulated T cells) in S-G2-M phase is shown (n=6 biological replicates from 2 donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right). Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Dunnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 8A] Functional responses of ORF-overexpressing T cells are shown. Quantitative expression of CD25 or CD154 after restimulation. A minimum of two donors were tested in triplicate per gene. Only genes that significantly increase T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. Means and SEMs are shown. CD4+ are shown in the top bar and CD8+ cells are shown in the bottom bar. [Figure 8B]Functional response of ORF-overexpressing T cells. Sensitivity to antigen dose. T cells were incubated with the indicated anti-CD3 antibody concentrations for 24 hours and the amount of secreted IFNγ was quantified. Representative dose-response curve fitting is shown (n=2 biological replicates). [Figure 8C] Functional responses of ORF-overexpressing T cells are shown. Sensitivity to antigen dose. T cells were incubated with the indicated anti-CD3 antibody concentrations for 24 hours and the amount of secreted IFNγ was quantified. IC50 determinations are shown (n=2 biological replicates). [Figure 8D] Functional responses of ORF-overexpressing T cells. Quantification of secreted IL-2 and IFNγ in T cells incubated alone or with CD3 / CD28 antibodies for 24 hours. Representative data from one of four donors (n=3 biological replicates) are shown. Resting cells are indicated by the bars on the left and +CD3 / CD28 are indicated on the right. [Figure 8E] Functional responses of ORF-overexpressing T cells. Multiplex quantification of selected secreted cytokines and chemokines by ORF-transduced T cells 24 hours after CD3 / CD28 stimulation. Mean z-scores (from independent samples) of duplicate measurements normalized to tNGFR are shown. [Figure 9A] OverCITE-seq identifying ORFs and their transcriptional effects. Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. [Figure 9B] OverCITE-seq identifying ORFs and their transcriptional effects are shown. Proportion of stimulated and resting T cells among cells assigned to each ORF. Chi-square test p-values ​​are shown for ORFs with significantly shifted (uneven) distribution of stimulated and resting cells. [Figure 9C]OverCITE-seq identifying ORFs and their transcriptional effects are shown. Cell cycle corrected scaled expression of overexpressed genes in cells transduced with the respective ORF and negative control (tNGFR). Two-tailed Wilcoxon test p-values ​​shown above the violin plots indicate statistical significance of gene expression levels between specific ORF- and tNGFR-transduced T cells. Boxes indicate 25-75th percentiles with a median line and whiskers extend to maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), 25 (FOSB), 119 (GPD1), 124 (GPN3), 199 (IFNL2), 60 (IL12B), 70 (IL1RN), 156 (ITM2A), 74 (LTBR), 88 (MRPL18), 167 (MRPL51), 107 (MS4A3), 69 (NFYB), 355 (NGFR), 261 (RAN), 182 (SLC10A7), and 56 (ZNF830) in single cells. [Figure 9D] OverCITE-seq identifying ORFs and their transcriptional effects. Expression of all ORF genes by cells assigned to each ORF. Each row is z-score normalized. [Figure 9E] OverCITE-seq identifying ORFs and their transcriptional effects are shown. Distribution of individual ORF frequencies within the clusters. Number of ORF cells and chi-square test residuals are displayed. Chi-square test p-values ​​indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution are shown above the plots. The percentage of stimulated and resting T cells in each cluster is shown below the cluster representation. [Figure 9F] OverCITE-seq identifying ORFs and their transcriptional effects. Spearman correlation between transcriptional profiles of selected ORFs cells in quiescent populations. [Figure 9G] OverCITE-seq identifying ORFs and their transcriptional effects. Spearman correlation between transcriptional profiles of selected ORFs cells in stimulated populations. [Figure 9H] OverCITE-seq identifying ORFs and their transcriptional effects are shown. Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. [Figure 9I] Shown is OverCITE-seq identifying ORFs and their transcriptional effects. Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). Shown is the log2 fold change of each ORF(stimulated) relative to tNGFR(resting) normalized to the log2 fold change of tNGFR(stimulated) relative to tNGFR(resting) for all genes. Genes of interest in each cluster are labeled. [Figure 9J] OverCITE-seq identifying ORFs and their transcriptional effects are shown. Average TCR clonotype diversity in ORF cells. [Figure 10A] Functional analysis of LTBR overexpression in T cells. LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate 25-75 percentiles with a median line. [Figure 10B] Functional analysis of LTBR overexpression in T cells. LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. [Figure 10C] Functional analysis of LTBR overexpression in T cells. Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single cell or bulk RNA-seq. [Figure 10D] 1 shows functional analysis of LTBR overexpression in T cells. TCF1 expression in LTBR or tNGFR transduced T cells. Representative histograms of TCF1 expression and the gate for TCF1+ cells (dashed line) are shown (n=3 biological replicates). [Figure 10E] Functional analysis of LTBR overexpression in T cells. TCF1 expression in LTBR or tNGFR transduced T cells. Quantification of TCF1+ cells is shown (n=3 biological replicates). [Figure 10F] Functional analysis of LTBR overexpression in T cells. ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histograms of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation from n=3 donors (CD8+) or n=4 donors (CD4+). [Figure 10G] Functional analysis of LTBR overexpression in T cells. ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Quantification of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n=3 donors (CD8+) or n=4 donors (CD4+) (n=3 biological replicates). [Figure 10H] Functional analysis of LTBR overexpression in T cells. ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Time course of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation (n=3 biological replicates). [Figure 10I] Figure 1 shows functional analysis of LTBR overexpression in T cells. Differentiation phenotype of NGFR and LTBR transduced T cells (n=4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROnegCCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector CD45ROnegCCR7neg). [Figure 10J]Functional analysis of LTBR overexpression in T cells. Representative dot plots of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. [Figure 10K] Functional analysis of LTBR overexpression in T cells. Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). [Figure 10L] Functional analysis of LTBR overexpression in T cells. LTBR and tNGFR cells were stimulated with excess CD3 / CD28 beads at 3:1 every 3 days for up to 3 rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression was measured in resting cells (n=3 biological replicates). Statistical significance in panels Fig. 10E, Fig. 10I, and Fig. 10K: two-tailed unpaired t-test, panel Fig. 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10M] Functional analysis of LTBR overexpression in T cells. LTBR and tNGFR cells were stimulated with excess CD3 / CD28 beads at 3:1 every 3 days for up to 3 rounds of stimulation. After repeated stimulation, IFNγ and IL2 secretion was measured in restimulated cells (n=3 biological replicates). Statistical significance in panels Fig. 10E, Fig. 10I, and Fig. 10K: two-tailed unpaired t-test, panel Fig. 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 11A] Expression of LTBR ligand and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. IL2 secretion after 24 h stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. [Figure 11B]Expression of LTBR ligand and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, then IL2 was measured. (n=3 biological replicates). From left to right, no stimulation, +CD3 / CD28, +LTA, and +LIGHT are shown. [Figure 11C] Expression of LTBR ligand and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibody or recombinant soluble LTA or LIGHT for 24 hours, then IFNγ was measured. (n=3 biological replicates). From left to right, no stimulation, +CD3 / CD28, +LTA, and +LIGHT are shown. [Figure 11D] Expression of LTBR ligand and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. Differentiation phenotype after restimulation of tNGFR and LTBR transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during culture (n=3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. [Figure 11E] Expression of LTBR ligand and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. Proliferation after restimulation of tNGFR and LTBR transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during culture (n=3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. [Figure 11F]Expression of LTBR ligands and LTBR via mRNA or with deletions and point mutations is shown. Transient LTBR or tNGFR expression via mRNA nucleofection. T cells were nucleofected with LTBR or tNGFR mRNA (n=3 biological replicates) and surface expression of LTBR (FIG. 11G), tNGFR (FIG. 11H), or the four genes upregulated in LTBR cells (FIG. 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. [Figure 11G] Expression of LTBR ligand and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. T cells were nucleofected with LTBR or tNGFR mRNA (n=3 biological replicates) and surface expression of LTBR was monitored over a 21 day period. At each time point, expression of the target gene was normalized to the appropriate tNGFR control. [Figure 11H] Expression of LTBR ligand and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. T cells were nucleofected with LTBR or tNGFR mRNA (n=3 biological replicates) and surface expression of tNGFR was monitored over a 21 day period. At each time point, expression of the target gene was normalized to the appropriate tNGFR control. [Figure 11I] Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. T cells were nucleofected with LTBR or tNGFR mRNA (n=3 biological replicates) and surface expression of the four genes upregulated in LTBR cells was monitored over a 21 day period. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. [Figure 11J] Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations. Schematic diagram of FLAG-tagged LTBR mutants. [Figure 11K]Expression of LTBR ligand and LTBR via mRNA or with deletion and point mutations. LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 12A] Figure 2 shows chromatin accessibility in LTBR T cells. Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFR T cells either resting or stimulated with CD3 / CD28 for 24 h. [Figure 12B] Chromatin accessibility in LTBR T cells. Differentially accessible chromatin regions between stimulated tNGFR and resting tNGFR, stimulated LTBR and resting LTBR, ​​resting LTBR and resting tNGFR, and stimulated LTBR and stimulated tNGFR. Numbers of peaks gained / lost are shown (using an absolute log2 fold change of 1 and adjusted p-value <0.1 as cutoffs). [Figure 12C] Figure 1. Chromatin accessibility in LTBR T cells. Changes in chromatin accessibility for differentially expressed (regulated p<0.05) genes. Two-tailed t-test p-values ​​are shown. Boxes indicate 25-75th percentiles with a median line and whiskers extend to 1.5x interquartile range. N=614 genes. [Figure 12D] Chromatin accessibility in LTBR T cells. Changes in gene expression for differentially accessible (regulated p<0.05) regions. Two-tailed t-test p-values ​​are shown. Boxes indicate 25-75th percentiles with a median line and whiskers extend to 1.5× interquartile range. N=614 genomic regions. [Figure 12E]Chromatin accessibility in LTBR T cells. Chromatin accessibility profile at more loci in LTBR compared to tNGFR cells resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0-860 for BATF3, 0-1950 for IL13, 0-1230 for TRAF1, 0-1000 for TNFSF4, 0-300 for PDCD1, 0-2350 for LAG3). [Figure 12F] Chromatin accessibility in LTBR T cells. Chromatin accessibility profile at less open loci in LTBR compared to tNGFR cells, resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0-860 for BATF3, 0-1950 for IL13, 0-1230 for TRAF1, 0-1000 for TNFSF4, 0-300 for PDCD1, 0-2350 for LAG3). [Figure 12G] Chromatin accessibility in LTBR T cells. Chromatin accessibility in resting or stimulated LTBR and tNGFR cells. Each row represents a peak that was significantly enriched in LTBR over the corresponding tNGFR control (log2 fold change >1, DESeq2 adjusted p-value <0.05). Peaks were clustered using k-means clustering and selected genes at or near the peak from each cluster are shown. [Figure 12H] Chromatin accessibility in LTBR T cells. Correlation of each ATAC sample (biological replicates) based on bias-corrected deviations. [Figure 12I] Figure 2. Chromatin accessibility in LTBR T cells. Top transcription factor (TF) motifs enriched at differentially accessible chromatin regions in resting LTBR cells compared to resting tNGFR cells. [Figure 13A] Proteomic and functional genomic assays of NF-κB activation. Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identification of phospho-RELA+ cells is shown. [Figure 13B] Proteomic and functional genomic assays of NF-κB activation. Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 min. A representative gel is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. [Figure 13C] Proteomic and functional genomic assays of NF-κB activation. Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 min. Quantification of band intensity relative to GAPHD is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. [Figure 13D] Proteomic and functional genomic assays of NF-κB activation. Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). [Figure 13E] Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with lentiviral vectors co-expressing single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. [Figure 13F] Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with lentiviral vectors co-expressing single-stranded guide RNA (sgRNA) and the LTBR ORF. Representative expression of target genes in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, essential components of the αβTCR. [Figure 13G]Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with lentiviral vectors co-expressing single-stranded guide RNA (sgRNA) and the LTBR ORF. Quantification of IFNγ after restimulation (n=3 sgRNAs). [Figure 13H] Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. Representative protein level-based quantification of gene knockout efficiency. Representative histograms of LTA, LIGHT, and RELA are shown (n=3 sgRNAs). Dashed lines represent the gates used to enumerate cells expressing a given protein. [Figure 13I] Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. Representative protein level-based quantification of gene knockout efficiency. Quantification of relative expression levels of LTA, LIGHT, and RELA are shown (n=3 sgRNAs). Dashed lines represent the gates used to enumerate cells expressing a given protein. [Figure 13J] Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. Representative protein level-based quantification of gene knockout efficiency. Representative histograms of LTA, LIGHT, and RELA are shown (n=3 sgRNAs). Dashed lines represent the gates used to enumerate cells expressing a given protein. [Figure 13K] Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. Representative protein level-based quantification of gene knockout efficiency. Quantification of relative expression levels of LTA, LIGHT, and RELA are shown (n=3 sgRNAs). Dashed lines represent the gates used to enumerate cells expressing a given protein. [Figure 13L]Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. Representative protein level-based quantification of gene knockout efficiency. Representative histograms of LTA, LIGHT, and RELA are shown (n=3 sgRNAs). Dashed lines represent the gates used to enumerate cells expressing a given protein. [Figure 13M] Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. Representative protein level-based quantification of gene knockout efficiency. Quantification of relative expression levels of LTA, LIGHT, and RELA are shown (n=3 sgRNAs). Dashed lines represent the gates used to enumerate cells expressing a given protein. [Figure 13N] Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. Representative protein level-based quantification of gene knockout efficiency. Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel is shown (n=3 sgRNA for RELB and n=2 non-targeting control sgRNA). [Figure 13O] Proteomic and functional genomic assays of NF-κB activation. Simultaneous gene knockout via CRISPR and ORF overexpression. Representative protein level-based quantification of gene knockout efficiency. Dashed lines represent gates used to enumerate cells expressing a given protein. Quantification of RELB expression is shown (n=3 sgRNA for RELB and n=2 non-targeting control sgRNA). [Figure 13P] Proteomic and functional genomic assays of NF-κB activation. Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR versus the corresponding tNGFR control ("Core LTBR" genes). Error bars indicate SEM. [Figure 14A]Co-delivery of ORF with CD19-targeted CAR. Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). [Figure 14B] Co-delivery of ORF with CD19-targeted CAR. CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). [Figure 14C] Co-delivery of ORF with CD19-targeted CAR is shown. CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Quantification of CAR expression relative to tNGFR is shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). [Figure 14D] Showing co-delivery of ORF with CD19-targeted CAR. Expansion curves of CAR+ORF transduced T cells (n=4 biological replicates). [Figure 14E] Co-delivery of ORF with CD19-targeted CAR. LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. [Figure 14F] Co-delivery of ORF with CD19-targeted CAR is shown. Expression of ICAM-1 by T cells transduced with LTBR ORF alone, CAR+LTBR or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. [Figure 14G] Co-delivery of ORF with CD19-targeting CAR is shown. Expression of CD70 by T cells transduced with LTBR ORF alone, CAR+LTBR or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. [Figure 14H]Co-delivery of ORF with CD19-targeted CAR is shown. Expression of CD74 by T cells transduced with LTBR ORF alone, CAR+LTBR or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. [Figure 14I] Co-delivery of ORF with CD19-targeted CAR is shown. Expression of MHC-II by T cells transduced with LTBR ORF alone, CAR+LTBR or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. [Figure 14J] Co-delivery of ORF with CD19-targeted CAR is shown. Expression of exhaustion marker PD-1 in CAR+ORF T cells. CD8 left bar, CD4 right bar. [Figure 14K] Co-delivery of ORF with CD19-targeted CAR. Expression of exhaustion marker TIM-3 in CAR+ORF T cells. CD8 left bar, CD4 right bar. [Figure 14L] Co-delivery of ORF with CD19-targeted CAR. Expression of exhaustion marker LAG-3 in CAR+ORF T cells. CD8 left bar, CD4 right bar. [Figure 14M] Co-delivery of ORF with CD19-targeted CAR is shown. Expression of exhaustion marker CD39 in CAR+ORF T cells. CD8 left bar, CD4 right bar. [Figure 14N] Co-delivery of ORF with CD19-targeted CAR is shown. Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (Naive: CD45ROnegCCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, Effector CD45ROnegCCR7neg). [Figure 14O]Shows co-delivery of ORF with CD19-targeted CAR. Expression of activation marker CD25 in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14P] Figure 1 shows co-delivery of ORF with CD19-targeted CAR. Expression of activation marker CD69 in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 15A] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Co-delivery of anti-CD19 CAR and ORF to T cells from healthy donors. IFNγ secretion after overnight co-incubation of CD4+ T cells with Nalm6 cells at a 1:1 ratio (n=3 biological replicates, representing 2 donors). [Figure 15B] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Co-delivery of anti-CD19 CAR and ORF to T cells from healthy donors. IL2 secretion after overnight co-incubation of CD4+ T cells with Nalm6 cells at a 1:1 ratio (n=3 biological replicates, representing 2 donors). [Figure 15C] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. IFNγ secretion by CAR+ORF or ORF only T cells alone or co-incubated with Nalm6 cells for 24 hours. [Figure 15D] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. IL-2 secretion by CAR+ORF or ORF only T cells alone or co-incubated with Nalm6 cells for 24 hours. [Figure 15E] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Cytotoxicity of 19-BBz CAR T cells expressing tNGFR or LTBR ORFs after co-incubation with Nalm6 GFP cells. [Figure 15F] Figure 1 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Quantification of Nalm6 clearance in CAR+ORF or ORF only T cells at different effector:target ratios (compared to Nalm6 co-incubated with non-transduced T cells). Independent two-tailed t-test p-values: 0.011, 1.3x10-4, 0.072, 0.02, 0.021, 0.52, 0.087, 1, 0.51 (from left to right). [Figure 15G] Figure 1 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Representative images of T cells transduced with 19-28z CAR and NGFR or LTBR and co-incubated with CD19+Nalm6 GFP cells at a 1:1 ratio for 48 hours. Scale bar: 200 μm. [Figure 15H] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Repeated stimulation of CAR+ORF T cells with Nalm6 cells. [Figure 15I] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Repeated stimulation of CAR+ORF T cells with Nalm6 cells. IL-2 secretion by 19-BBZ CAR LTBR or tNGFR T cells reloaded with Nalm6 after repeated stimulation with Nalm6 cells every 3 days for up to 3 rounds of stimulation. [Figure 15J] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Repeated stimulation of CAR+ORF T cells with Nalm6 cells. Nalm6 survival with 19-BBZ CAR LTBR or tNGFR T cells reloaded with Nalm6 after repeated stimulation with Nalm6 cells every 3 days for up to 3 rounds of stimulation. [Figure 15K]Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Secretion of cytokines IL2 and IFNγ by CAR / LTBR or CAR / tNGFR T cells from two patients with DLBCL after overnight incubation with Nalm6 target cells. Two-tailed paired t-test p-values ​​are shown. [Figure 15L] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Representative staining of ORF-transduced T cells endogenously expressing the Vy9V52 TCR. [Figure 15M] Figure 1 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. Quantification of ORF-transduced T cells expressing Vγ9Vδ2 TCR. [Figure 15N] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. IL2 secretion following 24 h co-incubation of ORF-transduced Vy9V52 T cells with leukemia cell lines. [Figure 15O] Figure 2 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. IFNγ secretion following 24 h co-incubation of ORF-transduced Vγ9Vδ2 T cells with leukemia cell lines. [Figure 15P] Figure 15 shows that the top ranked genes from the ORF screen promote antigen-specific T cell responses. IL2 or IFNγ secretion after 24 h co-incubation of ORF-transduced Vy9V52 T cells with pancreatic ductal adenocarcinoma cell line, BxPC3. Cell lines in panels (Figure 15N-P) were pre-treated with zoledronic acid prior to co-incubation. Error bars indicate SEM. N=3 biological replicates are shown unless otherwise indicated. [Figure 16A] Showing the top-ranked genes that improve antigen-specific CAR T cell responses in solid tumors. Co-delivery of anti-mesothelin CAR and ORF to T cells from healthy donors. [Figure 16B]Shown are the top ranked genes that improve antigen-specific CAR T cell responses in solid tumors. Secretion of cytokines IFNγ and IL2 by CD4+ and CD8+ T cells co-transduced with anti-mesothelin CAR and ORF after overnight co-incubation with the mesothelin-high cell line Capan-2. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. The dashed line indicates the level of cytokine secretion in regular CAR T cells (i.e., co-expressing tNGFR). [Figure 16C] Shown are the top ranked genes that improve antigen-specific CAR T cell responses in solid tumors. Secretion of cytokines IFNγ and IL2 by CD4+ and CD8+ T cells co-transduced with anti-mesothelin CAR and ORF after overnight co-incubation with the mesothelin-high cell line Capan-2. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. The dashed line indicates the level of cytokine secretion in regular CAR T cells (i.e., co-expressing tNGFR). [Figure 16D] Shown are the top ranked genes that improve antigen-specific CAR T cell responses in solid tumors. Secretion of cytokines IFNγ and IL2 by CD4+ and CD8+ T cells co-transduced with anti-mesothelin CAR and ORF after overnight co-incubation with the mesothelin-low cell line BxPC3. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. The dashed line indicated the level of cytokine secretion in regular CAR T cells (i.e., co-expressing tNGFR). [Figure 16E]Shown are the top-ranked genes that improve antigen-specific CAR T cell responses in solid tumors. Killing of GFP+mesothelin-high Capan-2 or mesothelin-low BxPC3 after 48 hours of co-incubation with engineered CAR T cells at a 1:2 T cell to cancer cell ratio. Cancer cell killing was normalized by dividing the integrated GFP signal in wells containing regular CAR T cells (i.e., co-expressing tNGFR) by the integrated GFP signal in a particular sample. A ratio above 1 indicates higher killing, i.e., lower GFP signal (and therefore lower number of cancer cells) in a particular sample than the corresponding CAR control (CAR+tNGFR). WT or no CAR = non-transduced T cells. [Figure 16F] Shown are the top-ranked genes that improve antigen-specific CAR T cell responses in solid tumors. Killing of GFP+mesothelin-high Capan-2 or mesothelin-low BxPC3 after 48 hours of co-incubation with engineered CAR T cells at a 1:2 T cell to cancer cell ratio. Cancer cell killing was normalized by dividing the integrated GFP signal in wells containing regular CAR T cells (i.e., co-expressing tNGFR) by the integrated GFP signal in a particular sample. A ratio above 1 indicates higher killing, i.e., lower GFP signal (and therefore lower number of cancer cells) in a particular sample than the corresponding CAR control (CAR+tNGFR). WT or no CAR = non-transduced T cells. [Figure 17A] Showing that the top ranked genes improve antigen-specific TCR T cell responses in solid tumors. Co-delivery of anti-NY-ESO-1 TCR and ORF to T cells from healthy donors. [Figure 17B]Shows that top ranked genes improve antigen-specific TCR T cell responses in solid tumors. Secretion of cytokines IFNγ and IL-2 by CD8+ T cells co-transduced with anti-NY-ESO-1 TCR and ORF after overnight co-incubation with melanoma cell line A375. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. Dashed line indicates levels of cytokine secretion in conventional TCR T cells (i.e., co-expressing tNGFR). [Figure 17C] Shows that top ranked genes improve antigen-specific TCR T cell responses in solid tumors. Secretion of cytokines IFNγ and IL-2 by CD8+ T cells co-transduced with anti-NY-ESO-1 TCR and ORF after overnight co-incubation with melanoma cell line A375. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. Dashed line indicates levels of cytokine secretion in conventional TCR T cells (i.e., co-expressing tNGFR). [Figure 17D] Shows that top ranked genes improve antigen-specific TCR T cell responses in solid tumors. Secretion of cytokines IFNγ and IL-2 by CD8+ T cells co-transduced with anti-NY-ESO-1 TCR and ORF after overnight co-incubation with melanoma cell line A375. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. Dashed line indicates levels of cytokine secretion in conventional TCR T cells (i.e., co-expressing tNGFR). [Figure 17E] Shows that top ranked genes improve antigen-specific TCR T cell responses in solid tumors. Killing of GFP+ A375 cells co-incubated with engineered TCR T cells for 48 hours at a 1:1 T cell to cancer cell ratio. Cancer cell killing was normalized by subtracting the integrated GFP signal in wells containing only A375 cells and no T cells from the integrated GFP signal in a particular sample. TCR = non-transduced T cells. [Figure 17F]Shows that top ranked genes improve antigen-specific TCR T cell responses in solid tumors. Killing of GFP+ A375 cells co-incubated with engineered TCR T cells for 48 hours at a 1:1 T cell to cancer cell ratio. Cancer cell killing was normalized by subtracting the integrated GFP signal in wells containing only A375 cells and no T cells from the integrated GFP signal in a particular sample. TCR = non-transduced T cells. [Figure 18A] Provides an overview of OverCITE-seq. [Figure 18B] Provides an overview of OverCITE-seq. [Figure 18C] Provides an overview of OverCITE-seq. [Figure 18D] Provides an overview of OverCITE-seq. [Figure 18E] Provides an overview of OverCITE-seq. [Figure 18F] Provides an overview of OverCITE-seq. [Figure 18G] Provides an overview of OverCITE-seq. [Figure 19] A listing of clinical trials involving chimeric antigen receptors available on clinicaltrials.gov. [Figure 20] A listing of clinical trials related to T cell receptors available on clinicaltrials.gov. [Figure 21] 1 provides exemplary antibody sequences for construction of chimeric antigen receptors. [Figure 22A] Demonstrate the in vivo efficacy of 19-BB-z CAR T cells co-expressing LTBR against a disseminated leukemia model in NSG mice. Experimental design. Female NSG mice (n=4 per group) were inoculated with 5x105 Nalm6-luc cells in the tail vein. After 4 days, mice were staged using bioluminescence (BLI) measurements to ensure that each group had the same median tumor burden. The next day, non-transduced or CAR-transduced T cells (CD4:CD8, 1:1) were injected into the tail vein. [Figure 22B]Demonstrating the in vivo efficacy of 19-BB-z CAR T cells co-expressing LTBR against a disseminated leukemia model in NSG mice. Survival of mice over the study period. Log-rank Mantel-Cox p-values ​​are shown. [Figure 22C] Demonstrating the in vivo efficacy of 19-BB-z CAR T cells co-expressing LTBR against a disseminated leukemia model in NSG mice. Dorsal and ventral whole body BLI signals. Individual values ​​are shown for all surviving mice. Lines connect the medians of each group. One-way ANOVA with post-hoc Sidak's multiple comparisons test p-values ​​between LTBR and tNGFR groups are shown. ****p<0.0001. [Figure 22D] Demonstrating the in vivo efficacy of 19-BB-z CAR T cells co-expressing LTBR against a disseminated leukemia model in NSG mice. Change in body weight compared to starting weight (d=0) for each mouse. [Figure 23] Demonstrate the viability of LTBR CAR T cells in the absence of IL2. Transduced and selected T cells were expanded and cultured in the presence of IL2 as described above. 14 days after transduction, CAR+LTBR or CAR+tNGFR T cells were washed and divided into two conditions, with and without IL2. Cell viability was then assessed three times a week by direct cell counting using trypan blue exclusion (until day 23) or flow cytometry with a viability dye (from day 23 onwards). At each time point, the number of viable cells in the condition without IL2 was compared to the number of viable cells in the +IL2 condition to determine viability. N=3 [Figure 24A] Demonstrating LTBR phenotype and function in different media. CD4 and CD8 T cells from healthy donors were cultured in given media throughout the experiment, including activation, lentiviral transduction, selection, and culture. 14 days after transduction, T cells were resuspended in the respective media without IL2 and stimulated overnight to induce cytokine secretion. The amount of secreted IFNγ (a) and IL2 (b) was measured by ELISA. The average amount of secreted cytokines in all relevant conditions is shown in c). [Figure 24B]Demonstrating LTBR phenotype and function in different media. CD4 and CD8 T cells from healthy donors were cultured in given media throughout the experiment, including activation, lentiviral transduction, selection, and culture. 14 days after transduction, T cells were resuspended in the respective media without IL2 and stimulated overnight to induce cytokine secretion. The amount of secreted IFNγ (a) and IL2 (b) was measured by ELISA. The average amount of secreted cytokines in all relevant conditions is shown in c). [Figure 24C] Demonstrating LTBR phenotype and function in different media. CD4 and CD8 T cells from healthy donors were cultured in given media throughout the experiment, including activation, lentiviral transduction, selection, and culture. 14 days after transduction, T cells were resuspended in the respective media without IL2 and stimulated overnight to induce cytokine secretion. The amount of secreted IFNγ (a) and IL2 (b) was measured by ELISA. The average amount of secreted cytokines in all relevant conditions is shown in c). [Figure 24D] Demonstrating LTBR phenotype and function in different media. Expression of CD54 and CD74 in CD4 and CD8 T cells transduced with tNGFR or LTBR normalized to non-transduced controls. [Figure 24E] Demonstrating LTBR phenotype and function in different media. Ratio of central memory (CM) to effector T cells in CD4 and CD8 T cells transduced with tNGFR or LTBR. CM: CD45RO+CCR7+, effector: CD45RO+ / -CCR7-. Figure 24F) PD1 expression in CD4 and CD8 T cells transduced with tNGFR or LTBR. **p<0.01 [Figure 25A] 14 demonstrates overexpression of TNFRSF members in primary T cells. Surface expression of selected TNFRSF members in ORF-transduced and non-transduced T cells. [Figure 25B]Demonstrating overexpression of TNFRSF members in primary T cells. CD8 left bar, CD4 right bar. Proliferation of T cells transduced with TNFRSF members 4 days after stimulation with CD3 / CD28 normalized to tNGFR. [Figure 25C] Demonstrating overexpression of TNFRSF members in primary T cells. IFNγ secretion by T cells transduced with TNFRSF members after 24 h stimulation with CD3 / CD28 normalized to tNGFR. CD8 left bar, CD4 right bar. [Figure 26A] Demonstrating overexpression of a constitutively active positive regulator of the NFκB pathway. IFNγ (a) and IL2 (b) secretion after overnight stimulation of CD3 / CD28-transduced T cells. Absolute amounts of secreted cytokines are normalized to LTBR. [Figure 26B] Demonstrating overexpression of a constitutively active positive regulator of the NFκB pathway. IFNγ (a) and IL2 (b) secretion after overnight stimulation of CD3 / CD28-transduced T cells. Absolute amounts of secreted cytokines are normalized to LTBR. [Figure 26C] Demonstrating overexpression of constitutively active positive regulators of the NFκB pathway. Surface expression of representative markers upregulated in LTBR T cells. Expression levels are normalized to LTBR. [Figure 26D] Demonstrating overexpression of constitutively active positive regulators of the NFκB pathway. Surface expression of representative markers upregulated in LTBR T cells. Expression levels are normalized to LTBR. [Figure 26E] Demonstrating overexpression of constitutively active positive regulators of the NFκB pathway. Surface expression of representative markers upregulated in LTBR T cells. Expression levels are normalized to LTBR. [Figure 26F] Demonstrating overexpression of constitutively active positive regulators of the NFκB pathway. Surface expression of representative markers upregulated in LTBR T cells. Expression levels are normalized to LTBR. [Figure 26G]Demonstrating overexpression of a constitutively active positive regulator of the NFκB pathway. Heatmap summary of the phenotype induced by a constitutively active positive regulator of the NFκB pathway in comparison to LTBR. tNGFR is used as an irrelevant gene. [Figure 27A] Demonstrating knockout of negative regulators of the NFκB pathway: NT, TNFAP3, and NFKBIA (left to right). IFNγ (a) and IL2 (b) secretion after overnight stimulation of CD3 / CD28-transduced T cells. Absolute amounts of secreted cytokines are normalized to LTBR co-expressing the NT sgRNA. Each dot represents an individual sgRNA. [Figure 27B] Demonstrating knockout of negative regulators of the NFκB pathway: NT, TNFAP3, and NFKBIA (left to right). IFNγ (a) and IL2 (b) secretion after overnight stimulation of CD3 / CD28-transduced T cells. Absolute amounts of secreted cytokines are normalized to LTBR co-expressing the NT sgRNA. Each dot represents an individual sgRNA. [Figure 27C] Demonstrating knockout of a negative regulator of the NFκB pathway. Surface expression of representative markers upregulated in LTBR T cells. Expression levels are normalized to LTBR co-expressing NT sgRNA. Each dot represents an individual sgRNA. [Figure 27D] Demonstrating knockout of a negative regulator of the NFκB pathway. Surface expression of representative markers upregulated in LTBR T cells. Expression levels are normalized to LTBR co-expressing NT sgRNA. Each dot represents an individual sgRNA. [Figure 27E] Demonstrating knockout of a negative regulator of the NFκB pathway. Surface expression of representative markers upregulated in LTBR T cells. Expression levels are normalized to LTBR co-expressing NT sgRNA. Each dot represents an individual sgRNA. [Figure 27F]Demonstrating knockout of a negative regulator of the NFκB pathway. Surface expression of representative markers upregulated in LTBR T cells. Expression levels are normalized to LTBR co-expressing NT sgRNA. Each dot represents an individual sgRNA. [Figure 27G] Demonstrating knockout of a negative regulator of the NFκB pathway. Heatmap summary of the phenotype induced by knockout of a negative regulator of the NFκB pathway in comparison to LTBR co-expressing NT sgRNA. [Figure 28A] 14. Demonstrating transgene positioning for co-expression of LTBR and CAR. Schematic diagram of the vectors used. [Figure 28B] Demonstrating transgene positioning for co-expression of LTBR and CAR. Expression of LTBR or tNGFR normalized to the corresponding CAR-puro-gene vector in CD4 and CD8 T cells. [Figure 28C] Demonstrating transgene positioning for co-expression of LTBR and CAR. Cytokine secretion upon overnight co-incubation of CAR T cells with CD19+ target cells Nalm6. [Figure 28D] Demonstrating transgene positioning for co-expression of LTBR and CAR. Cytokine secretion upon overnight co-incubation of CAR T cells with CD19+ target cells Nalm6. [Figure 28E] Demonstrating transgene positioning for co-expression of LTBR and CAR. Cytokine secretion upon overnight co-incubation of CAR T cells with CD19+ target cells Nalm6. [Figure 28F] Demonstrating transgene positioning for co-expression of LTBR and CAR. Cytokine secretion in response to target cells, normalized to the corresponding CAR-puro-gene vector, in CD4 and CD8 T cells. [Figure 29A] Demonstrating inducible transgene expression in T cells. Vector design. [Figure 29B]Demonstrating inducible transgene expression in T cells. Expression of LTBR (b) and tNGFR (c) in CD4 and CD8 T cells transduced with the vectors shown in a. T cells were left no stim or stimulated with CD3 / CD28 antibodies for 24 hours. Transgene expression is normalized to staining intensity in T cells transduced with the promoterless vector. [Figure 29C] Demonstrating inducible transgene expression in T cells. Expression of LTBR (b) and tNGFR (c) in CD4 and CD8 T cells transduced with the vectors shown in a. T cells were left no stim or stimulated with CD3 / CD28 antibodies for 24 hours. Transgene expression is normalized to staining intensity in T cells transduced with the promoterless vector. [Figure 29D] 14 demonstrates inducible transgene expression in T cells. Transgene expression in T cells transduced with an NFκB promoter vector compared to expression in T cells transduced with an EFS promoter vector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Engineering a patient's own T cells for adoptive cell therapy is revolutionizing the treatment of several types of cancer. 1 However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screening has been used to identify negative regulators of T cell function. 2-4 These have been limited to 100% and raise safety concerns due to permanent genome modification. Here, we identify positive regulators of T cell function through overexpression of approximately 12,000 barcoded human open reading frames (ORFs). The top ranked genes are primary human CD4 + and CD8 +It increased T cell proliferation and activation and their secretion of key cytokines such as interleukin-2 and interferon-γ. In addition, we developed a single-cell genomics method, OverCITE-seq, for high-throughput quantification of transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF - lymphotoxin-β receptor (LTBR) - is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells, LTBR induces extensive transcriptional and epigenomic remodeling, leading to increased T cell effector function and resistance to exhaustion in a long-term stimulation setting through constitutive activation of the canonical NF-κB pathway. LTBR and other highly ranked genes improved antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cross-cancer therapy. 5 Our results provide several strategies to improve next-generation T-cell therapy through the induction of synthetic cell programs.

[0026] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published documents which provide such persons with general guidance to many of the terms used in this application.

[0027] As used throughout this specification and claims, the terms "comprising," "containing," "including," and variations thereof are inclusive of other components, elements, integers, steps, etc. Conversely, the term "consisting" and variations thereof excludes other components, elements, integers, steps, etc.

[0028] It should be noted that the terms "a" or "an" refer to one or more, e.g., "a T cell" is understood to refer to one or more T cells. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0029] As used herein, the term "about" means a plus or minus 10% variation from a given reference, unless otherwise specified.

[0030] Furthermore, the term "and / or" as used herein should be understood as a specific disclosure of each of the two particular features or components with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include A and B, A or B, A (alone), and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0031] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Qhtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolim et af., Mol. Cell. Probes 8:91-98 (1994)).

[0032] The terms "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence" are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can be either single- or double-stranded DNA or RNA, e.g., mRNA.

[0033] The nucleic acids described herein can be cloned using routine molecular biology techniques or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies with operations in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Life Technologies, Eurofins). The nucleic acid sequences encoding aspects of the CRISPR-Cas editing system described herein can be assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., for example, to generate a non-viral delivery system (e.g., RNA-based systems, naked DNA, etc.) or to generate a viral vector in a packaging host cell and / or to deliver to a host cell of interest, and introduce the sequences carried therein into the host cell. In certain embodiments, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. Methods used to generate such engineered constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0034] "Variants" of proteins or peptides as defined in the context of the present invention can be generated, which have an amino acid sequence that differs from the original sequence by one or more mutations, such as one or more substitutions, insertions, and / or deleted amino acid(s). Preferably, these fragments and / or variants have the same biological function or specific activity, e.g. its specific inhibitory properties, compared to the full-length native protein. "Variants" of proteins or peptides as defined in the context of the present invention can contain conservative amino acid substitution(s) compared to their native, i.e. non-mutated physiological sequence. Substitutions in which an amino acid is exchanged with another amino acid from the same class are called conservative substitutions. In particular, these are amino acids with aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chain or amino acid, side chains that can enter into hydrogen bonds, e.g. side chains with hydroxyl functions. This means, for example, that an amino acid with a polar side chain is replaced by another amino acid that also has a polar side chain, or, for example, that an amino acid characterized by a hydrophobic side chain is replaced by another amino acid that also has a hydrophobic side chain (e.g., threonine (serine) by serine (threonine) or isoleucine (leucine) by leucine (isoleucine). Insertions and substitutions are possible, in particular, at those sequence positions that do not cause changes in the three-dimensional structure or affect the binding region. Insertion(s) or Deletion(s) The alterations to the three-dimensional structure due to the addition of an amino acid can be easily determined, for example, using CD spectroscopy (circular dichroism spectroscopy) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam). Variants can also include unnatural amino acids.

[0035] A "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75, 100 or more amino acids of such protein or peptide, or over the entire length of the protein or peptide.

[0036] The term "gene" can refer to a segment of DNA involved in producing or encoding a polypeptide chain. It can include regions preceding and following the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons).

[0037] As used herein, the terms "coding region" and "coding region," and grammatical variations thereof, refer to an open reading frame (ORF) within a polynucleotide which, upon expression, results in a polypeptide or protein.

[0038] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the term encompasses amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0039] The term "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0040] Unless otherwise specified, a "nucleic acid sequence encoding an amino acid sequence" includes all nucleic acid sequences that are degenerate forms of each other and that encode the same amino acid sequence. A nucleic acid sequence or RNA that encodes a protein may also contain introns, to the extent that a nucleotide sequence encoding a protein may contain an intron(s) in some forms.

[0041] The term "expression" is used herein in the broadest sense and includes production of RNA, production of protein, or production of both RNA and protein. Expression can be transient or stable.

[0042] The terms "expressing" and "overexpression" refer to increasing expression of a gene or protein. The terms refer to an increase in expression, e.g., an increase of at least about 10%, or at least about 20%, or at least about 40%, in the amount of mRNA or expressed protein in a T cell, other lymphocyte, or host cell, compared to the level of a reference control. "expression" refers to an increase of 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%. Various methods for expression and / or overexpression are known to those skilled in the art and include, but are not limited to, stably or transiently introducing a heterologous polynucleotide (i.e., a gene shown in Table 1) encoding a protein to be expressed and / or overexpressed in the cell, or inducing expression or overexpression of an endogenous gene encoding a protein in the cell. It is understood that one or more genes shown in Table 1 can be expressed and / or overexpressed in the cell. It is also understood that two or more genes to be expressed and / or overexpressed in the cell can be selected from one or more of the genes shown in Table 1.

[0043] The term "autologous" refers to any material originating from the same subject into which it is later reintroduced.

[0044] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0045] The term "expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), that incorporate a recombinant polynucleotide.

[0046] As used herein, "expression cassette" refers to a nucleic acid molecule encoding one or more ORFs or genes, such as an effector enhancement gene, or a CAR or TCR or a component thereof. The expression cassette may also contain a promoter and additional regulatory elements that control the expression of one or more elements of the gene editing system in a host cell. In one embodiment, the expression cassette may be packaged into a viral vector capsid (e.g., a viral particle). In one embodiment, such expression cassettes for generating the viral vectors described herein are flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.

[0047] The term "regulatory element" or "regulatory sequence" refers to expression control sequences that are contiguous with a nucleic acid sequence of interest, and that act in trans or at a distance to control the nucleic acid sequence of interest. As described herein, regulatory elements include, but are not limited to, promoters, enhancers, transcription factors, transcription terminators, efficient RNA processing signals such as splicing and polyadenylation signals (polyA), sequences that stabilize cytoplasmic mRNA, e.g., Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE), sequences that improve translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and sequences that enhance secretion of the encoded product, if desired. See also Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleic acid sequence in many types of target cells, and those that direct expression of a nucleic acid sequence only in specific target cells (e.g., tissue-specific regulatory sequences).

[0048] A "promoter" is defined as one or more nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, e.g., in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which may be located as far away as several thousand base pairs from the start site of transcription. The term "constitutive" when referring to a promoter specifies a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a cell to produce a gene product under most or all physiological conditions of the cell. The term "inducible" or "regulatable" when referring to a promoter specifies a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a cell to produce a gene product substantially only when an inducer corresponding to the promoter is present in the cell. In certain embodiments, an inducible promoter is activated in response to T cell stimulation. In certain embodiments, the promoter is an NFAT, AP1, NFκB, or IRF4 promoter. The term "tissue-specific" when referring to a promoter specifies a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter. Additional promoter elements, e.g., enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, such that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decrease. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription. Exemplary promoters include the CMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) promoters.

[0049] The term "operably linked" refers to a functional link between one or more regulatory sequences and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, if necessary, in the same reading frame to link two protein coding regions.

[0050] The term "lentivirus" refers to a genus of the Retroviridae family.Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and because they can deliver a significant amount of genetic information to the DNA of host cells, they are one of the most efficient methods of gene delivery vectors.HIV, SIV, and FIV are all examples of lentiviruses.

[0051] In certain embodiments, the one or more genes are encoded by a nucleic acid sequence delivered to the host cell by a vector or viral vector, many of which are known and available in the art. In one embodiment, a vector is provided that includes an expression cassette as described herein. In one embodiment, the vector is a non-viral vector. In another embodiment, the vector is a viral vector. "Viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a nucleic acid sequence of interest is packaged in a viral capsid or envelope. Examples of viral vectors include, but are not limited to, lentiviruses, adenoviruses, retroviruses (gamma-retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAV), baculoviruses, and herpes simplex viruses. In one embodiment, the viral vector is replication-deficient. "Replication-defective virus" refers to a viral vector in which any viral genomic sequences packaged within the viral capsid or envelope are replication-defective, i.e., they are unable to produce progeny virions, but retain the ability to infect cells.

[0052] The term "lentiviral vector" refers to a vector derived from at least a portion of the lentiviral genome, and specifically includes the self-inactivating lentiviral vectors provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX™ vector system, and the like. Non-clinical types of lentiviral vectors are also available and would be known to one of skill in the art.

[0053] In certain embodiments, the vectors are non-viral plasmids that contain the expression cassettes described herein, e.g., naked DNA, naked plasmid DNA, RNA, and mRNA, and are associated with various compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, poly-glycan compositions and other polymers, lipid and / or cholesterol-based-nucleic acid conjugates, and other constructs such as those described herein. See, e.g., X. Su et al, Mol. Pharmaceutics, 2011, 8(3), pp 774-787; web publication: March 2013; 21, 2011, WO2013 / 182683, WO2010 / 053572, and WO2012 / 170930, all of which are incorporated by reference herein.

[0054] The plasmids, other cloning and expression vectors, their properties, and their construction / manipulation methods that can be used according to the present invention are readily apparent to those skilled in the art. In one embodiment, the expression cassettes described herein are engineered into suitable genetic elements (vectors), such as naked DNA, phages, transposons, cosmids, episomes, etc., that transfer the sequences carried therein, useful for generating viral vectors and / or for introduction into host cells. The selected vector can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. The methods used to create such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0055] The term "transfected" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" cell is one that has been transfected with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0056] As used herein, "transient" refers to expression of a transgene that is not integrated for a period of hours, days, or weeks, which period of expression is less than the period of expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0057] RNA or DNA can be transfected using, for example, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), or electroporation using an ECM830 (BTX) (Harvard Instruments, Boston Scientific). The vector can be introduced into the target cell using any of a number of different methods, including, but not limited to, commercially available methods, including cationic liposome-mediated transfection using a Multiporator (Eppendorf, Hamburg Germany), lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0058] As used herein, the term "subject" means a mammal, including humans, veterinary or agricultural animals, domestic or pet animals, or animals commonly used in clinical research. In one embodiment, the subject of these methods and compositions is a human. Still other suitable subjects include, but are not limited to, mice, rats, dogs, cats, pigs, cows, sheep, non-human primates, and the like. As used herein, the term "subject" is used interchangeably with "patient."

[0059] composition Provided herein are compositions comprising nucleic acids, expression cassettes, and / or lymphocytes comprising sequences encoding genes shown to enhance T cell survival, proliferation, and / or effector function (collectively referred to herein as "effector-enhanced genes"). In certain embodiments, the effector-enhanced genes comprise any of the genes identified in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0060] Expression cassette and cells containing same An expression cassette comprising a nucleic acid sequence encoding one or more effector-enhancing genes is provided according to the present invention. In certain embodiments, the gene comprises any of the genes identified in Table 1 above, or fragments or variants thereof. In other embodiments, the gene comprises any of the genes identified in Table 2 below, or fragments or variants thereof. In certain embodiments, the expression cassette comprises two or more effector-enhancing genes, or fragments or variants thereof. Also provided are host cells containing the nucleic acids and expression cassettes described herein. In certain embodiments, the host cell is a lymphocyte. As used herein, when reference is made to a particular gene in Table 1 or Table 2, it is intended that the use of the coding sequence for the full-length protein, a fragment having a deletion or truncation, or a variant having one or more substitutions in an amino acid is intended. For example, in certain embodiments, the nucleic acid encodes a protein sequence having a deletion or truncation at the N-terminus. In certain embodiments, the nucleic acid encodes a protein sequence having a deletion or truncation at the C-terminus. In one embodiment, the nucleic acid encodes a protein having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, or at least 125 amino acids.

[0061] In one embodiment, the effector-enhancing gene is LTBR. LTBR, ​​a receptor endogenously expressed by professional antigen-presenting cells but not lymphocytes, has been identified as a potent synthetic driver of both T cell proliferation and secretion of key cytokines: IL-2 and IFNγ. Using a multimodal single-cell sequencing approach, it has been shown that LTBR induces profound transcriptional changes when overexpressed in T cells, activating cellular programs involved in antigen presentation and prevention of apoptosis. As described herein, a gene of interest (e.g., LTBR) can be linked to CRISPR targeting other genes. A platform was developed to test combinatorial perturbations in T cells by co-expressing sgRNAs to map signaling networks in T cells. Also, mRNA delivery of LTBR as an alternative to constitutive lentiviral expression is demonstrated herein, highlighting the transferability of our screening approach.

[0062] In certain embodiments, the expression cassette comprises a nucleic acid encoding LTBR, ​​or a fragment thereof. LTBR (lymphotoxin-beta receptor), encoding tumor necrosis factor receptor superfamily member 3, is essential for the development and organization of secondary lymphoid tissues and chemokine release. A representative nucleic acid sequence of LTBR can be found in Accession ID NM_002342.3, SEQ ID NO: 1. The full-length amino acid sequence of LTBR is shown in SEQ ID NO: 2.

[0063] The LTBR protein can be divided into three regions or domains: the extracellular domain (amino acids 31-227 of SEQ ID NO:2), the transmembrane (or helical) domain (amino acids 228-248 of SEQ ID NO:2), and the cytoplasmic (or intracellular) domain (amino acids 249-435 of SEQ ID NO:2). The signal peptide of the immature protein is located at amino acids 1-30 of SEQ ID NO:2.

[0064] In certain embodiments, the expression cassette comprises a nucleic acid encoding a fragment of LTBR. In certain embodiments, the nucleic acid encodes a protein sequence having a deletion of amino acids 2-31, 32-41, 32-151, 32-180, 393-435, 377-435, 324-377, 297-435, or 262-435 compared to the native protein (SEQ ID NO:2). In certain embodiments, the LTBR has a deletion of 378-435, 379-435, 380-435, 381-435, 382-435, 383-435, 384-435, 385-435, 386-435, 387-435, 388-435, 389-435, 390-435, 391-435, 392-435, 393-435, 394-435, 395-435, 396-435, 397-435, 398-435, 399-435, 398-435, 399-435, 390-435, 391-435, 392-435, 393-435, 394-435, 395-435, 396-435, 397-435, 398-435, 39 ...400-4 In certain embodiments, the nucleic acid encodes a protein sequence having a deletion at the N-terminus. In certain embodiments, the nucleic acid encodes a protein sequence having a deletion at the C-terminus. In one embodiment, the LTBR has a deletion of residues 393-435. In certain embodiments, the LTBR has a deletion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, or at least 125 amino acids.

[0065] In certain embodiments, the expression cassette comprises a nucleic acid encoding a fragment that is a domain of LTBR. In certain embodiments, the nucleic acid encodes the extracellular domain of LTBR (amino acids 31-227 of SEQ ID NO:2). In certain embodiments, the nucleic acid encodes the transmembrane domain of LTBR (amino acids 228-248 of SEQ ID NO:2). In certain embodiments, the nucleic acid encodes the cytoplasmic (or intracellular) domain of LTBR (amino acids 249-435 of SEQ ID NO:2). In other embodiments, the domain is a variant of one of the LTBR domains, including a variant having a deletion. Preferred variants of the cytoplasmic domain include those that contain all of amino acids 249-378, 249-379, 249-380, 249-381, 249-382, 249-383, 249-384, 249-385, 249-386, 249-387, 249-388, 249-389, 249-390, 249-391, or 249-392 of SEQ ID NO:2. Further desirable variants include those comprising all of amino acids 249-378, 249-379, 249-380, 249-381, 249-382, 249-383, 249-384, 249-385, 249-386, 249-387, 249-388, 249-389, 249-390, 249-391, or 249-392 of SEQ ID NO:2, which have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to SEQ ID NO:2.

[0066] In other embodiments, the expression cassette comprises a nucleic acid encoding two or more domains of LTBR or fragments thereof. In one embodiment, the nucleic acid encodes the cytoplasmic domain (or a fragment thereof) and the transmembrane domain of LTBR. In another embodiment, the nucleic acid encodes the cytoplasmic domain (or a fragment thereof), the transmembrane domain, and the extracellular domain of LTBR.

[0067] In another embodiment, the expression cassette comprises a nucleic acid encoding AHCY. AHCY encodes the enzyme S-adenosylhomocysteine ​​hydrolase, which catalyzes the reversible hydrolysis of S-adenosylhomocysteine ​​(AdoHcy) to adenosine (Ado) and L-homocysteine ​​(Hcy). A representative nucleic acid sequence for AHCY can be found in accession ID XM_011528656.3.

[0068] In another embodiment, the expression cassette comprises a nucleic acid encoding DUPD1. DUPD1 encodes the enzyme dual specificity phosphatase and proisomerase domain containing 1 (also called DUSP29 - dual specificity phosphatase 29), which can dephosphorylate phosphotyrosine, phosphoserine, and phosphothreonine residues in the same substrate. A representative nucleic acid sequence for DUPD1 can be found in accession ID XM_011539747.2.

[0069] In another embodiment, the expression cassette comprises a nucleic acid encoding AKR1C4, which encodes the enzyme aldo-keto reductase family 1 member C4 (3-alpha-HS D1, CDR, and DD-4) and can catalyze the NADH- and NADPH-dependent reduction of ketosteroids to hydroxysteroids. A representative nucleic acid sequence for AKR1C4 can be found in Accession ID NM_001818.5.

[0070] In another embodiment, the expression cassette comprises a nucleic acid encoding ATF6B. ATF6B encodes activating transcription factor 6 beta (also called cyclic AMP-dependent transcription factor ATF-6 beta). The processed form of ATF-6 beta acts in the unfolded protein response pathway by activating UPR target genes induced by ER stress. A representative nucleic acid sequence of ATF6B can be found in accession ID NM_004381.5.

[0071] In another embodiment, the expression cassette comprises a nucleic acid encoding ITM2A. ITM2A encodes integral membrane protein 2A (also called protein E25), which binds to amyloid-beta. A representative nucleic acid sequence of ITM2A can be found in accession ID NM_004867.5.

[0072] In another embodiment, the expression cassette comprises a nucleic acid encoding AHNAK. AHNAK encodes the neuroblast differentiation associated protein AHNAK (also called AHNAK nuclear protein). The encoded protein may play a role in diverse processes such as blood-brain barrier formation, cell structure and migration, cardiac calcium channel regulation, and tumor metastasis. A representative nucleic acid sequence for AHNAK can be found in accession ID XM_017018270.1.

[0073] In another embodiment, the expression cassette comprises a nucleic acid encoding BATF. BATF encodes basic leucine zipper transcription factor ATF-like (also called B-cell activating transcription factor (B-ATF) and SF-HT activating gene 2 protein (SFA-2)), an AP-1 family transcription factor that controls the differentiation of lineage-specific cells in the immune system. A representative nucleic acid sequence for BATF can be found in accession ID NM_006399.5.

[0074] In another embodiment, the expression cassette comprises a nucleic acid encoding GPD1. GPD1 encodes glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic (also called GPD-C and GPDH-C). A representative nucleic acid sequence for GPD1 is provided in Accession ID No. It can be found in NM_005276.4.

[0075] In another embodiment, the expression cassette comprises a nucleic acid encoding GPN3. GPN3 encodes GPN-loop GTPase3 (also called ATP-binding domain 1 family member C), a small GTPase required for proper localization of RNA polymerase II. A representative nucleic acid sequence of GPN3 can be found in accession ID XM_017019394.1.

[0076] In another embodiment, the expression cassette comprises a nucleic acid encoding MRPL51. MRPL51 encodes a small GTPase, GPN-loop GTPase3 (also called ATP-binding domain 1 family member C), which is required for proper localization of RNA polymerase II. A representative nucleic acid sequence for MRPL51 can be found in accession ID NM_016497.4.

[0077] In another embodiment, the expression cassette comprises a nucleic acid encoding DBI. DBI encodes diazepam binding inhibitor (also called ACBD1, ACBP, CCK-RP, EP), a protein that is regulated by hormones and is involved in lipid metabolism and displacement of beta-carbolines and benzodiazepines. A representative nucleic acid sequence for DBI is provided in Accession ID N It can be found at M_001282635.3.

[0078] In another embodiment, the expression cassette comprises a nucleic acid encoding CALML3. CALML3 encodes calmodulin-like 3 (also called CLP), a protein that enhances myosin-10 translation. A representative nucleic acid sequence for CALML3 can be found in accession ID NM_005185.4.

[0079] In another embodiment, the expression cassette comprises a nucleic acid encoding IL12B, which encodes interleukin 12B (also known as CLMF, CLMF2, IL-12B, IMD28, IMD29, NKSF, NKSF2), a cytokine that acts on T cells and natural killer cells and has a broad range of biological activities. A representative nucleic acid sequence for IL12B can be found in Accession ID NM_002187.3.

[0080] In another embodiment, the expression cassette comprises a nucleic acid encoding IFNL2. IFNL2 encodes interferon lambda 2 (also called IL28A, IFNL2a, IFNL3a, IL-28A). This gene, interleukin 28B (IL28B), and interleukin 29 (IL29) are three closely related cytokine genes that form a cytokine gene cluster in a chromosomal region mapped to 19q13. Expression of the cytokines encoded by the three genes can be induced by viral infection. All three cytokines have been shown to interact with a heterodimeric class II cytokine receptor consisting of interleukin 10 receptor, beta (IL10RB) and interleukin 28 receptor, alpha (IL28RA). A representative nucleic acid sequence for IFNL2 can be found in accession ID NM_172138.2.

[0081] In another embodiment, the expression cassette comprises a nucleic acid encoding ADA. ADA encodes adenosine deaminase (also called ADA1, IFNL2a, IFNL3a, IL-28A). This gene encodes an enzyme that catalyzes the hydrolysis of adenosine to inosine in the purine catabolic pathway. A representative nucleic acid sequence of ADA can be found in accession ID NM_000022.4.

[0082] Various isoforms of the genes identified above are known in the art. Some are listed in Table 2 below. In another embodiment, an expression cassette is provided that includes the coding sequence of any of the alternative isoforms. Alternative coding sequences that account for the degeneracy of the genetic code, including codon-optimized coding sequences, for these genes can be identified by one of skill in the art and utilized as alternative embodiments of the compositions and methods described herein. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0083] In other embodiments, the expression cassette comprises a nucleic acid encoding a gene selected from the genes in Table 1.

[0084] Engineered T cell receptors The present disclosure provides nucleic acid sequences encoding engineered T cell receptors, e.g., T cell receptors (TCRs), modified TCRs as described herein, and chimeric antigen receptors (CARs), for expression in T cells having nucleic acid sequences encoding genes that alter T cell effector function. Components of TCRs and CARs are further described herein.

[0085] TCRs are disulfide-linked membrane-anchored heterodimers present in T cell lymphocytes, and the majority of T cells are αβ T cells, with TCRs consisting of an alpha (α) chain and a beta (β) chain. Each chain contains a variable (V) and a constant (C) domain, with the variable domain recognizing an antigen, or an MHC-presented peptide. TCRα and TCRβ chains with known specificity or affinity for a particular antigen, e.g., a tumor antigen as described herein, can be introduced into T cells using the methods described herein. TCRα and TCRβ chains with desired, e.g., increased specificity or affinity for a particular antigen can be isolated using standard molecular cloning techniques known in the art. Other modifications that increase the specificity, affinity, or function of a TCR or an engineered T cell expressing a TCR, e.g., promoter selection for regulated expression, mutations in the antigen-binding regions of the TCRα and TCRβ chains, can be readily envisioned by one of skill in the art. Any isolated or modified TCRα and TCRβ chains can be operably linked to or associated with one or more intracellular signaling domains as described herein. Signal transduction can be mediated through interactions between the antigen-binding αβ heterodimer and a CD3 chain molecule, such as CD3 zeta (ζ).

[0086] A smaller subset of T cells expresses TCRs with gamma (γ) chains and delta (δ) chains. Gamma-delta (γδ) T cells constitute 3-10% of circulating lymphocytes in humans, and the Vδ2+ subset can account for up to 95% of γδ T cells in the blood. Vδ2+ cells recognize non-peptide epitopes and do not require antigen presentation by major histocompatibility complexes ("MHC") or human leukocyte antigens ("HLA"). Most Vδ2+ T cells also express Vγ9 chains and are stimulated by exposure to 5-carbon pyrophosphate compounds that are intermediates in the mevalonate and non-mevalonate sterol / isoprenoid synthesis pathways. Responses to isopentenyl pyrophosphate (5-carbon) are universal among healthy humans. Another subset of γδ T cells, Vδ1+, constitutes a much smaller percentage of T cells circulating in the blood, but is commonly found in epithelial mucosa and skin. γδ T cells have several functions, including killing tumor cells and pathogen-infected cells. Stimulation through γδ TCR improves the capacity for cytotoxicity, cytokine secretion, and other effector functions. The TCR of γδ T cells has unique specificity, and the cells themselves occur at high clonal frequency, thus enabling rapid innate responses against tumors and pathogens. See, e.g., Park and Lee, Exp Mol Med. 2021 Mar;53(3):318-327, incorporated herein by reference.

[0087] In certain embodiments, the T cell comprises a nucleic acid sequence encoding a TCR, e.g., a modified TCR that targets a tumor antigen as described herein, and a nucleic acid sequence encoding a gene. In any of the embodiments described herein, the TCR can be replaced with a CAR as described herein to generate the T cell. The engineered TCR as described herein can be replaced with a CAR in any of the embodiments described herein. In certain embodiments, an engineered TCR that targets NY-ESO-1 (SEQ ID NOs: 23 and 24) (see, e.g., Thomas et al., NY-ESO-1 Based Immunotherapy of Cancer: Current Perspectives, Front. Immunol., 01 May 2018, incorporated herein by reference).

[0088] In certain embodiments, the T cells comprise a TCR identified in Figure 20. In certain embodiments, the TCR targets MART-1. Chodon T, et al, Adoptive transfer of MART -1 T - cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma. Clin Cancer Res. 2014 May 1; 20(9):2457-65. doi:10.1158 / 1078-0432. CCR-13-3017. Epub 2014 Mar 14. PMID:24634374; PMCID:PMC4070853. In another embodiment, the TCR targets MAGE A4. Hong et al, Phase I dose escalation and expansion trial to assess the safety and efficacy of ADP-A2M4 SPEAR T cells in advanced solid tumors. ASCO Meeting Library, 2020 ASCO Virtual Scientific Program, J Clin Oncol 38:2020(suppl;abstr 102). In another embodiment, the TCR targets WT1. Chapuis AG, et al. T cell receptor gene therapy targeting WT1 prevents acute myeloid leukemia relapse post-transplant. Nat Med. 2019 Jul;25(7):1064-1072. doi:10.1038 / s41591-019-0472-9. Epub 2019 Jun 24. PMID:31235963; PMCID:PMC6982533. In other embodiments, the TCR targets MR1. Crowther, MD, Dolton, G., Legut, M. et al. Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1. Nat Immunol 21, 178-185 (2020). https: / / doi.org / 10.1038 / s41590-019-0578-8. In other embodiments, the TCR targets E6. In other embodiments, the TCR targets E7. In other embodiments, the TCR targets KK-LC-1. In other embodiments, the TCR targets NY-ESO-1.In another embodiment, the TCR targets MAGE A3. In another embodiment, the TCR targets GD-2. In another embodiment, the TCR targets P53. In another embodiment, the TCR targets LAGE-A1. In another embodiment, the TCR targets GP100.

[0089] Chimeric antigen receptors (CARs) The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct that comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to as an intracellular signaling domain) that comprises a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the stimulatory molecule is TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, 4-1BB, or CD3 zeta. In certain embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In certain embodiments, the stimulatory molecule is 4-1BB. In certain embodiments, the stimulatory molecule is CD28. In certain embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains (also referred to as "costimulatory signaling domains") derived from at least one costimulatory molecule as defined below. In certain embodiments, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD11a / CD18), ICOS, and 4-1BB (CD137), or any combination thereof. In certain embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain (primary signaling domain) derived from a stimulatory molecule. In certain embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain (primary signaling domain) derived from a costimulatory molecule. In certain embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule (primary signaling domain). In certain embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In certain embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In certain embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In certain embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, which is optionally cleaved from the scFv domain during cell processing and localization of the CAR to the cell membrane.

[0090] The present disclosure provides a nucleic acid sequence, e.g., a DNA or RNA construct, encoding a CAR, the CAR comprising an antibody fragment that binds to a disease-associated antigen. In certain embodiments, the sequence encoding the antibody fragment is contiguous with and in the same reading frame as the nucleic acid sequence encoding the intracellular domain. The intracellular domain comprises a costimulatory signaling region and / or a zeta chain. The costimulatory signaling region refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule.

[0091] In certain embodiments, the CAR construct comprises an optional leader sequence, an extracellular antigen binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain. In certain embodiments, the CAR construct comprises an optional leader sequence, an extracellular antigen binding domain, a hinge, a transmembrane domain, an intracellular costimulatory domain, and an intracellular stimulatory domain.

[0092] In certain embodiments, the expression cassette includes one encoding one or more components of a chimeric antigen receptor in addition to an effector-enhanced gene. For example, in one embodiment, a single expression cassette is provided that includes a coding sequence for an effector-enhanced gene and a coding sequence for a chimeric fusion protein that includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0093] In certain embodiments, the CAR targets CD19. In one embodiment, the CAR is axicabtagene siloreucel. In another embodiment, the CAR is brexcabtagene outeucel. In another embodiment, the CAR is tisagenlecleucel. In another embodiment, the CAR is lysocabtagene maraleucel. In another embodiment, the CAR is idecabtagene biculeucel.

[0094] In one embodiment, an expression cassette is provided comprising the coding sequence for LTBR and axicabtagene siloreucel. In another embodiment, an expression cassette is provided comprising the coding sequence for LTBR and brexcabtagene outelucel. In another embodiment, an expression cassette is provided comprising the coding sequence for LTBR and tisagenlecleucel. In another embodiment, an expression cassette is provided comprising the coding sequence for LTBR and lysocabtagene maraleucel. In another embodiment, an expression cassette is provided comprising the coding sequence for LTBR and idecabtagene biculeucel.

[0095] In another embodiment, an expression cassette is provided that includes the coding sequence of AHCY, DUPD1, AKR1C4, ATF6B, ITM2A, AHNAK, BATF, GPD1, GPN3, MRPL51, DBI, CALML3, IL12B, IFNL2, or ADA, and axicabtagene siloleucel. In another embodiment, an expression cassette is provided that includes a coding sequence for AHCY, DUPD1, AKR1C4, ATF6B, ITM2A, AHNAK, BATF, GPD1, GPN3, MPRL51, DBI, CALML3, IL12B, IFNL2, or ADA, and brexcavtagene outrousel. In another embodiment, an expression cassette is provided that includes a coding sequence for AHCY, DUPD1, AKR1C4, ATF6B, ITM2A, AHNAK, BATF, GPD1, GPN3, MRPL51, DBI, CALML3, IL12B, IFNL2, or ADA, and tisagenlecleucel. In another embodiment, an expression cassette is provided that includes the coding sequence of AHCY, DUPD1, AKR1C4, ATF6B, ITM2A, AHNAK, BATF, GPD1, GPN3, MRPL51, DBI, CALML3, IL12B, IFNL2, or ADA, and lysocabtagene maraleucel.In another embodiment, an expression cassette is provided that includes the coding sequence of AHCY, DUPD1, AKR1C4, ATF6B, ITM2A, AHNAK, BATF, GPD1, GPN3, MRPL51, DBI, CALML3, IL12B, IFNL2, or ADA, and idecbutagen biculeucel.

[0096] In another embodiment, an expression cassette is provided that includes any of the genes in Table 1 and a coding sequence for axicabtagene siloreucel. In another embodiment, an expression cassette is provided that includes any of the genes in Table 1 and a coding sequence for brexcabtagene outeucel. In another embodiment, an expression cassette is provided that includes any of the genes in Table 1 and a coding sequence for tisagenlecleucel. In another embodiment, an expression cassette is provided that includes any of the genes in Table 1 and a coding sequence for lysocabtagene malareucel. In another embodiment, an expression cassette is provided that includes any of the genes in Table 1 and a coding sequence for idecabtagene biclueucel.

[0097] In certain embodiments, the CAR targets mesothelin. In certain embodiments, the CAR targets ROR1. In certain embodiments, the CAR targets B7-H3. In certain embodiments, the CAR targets CD33. In certain embodiments, the CAR targets EGFR806. In certain embodiments, the CAR targets IL13Rα2. In certain embodiments, the CAR targets GD2. In certain embodiments, the CAR targets HER2. In certain embodiments, the CAR targets glypican 3. In certain embodiments, the CAR targets CD7. In certain embodiments, the CAR targets NY-ESO-1. In certain embodiments, the CAR targets CD30. In certain embodiments, the CAR targets MAGE-A1. In certain embodiments, the CAR targets LMP2. In certain embodiments, the CAR targets PD1. In certain embodiments, the CAR targets mutant KRAS G12V. In certain embodiments, the CAR targets CD20. In certain embodiments, the CAR targets CD22. In certain embodiments, the CAR targets CD171. In certain embodiments, the CAR targets CD123. In certain embodiments, the CAR targets CD38. In certain embodiments, the CAR targets CD10. In certain embodiments, the CAR targets BAFFR. In certain embodiments, the CAR targets PSMA. In certain embodiments, the CAR targets mucin (TnMUC1). Posey AD Jr, et al, Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma. Immunity. 2016 Jun 21; 44(6): 1444-54. doi: 10.1016 / j.immuni.2016.05.014. PMID: 27332733; PMCID: PMC5358667. In certain embodiments, the CAR targets CD70.Srinivasan et al,1972 Investigation of ALLO-316:A Fratricide-Resistant Allogeneic CAR T Targeting CD70 As a Potential Therapy for the Tr. Food intake of AML,62 nd See ASH Annual Meeting and Exposition, Dec. 5-8, 2020. In certain embodiments, the CAR targets TRIB1C. Maciocia PM, et al, Targeting the T cell receptor β-chain constant region for immunotherapy of T cell malignancies. Nat Med. 2017 Dec;23(12):1416-1423. doi:10.1038 / nm.4444. Epub 2017 Nov 13. PMID:29131157.

[0098] A variety of other chimeric antigen receptors are known in the art or can be designed by one of skill in the art. Such CARs include those currently being clinically tested, such as those identified in Figure 19. Clinical trial information can be found at ClinicalTrials.gov using the NCT numbers provided. In an alternative embodiment, an expression cassette is provided that includes the coding sequence of any of the genes in Table 1 and the CAR identified in Figure 19. In another embodiment, an expression cassette is provided that includes the coding sequence of the LTBR and the CAR identified in Figure 19.

[0099] Other chimeric antigen receptors include those useful for the treatment of autoimmune diseases, such as chimeric autoantigen receptors (CAARs). Such CAARs include DSG3-CAART and MuSK-CAART. Others may be known in the art or may be designed by one of skill in the art. In an alternative embodiment, an expression cassette is provided that includes any of the genes in Table 1 and a coding sequence for CAAR. In another embodiment, an expression cassette is provided that includes a coding sequence for LTBR and CAAR. In another embodiment, an expression cassette is provided that includes a coding sequence for AHCY, DUPD1, AKR1C4, ATF6B, ITM2A, AHNAK, BATF, GPD1, GPN3, MRPL51, DBI, CALML3, IL12B, IFNL2, or ADA, and a CAAR.

[0100] Exemplary sequences of the CARs and TCRs described herein are provided below in Examples 2, 3, and 4. Other exemplary antibody sequences useful for constructing CARs are provided in FIG.

[0101] In certain instances, the expression cassette includes the coding sequence for a gene in Table 1 and a follicle stimulating hormone immunoreceptor, such as that described by Powell et al., WO2016 / 073456, incorporated herein by reference.

[0102] In certain embodiments, the expression cassette comprises one or more components including an engineered T cell receptor (TCR) in addition to an effector enhancement gene. For example, in one embodiment, a single expression cassette is provided that comprises a coding sequence for an effector enhancement gene and a coding sequence for an engineered TCR including TCR alpha and beta chains. In one embodiment, an expression cassette is provided that comprises a coding sequence for an LTBR and a TCR. In another embodiment, an expression cassette is provided that comprises a coding sequence for AHCY, DUPD1, AKR1C4, ATF6B, ITM2A, AHNAK, BATF, GPD1, GPN3, MRPL51, DBI, CALML3, IL12B, IFNL2, or ADA, and a TCR. In another embodiment, an expression cassette is provided that comprises a coding sequence for any of the genes in Table 1 and a TCR.

[0103] A variety of other engineered T cell receptors are known in the art or can be designed by one of skill in the art. Such TCRs include those currently being clinically tested, such as those identified in FIG. 20. In an alternative embodiment, an expression cassette is provided that includes the coding sequence for any of the genes in Table 1 and a TCR identified in FIG. 20. In another embodiment, an expression cassette is provided that includes the coding sequence for an LTBR and a TCR identified in FIG. 20. It will be offered.

[0104] In certain embodiments of the nucleic acids provided herein, the effector enhancement gene is provided in an expression cassette together with the components for the CAR or TCR. In other embodiments, the effector enhancement gene is provided in a separate expression cassette from the CAR or TCR components.

[0105] In certain embodiments, it is desirable to downregulate or silence certain other genes in conjunction with the expression of the effector enhancing gene and CAR or TCR. Such genes include, for example, genes of the NFκB pathway, such as TNFAIP3 and NFKBIA. Compositions and methods for downregulating or silencing genes are known in the art, and include, for example, siRNA, miRNA, CRISPR / CAS, etc. In certain embodiments, sgRNA is provided that targets the gene of interest in conjunction with the delivery of CAS protein, as described in Example 10.

[0106] In another embodiment, a composition is provided that includes a nucleic acid encoding an effector-enhancing gene and a nucleic acid encoding a viral protein. Desirable viral proteins include glycoproteins such as spike protein, E2 protein, E1 protein, and haemaglutinin. In one embodiment, the viral protein is a coronavirus spike protein. There are at least 16 different HAs, including subtypes H1-H16. H1, H2, and H3 are found in human influenza viruses. Another HA of interest is H5, found in avian influenza virus H5N1. For example, the viral protein may include any HA of subtypes H1-H16. Other suitable viral glycoproteins include, but are not limited to, dengue virus envelope glycopolypeptide, hepatitis C virus envelope glycopolypeptide E1, hepatitis C virus envelope glycopolypeptide E2, hantavirus envelope glycopolypeptide G1, hantavirus envelope glycopolypeptide G2. The hantavirus envelope glycopolypeptides G1 and G2 are optionally from the Andean, Hantan, or Sin Nombre strains of hantavirus. Viral glycopolypeptides also include human cytomegalovirus glycopolypeptide B, human cytomegalovirus glycopolypeptide H, human herpesvirus-8 glycopolypeptide B, human herpesvirus-8 glycopolypeptide H, human metapneumovirus glycopolypeptide F, human metapneumovirus glycopolypeptide G, human parainfluenza virus humaglutinin-neuraminidase, human parainfluenza virus fusion glycopolypeptide, Nipah virus glycopolypeptide F, Nipah virus glycopolypeptide G, respiratory syncytial virus glycopolypeptide F, respiratory syncytial virus glycopolypeptide G, severe acute respiratory syndrome (SARS) virus spike glycopolypeptide, Westinal virus envelope glycopolypeptide, and HIV-1 envelope glycopolypeptide.The HIV-1 envelope glycopolypeptide is optionally YU2 Env, SF162 Env, Env from HIV-1 strain B, Env from HIV-1 strain C, and Env from HIV-1 strain M. In certain embodiments, the coding sequence for the effector-enhancing gene and / or viral protein is provided as mRNA.

[0107] As used herein, the term "expression cassette" refers to a nucleic acid molecule that encodes one or more biologically useful nucleic acid sequences (e.g., genes, cDNAs encoding proteins, enzymes, or other useful gene products, mRNAs, etc.) and, operably linked thereto, regulatory sequences that direct or regulate the transcription, translation, and / or expression of the nucleic acid sequence(s) and its gene product(s). Such regulatory sequences typically include, for example, promoters, enhancers, introns, Kozak sequences, polyadenylation sequences, and TATA signals. The expression cassette may contain, among other elements, one or more of a regulatory sequence upstream (5') of the gene sequence, such as one or more of a promoter, enhancer, intron, etc., and an enhancer, or one or more regulatory sequences downstream (3') of the gene sequence, such as one or more 3' untranslated regions including a polyadenylation site. Thus, in addition to the coding sequence of the effector-enhanced gene (and / or CAR or TCR), the expression cassette may also include expression control sequences.

[0108] The expression control sequence includes a promoter. In some embodiments, it is desirable to utilize a promoter with high transcriptional activity, among others. Certain strong constitutive promoters are known in the art, including, but not limited to, CMV promoter, EF-1α promoter, EFS promoter, CBG promoter, CB7 promoter, hPGK, RPBSA, WAS promoter, and the like. Alternatively, other promoters, such as regulatable (inducible) promoters [see, for example, WO2011 / 126808 and WO2013 / 049493, which are incorporated by reference herein], or promoters that respond to physiological cues, can be utilized. In certain embodiments, it is desirable to utilize a system in which LTBR (or other effector-enhanced genes) are expressed only after the T cell encounters its target cell and receives a signal via the antigen receptor (CAR or TCR); once the target cell is removed and the antigen receptor no longer transmits a signal, effector-enhanced gene expression decays back to background levels. Thus, in certain embodiments, it is desirable to utilize a promoter that responds to T cell activation. Such promoters include, but are not limited to, NFAT, NFκB, and AP1 promoters.

[0109] The expression cassette may also, in certain embodiments, include one or more IRES or 2A sequences, allowing expression of multiple coding sequences from the same expression cassette. As exemplified herein, in one embodiment, a CAR directed to CD19 is provided with an ORF directed to one of the genes identified in Table 1, e.g., LTBR. For a lentiviral vector including a 2A sequence, see FIG. 5A. An exemplary P2A sequence is shown in SEQ ID NO: 59. The construction of such cassettes and vectors is known in the art and described in the Examples herein. See, e.g., Sack et al. Profound Tissue Specificity in Proliferation Control Underlies Cancer Drivers and Aneuploidy Patterns. Cell. 2018 Apr 5; 173(2): 499-514. e2, and Yang et al. A public genome-scale lentiviral expression library of human ORFs, Nat Methods. 2011 Aug; 8(8): 659-661, which are incorporated herein by reference.

[0110] The arrangement of the coding sequences of the various components of the construct can be varied. For example, in certain aspects, it is desirable to place the effector enhancement gene coding sequence upstream of the CAR coding sequence. In other embodiments, it is desirable to place the effector enhancement gene coding sequence downstream of the CAR coding sequence. In other embodiments, a selectable marker gene is included in the construct.

[0111] In certain aspects, provided herein are compositions comprising modified lymphocytes comprising a nucleic acid and / or expression cassette as described herein. In one embodiment, the host lymphocyte is a T cell. In another embodiment, the host lymphocyte is a natural killer (NK) cell. In certain embodiments, the composition comprises a cell population comprising a mixed population of lymphocytes (e.g., alpha beta T cells and NK T cells). In other embodiments, the composition comprises cells comprising a population enriched for a particular lymphocyte population.

[0112] As used herein, the phrase "T cells" refers to lymphocytes that express T cell receptor molecules. T cells include human alpha beta (αβ) T cells and human gamma delta (γδ) T cells. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells, cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subpopulations thereof. T cells can be CD4+, CD8+, or CD4+ and CD8+. T cells can also be CD4-, CD8-, or CD4- and CD8-. T cells can be helper cells, such as T H 1. T H 2. T H 3. T H 9. T H 17 or T FH The T cells can be of the CD4+CD25 type. The T cells can be cytotoxic T cells. The T cells can also be regulatory T cells. Regulatory T cells (Treg) can be FOXP3+ or FOXP3-. The T cells can be alpha / beta T cells or gamma / delta T cells. In some cases, the T cells are CD4+CD25 hi CD127 lo In some cases, the T cells are type 1 regulatory (Tr1), T H In some embodiments, the T cells are regulatory T cells selected from the group consisting of FOXP3, CD8+CD28-, Treg17, and Qa-1 restricted T cells, or combinations or subpopulations thereof.+ T cells. In some cases, the T cells are CD4 + CD25 lo CD127 hi Optionally, the T cell is a CD4 + CD25 lo CD127 hi CD45RA hi The T cells are CD45RO- naive T cells. In certain embodiments, the T cells are Vy9V52 T cells. In some embodiments, the T cells express a viral antigen. In other embodiments, the T cells express a cancer antigen. The T cells can be recombinant T cells that have been genetically engineered.

[0113] As used herein, the term "primary" in the context of primary cells refers to cells that have not been transformed or immortalized. Such primary cells can be cultured, subcultured, or passaged a limited number of times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some cases, primary cells are adapted to in vitro culture conditions. In some cases, primary cells are isolated from an organism, system, organ, or tissue, optionally selected, and utilized directly without culturing or subculturing. In some cases, primary cells are stimulated, activated, or differentiated. For example, primary T cells can be activated by contact with (e.g., cultured in the presence of) CD3, CD28 agonist, IL-2, IFN-γ, or a combination thereof.

[0114] method Methods for Producing Engineered Host Cells Also provided herein are methods for producing modified cells as described herein and compositions containing modified cells.Methods for modifying cells, such as lymphocytes, to introduce exogenous sequences, such as expression cassettes or expression vectors that contain the coding sequences of effector-enhancing genes, CARs, or TCRs, or two or more of these sequences, are known in the art.See, for example, WO2016 / 109410A2, which is incorporated herein by reference.In certain embodiments, two or more exogenous sequences are introduced.

[0115] By the term "modified" as used herein is meant an altered state or structure of a molecule or cell of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids. Modifying can refer to, for example, changing the expression of a gene in a lymphocyte by introducing an exogenous nucleic acid encoding the gene.

[0116] The lymphocytes provided herein can be genetically modified, e.g., by transfection, transduction, or electroporation, to express a nucleic acid sequence encoding a gene, as described herein. Depending on the clinical situation, e.g., the patient's condition or the condition to be treated, long-term or permanent expression of the gene and / or, e.g., robust and long-lasting CAR activity, e.g., anti-tumor activity, may be desired. In such embodiments, the lymphocytes are genetically modified, e.g., transduced, e.g., virally transduced, using a vector comprising a nucleic acid sequence encoding a gene disclosed herein to confer the desired effector function. In other embodiments, transient expression of the gene is desired. In such embodiments, the use of, e.g., mRNA or a regulatable promoter to express the effector-enhancing gene may be used.

[0117] Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into a host cell, such as a mammalian, bacterial, yeast, or insect cell, by any known method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0118] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0119] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc.

[0120] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of targeted delivery of nucleic acid are available, such as delivery of polynucleotides by targeted nanoparticles or other suitable submicron-sized delivery systems. The use of lipid formulations is contemplated for the introduction of nucleic acid into host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acid can be associated with lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that is associated with both the liposome and the polynucleotide, entrapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist as bilayer structures, micelles, or in "collapsed" structures. They may also simply be interspersed in the solution, or in some cases may form aggregates that are not uniform in size or shape. The lipid may be a fatty lipid, which may be a naturally occurring lipid or a synthetic lipid. Substances. For example, lipids include the lipid droplets that naturally occur in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Lipofectamine-nucleic acid complexes are also contemplated.

[0121] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose the cells to an inhibitor of the invention, various assays can be performed to confirm the presence of recombinant nucleic acid sequences within the host cell. Such assays include, for example, Southern and Northern blotting, RT-PCR and PCR, biochemical assays such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blots).

[0122] In certain embodiments, an expression vector is provided that includes a coding sequence of an effector-enhancing gene. In other embodiments, the expression vector includes a coding sequence of one or more components of a CAR or TCR. In other embodiments, a separate expression vector is provided that includes a coding sequence of one or more components of a CAR or TCR. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides. In one embodiment, the expression vector is a lentivirus. When more than one expression vector is utilized, each expression vector can be individually selected from those known in the art.

[0123] Provided herein is a method for generating a population of immune effector cells (e.g., T cells, NK cells) that are modified to express the effector-enhancing genes described herein, and optionally to express a CAR or TCR. The method for generating such immune cells includes introducing an exogenous nucleic acid into cells that encodes a gene selected from those in Table 1. In the examples described below, for convenience, a method for generating modified T cells is described. However, alternative embodiments are envisioned to use other types of immune cells, such as NK T cells or NK cells. Suitable methods are known in the art.

[0124] Briefly, an exemplary method includes providing a population of immune effector cells (e.g., T cells) and, optionally, removing T regulatory cells, e.g., CD25+ T cells, from the population. In certain embodiments, the population of immune effector cells is autologous to the subject to whom the cells are administered for therapy. In certain embodiments, the population of immune effector cells comprises autologous Vy9V52 T cells. In certain embodiments, the population of immune effector cells is allogeneic to the subject to whom the cells are administered for therapy. In one embodiment, the T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody, or a fragment thereof, or a CD25 binding ligand, e.g., IL-2. In one embodiment, the anti-CD25 antibody, or a fragment thereof, or a CD25 binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated to a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, or a fragment thereof, is conjugated to a substrate as described herein. In one embodiment, T regulatory cells, e.g., CD25+ T cells, are depleted from the population using an anti-CD25 antibody molecule, or a fragment thereof. In another embodiment, the CD25+ cells are not depleted.

[0125] Another exemplary method includes providing a population of immune effector cells (e.g., T cells) and enriching the population for CD8+ and / or CD4+ cells. In one embodiment, the population is enriched for CD8+ and / or CD4+ cells using anti-CD8 and / or anti-CD4 antibodies, or fragments thereof, or CD8-binding ligands and / or CD4-binding ligands. In one embodiment, the anti-CD4 and / or anti-CD8 antibodies or fragments thereof, or anti-CD4 and / or anti-CD8 binding ligands are conjugated to or otherwise coated on a substrate, such as a bead.

[0126] In certain embodiments, the method further comprises delivering to the cells one or more vectors comprising a gene selected from those in Table 1, e.g., LTBR, ​​and optionally a nucleic acid encoding a CAR or TCR. In certain embodiments, the vector is selected from DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In certain embodiments, cells from a population of T cells are transduced once with the vector, e.g., within one day after a population of immune effector cells is obtained from a blood sample from a subject, e.g., by apheresis. In certain embodiments, the method further comprises generating a population of RNA-engineered cells that transiently express an exogenous RNA from the population of T cells. The method comprises introducing in vitro transcribed RNA or synthetic RNA into cells from the population, the RNA comprising a nucleic acid encoding a gene in Table 1, e.g., LTBR. For example, in certain embodiments, a population of T cells can be transduced with a vector comprising a nucleic acid encoding a CAR, and then the same cell population can be transduced with an mRNA encoding a gene in Table 1, e.g., LTBR.

[0127] In one embodiment, the modified cells described herein are expanded. In one embodiment, the cells are expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In one embodiment, the cells are expanded in culture for 3 or 4 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. Potency can be defined, for example, by various T cell functions, such as proliferation, target cell killing, cytokine production, activation, migration, or a combination thereof.

[0128] Treatment method Also provided herein in certain aspects is a method of defeating cancer in a subject. In certain embodiments, the method comprises administering to the subject a cell expressing an effector-enhancing gene as described herein, thereby defeating cancer in the subject. In certain embodiments, the cell further expresses a CAR. In other embodiments, the cell further expresses a TCR. When used herein to describe the method of defeating, LTBR is used as an exemplary effector-enhancing gene for convenience. However, in alternative embodiments, other genes in Table 1 are used. In certain embodiments, the method comprises obtaining cells from a patient, modifying the cells as described herein, and administering the cells to the patient.

[0129] An example of a cancer treatable by effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells) is a hematological cancer. In certain embodiments, hematological cancers include, but are not limited to, malignant lymphoproliferative conditions including leukemias (such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, and myelodysplastic syndromes) and lymphomas (such as multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and small cell and large cell follicular lymphoma). In other embodiments, hematological cancers can include, for example, minimal residual disease, MRD, of leukemias, such as AML or MDS. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is pancreatic cancer, melanoma, multiple myeloma, sarcoma, or lung cancer. In another embodiment, the cancer is a follicle-stimulating hormone-associated cancer. Such cancers include breast cancer, lung cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, kidney cancer, cervical cancer, These include liver cancer, ovarian cancer, and testicular cancer. In another embodiment, the cancer is a cancer listed in FIG. 19 or FIG.

[0130] In certain embodiments, the CAR is selected from axicabtagene siloreucel (Yescarta®), brexcabtagene outelucel (Tecartus™), idecabtagene bicurucel (Abecma™), lysocabtagene maraleucel (Breyanzi®), and tisagenleucel (Kyrmriah®).

[0131] In another embodiment, the subject has a virally driven cancer. In a particular embodiment, the virally driven cancer is selected from the following: [Table 3]

[0132] In another aspect, a method for treating autoimmune disease in a subject is provided. Autoimmune diseases are conditions resulting from abnormal immune attacks on the body, and they substantially increase morbidity, mortality, and medical costs worldwide. As T cells play a key role in the process of autoimmune disease, engineered T cell therapy has emerged and is considered as a potential approach to overcome the current obstacles in the treatment of autoimmune disease. Either autoreactivity or autoantibodies play a major role in the process of autoimmune disease. Therefore, engineering T cells to express chimeric autoantibody receptors (CAARs) is a strategy for the treatment of autoimmune disease. In one embodiment, the CAR comprises a CAAR. See, e.g., Zhang et al, Chimeric antigen receptor T-cell therapy beyond cancer: current practice and future prospects, Immunotherapy, 2020 Sep;12(13):1021-1034. doi:10.2217 / imt-2020-0009. Epub 2020 Jul 30, which is incorporated herein by reference. Autoimmune diseases include pemphigus vulgaris (PV) (e.g., DSG3-CAAR-T) and lupus (e.g., MuSK-CAAR-T). Other autoimmune diseases include type 1 diabetes, autoimmune thyroid disease, rheumatoid arthritis (RA), inflammatory bowel disease, colitis, systemic lupus erythematosus, and multiple sclerosis (MS). See, e.g., Chen et al, Immunotherapy Deriving from CAR-T Cell Treatment in Autoimmune Diseases, Journal of Immunology Research Volume 2019, December 31, 2019, which is incorporated herein by reference.

[0133] In one aspect, the method includes administering to a subject in need thereof an effective amount of an effector-enhanced gene-expressing cell (e.g., an LTBR CART or LTBR TCR-T cell) described herein. , including administration in combination with an effective amount of another therapy. As used herein, administered "in combination" means that two (or more) different therapies are delivered to a subject during the course of the subject's suffering from a disorder, e.g., two or more therapies are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated or the treatments are discontinued for other reasons. In some embodiments, the delivery of one treatment has already occurred when the delivery of the second begins, so there is an overlap in terms of administration. This may be referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments in either case, the treatments are more effective due to the combination administration. For example, the second treatment is more effective than would be observed if the second treatment were administered in the absence of the first treatment, or a similar situation would be observed with the first treatment, e.g., a comparable effect is observed with less of the second treatment, or the second treatment reduces symptoms to a greater extent. In some embodiments, delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed when one treatment is delivered in the absence of the other treatment. The effect of the two treatments can be partially additive, fully additive, or greater than additive. Delivery can be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0134] The effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells) and at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions, or sequentially. In sequential administration, the effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells) described herein can be administered first and the additional agent can be administered second, or the order of administration can be reversed.

[0135] The effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells) and / or other therapeutic agents, treatments, or modalities can be administered during periods of active disorder or during periods of remission or less active disease. The effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells) can be administered prior to, concurrently with, or after other treatments, or during remission of the disorder.

[0136] When administered in combination, the effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells) and additional agents (e.g., second or third agents), or all, can be administered in amounts or dosages that are higher, lower, or the same as the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of the effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells), additional agents (e.g., second or third agents), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy. In other embodiments, the amount or dosage of the effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells), additional agent (e.g., second or third agent), or all that results in a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent used individually, e.g., as monotherapy, required to achieve the same therapeutic effect.

[0137] In further aspects, the effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells) described herein can be treated with surgery, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, or may be used in a treatment regimen in combination with other antibody therapies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, radiation, or peptide vaccines as described in Izumoto et al. 2008 J Neurosurg 108:963-971.

[0138] In certain cases, the effector-enhanced gene-expressing cells described herein (e.g., LTBR CART or LTBR TCR-T cells) are used in combination with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, antiemetic (or anti-emetic) agents, analgesics, cytoprotective agents, and combinations thereof.

[0139] In certain embodiments, the effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells) herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), antimetabolites (e.g., antifolates, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine, (including cyclosporine), mTOR inhibitors, TNFR glucorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), immunomodulators such as thalidomide or thalidomide derivatives (e.g., lenalidomide), or immune checkpoint inhibitors (e.g., PD-1 or PD-L1 inhibitors, e.g., pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi)).

[0140] Common chemotherapy agents considered for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraptidine), and rifampicin (Ricinib). cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), cyclophosphamide (Cytoxan® or Neosar®), cyclophosphamide (Cytoxan®), cyclophosphamide (Cytosar-U ... (registered trademark)), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine (registered trademark)), daunorubicin citrate liposome injection (DaunoXome (registered trademark)), dexamethasone, docetaxel (Taxotere (registered trademark)), doxorubicin hydrochloride (Adriamycin (registered trademark), Rubex (registered trademark)), etoposide (Vepesid (registered trademark)), fludarabine phosphate (Fludara (registered trademark)), 5-fluridan Olouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Al keran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), Milotarg, paclitaxel (Taxol®), Phoenix (Yttrium 90 / MX-DTPA), pentostatin, polipheprosan 20 implant with carmustine (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0141] Treatment with a combination of a chemotherapeutic agent and an effector-enhanced gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) as described herein can be used to treat a hematological cancer, e.g., AML, as described herein. In embodiments, the combination of a chemotherapeutic agent and an effector-enhanced gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) is useful for targeting, e.g., killing, cancer stem cells, e.g., leukemia stem cells, e.g., in a subject with AML. In embodiments, the combination of a chemotherapeutic agent and an effector-enhanced gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell) is useful for treating minimal residual disease (MRD). MRD refers to a small number of cancer cells that remain in a subject during treatment, e.g., during chemotherapy, or after treatment. MRD is often the main cause of recurrence. The present invention provides methods for treating cancer, e.g., MRD, comprising administering a chemotherapeutic agent in combination with an effector-enhanced gene-expressing cell (e.g., LTBR CART or LTBR TCR-T cell), e.g., as described herein.

[0142] In certain embodiments, the chemotherapeutic agent is administered prior to administration of the effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells). In chemotherapy regimens in which more than one administration of the chemotherapeutic agent is desired, the chemotherapy regimen is initiated or completed prior to administration of the effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells). In embodiments, the chemotherapeutic agent is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 days prior to administration of the effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells). In embodiments, the chemotherapy regimen is initiated or completed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 days prior to administration of the effector-enhanced gene-expressing cells (e.g., LTBR CART or LTBR TCR-T cells).

[0143] When an "immunologically effective amount", "antitumor effective amount", "tumor inhibiting effective amount", or "effective amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in the age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). 4 ~10 9 / kg body weight, in some cases, 10 cells 5 ~10 6It can be generally stated that T cell compositions may be administered in dosages ranging from 100 to 1500 cells / kg body weight, including all integer values ​​within those ranges. The T cell compositions may also be administered multiple times at these dosages. The cells may be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). As used herein, the term "effective amount" of an agent is an amount sufficient to bring about a beneficial or desired result, e.g., a clinical result, and thus, an "effective amount" depends on the context in which it is applied. For example, in the context of administering an agent to treat a tumor, an effective amount The amount of the agent is sufficient to, for example, reduce or decrease the size of a tumor or inhibit the growth of a tumor, compared to the response obtained without administration of the agent. The term "effective amount" can be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose."

[0144] Also provided herein is a method of vaccinating a subject with a combination vaccine comprising at least two nucleic acid sequences encoding at least one effector-enhanced gene and at least one viral protein. In one embodiment, the effector-enhanced gene is LTBR. In another embodiment, the viral protein is a coronavirus spike protein. Some embodiments provide a vaccine comprising an RNA polynucleotide having an open reading frame encoding an effector-enhanced gene, an RNA polynucleotide having an open reading frame encoding an effector-enhanced gene, a viral protein, and a pharma- ceutically acceptable carrier or excipient, formulated in a cationic lipid nanoparticle (LNP). The vaccines described herein (e.g., LNP-encapsulated mRNA vaccines) produce prophylactically and / or therapeutically effective levels, concentrations, and / or titers of antigen-specific antibodies in the blood or serum of a vaccinated subject. See, e.g., US2018 / 0311336A1, which is incorporated herein by reference in its entirety.

[0145] As used herein, the term "treatment" and its variants, such as "treat" or "treating", refer to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing or reducing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or mitigating the disease state, and remission or improved prognosis. In certain embodiments, the compositions described herein are used to delay the onset of disease or to slow the progression of disease.

[0146] Similarly, as used herein, the term "treatment of cancer" or "treating cancer" can be described by a number of different parameters, including, but not limited to, a reduction in tumor size in an animal with cancer, a reduction in tumor growth or proliferation in an animal with cancer, preventing metastasis or reducing the extent of metastasis, and / or prolonging the survival of an animal with cancer compared to a control. In certain embodiments, treatment results in a reduced risk of distant recurrence or metastasis.

[0147] The term "cancer" as used herein refers to any disease, condition, trait, genotype, or phenotype characterized by unregulated cell growth or replication. In certain embodiments, administration of the compositions disclosed herein treats cancer, for example, according to the methods disclosed herein. In certain embodiments, the cancer is adrenocortical carcinoma, advanced cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain tumor, brain cancer, breast cancer, childhood cancer, cancer of unknown etiology, Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing's tumor, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myelomonocytic ... Leukemia, Liver Cancer, Hepatocellular Carcinoma (HCC), Non-Small Cell Lung Cancer, Small Cell Lung Cancer, Pulmonary Carcinoid Tumor, Lymphoma of the Skin, Malignant Mesothelioma, Multiple Myeloma, Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumor, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Adult Soft Tissue Sarcoma, Basal and Squamous Cell Carcinoma, Melanoma, Small Intestine Cancer, Gastric Cancer, Testicular Cancer, Pharyngeal Cancer, Thymic Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldens Cancer In certain embodiments, the cancer is selected from the group consisting of Strom's macroglobulinemia, Wilms' tumor, a secondary cancer caused by cancer therapy, and any combination thereof. In certain embodiments, the cancer is one of those listed in Figure 19 or Figure 20.

[0148] Screening Methods Disclosed herein is a method for performing gain-of-function screening to identify genes that alter lymphocyte activity. Genes include, for example, those that cannot be typically expressed by lymphocytes, or those that are expressed only in certain lymphocyte populations or situations (e.g., after exposure to antigen). The ability to identify genes whose expression is altered by stimuli or that are not typically expressed has certain advantages of methods that rely on regulated biased libraries, including those based on RNA sequencing. In particular, the disclosed method facilitates the transfer of gain-of-function studies to many different physiological and pathological situations and modifies difficult-to-manipulate cell types.

[0149] In certain embodiments, a method is provided for identifying a gene that, when exogenously expressed in an engineered lymphocyte, alters the therapeutic function of the engineered lymphocyte, the method comprising: (a) obtaining a lymphocyte population; (b) transducing the lymphocyte population with a plurality of viral vectors, each viral vector encoding a gene linked to a barcode; (c) stimulating the transduced lymphocytes to induce activation, proliferation, and / or effector function; (d) isolating the transduced lymphocytes from the lymphocyte population of (c); and (e) detecting the presence of the gene and / or the linked barcode in the isolated lymphocytes. The gene identified according to the method is effective to alter the therapeutic function of the engineered lymphocyte expressing the gene. Provided herein are lymphocytes expressing the gene and methods for delivering the lymphocytes to a subject.

[0150] The screening methods exemplified herein utilize lentiviral-mediated delivery of open reading frame (ORF) libraries (see Sack et al. Cell. 2018 Apr 5;173(2):499-514.e2, which is incorporated herein by reference), however, the methods extend to approaches to screen expression of non-coding sequences such as non-coding RNAs (e.g., microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)), as well as alternative ORFs, microORFs, upstream ORFs, etc. (i.e., small protein-coding elements in the genome). Thus, as used herein, the term "gene" refers to sequences that code for both proteins and those that do not.

[0151] The term "barcode" or "barcode sequence" as used herein refers to a nucleotide sequence that corresponds to and allows for the detection and / or identification of an expressed gene. A barcode typically comprises 4 or more nucleotides. In some embodiments, a barcode comprises 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, or 15 nucleotides. In some embodiments, a barcode comprises 8-15 nucleotides. As used herein, the terms "barcoded gene", "barcoded ORF", and the like refer to a nucleic acid having a barcoded sequence appended thereto, either the barcode being directly linked to the 5' or 3' end of the ORF, or separated by one or more nucleotides at the 5' or 3' end of the ORF.

[0152] Although certain aspects are described herein with respect to T cells, the disclosed methods can identify altered functional responses in other lymphocyte populations, including, but not limited to, NK T cells, NK cells, B cells, γδ T cells, and combinations and subpopulations thereof. In certain embodiments, lymphocyte populations are enriched for one or more of T cells, B cells, NK T cells, NK cells, Vγ9V52 T cells, or subpopulations thereof. The lymphocyte population is a cell population that has been identified. The cells may be obtained from a biological sample (blood, tissue, etc.) from one or more subjects using a variety of isolation or purification methods known in the art for obtaining and / or enriching a cell sample having lymphocytes or a population / subpopulation of lymphocytes, including, for example, positive and negative selection techniques, either by Ficoll gradient separation, magnetic separation using antibodies, tetramers, etc., or flow cytometry. In certain embodiments, the lymphocyte population is enriched for one or more of CD4+ T cells, CD8+ T cells, αβ T cells, and γδ T cells. In certain embodiments, the screening method includes lymphocytes having functional antigen-specific receptors expressed on the lymphocyte surface. In certain embodiments, the lymphocyte population includes CAR-expressing or engineered TCR-expressing lymphocytes. In certain embodiments, the lymphocyte population is an immortalized cell line. In further embodiments, the cell line has been modified to express a CAR or an engineered TCR. In certain embodiments, the lymphocyte population is an NK cell line (e.g., NK-92 or an engineered variant). In certain embodiments, the lymphocyte population is derived from a subject or subjects with a particular disease or demographic profile, including cancer, such as those described herein, or an infectious disease.

[0153] The screening method disclosed herein involves expressing a collection of barcoded genes in a lymphocyte population. Methods for introducing nucleic acids into cells include viral and non-viral mediated. In certain cases, the method utilizes a barcoded ORF library that is delivered to a cell population using a suitable retroviral or lentiviral vector (see, e.g., Sack et al. Cell. 2018 Apr 2018). 5;173(2):499-514.e2 and Yang et al. Nat Methods. 2011 Jun 26;8(8):659-61, which are incorporated herein by reference). In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the lymphocyte population is transduced with a vector delivering the gene to be screened. Although a higher efficiency of transduction is advantageous, especially when analyzing smaller or rarer lymphocyte populations, in certain embodiments, the method includes one or more selection steps to further enrich the cell population for cells expressing the gene to be screened. For example, a drug resistance gene (e.g., a puromycin resistance gene) can be included in the gene delivery vector to facilitate selection of transduced cells. In certain embodiments, the gene-encoding construct includes a selection gene (e.g., a fluorescent protein, GFP) that facilitates further isolation or enrichment of the transduced cells using flow cytometry.

[0154] In certain embodiments, the method includes delivery of a barcoded gene operably linked to a promoter and / or other regulatory elements. The choice of promoter may depend on factors such as the target cell type (i.e., lymphocyte population), the desired level of gene expression, and / or duration of expression. Suitable primers are provided herein. In certain embodiments, the choice of promoter determines the efficiency or effectiveness of the method and confers surprising advantages. In certain embodiments, the promoter is an elongation factor-1 alpha short (EFS) promoter. In certain embodiments, the promoter is an elongation factor-1 alpha short (EFS) promoter. In certain embodiments, the promoter is a cytomegalovirus (CMV) promoter. In certain embodiments, the CMV promoter is preferred as it provides a higher level of expression than the EFS promoter. In certain embodiments, the promoter is a phosphoglycerate kinase-1 (PGK) promoter. In still further embodiments, the promoter is an inducible promoter.

[0155] In certain embodiments, the methods provided entail assessment of one or more effector functions following stimulation of the lymphocyte population, and changes in effector function(s) are determined by screening. The present invention shows an altered response due to the expression of an exogenous gene contained in the antigen binding fragment. As used herein, the term "stimulation" refers to a primary response induced by a stimulatory molecule of a lymphocyte binding to its cognate ligand and thereby mediating even signal transduction. In certain embodiments, the method includes a stimulation that induces signal transduction through the TCR / CD3 complex. The stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β and / or rearrangement of cytoskeletal structures.

[0156] The term "stimulatory molecule" refers to a molecule expressed by an immune cell, e.g., a T cell, a NK cell, or a B cell, that provides a cytoplasmic signaling sequence(s) that regulates activation of the immune cell in a stimulatory manner for at least some aspect of the immune cell signaling pathway. In one aspect, the signal is a primary signal initiated by binding of the TCR / CD3 complex with an MHC molecule, e.g., to which a peptide or polyclonal crosslinker has been added, resulting in a response that includes, but is not limited to, proliferation, activation, differentiation, and resistance to apoptosis.

[0157] The disclosed methods can be adapted to stimulate cells according to a variety of means, including the use of polyclonal and non-specific stimulation. In certain embodiments, cells are stimulated using bound or soluble antibodies specific for epitopes on the lymphocyte cell surface. In certain embodiments, the antibodies bind and crosslink antigen receptors on the surface of the cells. In certain embodiments, lymphocytes are stimulated in an antigen-specific manner. In certain embodiments, the method includes stimulating the lymphocyte population with one or more of culturing the lymphocytes with one or more of antibodies, cytokines, antigens, superantigens, antigen-presenting cells, cancer cells, and cancer cell lines. In certain embodiments, the lymphocyte population includes T cells that are activated via incubation with anti-CD3 and anti-CD28 antibodies. The lymphocytes may also be cultured with cytokines that promote activation, proliferation, differentiation, apoptosis, and / or survival.

[0158] The term "antigen-presenting cells" refers to immune system cells such as accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells. In certain embodiments, antigen-presenting cells induce responses in lymphocytes expressing CARs or engineered TCRs. In certain embodiments, antigen-presenting cells are cancer cell lines.

[0159] In certain embodiments, stimulation of lymphocytes identifies cells that have an altered response as a result of expression of the exogenous gene. Altered responses include, but are not limited to, changes in the degree of proliferation, survival, apoptosis, phenotypic changes (e.g., surface markers, size), cytokine or chemokine production and / or secretion, and cytotoxic potential. In certain embodiments, lymphocyte proliferation is determined by labeling the cells with a dye (e.g., CFSE or CellTrace) prior to stimulation. In certain embodiments, expression of markers on lymphocytes, including one or more of CD69, CD25, OX40L (CD154), ICAM-1, CD70, CD74, CD54, MHC-II, CD137, CD44, CD62L, CCR7, CD107a, PD1, TIM3, LAG3, CD80, CD86, TIGIT, VISTA, B7-H3, BTLA, and SIGLEC15, is determined to identify and / or isolate stimulated cells. In certain embodiments, IL-2, IL-12, IL-23, IFNγ, TNFα, GM-CSF, IL7, IL15, IL12, IL18, IL21, IL23, LTA, IL4, IL5, IL6, IL10, IL13, TGFb eta, IL17, LTA, LIGHT, CCL3, CCL4, CCL5, MCP-3, CXCL9, MIP1α, IL8, PDGF-AA, IP10, IL22, IL3, MCP-1, IL9, MDC, sCD40L, and MC Production and / or secretion of cytokines or chemokines, including one or more of the SFs, are determined to identify and / or isolate stimulated cells. In certain embodiments, expression of markers, cytokines, and / or chemokines is determined by flow cytometry, which facilitates sorting of cells based on expression, including relative levels of expression. In certain embodiments, lymphocytes express proteins indicative of cytotoxic potential. In certain embodiments, expression of perforin and / or granzymes is determined.

[0160] Following isolation of stimulated lymphocytes, the identity of the expressed genes is determined by PCR amplification of the gene and / or barcode sequences. In certain embodiments, PCR is performed on genomic DNA (gDNA) obtained from the lymphocytes. In certain embodiments, a reverse transcription step is performed to generate cDNA from the cellular transcriptome and / or from the exogenous genes and barcode mRNA transcripts. The amplified DNA products are then sequenced to identify the exogenous genes expressed in the isolated lymphocytes and / or to quantify the relative expression of the exogenous genes in a population of isolated lymphocytes. In certain embodiments, the disclosed screening methods include RNA and / or DNA sequencing of the isolated lymphocytes using techniques including, but not limited to, whole transcriptome analysis, whole genome analysis, barcoded sequencing of whole or targeted regions of the genome, and combinations thereof. In certain embodiments, RNA and / or DNA sequencing is performed in combination with proteome analysis. In certain embodiments, the methods include detection of cell surface or intracellular proteins, for example, using flow cytometry.In certain embodiments, the methods include detection or identification of barcoded genes in combination with profiling additional molecular modalities using methods reported in the art, including, for example, single cell sequencing analysis (e.g., 10X Genomics multi-ome platform), single cell RNA sequencing (scRNA-seq) (see, e.g., Haque et al. A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications, Genome Medicine, 9, Article number:75(2017); Hwang et al. Single-cell RNA sequencing technologies and bioinformatics pipelines. Exp Mol Med. 2018 Aug 7;50(8):96), cell hashing (see, e.g., Stoeckius et al. Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol. 2018;19:224), Perturb-Seq. (see, e.g., Dixit et al. al.Perturb-seq:Dissecting molecular. circuits with scalable single cell RNA profiling of pooled genetic screens. Cell. 2016 Dec 15;167(7):1853-1866.e17), CROP-seq (e.g., Datlinger et al. Pooled CRISPR screening with single-cell transcriptome readout Nat Methods.2017 Mar;14(3):297-301), CRISPR-seq (e.g., Jaitin et al.Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq Cell. 2016 Dec 15;167(7):1883-1896.e15), and extended CRISPR-compatible CITE-seq (ECCITE-seq) (see, e.g., Mimitou et al. Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells.Na t Methods. 2019 May;16(5):409-41), and Cellular Indexing of Transcriptome and Epitope-seq (CITE-seq) (see, e.g., Stoeckius et al. Simultaneous epitope and transcriptome measurement in single cells. Nat Methods. 2017 Sep;14(9):865-868).

[0161] OverCITE-seq To obtain a more comprehensive view of the mechanism of action of individual genes and to provide a multidimensional description of the phenotypic changes they induce, a single-cell sequencing strategy using direct ORF capture is described herein. An overview of an embodiment of this aspect is provided in FIG. 18A. As referred to herein, the term "open reading frame mRNA" or ORF mRNA refers not only to the coding sequence of the gene of interest, but also includes downstream sequences that may, in some embodiments, include barcodes and / or selection markers, e.g., puromycin. This approach, termed OverCITE-seq (Overexpression-Compatible CITE-seq), builds on previous approaches developed to quantify surface antigens and CRISPR perturbations, allowing high-throughput, single-cell analysis of pools of T cells bearing different ORFs. Briefly, mRNA from an ORF incorporated into a lentivirus is specifically reverse transcribed by a primer that binds to a constant sequence of the transcript downstream of the ORF and barcoded along with the cellular transcriptome during template switching. The resulting cDNA pool is then split for separate construction of gene expression and ORF expression libraries.

[0162] Thus, in another aspect, a method is provided for analyzing the effect of overexpression of an ORF of interest on an individual cell. The method includes introducing an expression cassette containing a nucleic acid encoding the ORF of interest into a cell and overexpressing the ORF in the cell. Overexpression of the ORF can be achieved by using a strong promoter such as CMV, EF-1a, CAG, PKG, etc. Such promoters are known in the art.

[0163] A first set of nucleic acids from individual cells are provided in separate fractions together with oligonucleotides having a common barcode sequence, and the oligonucleotides are bound to beads. The barcode sequence provides a unique identifier so that upon characterization of these nucleic acids, they can be attributed as being from the same cell. That is, the oligonucleotides are fractionated such that the nucleic acid barcode sequence contained therein is the same between the oligonucleotides in a given fraction, but between different fractions, the oligonucleotides have different barcode sequences. The nucleic acid barcode sequence can include from 6 to about 20 or more nucleotides within the sequence of the oligonucleotide. In some cases, the length of the barcode sequence can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some cases, the length of the barcode sequence can be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some cases, the length of the barcode sequence can be up to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or fewer nucleotides.

[0164] The first set of nucleic acids includes both endogenous transcriptome mRNA and ORF mRNA. In some embodiments, the nucleic acids are released from individual cells in separate fractions. In some embodiments, the nucleic acids include ribonucleic acid (RNA), such as, for example, messenger RNA (mRNA). As used herein, in some embodiments, a fraction refers to a container or vessel (e.g., a well, a microwell, a tube, a through port in a nanoarray substrate, e.g., a BioTrove nanoarray, or other container). However, in some embodiments, the compartments or fractions include fractions that are flowable within the fluid stream. These fractions may be comprised of, for example, microcapsules or micro-vesicles with an outer barrier surrounding an inner fluid center or core, or they may be porous matrices that can entrap and / or retain material within their matrix. However, in some embodiments, these fractions include droplets of aqueous fluid within a non-aqueous continuous phase, e.g., an oil phase. See, for example, US2015 / 0376609A1, which is incorporated herein by reference in its entirety. In some embodiments, cells may be fractionated with a lysis reagent to release the contents of the cells within the fraction. In such cases, the lysis agent may be contacted with the cell suspension simultaneously with or immediately prior to the introduction of the cells to the fraction junction / droplet generation zone. In addition to the lysis agents co-fractionated with the cells described above, other reagents may also be co-fractionated with the cells, including, for example, DNase and RNase inactivators or inhibitors such as proteinase K, chelating agents such as EDTA, and other reagents used to remove or otherwise reduce the negative activity or effect of different cell lysate components on subsequent processing of nucleic acids.

[0165] Additional reagents are co-fractionated with the cells, such as an endonuclease that fragments the cell's DNA, a DNA polymerase enzyme that is used to amplify the cell's nucleic acid fragments and attach barcode oligonucleotides to the amplified fragments, and dNTPs. The additional reagents may also include reverse transcriptase, primers and oligonucleotides, including enzymes with terminal transferase activity, as well as switch oligonucleotides (also referred to herein as "switch oligos") that can be used for template switching. In some cases, template switching can be used to increase the length of the cDNA. In one example of template switching, a cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, and a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g., polyC, to the cDNA that are not encoded by the template, such as at the ends of the cDNA. The switch oligo can include a sequence complementary to the additional nucleotide, e.g., polyG. Additional nucleotides in the cDNA (e.g., polyC) can hybridize to sequences complementary to additional nucleotides in the switch oligo (e.g., polyG), such that the switch oligo can be used as a template by reverse transcriptase to further extend the cDNA. The switch oligo can comprise deoxyribonucleic acid, ribonucleic acid, modified nucleic acid including locked nucleic acid (LNA), or any combination. Such reagents are known in the art. See, for example, Chromium Next GEM Single Cell See 5' Reagent Kits v2 User Guide (available at assets.ctfassets.net / an68im79xiti / 1Ap6qQysq80oQAGacem7RD / 6f895796e18c38a14ef1e8b8ff79a82c / CG000331_ChromiumNextGEMSingleCell5-v2_RevB.pdf), which is incorporated by reference in its entirety.

[0166] The method further comprises performing RT-PCR to generate a second set of nucleic acids derived from the first set of nucleic acids comprising endogenous transcriptome cDNA and ORF cDNA, the second set of nucleic acids in the fraction being bound to an oligonucleotide comprising a common nucleic acid barcode sequence. RT-PCR reagents comprising an oligonucleotide primer that specifically anneals to a sequence in the ORF mRNA that is not a polyA sequence. In some embodiments, the oligonucleotide primer that specifically anneals to a sequence in the ORF mRNA anneals to an mRNA at a portion of the transcript that is common to sequences in the library, e.g., a coding sequence for a resistance marker, e.g., puromycin. This allows for the amplification of many ORF sequences from the ORF library using a common set of reagents. In some embodiments, generating one or more second nucleic acid sequences comprises subjecting the nucleic acid to reverse transcription under conditions that result in one or more second nucleic acid sequences. In some embodiments, the reverse transcription is performed using a common set of reagents. The reverse transcription occurs in separate fractions. In some embodiments, the oligonucleotides are provided in separate fractions and include a poly-T sequence. In some embodiments, the reverse transcription comprises hybridizing the poly-T sequence to at least a portion of each of the nucleic acids and extending the poly-T sequence in a template-directed manner. In some embodiments, the oligonucleotides comprise an anchor sequence that promotes hybridization of the poly-T sequence. In some embodiments, the oligonucleotides comprise a random priming sequence, which can be, for example, a random hexamer. In some embodiments, the reverse transcription comprises hybridizing the random priming sequence to at least a portion of each of the nucleic acids and extending the random priming sequence in a template-directed manner.

[0167] The method further includes amplifying the second set of nucleic acids using PCR reagents that include a second primer that specifically anneals to a sequence on the ORF cDNA that is not the polyA sequence to generate a third set of nucleic acids.

[0168] In certain embodiments, the method includes obtaining a portion of the third set of nucleic acids and amplifying the ORF cDNA using a second set of PCR reagents that includes a third primer that specifically anneals to a sequence on the ORF cDNA that is not a polyA sequence to generate a fourth set of nucleic acids.

[0169] In certain embodiments, the method further comprises amplifying the ORF cDNA in the fourth set of nucleic acids using a third set of PCR reagents comprising a fourth primer that specifically anneals to a sequence on the ORF cDNA that is not a polyA sequence to generate a fifth set of nucleic acids.

[0170] The third and optionally fifth set of nucleic acids are then fragmented, adapters are attached to both ends of the fragments, and subjected to next generation sequencing (NGS) using standard techniques known in the art. See, e.g., Kanzi et al, Next Generation Sequencing and Bioinformatics Analysis of Family Genetic Inheritance, Front. Genet., 23 October 2020 | https: / / doi.org / 10.3389 / fgene.2020.544162, which is incorporated herein by reference. See also US2018 / 0251825A1 and US2015 / 0376609, both of which are incorporated herein by reference.

[0171] In certain embodiments, the method includes detection or identification of barcoded sequences in combination with profiling additional molecular modalities using methods reported in the art, including, for example, single-cell sequencing analysis (e.g., 10X Genomics Multi-Ome Platform), single-cell RNA sequencing (scRNA-seq) (e.g., Haque et al. A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications, Genome Medicine, 9, Article number: 75 (2017); Hwang et al. al.Single-cell RNA sequencing technologies and bioinformatics pipelines.Exp Mol Med. 2018 Aug 7;50(8):96), cell hashing (see, e.g., Stoeckius et al. Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol. 2018;19:224), Perturb-Seq. (see, e.g., Dixit et al. Perturb-seq: Dissecting ng molecular circuits with scalable single cell RNA profiling of pooled genetic screens. Cell. 2016 Dec 15;167(7):1853-1866.e17), CROP-seq (see, e.g., Datlinger et al. Pooled CRISPR screening with single-cell transcriptome readout Nat Methods. 2017 Mar;14(3):297-301), CRISP-seq (see, e.g., Jaitin et al. Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq Cell. 2016 Dec 15;167(7):1883-1896.e15), Extended CRISPR-Compatible CITE-seq (ECCITE-seq) (see, e.g., Mimitou et al. Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells. Nat Methods. 2019 May;16(5):409-41), and Cellular Indexing of Transcriptomes and Epitopes-seq (CITE-seq) (see, e.g., Stoeckius et al. Simultaneous epitope and transcriptome measurement in single cells. Nat Methods. 2017 Sep;14(9):865-868).

[0172] With respect to the description of the invention provided herein, it is contemplated that each of the compositions described herein is, in separate embodiments, useful in the methods of the invention. In addition, it is contemplated that each of the compositions described herein that is useful in the methods is, in separate embodiments, itself an embodiment of the invention.

[0173] Specific embodiments 1. A modified lymphocyte comprising an exogenous nucleic acid encoding LTBR. 2. The modified lymphocyte of embodiment 1, wherein the nucleic acid encoding the LTBR encodes the intracellular domain, or a fragment or variant thereof. 3. The modified lymphocyte of embodiment 2, wherein the LTBR intracellular domain comprises amino acids 249-435 of SEQ ID NO:2, or a fragment, deletion, or variant thereof. 4. The modified lymphocyte of embodiment 2 or 3, wherein the LTBR intracellular domain has a deletion at least at amino acids 393-435. 5. The modified lymphocyte of any one of embodiments 1 to 4, wherein the lymphocyte comprises an expression cassette comprising an expression control sequence and a nucleic acid encoding LTBR. 6. The modified lymphocyte of any one of embodiments 1 to 5, wherein the lymphocyte further comprises a nucleic acid encoding a chimeric antigen receptor (CAR). 7. The modified lymphocyte of embodiment 5, wherein the expression cassette further comprises a nucleic acid encoding a CAR. 8. The modified lymphocyte of embodiment 1 or 5, wherein the lymphocyte further comprises a nucleic acid encoding a T cell receptor (TCR). 9. The modified lymphocyte of embodiment 8, wherein the expression cassette further comprises a nucleic acid encoding a TCR. 10. The modified lymphocyte of any one of embodiments 1-9, wherein the expression control sequence comprises an EF-1α, EFS, or CMV promoter. 11. The modified lymphocyte of embodiment 1, wherein the exogenous nucleic acid encoding LTBR is mRNA. 12. The modified lymphocyte of any one of embodiments 1-11, wherein the lymphocyte is a T cell. 13. The modified lymphocyte of any one of embodiments 1-12, wherein the lymphocyte is an alpha beta T cell or a gamma delta T cell, optionally a Vγ9Vδ2 T cell. 14. The modified lymphocyte of any one of embodiments 1 to 12, wherein the lymphocyte is a NK cell. 15. The modified lymphocyte of any one of embodiments 1-12, wherein the lymphocyte is a NK T cell. 16. The modified lymphocyte of any one of embodiments 6, 7, 10, or 12-15, wherein the CAR is axicabtagene siloreucel (Yescarta®), brexcabtagene outelucel (Tecartus™), idecabtagene bicurucel (Abecma™), lysocabtagene maraleucel (Breyanzi®), tisagenlecleucel (Kyrmriah®), or one of those found in FIG. 19. 17. A vaccine composition comprising a nucleic acid encoding an LTBR and a nucleic acid encoding a viral protein. 18. The vaccine composition of embodiment 17, wherein the nucleic acid encoding LTBR encodes the LTBR intracellular domain, or a fragment or variant thereof. 19. The modified lymphocyte of embodiment 18, wherein the LTBR intracellular domain comprises amino acids 249-435 of SEQ ID NO:2, or a fragment, deletion, or variant thereof. 20. The modified lymphocyte of embodiment 18 or 19, wherein the LTBR intracellular domain has a deletion at least at amino acids 393-435. 21. A vaccine composition according to any one of embodiments 17 to 20, wherein the viral protein is a glycoprotein. 22. The vaccine composition of embodiment 21, wherein the glycoprotein is a viral spike protein. 23. The vaccine composition of embodiment 22, wherein the viral spike protein is a coronavirus spike protein. 24. A vaccine composition according to any one of embodiments 17 to 23, wherein the nucleic acid encoding the LTBR is mRNA or the nucleic acid encoding the viral spike protein is mRNA, or both. 25. An expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid encoding an LTBR. 26. An expression cassette comprising a nucleic acid encoding a T cell receptor and a nucleic acid encoding an LTBR. 27. An expression cassette comprising a nucleic acid encoding a viral protein and a nucleic acid encoding an LTBR. 28. An expression cassette according to any one of embodiments 25 to 27, wherein the nucleic acid encoding LTBR encodes the LTBR intracellular domain, or a fragment or variant thereof. 29. The expression cassette of embodiment 28, wherein the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO:2, or a fragment, deletion, or variant thereof. 30. An expression cassette according to embodiment 28 or 29, wherein the LTBR intracellular domain has a deletion at least at amino acids 393 to 435. 31. A composition comprising a modified lymphocyte comprising an expression cassette according to any one of embodiments 25 to 30. 32. A method for producing modified lymphocytes, comprising introducing into a cell an exogenous nucleic acid encoding an LTBR. 33. The method of embodiment 32, wherein the nucleic acid encoding LTBR encodes the LTBR intracellular domain, or a fragment or variant thereof. 34. The method of embodiment 32 or 33, wherein the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof. 35. The LTBR intracellular domain contains a deletion at least in amino acids 393-435. The method according to any one of embodiments 32 to 34, comprising: 36. The method of any one of embodiments 32 to 35, wherein the lymphocyte comprises an expression cassette comprising an expression control sequence and a nucleic acid encoding LTBR. 37. The method of any one of embodiments 32 to 36, wherein the lymphocyte further comprises a nucleic acid encoding a chimeric antigen receptor (CAR). 38. The method of embodiment 37, wherein the expression cassette further comprises a nucleic acid encoding a CAR. 39. The method of any one of embodiments 32 to 36, wherein the lymphocytes further comprise a nucleic acid encoding an engineered T cell receptor (TCR). 40. The method of embodiment 39, wherein the expression cassette further comprises a nucleic acid encoding a TCR. 41. The method of any one of embodiments 36 to 40, wherein the expression control sequence comprises an EF-1α (full length or truncated) or CMV promoter. 42. The method according to any one of embodiments 32 to 42, wherein the exogenous nucleic acid encoding LTBR is mRNA. 43. The method according to any one of embodiments 32 to 42, wherein the lymphocytes are T cells, optionally CD4+ T cells, CD8+ T cells, or Treg cells. 44. The method of any one of embodiments 32-43, wherein the lymphocytes are alpha beta T cells. 45. The method of any one of embodiments 32 to 43, wherein the lymphocytes are gamma delta T cells, optionally Vγ9Vδ2 T cells. 46. ​​The method of any one of embodiments 32 to 42, wherein the lymphocytes are NK cells. 47. The method of any one of embodiments 32 to 42, wherein the lymphocytes are NK T cells. 48. The method of any one of embodiments 37, 38, or 41-47, wherein the CAR is axicabtagene siloreucel (Yescarta®), brexcabtagene outelucel (Tecartus™), idecabtagene bicurucel (Abecma™), lysocabtagene maraleucel (Breyanzi®), tisagenlecleucel (Kyrmriah®), a chimeric autoantibody receptor, or one of those found in FIG. 19. 49. The method according to any one of embodiments 32 to 48, wherein the lymphocytes are nucleofected with mRNA encoding LTBR. 50. A method of treating cancer in a subject in need of such treatment, the method comprising administering to the subject a modified lymphocyte of any one of embodiments 1-16, an expression cassette of any one of embodiments 25-30, or a composition of embodiment 31. 51. The method of embodiment 50, wherein the subject has lymphoma. 52. The method of embodiment 50, wherein the subject has a solid tumor. 53. The method of embodiment 50, wherein the subject has leukemia. 54. The method of embodiment 50, wherein the subject has multiple myeloma. 55. The method of embodiment 50, wherein the subject has a virally-driven cancer. 56. The method of embodiment 55, wherein the subject has HPV. 57. The method of embodiment 50, wherein the subject has a cancer that is Burkitt's lymphoma, liver cancer, Kaposi's sarcoma, cervical cancer, head and neck cancer, anal cancer, oral cancer, pancreatic cancer, ovarian cancer, melanoma, pharyngeal cancer, penile cancer, adult T-cell lymphoma, or Merkel cell carcinoma. 58. A method for defeating a viral disease in a subject in need thereof, the method comprising administering to the subject a composition described in any one of embodiments 1-16. 59. The method of embodiment 58, wherein the disease is HIV or HPV. 60. A method of treating an autoimmune disorder in a subject in need of such treatment, the method comprising administering to the subject a modified lymphocyte of any one of embodiments 1-16, an expression cassette of any one of embodiments 25-30, or a composition of embodiment 31. 61. A method for increasing T cell effector function, including proliferation, or cytokine production and / or secretion, the method comprising introducing into a T cell an expression cassette described in any one of embodiments 25 to 30. 62. The method of embodiment 61, wherein the T cells are obtained from a human before the T cells are outcompeted to overexpress LTBR, ​​and the treated T cells are reintroduced into the human. 63. A method for increasing a response to a vaccine composition, comprising co-administering to a subject a vaccine comprising a nucleic acid encoding LTBR. 64. The method of embodiment 63, wherein the nucleic acid encoding LTBR encodes the LTBR intracellular domain, or a fragment or variant thereof. 65. The method of embodiment 64, wherein the LTBR intracellular domain comprises amino acids 249-435 of SEQ ID NO:2, or a fragment, deletion, or variant thereof. 66. The method of embodiment 64 or 65, wherein the LTBR intracellular domain has a deletion at least at amino acids 393 to 435. 67. The method of any one of embodiments 63 to 66, wherein expression of LTBR is transient. 68. A modified lymphocyte comprising an exogenous nucleic acid encoding a gene in Table 1. 69. The modified lymphocyte of embodiment 68, wherein the lymphocyte comprises an expression cassette comprising an expression control sequence and a nucleic acid encoding a gene of Table 1. 70. The modified lymphocyte of embodiment 68 or 69, wherein the lymphocyte further comprises a nucleic acid encoding a chimeric antigen receptor (CAR). 71. The modified lymphocyte of embodiment 70, wherein the expression cassette further comprises a nucleic acid encoding a CAR. 72. The modified lymphocyte of embodiment 68 or 69, wherein the lymphocyte further comprises a nucleic acid encoding a T cell receptor (TCR). 73. The modified lymphocyte of embodiment 72, wherein the expression cassette further comprises a nucleic acid encoding a TCR. 74. The modified lymphocyte of any one of embodiments 68-73, wherein the expression control sequence comprises an EF-1α, EFS, or CMV promoter. 75. The modified lymphocyte of embodiment 68, wherein the exogenous nucleic acid encoding a gene of Table 1 is mRNA. 76. The modified lymphocyte of any one of embodiments 68-75, wherein the lymphocyte is a T cell. 77. The modified lymphocyte of any one of embodiments 68-75, wherein the lymphocyte is an alpha beta T cell. 78. The modified lymphocyte of any one of embodiments 68-75, wherein the lymphocyte is a gamma delta T cell, optionally a Vγ9Vδ2 T cell. 79. The modified lymphocyte of any one of embodiments 68 to 75, wherein the lymphocyte is a NK cell. 80. The modified lymphocyte of any one of embodiments 68-75, wherein the lymphocyte is a NK T cell. 81. The method of any one of embodiments 70, 71, or 74-80, wherein CAR is axicabtagene siloreucel (Yescarta®), brexcabtagene outrucel (Tecartus®), idecabtagene bicurucel (Tecartus®), lysocabtagene maraleucel (Breyanzi®), tisagenlecleucel (Kyrmriah®), or one of those found in FIG. 19. The modified lymphocytes. 82. A vaccine composition comprising a nucleic acid encoding a gene of Table 1 and a nucleic acid encoding a viral protein. 83. The vaccine composition of embodiment 82, wherein the viral protein is a glycoprotein. 84. The vaccine composition of embodiment 83, wherein the glycoprotein is a viral spike protein. 85. The vaccine composition of embodiment 84, wherein the viral spike protein is a coronavirus spike protein. 86. The vaccine composition according to any one of embodiments 82 to 85, wherein the nucleic acid encoding the gene of Table 1 is mRNA, or the nucleic acid encoding the viral spike protein is mRNA, or both. 87. An expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor and a nucleic acid encoding a gene in Table 1. 88. An expression cassette comprising a nucleic acid encoding a T cell receptor and a nucleic acid encoding a gene in Table 1. 89. An expression cassette comprising a nucleic acid encoding a viral protein and a nucleic acid encoding a gene in Table 1. 90. A composition comprising a modified lymphocyte comprising an expression cassette of any one of embodiments 87 to 89. 91. A method of producing a modified lymphocyte, comprising introducing into a lymphocyte an exogenous nucleic acid encoding a gene in Table 1. 92. The method of embodiment 91, wherein the lymphocyte comprises an expression cassette comprising an expression control sequence and a nucleic acid encoding a gene of Table 1. 93. The method of embodiment 91 or 92, wherein the lymphocyte further comprises a nucleic acid encoding a chimeric antigen receptor (CAR). 94. The method of embodiment 93, wherein the expression cassette further comprises a nucleic acid encoding a CAR. 95. The method of embodiment 91 or 92, wherein the lymphocyte further comprises a nucleic acid encoding an engineered T cell receptor (TCR). 96. The method of embodiment 95, wherein the expression cassette further comprises a nucleic acid encoding a TCR. 97. The method of any one of embodiments 91 to 97, wherein the expression control sequence comprises an EF-1α (full length or truncated) or CMV promoter. 98. The method of embodiment 91, wherein the exogenous nucleic acid encoding a gene of Table 1 is mRNA. 99. The method of any one of embodiments 91 to 98, wherein the lymphocyte is a T cell, optionally a CD4+ T cell, a CD8+ T cell, or a Treg cell. 100. The method of any one of embodiments 91-99, wherein the lymphocytes are alpha beta T cells. 101. The method of any one of embodiments 91-98, wherein the lymphocytes are gamma delta T cells. 102. The method of any one of embodiments 91 to 98, wherein the lymphocytes are NK cells. 103. The method of any one of embodiments 91-98, wherein the lymphocytes are NK T cells. 104. CAR is selected from axicabtagene siloreucel (Yescarta®), brexcabtagene outrucel (Tecartus®), idecabtagene bicurucel (trademark), lysocabtagene maraleucel (Breyanzi®), tisagenlecleucel (Kyrmriah®), or any of the compounds shown in FIG. The method of any one of embodiments 93, 94, or 97 to 103, wherein the method is one of the following: 105. The method of any one of embodiments 91-104, wherein the lymphocytes are nucleofected with mRNA encoding a gene of Table 1. 106. A method for treating cancer in a subject in need of such treatment, the method comprising administering to the subject an expression cassette described in any one of embodiments 68 to 81, embodiments 87 to 89, or a composition described in embodiment 90. 107. The method of embodiment 106, wherein the subject has lymphoma. 108. The method of embodiment 106, wherein the subject has a solid tumor. 109. The method of embodiment 106, wherein the subject has leukemia. 110. The method of embodiment 106, wherein the subject has multiple myeloma. 111. The method of embodiment 106, wherein the subject has a virally-driven cancer. 112. The method of embodiment 111, wherein the subject has HPV. 113. The method of embodiment 106, wherein the subject has a cancer that is Burkitt's lymphoma, liver cancer, Kaposi's sarcoma, cervical cancer, head cancer, neck cancer, anal cancer, oral cavity cancer, pharyngeal cancer, penile cancer, adult T-cell lymphoma, or Merkel cell carcinoma. 114. A method of treating a disease in a subject in need of such treatment, the method comprising administering to the subject a composition described in any one of embodiments 82-86. 115. The method of embodiment 114, wherein the disease is HIV. 116. The method of embodiment 114, wherein the disease is HPV. 117. The method of embodiment 114, wherein the disease is an autoimmune disorder. 118. A method for increasing T cell effector function, including proliferation, or cytokine production and / or secretion, the method comprising introducing into a T cell a composition described in any one of embodiments 87 to 89. 119. The method of embodiment 118, wherein the T cells are obtained from a human before treating the T cells to overexpress the genes of Table 1, and the treated T cells are reintroduced into the human. 120. A method of increasing a response to a vaccine composition, comprising co-administering a nucleic acid encoding a gene of Table 1 with the vaccine. 121. The method of any one of embodiments 118-120, wherein the expression of the genes of Table 1 is transient. 122. The genes in Table 1 are LTBR, ​​ADA, IFNL2, IL12B, CALML3 122. The modified lymphocyte, composition, expression cassette, or method of any one of embodiments 68-121, wherein the modified lymphocyte, composition, expression cassette, or method is MRPL51, DBI GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4. 123. A method for identifying a gene that, when exogenously expressed in an engineered lymphocyte, alters a therapeutic function of the engineered lymphocyte, the method comprising: (a) obtaining a population of lymphocytes; (b) transducing the lymphocyte population with a plurality of viral vectors, each viral vector encoding a gene that can be linked to one or more barcodes; (c) stimulating the transduced lymphocytes to induce activation, proliferation, and / or effector function; (d) isolating transduced lymphocytes from the lymphocyte population of (c); and (e) detecting the presence of the gene and / or the linked barcode in the isolated lymphocytes; The method, wherein the detected gene is effective to alter the therapeutic function of an engineered lymphocyte expressing the gene. 124. The method of embodiment 123, wherein the gene is a nucleotide sequence encoding an open reading frame (ORF) or a non-coding RNA, optionally a microRNA (miRNA) or a long non-coding RNA (lncRNA, long ncRNA). 125. The method of embodiment 123 or 124, comprising a cell population, wherein the lymphocyte population is enriched for one or more of T cells, B cells, NK T cells, NK cells, or subpopulations thereof, and optionally, the cells are human. 126. The method of embodiment 125, wherein the lymphocyte population is enriched for one or more of CD4+ T cells, CD8+ T cells, αβ T cells, and γδ T cells. 127. The method of any one of embodiments 123 to 126, wherein the lymphocyte population comprises CAR T cells. 128. The method of any one of embodiments 123 to 126, wherein the lymphocyte population comprises lymphocytes comprising an engineered TCR expressed on their surface. 129. The method of any one of embodiments 123 to 128, wherein the lymphocyte population comprises a cell line. 130. The method of any one of embodiments 123 to 129, wherein the plurality of viral vectors comprises a library of open reading frames (ORFs). 131. The method of any one of embodiments 123 to 130, wherein the viral vector comprises a retroviral vector or a lentiviral vector. 132. The method of any one of embodiments 123 to 131, wherein (b) comprises transducing at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the lymphocyte population. 133. The method according to any one of embodiments 123 to 132, wherein the viral vector comprises an expression cassette having an elongation factor-1 alpha short (EFS) promoter, a cytomegalovirus (CMV) promoter, or a phosphoglycerate kinase-1 (PGK) promoter. 134. The method according to any one of embodiments 123 to 133, wherein the viral vector comprises a nucleotide sequence encoding a selection gene or marker. 135. The method of any one of embodiments 123-134, wherein the modified lymphocytes are γδ T cells or αβ T cells. 136. The method of any one of embodiments 123-134, wherein the modified lymphocytes are T cells expressing Vγ9- or Vγ9Vδ2 TCR. 137. The method of any one of embodiments 123 to 136, wherein the modified lymphocytes are CAR T cells. 138. The method of any one of embodiments 123 to 136, wherein the modified lymphocytes are selected based on expression of an endogenous antigen receptor, optionally a TCR. 139. The method of any one of embodiments 123 to 138, wherein stimulating the transduced lymphocytes comprises culturing the lymphocytes with one or more of an antibody, a cytokine, an antigen, a superantigen, an antigen-presenting cell, a cancer cell, and a cancer cell line. 140. The method according to any one of embodiments 123 to 138, wherein stimulating the transduced lymphocytes comprises TCR stimulation, and optionally CD3 / CD28 stimulation. 141. The method of any one of embodiments 123 to 140, further comprising labeling the transduced lymphocytes with a cytoproliferative dye and isolating the progeny cells. 142. The method of any one of embodiments 123 to 141, wherein (d) comprises identifying cells expressing one or more cell surface markers and / or one or more effector functions and / or one or more secreted cytokines. 143. The method of embodiment 142, wherein the one or more cell surface markers comprise CD69, CD25, OX40L (CD154), ICAM-1, CD70, CD74, CD54, MHC-II, CD137, CD44, CD62L, CCR7, CD107a, PD1, TIM3, LAG3, CD80, CD86, TIGIT, VISTA, B7-H3, BTLA, and SIGLEC15. 144. The one or more effector functions include production and / or secretion of a cytokine or chemokine, optionally the cytokine or chemokine is IL-2, IL-12, The method of any one of embodiments 123 to 143, wherein the antibody is one or more of IL-23, IFNγ, TNF, GM-CSF, IL7, IL15, IL12, IL18, IL21, IL23, LTA, IL4, IL5, IL6, IL10, IL13, TGF beta, IL17, LTA, LIGHT, CCL3, CCL4, and CCL5. 145. The method of any one of embodiments 123-144, wherein the one or more effector functions include a cytotoxic potential, optionally determined by expression of perforin and / or granzyme. 146. The method of any one of embodiments 123 to 145, wherein (e) comprises obtaining genomic DNA from isolated lymphocytes and PCR amplification of gene and / or barcode sequences. 147. The method of any one of embodiments 123 to 146, wherein (e) further comprises single-cell transcriptome and / or proteome analysis. 148. The method of any one of embodiments 123 to 147, wherein (e) comprises flow cytometry analysis, cell hashing, single-cell sequencing analysis, single-cell RNA sequencing (scRNA-seq), Perturb-seq, CROP-seq, CRISP-seq, ECCITE-seq, or Cellular Indexing of Transcriptomes and Epitopes (CITE-seq). 149. A method for analyzing the effect of overexpression of an ORF of interest on an individual cell, comprising: (a) introducing into a cell an expression cassette comprising a nucleic acid encoding an ORF of interest, thereby overexpressing the ORF; (b) providing a first set of nucleic acids from an individual cell and a first oligonucleotide having a first barcode sequence to a separate fraction, the oligonucleotides being bound to beads, the first set of nucleic acids including endogenous transcriptome mRNA and ORF mRNA; (c) performing RT-PCR to generate a second set of nucleic acids derived from the first set of nucleic acids, wherein the second set of nucleic acids in the fraction have bound thereto a first oligonucleotide comprising a first nucleic acid barcode sequence, and the RT-PCR is performed using RT-PCR reagents comprising a primer that specifically anneals to a sequence on the ORF mRNA that is not a polyA sequence, generating the second set of nucleic acids comprising endogenous transcriptome cDNA and ORF cDNA; (d) amplifying the second set of nucleic acids using PCR reagents that include a second primer that specifically anneals to a sequence on the ORF cDNA that is not a polyA sequence to generate a third set of nucleic acids; (e) detecting and / or sequencing the barcode sequence, the transcriptome cDNA, and / or the ORF cDNA. 150.(d') Obtaining a portion of the third set of nucleic acids and an ORF that is not a polyA sequence. 150. The method of embodiment 149, further comprising amplifying the ORF cDNA using a second set of PCR reagents comprising a third primer that specifically anneals to a sequence on the cDNA to generate a fourth set of nucleic acids. 151. The method of embodiment 150, further comprising using a third set of PCR reagents comprising (d") to amplify a fourth primer that specifically anneals to a sequence on the ORF cDNA that is not the ORF cDNA polyA sequence in the fourth set of nucleic acids to generate a fifth set of nucleic acids. 152. The method of any one of embodiments 149 to 151, further comprising contacting the cells of (a) with a construct comprising an antibody or antibody fragment bound to the first oligonucleotide. 153. The method of any one of embodiments 149 to 152, further comprising single-cell transcriptome and / or proteome analysis. 154.(e) Flow cytometry analysis, cell hashing, single cell sequencing analysis, single cell RNA sequencing (scRNA-seq), Perturb-seq, CRO The method of any one of embodiments 149 to 153, comprising P-seq, CRISP-seq, ECCITE-seq, or Cellular Indexing of Transcriptomes and Epitopes (CITE-seq).

[0174] The following examples are provided for illustrative purposes only and should not be construed in any way to limit the invention to these examples, but rather to encompass any and all variations that become evident as a result of the teachings provided herein. EXAMPLES

[0175] Example 1 Materials and Methods Isolation and culture of primary human T cells Standard buffy coats containing peripheral blood from de-identified healthy donors were collected by the New York Blood Center, from which they were purchased under an IRB-waived protocol. All donors provided informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats using Lymphoprep (Stemcell) gradient centrifugation. For most assays, CD8 + and CD4 + were isolated consecutively from the same donor. First, CD8 + T cells were isolated by magnetic positive selection using the EasySep Human CD8 Positive Selection Kit II (Stemcell). + T cells were isolated using EasySep human CD4 + γδ T cells were isolated from the flow-through obtained by negative magnetic selection using a T cell isolation kit (Stemcell). γδ T cells were isolated by magnetic negative selection using the EasySep human gamma / delta T cell isolation kit (Stemcell). Immediately after isolation, T cells were cultured at 10 ng ml -1 They were resuspended in T cell media consisting of Immunocult-XF T cell expansion medium (Stemcell) supplemented with recombinant human IL-2 (Stemcell).

[0176] T cell activation was achieved with 10 cells / ml 6Immunocult human CD3 / CD28 T cell activator (Stemcell) was used, using 25 μl per cell. Typically, T cells were transduced with concentrated lentivirus 24 hours after isolation. In some experiments, T cells were electroporated with in vitro transcribed mRNA 24 hours after isolation or with Cas9 protein 48 hours after isolation. 72 hours after isolation, lentivirally transduced T cells were transduced with 2 μg ml -1 Selection was performed with puromycin.

[0177] Split T cells or replace the medium every 2-3 days and culture at 1 x 10 cells per ml. 6 ~2×10 6 A cell density of 1000–1000 cells was maintained. Lentivirally transduced T cells were cultured at 2 μg ml -1 T cells were maintained in medium containing puromycin. T cells were used for phenotypic or functional assays between 14 and 21 days after isolation or cryopreserved in Bambanker cell freezing medium (Bulldog Bio). γδ T cells were further purified before functional assays using anti-Vγ9 PE antibody (Biolegend) and anti-PE microbeads (Miltenyi Biotec) according to the manufacturer's recommendations in the presence of the protein kinase inhibitor dasatinib to prevent activation-induced cell death resulting from TCR crosslinking. 42 PBMCs from patients with diffuse large B-cell lymphoma were obtained from the Perlmutter Cancer Center according to a protocol (S14-02164) approved by the Perlmutter Cancer Center Institutional Review Board.

[0178] Vector design and molecular cloning All vectors used were cloned using Gibson Assembly (NEB). In the experiments shown in Figure 1A-B, the lentiviral backbone from the pHAGE plasmid was used. 14In all other experiments, the backbone from lentiCRISPRv2 (Addgene 52961) was used. ORFs were PCR amplified for cloning from the genome-scale library used in the screen.

[0179] After adding Gibson overhangs by PCR, the ORF and P2A-puro were inserted into XbaI- and EcoRI-cut lentiCRISPRv2. The sgRNA cassette was removed from lentiCRISPRv2 using PacI and NheI digestion. For LTBR overexpression and knockout experiments, the sgRNA cassette was not removed. CAR was synthesized as gBlocks (IDT). For CAR-ORF cloning, CAR-P2A-puro-T2A (partial) was first inserted into XbaI- and EcoRI-cut lentiCRISPRv2. For subsequent ORF insertion, the plasmid was cut with HpaI located within the partial T2A and EcoRI. The following vectors were deposited with Addgene: pOT_01 (lenti-EFS-LTBR-2A-puro, Addgene 181970), pOT_02 (lenti-EFS-tNGFR-2A-puro, Addgene 181971), pOT_03 (lenti-EFS-FMC6.3-28z-2A-puro-2A-LTBR, ​​Addgene 181972), pOT_04(lenti-EFS-FMC6.3-BBz-2A-puro-2A-LTBR, ​​Addgene 181973), pOT_05(lenti-EFS-FMC6.3-28z-2A-puro-2A-tNGFR, Addgene 181974), and pOT_06(lenti-EFS-FMC6.3-BBz-2A-puro-2A-tNGFR, Addgene 181975).

[0180] Nuclease and CRISPR guide RNA design All sgRNAs were designed using the GUIDES web tool. 43We selected guides that targeted the first protein-coding exon (with a preference for targeting protein family domains, enabled by GUIDES), minimized off-targets, and maximized on-target scores. For Cas9 nuclease nucleofection, we used purified sNLS-SpCas9-sNLS nuclease (Aldevron).

[0181] Preparation of ORF library plasmids for paired-end sequencing Using Endura electrocompetent cells (Lucigen), we generated a genome-scale ORF library as previously reported. 14 The identity of the ORF and the corresponding barcode was confirmed by paired-end sequencing. Briefly, the plasmid was first linearized with I-SceI meganuclease, which cuts downstream of the barcode. The linearized plasmid was then tagged using TnY transposase. 44 The fragmented plasmid was then amplified in a PCR reaction with a forward primer binding to the handle introduced by TnY and a reverse primer binding to the sequence downstream of the barcode. All transposon and PCR primer oligonucleotides were synthesized by IDT. The resulting amplicons were sequenced on a NextSeq500. Forward reads (containing the ORF) were mapped to the GRCh38.101 CDS transcriptome annotation using STAR v.2.7.3a (map quality ≥ 10). 45Paired-end reads were also used to capture 24-nucleotide barcodes downstream of stationary plasmid sequences. ORF-barcode combinations were tabulated and the table was further curated by eliminating any spurious pairs that may be due to sequencing or PCR errors. Specifically, a permutation test was performed to identify the maximum number of ORF-barcode combinations expected by random chance, and then only ORF-barcode combinations with numbers exceeding this maximum were retained. All non-coding elements were removed from the reference, and barcodes that were within a Levenshtein distance of less than 2 were then tabulated. This caused the collapse of the Do.

[0182] cell culture HEK293FT cells were obtained from Thermo Fisher Scientific and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Serum Plus-II (Thermo Fisher Scientific). Nalm6, Jurkat, and BxPC3 cells were obtained from ATCC and cultured in RPMI-1640 supplemented with 10% Serum Plus-II. Capan-2 cells were obtained from ATCC and cultured in McCoy's medium supplemented with 10% Serum Plus-II. For γδ co-incubation experiments, cell lines were pretreated with 50 μM zoledronic acid (Sigma) for 24 h. Cell lines were routinely tested for mycoplasma using MycoAlert Plus (Lonza) and found to be negative. Cell lines were not confirmed in this study.

[0183] Lentivirus production Third generation lentiviral transfer plasmids were transfected with the packaging plasmid psPAX2 (Addgene 12260) and the envelope plasmid pMD2.G (Addgene 12260) using polyethyleneimine linear MW25000 (Polysciences). Lentivirus was produced by co-transfecting HEK293FT cells with 12259 (Figure 1). After 72 h, the supernatants were collected, filtered through 0.45 μm Steriflip-HV filters (Millipore), and the virus was concentrated using lentivirus precipitation solution (Alstem). The concentrated lentivirus was resuspended in T cell medium containing IL-2 and stored at -80°C.

[0184] Pooled ORF library screening Pooled ORF library screening revealed that CD4 + and CD8 + T cells were cultured at least 500 × 10 6 lentivirus were isolated from PBMCs. The amount of lentivirus used for transduction was titrated to result in a transduction efficiency of 20-30% to minimize the chance of multiple ORFs being introduced into a single cell. Cells were maintained in T cell medium containing 2 μg ml-1 puromycin and counted every 2-3 days to obtain a concentration of 1 × 10 cells per ml. 6 ~2×10 6 At day 14 post-isolation, T cells were harvested, counted, labeled with 5 μM CFSE (Biolegend) and cultured at a density of 1 × 10 cells. 6 Cells were stimulated with 1.56 μl of CD3 / CD28 activator (Stemcell) per cell. An aliquot of cells representing 1,000× coverage of the library was frozen at this step to serve as a pre-stimulation control. Four days after stimulation, cells were harvested and an aliquot of cells representing 1,000× coverage of the library was frozen to serve as a pre-sorting control. The remaining cells were stained with LIVE / DEAD Violet cell viability dye (Thermo Fisher Scientific) and CFSE. 低 Cells (corresponding to the bottom 15% of the distribution) were sorted using a Sony SH800S cell sorter. Genomic DNA was isolated and subjected to two rounds of PCR to amplify the ORF barcodes and add Illumina adapters. 46 .

[0185] Pooled ORF screening analysis For most analyses, equal numbers of reads from all three donors were combined per bin prior to trimming and alignment. Barcodes were adapter trimmed using Cutadapt v.1.13 (-m24-e0.1--discard-untrimmed) and then mapped to the reference library using Bowtie v.1.1.2 (-v1-m1--best--strata). 47、48 All subsequent analyses were performed in RStudio v.1.1.419 with R4.0.0.2. To calculate individual barcode enrichment, barcode counts were normalized to the total number of reads per sample (pseudocounts were added) and log2 transformed. To calculate ORF enrichment, To do this, raw barcode counts were first collapsed to genes before normalization and log2 transformation.

[0186] Enrichment analysis was performed at both the barcode and gene levels. Statistical analysis of barcode enrichment was performed on CD4 + and CD8 + as replicates, and CFSE 低 Samples were compared with the corresponding inputs (before stimulation) to obtain MAGeCK 49 Statistical analysis of ORF enrichment was performed using DESeq2. 50 Raw gene counts were obtained by collapsing the barcodes into their corresponding genes. CFSE 低 The samples were + and CD8 + were used as replicates and compared with the corresponding inputs (both pre-stimulation and pre-sorting). adj The GO enrichment (biological process) of genes matching the GO criteria (<0.05) was analyzed using the topGO package 51 CFSE was used. 低 For genes enriched in the screening (DESeq2 analysis), These genes were overlapped with genes differentially expressed after CD3 / CD28 stimulation using data from the Database of Immune Cell eQTLs, Expression, Epigenomics (DICE; https: / / dice-database.org / ). 41 For differentially expressed genes, the following DICE datasets were used: T cells, CD4, naive vs. T cells, CD4, naive [activated], T cells, CD8, naive vs. T cells, CD8, naive [activated]. Significant differential expression was as obtained in the DICE dataset (P adj <0.05).

[0187] Proliferation assay Transduced T cells were harvested 14 days after isolation, counted, and plated in round-bottom 96-well plates at 2.5 x 10 cells per well in two sets of triplicate wells per transduction. 4 One set of triplicate wells was seeded with 10 ng ml -1 Cells were cultured in Immunocult-XF T cell expansion medium supplemented with 1.5 μl of IL-2 and another set of triplicate wells was further supplemented with 1.56 μl of CD3 / CD28 activator per ml of medium. Cells were cultured for 4 days and then harvested and stained with LIVE / DEAD Violet cell viability dye. Prior to flow cytometry acquisition, cells were resuspended in D-PBS supplemented with 10% v / v Precision Counting Beads (Biolegend). For quantification, the number of viable cell events was normalized to the number of bead events per sample. For each ORF, the normalized viable cell count in wells supplemented with CD3 / CD28 activator was then divided by the average number of viable cells in control wells to quantify T cell proliferation. Donor and CD4 + / CD8 + To allow comparisons between T cells, proliferation of T cells transduced with a given ORF was finally normalized to proliferation of the appropriate tNGFR control.

[0188] In addition to the counting bead assay, proliferation was also measured using a dye dilution assay. For this assay, transduced T cells were harvested 14 days after isolation, washed with D-PBS, and then labeled with 5 μM CellTrace Yellow (CTY) in D-PBS for 20 min at room temperature. Excess dye was removed by washing with a 5-fold excess of RPMI-1640 supplemented with 10% Serum Plus-II. The labeled cells were then plated at 2.5 × 10 cells per well. 4 One set of triplicate wells was cultured in supplemented Immunocult-XF T cell expansion medium (i.e., no IL-2) and another set of triplicate wells was cultured in 10 ng ml per ml medium. -1 The cells were supplemented with 1.56 μl of IL-2 and 1.56 μl of CD3 / CD28 activator. Cells were cultured for 4 days and then harvested and stained with LIVE / DEAD Violet cell viability dye. For quantification of the proliferation index, events were first gated on viable T cells with FlowJo (Treestar) and analyzed using the flowFit and flowCore packages. 52 The unstimulated cells were used to send for further analysis in R / RStudio using To account for differences in staining intensity between different samples, the size and location of the parent population were determined. These fitted parent population parameters were then used to fit the CTY profiles of the corresponding stimulated samples, modeled as Gaussian distributions assuming log2-spaced peaks as a result of cell division and dye dilution. The fitted CTY profiles were visually inspected for agreement with the original CTY profiles and used to calculate a proliferation index, which is defined as the sum of all generations of cells divided by the calculated number of parent cells present at the start of the assay.

[0189] Flow cytometry for cell surface and intracellular markers For CD25 (IL2RA) and CD154 (CD40L) quantification, the cells were incubated for 6 h before staining (CD8 + CD154 staining in the 24-h group) or 24 h prior (CD4 +and CD8 + CD25 staining in both + (CD154 staining in 1000 cells), T cells were treated with CD3 / CD28 activator (cells 10 6 For Ki-67 and 7-amino-actinomycin D (7-AAD) staining, T cells were rested overnight in Immunocult-XF T cell growth medium without IL-2, then stimulated with CD3 / CD28 activator (10 cells / mL). 6 In other cases, T cells were stained without stimulation. For detection of secreted proteins, T cells were stimulated with CD3 / CD28 activator (10 cells / mL). 6 The cells were stimulated with LTA (25 μl per cell) (LTA, LIGHT) for 24 h and treated with the protein transport inhibitor brefeldin A (5 μg ml -1 ) and monensin (2 μM) were included for the last 6 h of stimulation (IL12B, LTA, LIGHT).

[0190] First, cells were harvested, washed with D-PBS, and stained with LIVE / DEAD Violet cell viability dye for 5 min at room temperature in the dark, followed by surface antibody staining for 20 min on ice. After surface antibody staining (if applicable), cells were washed with PBS and acquired on a Sony SH800S cell sorter or harvested for intracellular staining. For intracellular staining, cells were resuspended in the appropriate fixation buffer. The following fixation buffers were used for specific protein detection: Fixation Buffer (Biolegend) for IL12B and MS4A3 staining; True-Nuclear Transcription Factor Fix (Biolegend) for BATF, TCF1, and FLAG staining; and FoxP3 / Transcription Factor Fixation for Ki-67. After resuspension in fixation buffer, cells were incubated for 1 h at room temperature in the dark. After incubation, cells were washed twice with the appropriate permeabilization buffer. The following permeabilization buffers were used: Intracellular Staining Permeabilization Wash Buffer (Biolegend) for IL12B and MS4A3 staining; True-Nuclear Perm Buffer (Biolegend) for BATF, TCF1 and FLAG staining; and FoxP3 / Transcription Factor Permeabilization Buffer (eBioscience) for Ki-67. After permeabilization, cells were stained with specific antibodies or isotype controls for 30 min at room temperature in the dark. Finally, cells were washed twice with the appropriate permeabilization buffer and acquired on a Sony SH800S flow cytometer. For cell cycle analysis, cells were stained with 0.5 μg ml -1 Further staining was performed with 7-AAD for 5 min. Gating was performed using appropriate isotype, fluorescence minus 1, and biological controls. Typically, 5,000-10,000 live events were recorded per sample.

[0191] Flow cytometric detection of phosphorylated proteins T cells were rested for 24 h in Immunocult-XF T cell expansion medium without IL-2, and phosphorylated proteins were detected. Resting cells were incubated with CD3 / CD28 activator (10 cells / mL) 6 Immediately after the stimulation period, cells were stimulated with 1:1 volumetric ratio of prewarmed Fixation Buffer (Bioleg). The cells were fixed with 1 ml of pre-chilled True-Phos Perm Buffer (Biolegend) at 37°C for 15 min and washed twice with cell staining buffer (D-PBS + 2% FBS). The cells were resuspended in the residual volume and permeabilized in 1 ml of pre-chilled True-Phos Perm Buffer (Biolegend) with vortexing according to the manufacturer's protocol. The cells were incubated in True-Phos Perm Buffer at -20°C for 60 min. After permeabilization, the cells were washed twice with cell staining buffer and stained with anti-CD4, anti-CD8, anti-RELA, and anti-phospho-RELA antibodies (or isotype control) for 30 min at room temperature. After staining, the cells were washed twice in cell staining buffer and acquired on a Sony SH800S cell sorter. Gating was performed on CD4 + or CD8 + Cells were assayed and levels of RELA and phospho-RELA were determined using appropriate isotype and biological controls.

[0192] Western blot detection of proteins and phosphorylated proteins They express tNGFR or LTBR and are either resting or stimulated with CD3 / CD28 activators (cells 10 6T cells stimulated with 25 μl of 100 mM Tris-HCl (pH 7.4) for 15 min were harvested, washed with 1×D-PBS, and lysed in TNE buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) in the presence of protease inhibitor cocktail (Bimake B14001) and phosphatase inhibitor cocktail (Cell Signaling Technologies 5872S) for 1 h on ice. Cell lysates were spun at 10,000 g for 10 min, and protein concentrations were determined by BCA assay (Thermo Fisher Scientific). Equal amounts of cell lysates (25 mg) were denatured in Tris-Glycine SDS sample buffer (Thermo Fisher Scientific) and loaded onto Novex 4-12 or 4-20% Tris-Glycine gels (Thermo Fisher Scientific). Protein sizes were determined using PageRuler prestain protein ladder (Thermo Fisher Scientific). Gels were run in 1× Tris-glycine-SDS buffer (IBI Scientific) for approximately 120 min at 120 V. Proteins were transferred onto nitrocellulose membranes (BioRad) in the presence of pre-chilled 1× Tris-glycine transfer buffer (Thermo Fisher Scientific) supplemented with 20% methanol at 100 V for 100 min.

[0193] Immunoblots were blocked with 5% nonfat milk in 1× PBS supplemented with 1% Tween-20 (PBST) and incubated with the following primary antibodies: rabbit anti-GAPDH (0.1 mg ml -1, Cell Signaling, 2118S), mouse anti-IKKα (1:1,000 dilution, Cell Signaling, 3G12), rabbit anti-IKKβ (1:1,000 dilution, Cell Signaling, D30C6), rabbit anti-NF-κB p65 (1:1,000 dilution, Cell Signaling, D14E12), rabbit anti-phospho-NF-κB p65 Ser536 (1:1,000 dilution, Cell Signaling, 93H1), mouse anti-IκBα (1:1,000 dilution, Cell Signaling, L35A5), rabbit anti-phospho-IκBα Ser32 (1:1,000 dilution, Cell Signaling, 14D4), rabbit anti-NF-κB p100 / p52 (1:1,000 dilution, Cell Signaling, Signaling, 4882), and rabbit anti-RELB (1:1,000 dilution, Cell Signaling, C1E4) separately at 4°C overnight. After the primary antibodies, the blots were incubated overnight with IRDye 680RD donkey anti-rabbit (0.2 mg ml -1 , LI-COR 926-68073) or IRDye 800CW donkey anti-mouse (0.2 mg ml -1 , LI-COR 926-32212). Blots were imaged using Odyssey CLx (LI-COR) and quantified using ImageJ v.1.52.

[0194] Quantification of cytokine secretion For the measurement of secreted IFNγ and IL-2, T cells were first harvested and incubated in the absence of IL-2. They were then counted and plated in round-bottom 96-well plates at 2.5 x 10 cells per well. 4 10 cells and treated with CD3 / CD28 activator (10 cells 6Cells were incubated for 24 h in IL-2-free medium with or without IL-2 (25 μl per cell). Cell supernatants were then harvested, diluted, and used for cytokine quantification using enzyme-linked immunosorbent assays (human IL-2 or IFNγ DuoSet, R&D Systems) using an Infinite F200 Pro (Tecan) plate reader. Multiplex quantification of secreted cytokines and chemokines in resting or stimulated T cells was performed using the Human Cytokine / Chemokine 48-Plex Discovery Assay Array (Eve Technologies).

[0195] T cell killing assay CD19 + Nalm6 cells were first transduced with a lentiviral vector encoding EGFPd2PEST-NLS and a puromycin resistance gene. 53 Transduced cells were kept under puromycin selection throughout culture to maintain stable EGFP expression, and puromycin was only removed from the medium prior to the killing assay. T cells were transduced with vectors encoding CARs specific for CD19 with either a CD28 stalk, a CD28 transmembrane, and a CD28 signaling domain, or a CD8 stalk and a CD8 transmembrane domain with a 4-1BB signaling domain, and a CD3ζ signaling domain. 54 Fourteen days after transduction, transduced T cells were cultured at 5 × 10 4 Nalm6 GFP + Cells were mixed in Immunocult media without IL-2 in triplicate at the indicated effector:target ratios. Wells were then imaged using an Incucyte SX1 using 20x magnification, acquiring 4 images per well every 2 hours for up to 120 hours. For each well, the incorporated GFP intensity was normalized to the 2 hour time point, which was allowed to settle completely after cells were seeded.

[0196] In vitro mRNA preparation Templates for in vitro transcription were generated by PCR from plasmids encoding LTBR or tNGFR, and the resulting amplicons contained a T7 promoter upstream of the ORF. Purified templates were then transfected with HiScribe T7 ARCA mRNA PCR kit. The kit was used for in vitro transcription with capping and polyA tailing using the Kit with Capping (NEB).

[0197] Primary T cell nucleofection Activated T cells were nucleofected with in vitro transcribed mRNA 24 hours after activation or with Cas9 protein 48 hours after activation. Cells were harvested, washed twice with PBS, and incubated at 5×10 cells per 20 μl. 5 Cells were resuspended in P3 Primary Cell Nucleofector Solution (Lonza) at 10x10 cells / ml. Immediately after resuspension, 1 μg of mRNA or 10 μg of Cas9 (Aldevron) was added (not to exceed 10% v / v of the reaction) and cells were nucleofected in a 4D-Nucleofector (Lonza) using the E0-115 program. After nucleofection, cells were resuspended in pre-warmed Immunocult medium with IL-2 and allowed to recover for 20 min at 37°C with 5% CO2. After recovery, cells were resuspended at 1x10 cells / ml. 6 were seeded at 100x and used for downstream assays.

[0198] OverCITE-seq sample preparation and sequencing Single cell sequencing revealed that CD8 + T cells were individually transduced with the ORFs and separately maintained under puromycin selection for 14 days. The transduced cells were then combined and split into two conditions, one in which IL-2 was only cultured for 24 hours, and the other in which 10 cells were cultured for 24 hours. 6 6 per piece After stimulation, cells were harvested, counted, and stained at 2×10 cells per ml in staining buffer (2% BSA+0.01% Tween-20 in PBS).7 Then, 10% (v / v) Human TruStain FcX Fc Receptor Blocking Solution (Biolegend) was added, and the cells were incubated at 4°C for 10 min. After Fc receptor blocking, the cell concentration was adjusted to 5 × 10 cells per ml. 6 The stimulated and unstimulated cells were each divided into four conditions. Each condition received a different oligonucleotide-conjugated (barcoded) cell hashing antibody to allow for pooling of different conditions in the same 10x Genomics Chromium lane. 23 After 20 min co-incubation on ice, cells were washed three times with staining buffer and counted using trypan blue exclusion. Cell viability was typically approximately 95%.

[0199] Cells stained with the different hashing antibodies were then pooled together in equal numbers and stained with the following oligonucleotide-conjugated (barcoded) antibodies for quantification of cell surface antigens: CD11c (0.1 μg), CD14 (0.2 μg), CD16 (0.1 μg), CD19 (0.1 μg), CD56 (0.2 μg), CD3 (0.2 μg), CD45 (0.01 μg), CD45RA (0.2 μg), CD45RO (0.2 μg), CD4 (0.1 μg), CD8 (0.1 μg), CD25 (0.25 μg), CD69 (0.25 μg), and NGFR (0.25 μg) (TotalSeq-C, Biolegend). Cells were stained for 30 min on ice, washed three times with staining buffer, resuspended in PBS, and filtered through a 40 μm cell strainer. Cells were then counted and the concentration was adjusted to 1 × 10 6 pieces ml -1 For loading into 10x Genomics Chromium, 3x10 cells were used. 4 Each was supplemented with a custom reverse primer that binds to a puromycin resistance cassette to facilitate ORF transcript capture during the reverse transcription step. It was combined with Next GEM Single Cell 5' v2 Master Mix (10x Genomics). The custom reverse primer was added at a 1:3 ratio to the poly-dT primer included in the master mix.

[0200] For cDNA amplification, additional primers for sample hashing and amplification of surface antigen barcodes 23 , and a nested reverse primer that binds to a puromycin resistance cassette downstream of the ORF. After cDNA amplification, SPRI beads were used to size select the resulting PCR products: small size (<300 bp) and physically separate the sample hashing and surface antigen barcodes from the larger cDNA and ORF amplicons for downstream processing. The sample hashing and surface antigen barcodes were also processed 22 The amplified cDNA was then split into three conditions for the construction of gene expression libraries, αβTCR libraries, and ORF libraries. The ORF library was treated similarly to the αβTCR library, using a nested reverse primer that binds downstream of the ORF. The quality of the generated libraries was verified on a BioAnalyzer using a high sensitivity DNA kit (Agilent). The libraries were sequenced on a NextSeq500. The gene expression library generated more than 25,000 reads per cell. The other libraries generated more than 5,000 reads per cell.

[0201] OverCITE-seq data analysis Gene expression unique molecular identifier (UMI) count matrices and TCR clonotypes were derived using 10x Genomics Cell Ranger 3.1.0. Hashtag oligo (HTO) and antibody UMI count matrices were generated using kallisto v.0.46.0. 55 and bustool v.0.39.3 56 ORF reads were generated using Bowtie2 v.2.2.8. 57The plasmids were first aligned to the plasmid reference and indexed to the relevant ORFs using , and then a UMI count matrix was generated using Callisto and Bastool. Darity was normalized using a centered log-ratio (CLR) transformation. Cell doublets and negatives were analyzed using HTODemux. 58 function and then excluded from downstream analysis. The UMI cutoff quantile in HTODemux was optimized to maximize singlet recovery using a grid search with values ​​between 0 and 1. ORF singlets were identified using the MULTIseqDemux 59 Cells with low quality gene expression metrics were then filtered out, and cells with fewer than 200 unique RNA features or more than 5% of reads mapping to the mitochondrial transcriptome were filtered out.

[0202] The count matrix was then run in Seurat v.4.0.1 60 The gene expression data was loaded into and analyzed using CellCycleScoring function with default genes for cell cycle correction and scaling, followed by data scaling using the ScaleData function. Principal component (PC) optimization of the scaled and corrected data was then performed using JackStraw 61 and selected all PCs up to the first non-significant PC to be used for clustering. Cell clustering was performed using a shared nearest neighbor (SNN)-based clustering algorithm and UMAP dimensionality reduction. 62 Cluster PCs were visualized using and projected into 2D space. Cluster marker analysis was performed using the FindAllMarkers function with a hypothesis set defined as positive and negative markers present in at least 25% of clustered cells and a log2-transformed fold change threshold of 0.25 compared to non-clustered cells. Differential expression analysis of ORFs was performed using DESeq2. 50The differential expression was determined using the Storey method to identify genes that were up- and down-regulated in ORF-expressing cells compared to NGFR (control) cells. 63 was defined as having a q<0.1, calculated using

[0203] Bulk RNA-seq and analysis CD4 + and CD8 + LTBR-transduced or tNGFR-transduced T cells were incubated with CD3 / CD28 activators (10 cells / 6 Cells were stimulated with 25 μl of PBS (per cell) or left unstimulated for 24 h (n = 3 biological replicates). Total RNA was extracted using the Direct-zol RNA purification kit (Zymo). 3' enriched RNA-seq libraries were prepared as previously reported. 64 Briefly, RNA was reverse transcribed using SMARTScribe reverse transcriptase (Takara Bio) and a poly(dT) oligo containing a partial Nextera handle. The resulting cDNA was then PCR amplified for three cycles using OneTaq polymerase (NEB) and tagged with a homemade transposase TnY for 5 min at 55 °C. 44 Immediately after, tagged DNA was purified with MinElute columns (Qiagen) and PCR amplified for 12 cycles using OneTaq polymerase and barcoded primers. PCR products were purified using dual (0.5×–0.8×) SPRI cleanup (Agencourt) and size distribution was determined using Tapestation (Agilent). Samples were sequenced on a NextSeq 500 (Illumina) using the v2.5 75 cycle kit (paired end). Paired-end reads were analyzed using Callisto v.0.46.0 55 Using the transcriptome (Human Ensemble See v.96 65 ) and the tximport package 66 Loaded into RStudio1.1.419 with R 4.0.0.2 using DESeq250 Differential gene expression analysis was performed using the topGO package. GO enrichment (biological processes) of genes meeting DESeq2 criteria (log2-transformed fold change >1, Padj <0.05) was analyzed using the topGO package. 51 This was done using

[0204] ATAC-seq library preparation CD8 + LTBR T cells and tNGFR T cells were treated with CD3 / CD28 activators (10 cells / 6 The cells were stimulated with 25 μl of 1000 μl / cell for 24 hours or left unstimulated (n=2). (biological replicates). Bulk ATAC-seq was performed as previously reported. 44 Briefly, cell membranes were dissolved in fresh RSB buffer (10 mM Tris-HCL, pH 7.4, 3 mM MgCl2, 10 mM NaCl) supplemented with 0.1% IGEPAL. After pipetting up and down, nuclei were isolated by centrifugation at 500g for 5 min at 4°C. After discarding the supernatant, the homemade transposase TnY protein was added to the nuclei. 44 Tagmentation DNA (TD) buffer containing 44 Nuclei were resuspended in 0.5% CO and incubated at 37° C. for 30 min. After purification on a MinElute column (Qiagen), the tagged DNA was purified using a homemade Pfu X7 DNA polymerase 44 and barcoded primers were used for PCR amplification for 12 cycles. PCR products were purified via 1.5x SPRI cleanup (Agencourt) and checked for characteristic nucleosome banding patterns using TapeStation (Agilent). Samples were sequenced on a NextSeq500 (Illumina) using the v2.5 75 cycle kit (single-end).

[0205] ATAC-seq analysis Single-ended reads with Bowtie2 v.2.4.4 57 Using Gencode hg38 primary assembly 67Next, the alignment was performed using SAMtools v.1.9 68 Filter alignments with low mapping quality (MAPQ<30) using the following and subsequently filter the BAM files: 68 The reads were sorted and indexed using Picard v.4.1.8.1. 69 Peaks were removed using MACS3 v.3.0.0 with default parameters (-g2.7e9 -q0.05). 70 Called using.

[0206] To construct the union feature space ("unionPeak") used for many of the downstream analyses, we used BEDTools v.2.29.0 (using bedtools intersect) to perform an intersection on pairs of narrowPeak files from biological replicates, and then replicated both replicates. 71 Only those peaks that were recognized in the replicates were kept. After marking the shared peaks between the replicates, Bedtools merge was used. The biological replicates in each shared peak (at least 1 bp overlap) were merged using merge. In this new peakBED file, each shared peak contains all sequences found under the peak in any of the biological replicates. We then took the union of each of these peak files (LTBR resting, LTBR stimulated, tNGFR resting, tNGFR stimulated) and combined any peaks with at least 1 bp overlap. The union peaks were used to generate a peak read count matrix (union peak x ATAC sample), where each entry in the matrix corresponds to the number of reads that overlap with that peak in a given sample - we call this the ATAC matrix per peak. The overlapping reads are taken directly from the BAM file (converted to BED) that provides the alignment for each sample. Therefore, the matrix contains a column for each biological replicate. Although the samples had minimal differences in aligned reads, we normalized each entry in the matrix by the number of reads that overlapped the TSS region in each sample. In this way, any differences in reads or alignment depth between samples are properly normalized. In addition to the peak-by-peak ATAC matrix, a gene-by-gene ATAC matrix was also constructed as follows: for all peaks within 3 kb of a gene's start or end coordinate, the gene's total ATAC reads were assigned as the sum of normalized reads from the peak-by-peak ATAC matrix.

[0207] These two ATAC matrices (peak-wise and gene-wise) were imported into R v.4.1.1 for gene and peak enrichment analysis using DESeq2 v.1.32.0. For comparisons between ATAC-seq and RNA-seq, statistical thresholds of adjusted P-values ​​<0.05 and log2-transformation fold change >0 (increases in ATAC or RNA) were used. Either log2-transformation fold change <0 (increase in ATAC or RNA) or log2-transformation fold change <0 (increase in ATAC or RNA) were used. For transcription factor-motif analysis, Chrom-VAR v.1.14.072 was used as follows: Summarized Experiment objects were constructed using the peak-by-peak matrix columns and sample subsets and the union feature space for each of the test versus control conditions. Transcription factor motifs were annotated using the matchMotifs function. Enrichment deviations between test and control conditions were calculated using the computeDeviations function.

[0208] To generate read pile-up tracks at specific genomic loci, deduplicated reads from biological replicates (BAMs) were pooled using samtools merge. These pooled replicate BAM files were converted to bigWig files by using the bamCoverage function from deeptools v.3.4.2, with scaleFactor set to the relative number of TSSs found in the pooled biological replicate compared to all other sample populations. The bigWig files were used to analyze the results using pyGenomeTracks v.3.6. 74 The lead pile-up was plotted using

[0209] Finally, k-means clustering was performed on ATAC peaks near genes with increased chromatin accessibility. First, DEseq2 was used on the gene-by-gene ATAC matrix to identify genes with a log2-transformation fold change >1 and an adjusted P-value <0.05 (i.e., genes with increased chromatin accessibility) in one of two comparisons: (1) LTBR stimulation vs. tNGFR stimulation, or (2) LTBR quiescence vs. tNGFR quiescence. After identifying these genes, we isolated all accessibility peaks in the peak-by-peak ATAC matrix within 3 kb of the gene body, and this subset of peaks from the peak-by-peak ATAC matrix was used as input for clustering. Next, k-means clustering was performed on this subset of ATAC peaks using deep tools (computeMatrix and plotHeatmap functions) with k=4 clusters and a 6 kb read window.

[0210] statistical analysis Data between two groups were compared using a two-tailed unpaired Student's t-test or Mann-Whitney test, as appropriate for the type of data (depending on the normality of distribution). Unless otherwise indicated, P values ​​of 0.05 or less were considered statistically significant for all analyses and were not corrected for multiple comparisons. When multiple comparison correction was required, P values ​​were adjusted using the Benjamini-Hochberg method. Unless otherwise stated, results for all groups are expressed as mean ± sem. Statistical analyses were performed in Prism (GraphPad) and RStudio (RStudio PBC). Flow cytometry data were analyzed using FlowJo v.10.7.1 (Treestar).

[0211] Example 2 Genome-scale screening for synthetic drivers of T cell proliferation Using a lentiviral library of barcoded human ORFs, primary human CD4 + and CD8 +We performed a genome-scale gain-of-function screen in T cells. We show that T cells with the strongest proliferative phenotypes are enriched for both known and unknown regulators of immune responses, many of which are not typically expressed by peripheral T cells. We validate the top-ranked ORFs in cells from donors unrelated to the screen and further demonstrate that these ORFs not only drive T cell proliferation but also increase expression of activation markers and secretion of key pro-inflammatory cytokines. To gain more comprehensive insight into the mechanism of action of these genes, we use a single-cell sequencing approach coupled with direct ORF capture. We develop a novel approach to identify LTBR - one of the top-ranked ORFs not expressed by lymphocytes - as a key driver of profound transcriptional and epigenetic remodeling via increased NF-κB signaling, which leads to a marked increase in the secretion of proinflammatory cytokines and resistance to apoptosis. Finally, we show that the top-ranked ORF enhances antigen-specific T cell function in the context of CD19-directed CAR T cells and a broad range of tumor-reactive γδ T cells from healthy donors and patients with hematological cancers.

[0212] Genome-scale ORF screening in T cells To avoid relying on constitutive expression of large bacterial proteins or on chromatin accessibility in the vicinity of target genes 13 Therefore, it was decided to use a lentiviral library of human ORFs, which contains nearly 12,000 full-length genes with approximately 6 barcodes per gene. 14 (Figure 1A, Figure 6A-6F). To date, genome-scale loss-of-function screening in human T cells has focused on CD4 + or CD8 + However, the focus has been on either CD4 + and CD8 + Both T cell types are required for sustained tumor control in adoptive therapy. 15、16 , defined as 1:1 CD4 + and CD8+ As further exemplified by the FDA approval of anti-CD19 CAR T cells having a ratio of 1 Therefore, we used the ORF library to identify CD4 + We aimed to discover genes that promote the proliferation of both CD4+ and CD8+ T cells (Figures 1A, 6G-I).

[0213] The lentiviral ORF library was cloned into CD4 + and CD8 + We transduced T cells and restimulated the cells after a short period of culture (14 days) to identify drivers of proliferation in response to TCR stimulation. We were able to capture the majority of the individual ORF barcodes, and nearly all ORFs, including the largest (Figure 6J). Comparing the relative frequency of genes in the most proliferative cells to unsorted cells, we found enrichment for genes known to be involved in immune processes among the top-ranked ORFs (Figure 6K). MAPK3 (encoding ERK1), a key mediator of T cell function 17 , costimulatory molecule CD59 18 , the transcription factor BATF, and cytokines known to promote T cell proliferation, such as IL12B and IL23A. 19 Indeed, two recent studies showed that overexpression of IL12B and BATF promoted proliferation, cytotoxicity, and cytokine secretion in CAR T cells. 19、20 .

[0214] Each ORF in the library is linked to an average of six DNA barcodes (Figure 6B). To increase confidence in our top-ranked ORFs from the pooled screen, we used proliferating CD4 + and CD8 +The enrichment of individual barcodes corresponding to a given ORF in cells was assessed (Figure 6B,C). For the majority of ORFs, multiple individual barcodes in each gene were enriched in the highly expanded population, suggesting that the observed enrichment does not result from spurious clonal expansion or PCR bias. Surprisingly, the most significantly enriched gene was lymphotoxin-β receptor (LTBR), a gene that is broadly expressed in stromal and myeloid cells but completely absent in lymphocytes.

[0215] Overall, the enriched ORFs spanned diverse biological processes. Among the top enriched gene ontology (GO) biological processes were lymphocyte proliferation, interferon gamma (IFNγ) production, and NF-κB signaling (Figure 6L). We observed that the enriched ORFs showed only a slight preference for genes endogenously upregulated by T cells during stimulation with CD3 and CD28 (CD3 / CD28), and in fact, represented all classes of differential expression (Figure 6M). This result highlights the power of pooled ORF screening to discover genes that enable T cell proliferation but are not normally expressed during CD3 / CD28-mediated activation and proliferation. For subsequent validation, We decided to test a wide range of ORFs that function in diverse pathways related to T cell compatibility and displayed different intrinsic modes of regulation. Neutral genes (MHC-I complex and cell type specific differentiation markers) were included for comparison. Genes were identified based on differential expression in CD3 / CD28 stimulated and resting T cells (upregulated: IL1RN, NFYB, BATF, AHNAK, CLIC1, RAN, DBI, GPD1, GPN3, AHCY, HOMER1, MRPL18, MRPL51, LIG3, ZNF830, HLA-A; downregulated: FOSB, ATF6B, SLC10A7, CDK2, ADA, CD19; unchanged: CDK1, DCLRE1B, B2M; no expression: IFNL2, LTBR, ​​CXCL12, CRLF2, IL12B, CALML3, CYP27A1, AKR1C4, DUPD1, NGFR). 41 .

[0216] The top ORF enhances T cell function To validate the top-ranked ORFs and understand their effects on other relevant aspects of T cell function, 33 ORFs from the library were subcloned into a vector co-expressing a P2A-binding puromycin resistance gene from the same promoter, which may be involved in the regulation of T cell phenotype. 21 A truncated nerve growth factor receptor (tNGFR), which lacks the intracellular domain, was chosen as a control that has no effect on CD4 + and CD8 + Populations were isolated separately from several screen-independent healthy donors and transduced with individual ORFs (Figure 2A). Using flow cytometry on representative ORFs, we confirmed that they were stably and uniformly expressed in both subsets of T cells over the course of the experiment (Figures 7A,B).

[0217] Fourteen days after isolation, the cells were restimulated and the relative increase in cell number was measured. The 16 tested ORFs significantly improved cell proliferation compared to tNGFR, and the proliferation was significantly higher than that of CD4 + Cells and CD8 + We found that the expression of the top ORFs correlated well with T cell proliferation (Spearman's r = 0.61, P = 0.002) (Figures 2B, 2C, 7C-H). Having established that the top ORFs improve T cell proliferation, we next tested whether there were changes in other T cell phenotypes and functions, such as increased cell cycle entry, expression of activation markers IL2RA (CD25) and CD40L (CD154), and cytokine secretion. The majority of the ORFs tested showed no difference in cycle (Figures 2I, 2J), but showed higher expression of both CD25 and CD154 in T cells after stimulation (Figures 2D, 8A), further supporting their effect in improving the magnitude of T cell responses.

[0218] Finally, secretion of the cytokines interleukin-2 (IL-2) and IFNγ after restimulation with CD3 / CD28 was measured (Figure 2E, Figure 8B-E). Although the screen was not designed to identify genes regulating cytokine secretion, some ORFs were able to both improve T cell proliferation and promote IL-2 or IFNγ secretion (Figure 2F). The strongest effect was observed for LTBR, ​​followed by CD4 + and CD8 + It increased secretion of both of these cytokines in T cells by more than five-fold.

[0219] Single-cell analysis of ORF phenotypes Based on the quantification of how each ORF affects proliferation, activation, and cytokine release, we next sought to better understand the underlying mechanisms that drive these changes in cellular state. To gain a more comprehensive view of the mechanism of action of individual ORFs and to provide a multidimensional characterization of the phenotypic changes they induce, we developed a single-cell sequencing strategy with direct ORF capture. This approach, OverCITE-seq (Overexpression of Epitopes by Transcriptome and Sequencing-Compatible Cellular Indexing), allows for the identification of surface antigens. 22 and CRISPR perturbation 23 This work expands on a previous approach we are developing to quantify the expression of IL-1 in T cells harboring distinct ORFs, allowing for high-throughput, single-cell analysis of pools of T cells harboring distinct ORFs. Briefly, mRNA from an ORF integrated by a lentivirus is expressed as a primer that binds to constant sequences in the transcript downstream of the ORF. The cDNAs are reverse transcribed by PCR and barcoded along with the cellular transcriptome during template switching. The resulting cDNA pool is then partitioned for the separate construction of gene expression and ORF expression libraries (Figure 3A, Figure 3B, Figure 9A).

[0220] CD8 from healthy donors +We optimized and applied OverCITE-seq to a pool of approximately 30 ORFs that were transduced into T cells. The cell pools were either left unstimulated ("resting") or stimulated with CD3 / CD28 antibodies to mimic TCR activation. To gain confidence in how well ORFs were assigned to each single cell, we exploited the fact that the protein produced by a control gene, tNGFR, is expressed at the cell surface and can therefore be captured with a DNA-barcoded antibody. 23 The percentage of cells marked as tNGFR positive was consistent when measured by CITE-seq or flow cytometry (Figure 3C). Analysis of the entire ORF pool showed that single cells assigned with a given ORF had the strongest expression of the corresponding gene overall (Figures 9B-D), indicating that our ORF capture strategy reliably assigned genetic perturbations to each single cell.

[0221] Unsupervised clustering showed a clear separation for stimulated and resting T cells. Within these activation-driven superclusters, individual clusters could be observed that were associated with specific cellular states or functions, such as cell cycle (clusters 1 and 9), macromolecular biogenesis (cluster 2), type I IFN signaling (cluster 3), cytotoxicity (cluster 6), T cell activation and proliferation (cluster 10), and stress response and apoptosis (cluster 11) (Figure 3D). Although in many cases several ORFs contributed to a given cluster phenotype (Figure 9E), a striking enrichment was observed for two ORFs, CDK1 and CLIC1, in cluster 1, characterized by increased expression of genes involved in chromosome condensation in preparation for the cell cycle (Figure 3E). An even stronger enrichment was observed in cluster 10, which was almost exclusively composed of cells expressing LTBR.

[0222] To investigate the mechanisms of gene perturbations with the strongest transcriptional changes, we examined the transcriptional profiles of CD3 / CD28 stimulated ORF T cells compared to unstimulated control T cells (Figure 9F-I). This approach allowed us to identify gene modules that were shared between perturbations or were perturbation specific. For example, LTBR and CDK1 showed the strongest enrichment of genes involved in RNA metabolism and cell cycle (CDK4, HSPA8, and BTG3), as well as the tumor necrosis factor (TNF) signaling pathway (TNFAIP3, TRAF1, and CD70). FOSB appeared to drive an opposite program to LTBR in terms of genes involved in TCR signaling (CD3D, CD3E, LAPTM5, and LAT), cytokine responses (GATA3 and TNFRSF4), and the NF-κB pathway (NFKB2, NFKBIA, and UBE2N). Finally, we determined that the observed phenotype was the result of genetic perturbations rather than the expansion of a single clone, as virtually every single cell expressed a unique TCR clonotype (Figure 9J). This result highlights the utility of OverCITE-seq's multimodal capture approach, yielding transcriptome, clonotype, cell surface proteome, cell hashing (for treatment or stimulation conditions), and lentiviral ORF identity for each T cell.

[0223] LTBR improves multiple T cell functions Having identified LTBR as a potent driver of proinflammatory cytokine secretion (Figure 2E) and profound transcriptional remodeling (Figure 3D, Figure 3E), we decided to investigate its mechanism of action in more detail. LTBR belongs to the tumor necrosis factor receptor superfamily (TNFRSF) and is expressed in a variety of non-immune cell types and immune cells of myeloid origin, but is absent in lymphocytes (Figure 10A, Figure 10B). Using bulk RNA sequencing (RNA-seq), we characterized the expression of LTBR-transduced cells and tNGFR-transduced cells with or without TCR stimulation. We compared global gene expression between LTBR and LTBR-overexpressing cells (Figures 4A, 4B, and 10C). In addition to upregulation of MHC-I and II genes (HLA-C, HLA-B, HLA-DPB1, HLA-DPA1, and HLA-DRB6) and transcription factors required for MHC-II expression (RFX5 and CIITA), LTBR cells also expressed MHC-II invariant chain (encoded by CD74). In particular, CD74 has been shown to activate the pro-survival NF-κB pathway in B cells, particularly through upregulation of the anti-apoptotic genes TRAF1 and BIRC3, both of which are also upregulated in LTBR-overexpressing cells. 24 Similarly, LTBR cells express CD8 + BATF3, which has been shown to promote T cell survival 25 JUNB, a transcription factor involved in IL-2 production, was strongly upregulated. 26 , and TCF7 (encoding TCF1), a key transcription factor involved in T cell self-renewal. 27 We also observed upregulation of LTBR1, confirming the RNA-seq results at the protein level (Figures S10D-S10I). LTBR cells were also more resistant to activation-induced cell death and retained greater functionality after repeated stimulation (Figures 4C, S10D, S10J-S10M).

[0224] LTBR signaling in its endogenous context (in myeloid cells) is triggered either by the heterotrimer of lymphotoxin-α (LTA) and lymphotoxin-β (LTB) or by LIGHT (encoded by the TNFSF14 gene). Because LTA, LTB, and LIGHT are expressed by activated T cells, we attempted to determine whether the addition of exogenous LTA or LIGHT could modulate cytokine secretion, differentiation, or proliferation of CD3 / CD28-stimulated LTBR-overexpressing T cells, but no effect of exogenous ligands on LTBR T cell function was observed (Figures 11A-E). Thus, although LTBR can enhance TCR-driven T cell responses, it does not drive activation by itself, which would pose a potential safety issue and result in loss of antigen specificity of engineered T cell responses. It was also determined that constitutive expression of LTBR is required for maintenance of the phenotype, but there is a significant lag time between loss of detectable LTBR expression and loss of phenotype (Figures 11F-11I), indicating that transient expression of LTBR may be a safe avenue for therapeutic applications.

[0225] Finally, to identify the key domains of the LTBR protein that drive activity in T cells, a series of point or deletion mutants of LTBR were designed (Figure 4E, Figure S11J). In general, the N-terminus of LTBR was found to be less sensitive to deletions than the C-terminus. Similarly, partial reduction of the LTBR phenotype was achieved by introducing three alanine point mutations in key residues for LTA and LTB binding. 28 Using our C-terminal deletions, a mutant form of LTBR lacking residues 393-435 showed no difference compared to full-length LTBR, ​​whereas the deletion of residues 377-435 showed no difference compared to full-length LTBR, ​​likely due to loss of binding sites for TRAF2, TRAF3, or TRAF5. 29 We found that the self-association domain completely abrogated the LTBR phenotype, despite it being expressed at comparable, if not higher, levels (Fig. S11K). 30 Deletion of (324–377) also completely abrogated the phenotype.

[0226] LTBR acts through canonical NF-κB in T cells LTBR overexpression was shown to induce widespread transcriptomic changes in T cells accompanied by alterations in T cell function (Figure 4A,B). Therefore, utilizing an assay of transposase-accessible chromatin by sequencing (ATAC-seq), we sought to determine whether perturbations in gene expression in LTBR cells were accompanied by epigenetic changes (Figures S12A-S12G). Comparing the enrichment of specific transcription factor motifs in differentially accessible chromatin regions, we identified NF-κB p65(RELA) as the most enriched transcription factor in LTBR cells (Figure S12H,I). Notably, NF-κB p65 and NFAT-AP-1 were significantly downregulated in stimulated versus resting T cells (LTBR and tN), in line with their well-established role in T cell activation. These were the two transcription factors most highly enriched in open chromatin in both GFRs. 31 Only NF-κB p65 showed strong enrichment in LTBR cells, regardless of the presence or absence of stimulation (Figure 4F), suggesting that LTBR induces a partial T cell activation state but still requires signal 1 (TCR stimulation) for full activation.

[0227] We next decided to investigate changes in protein expression and / or phosphorylation of members of the NF-κB signaling pathway. We observed a more rapid phosphorylation of p65(RELA) and a strong increase in the phosphorylation of the NF-κB inhibitor, IκBα, which targets IκBα for degradation, both of which effects enhance NF-κB activation or transcription (Figure 4G, Figure 4H, Figure 13A-C). In addition to changes in the canonical NF-κB pathway, we also detected upregulation of RELB and NF-κB p52, key mediators of the non-canonical NF-κB pathway (Figure 4I, Figure 13B, Figure 13C).

[0228] Having established that LTBR activates both the canonical and non-canonical NF-κB pathways, we sought to determine the molecular basis of this phenomenon by perturbing key genes in the LTBR and NF-κB pathways by co-delivery of CRISPR constructs targeting LTBR or tNGFR and 11 genes involved in the LTBR signaling pathway. 32 (Figure 4J, Figures 13D-O). Knockout of LTB, TRAF2, and NIK (also known as MAP3K14) significantly reduced IFNγ secretion from LTBR cells but not (or to a lesser extent) from control (tNGFR) cells, whereas perturbation of LIGHT (also known as TNFSF14), ASK1 (also known as MAP3K5), and RELA had a stronger effect on control than LTBR cells. The effect of LTB loss on T cell activation in LTBR cells supports the observation that alanine mutagenesis of key residues involved in LTA or LTB binding (Figure 4E) partially reduced the LTBR phenotype. Notably, loss of either TRAF2 or TRAF3 was observed to promote IFNγ secretion only in tNGFR cells, in line with previous results that T cells from TRAF2 dominant negative mice were hyperresponsive to TCR stimulation. 33 .

[0229] To investigate the potential role of canonical versus non-canonical NF-κB signaling in LTBR T cells, we decided to analyze the global impact of RELA or RELB loss on LTBR-driven gene expression profiles. Using bulk RNA-seq in T cells overexpressing LTBR or tNGFR, we found that only loss of RELA significantly downregulated the expression of "core" LTBR genes, whereas loss of RELB had no effect (Figure 4K, Figure S13P).

[0230] The ORF enhances antigen-specific responses Thus far, we have shown that the top-ranked genes from the ORF screen improve T cell function using non-specific pan-TCR stimulation. We next sought to determine whether a similar improvement could be observed using antigen-specific stimulation (Figure 5A). To that end, we co-expressed several top-ranked genes with two FDA-approved CARs targeting the B cell marker CD19 (Figures 14A-D). Using LTBR as an example, we demonstrated that ORF expression is achievable with this tricistronic vector (Figures 14E-I). The sequence of the tricistronic vector is provided in the sequence listing (a schematic diagram is provided in Figure 5A): 19-28-z+LTBR protein: SEQ ID NO:3 19-28-z+LTBR DNA: SEQ ID NO:4 19-28-z+NGFR protein: SEQ ID NO:5 19-28-z+NGFR DNA: SEQ ID NO:6 19-BB-z+LTBR protein: SEQ ID NO:7 19-BB-z+LTBR DNA: SEQ ID NO:8 19-BB-z+NGFR protein: SEQ ID NO: 9 19-BB-z+NGFR DNA: SEQ ID NO: 10

[0231] Because both CARs use different costimulatory domains from CD28 or 4-1BB, we wanted to determine whether the top-ranked genes selected using CD28 costimulation could also function in the context of 4-1BB costimulation. With the exception of AKR1C4, nearly all of the top-ranked genes tested improved upregulation of CD25 and antigen-specific cytokine secretion, with no significant differences in the differentiated or exhausted phenotype (Figures 5B, 5C, 14J-P, 15A-D).

[0232] While IL-2 and IFNγ production are important for T cell clonal expansion and antitumor activity, another important component of tumor immune surveillance is direct cytotoxicity. The top-ranked genes had an overall stronger effect on the cytotoxicity of CD28 CAR T cells than 4-1BB CAR T cells (Figures 5D-E, 15E, 15F). Notably, we observed that CAR T cells co-expressing LTBR tended to form large cell clusters, which were typically absent in wells with control cells, consistent with the overall higher expression of adhesion molecules such as ICAM-1 in LTBR-expressing cells (Figure 15G). Another important feature of effective antitumor T cells is their ability to maintain function despite prolonged antigen exposure. In line with our previous results in the context of LTBR alone (Figure 4D), CAR T cells expressing LTBR showed superior functionality after repeated challenge with target cells than their CAR T cells expressing tNGFR (Figure 5F, Figures S15H-J).

[0233] While T cells from healthy donors are relatively easy to manipulate and rarely show signs of dysfunction in culture, autologous T cells in patients with cancer are often dysfunctional, exhibiting limited proliferation and effector function. 34 The top-ranked genes were shown to mediate CAR expression not only in healthy T cells but also in potentially dysfunctional T cells derived from patients. To examine whether T cell responses could be improved, CD19 CAR co-expressed with LTBR or a control gene was transduced into peripheral blood mononuclear cells (PBMCs) from patients with diffuse large B-cell lymphoma. +After co-incubation with target cells, we observed an increase in IL-2 and IFNγ secretion from LTBR CAR T cells similar to that shown in healthy donors, indicating that the identified ORFs can be successfully used to engineer T cells from patients with lymphoma ex vivo (Figure 5G, Figure 15K). Notably, there was no cytokine secretion in response to CD19- cells, indicating that overexpression of LTBR does not induce a false antigen-independent response.

[0234] Screening and subsequent validation were performed in αβ T cells, the predominant subset of T cells in human peripheral blood. Although αβ T cell-based immunotherapies have shown considerable clinical potential, γδ T cells offer an attractive alternative due to their lack of MHC restriction, their ability to target broadly expressed stress markers in a cross-cancer manner, and their more innate-like properties. 5 We therefore sought to determine whether the top genes validated in αβ T cells were transferred to γδ T cells. We observed an increase in IL-2 and IFNγ secretion from γδ T cells transduced with the top-ranked genes after co-incubation with leukemia or pancreatic ductal adenocarcinoma cells (Figure 5H, Figures S1L-S15P). Thus, the top-ranked genes from our screen can act on signaling pathways that are conserved even among highly divergent T cell subsets, highlighting their broad applicability in cancer immunotherapy.

[0235] Consideration In summary, here we developed a genome-scale gain-of-function screen in primary human T cells to examine in a massively parallel manner the effect of nearly 12,000 full-length genes on TCR-driven proliferation. The largest previously published gain-of-function study in primary T cells - to the best of our knowledge - included 36 constructs, including full-length genes and synthetic receptors. 35The approach relied on donor DNA and construct delivery via Cas9-mediated targeted insertion. Although the use of donor DNA for targeted gene delivery allows for greater flexibility in construct design, particularly in terms of engineering synthetic receptors, the method is less scalable and accessible in terms of cost and complexity than the lentiviral libraries used here. Thus, ORF-based gain-of-function screens are readily applicable to a multitude of T cell phenotypes and settings, offering opportunities for clinical translation. Indeed, all FDA-approved CAR therapies already rely on lentiviral or retroviral integration of the CAR transgene, and therefore the addition of ORFs to this system would not pose major manufacturing or regulatory challenges. The use of ORF-encoding mRNA delivered to CAR T cells prior to infusion is another route of translation, especially if there are safety concerns about the mode of action of a particular ORF.

[0236] Gain-of-function screening has the potential to reveal regulators that are tightly controlled, restricted to certain developmental stages, or expressed only in certain contexts. As shown herein, LTBR is not canonically present in cells of lymphoid origin, but due to the intact signaling pathway, it can have a synthetic role when introduced into T cells. Although constitutive activation of other TNFRSF members may result in similar phenotypes, one of the features that distinguishes LTBR (and likely leads to its enrichment in the screen, but not to the enrichment of other TNFRSF members) is the formation of an autocrine loop, whereby the receptor and its ligand are present in the same cell. It is particularly noteworthy that expression of LTBR promotes IL-2 secretion, a cytokine that is exclusively produced by T cells and not by cell types that endogenously express LTBR. In addition to promoting cytokine secretion, overexpression of LTBR promoted stemness (expression of TCF1), reduced activation-induced apoptosis, and provided a level of protection against phenotypic and functional features of T cell exhaustion - all features that are not recapitulated by cell types that endogenously express LTBR. Previous studies using overexpression of LTBR in cell lines have shown that LTBR has a proapoptotic role, in direct opposition to the phenotype observed in primary T cells. 36 Transcriptional and protein level analyses revealed that LTBR promotes constitutive activation of both the canonical and non-canonical NF-κB pathways. However, using epigenomic profiling and CRISPR-based functional perturbation, we showed that the phenotypic and functional changes resulting from LTBR expression are primarily mediated by activation of the canonical NF-κB pathway, whereas changes in the non-canonical pathway may not be essential for the observed phenotypes, in contrast to the well-established role of non-canonical NF-κB activation in cells endogenously expressing LTBR.

[0237] Gene overexpression has been used in a number of studies for preclinical enhancement of CAR T cell therapy. For example, armoring CAR T cells with cytokines such as IL-12 or IL-18, which are not typically produced by T cells but are known to improve T cell function when secreted by other cell types, has been shown to improve their antitumor activity. 38、39 Notably, previous studies found that CAR T cell exhaustion could be alleviated by overexpression of c-JUN, a transcription factor identified by RNA-seq as specifically depleted in exhausted cells. 40 Future studies adapting genome-wide gain-of-function screens to relevant immunotherapy models will lead to advanced target selection for engineered synthetic cell therapies capable of overcoming the immunosuppressive tumor microenvironment and eradicating established cancers.

[0238] Example 3 Improved CAR solid tumor responses We have shown that LTBR and several other top-ranked genes (ORFs, open reading frames) identified in the screen promote anti-tumor responses of anti-CD19 CARs in the context of B-cell leukemia. Here, we tested whether a similar improvement in activity could be shown in conjunction with two clinically tested anti-mesothelin CARs (using either the 4-1BB or CD28 costimulatory domains) in the context of pancreatic cancer. T cells co-expressing the CAR and ORF were tested against Capan-2, a pancreatic cancer line that expresses high levels of mesothelin, the CAR targets, and BxPC3, a pancreatic cancer line that expresses low levels of mesothelin (Figure 16A). The sequence of the tricistronic vector is provided in the sequence listing (a schematic diagram is provided in Figure 16A): SS1-28-z+LTBR protein: SEQ ID NO: 11 SS1-28-z+LTBR DNA: SEQ ID NO: 12 SS1-BB-z+LTBR protein: SEQ ID NO: 13 SS1-BB-z+LTBR DNA: SEQ ID NO: 14

[0239] After overnight co-incubation, it was determined that all but one of the ORFs tested (i.e., AHNAK, BATF, IFNL2, IL12B, and LTBR) promoted antigen-specific secretion of IFNγ when used with CAR (either 41BB or CD28) against the mesothelin-high cell line Capan-2 (Figure 16B). With regard to promotion of IL-2 secretion, a marked improvement over the negative (tNGFR) control was observed with LTBR and to a lesser extent with AHNAK (Figure 16C). With regard to responsiveness to the mesothelin-low cell line BxPC3, improvement over the negative control was observed primarily in T cells overexpressing IL12B or LTBR (Figure 16D).

[0240] Cytokine secretion is one aspect of a productive antitumor response, another being direct cytotoxicity. Therefore, the ability of CAR T cells co-expressing the top genes to kill GFP+Capan2 or BxPC3 cells was tested (Figure 16E, Figure 16F). The increased cytotoxicity against mesothelin-high Capan-2 shown by CAR T cells overexpressing any of the six top genes tested (including GPD1) was expected given the improvement in cytokine secretion, but increased cytotoxicity against BxPC3 cells was also observed. Thus, we conclude that the top ranked genes identified in the screen (including but not limited to AHNAK, BATF, GPD1, IFNL2, IL12B, and LTBR) were able to drive reactivity of diverse CARs (anti-CD19 as previously shown, anti-mesothelin as shown here) using different costimulatory domains (CD28 or 4-1BB) in different cancer types (including liquid tumors such as B cell leukemia and solid tumors such as pancreatic cancer) and with different target antigen densities (mesothelin-high and mesothelin-low cell lines).

[0241] Example 4 Improved activity of TCRs in solid tumors T cell therapy can rely on redirecting cells to a given tumor target using either CAR or TCR. The former has the advantage of being able to target different patients' tumors, regardless of their HLA haplotype, while the latter can also target antigens that are present inside the cell (as epitopes from all cellular proteins are sampled and presented by HLA molecules). Here, we used a clinically tested TCR directed against an epitope from NY-ESO-1, which is commonly expressed in many cancer histologies, including but not limited to melanoma, multiple myeloma, sarcoma, and lung cancer. Due to size limitations, the TCR and genes (ORF, open reading frame) were included in two separate lentiviruses used to co-transduce the T cells. The doubly transduced T cells were then selected using puromycin (only T cells transduced with the ORF lentivirus survive) and antibody-based selection of NY-ESO-1 TCR positive cells (in the presence of dasatinib, which prevents T cell activation and thus activation-induced cell death during the selection process).

[0242] The engineered CD8+ T cells were then tested against the HLA-A2+NY-ESO-1+ melanoma line A375. Most of the genes tested increased the secretion of one or both of the cytokines IFNγ and IL2 (Figure 17B, Figure 17C). We also measured direct cytotoxicity against A375 cells and demonstrated that all genes tested exhibited superior cytotoxicity to TCR-transduced T cells co-expressing the negative control gene tNGFR (Figure 17D). Therefore, we concluded that the top-ranked genes identified in the screen (including but not limited to AHNAK, BATF, GPD1, IFNL2, IL12B, and LTBR) were able to promote the reactivity of cancer-specific TCR-engineered T cells.

[0243] Example 5 LTBR codelivery improves anti-CD19 4-1BB-z CAR activity in vivo. We have previously shown that co-delivery of anti-CD19 CAR and LTBR in the same lentiviral vector results in superior anti-tumor activity in vitro (Legut et al., Nature 2022). Here, we show that the co-delivery of anti-CD19 CAR (FMC6.3 The efficacy of CAR T cells expressing LTBR (19-BB-z, CD8 stalk and transmembrane domains, 4-1BB and CD3z signaling domain) was tested in a Nalm6 immunodeficient (NSG) mouse model of disseminated leukemia with or without LTBR (Figure 22A). Mice treated with non-transduced T cells (CD4 and CD8, 1:1 ratio) survived a median of 19 days after tumor inoculation, whereas mice treated with CAR T cells co-expressing the unrelated gene tNGFR extended their survival to a median of 23 days (20% increase over non-transduced T cells). In contrast, mice treated with CAR T cells co-expressing LTBR survived a median of 31 days (63% increase over non-transduced T cells and 35% increase over control CAR T cells) (Figure 22B). Furthermore, LTBR CAR T cells significantly reduced tumor burden in treated mice compared to control CAR T cells (Figure 22C). Finally, no specific toxicity was observed in mice treated with either CAR T cell product as determined by gross pathology examination at termination, and the observed weight loss is typical for this model and attributable to tumor burden. In line with the improved antitumor efficacy of LTBR CAR T cells, the weight loss was significantly greater in treated mice treated with LTBR CAR T cells compared to non-transduced or control CAR T cells. was substantially delayed in mice treated with CAR T cells (Figure 22D).

[0244] Example 6 LTBR codelivery improves anti-CD19 4-1BB-z CAR survival in the absence of IL2 but does not result in leukemic transformation. We have already shown that LTBR promotes T cell proliferation and redu...

Claims

1. A modified lymphocyte comprising an exogenous nucleic acid sequence encoding LTBR.

2. The modified lymphocyte according to claim 1, wherein the exogenous nucleic acid sequence encoding LTBR encodes an intracellular domain, or a fragment or variant thereof.

3. The modified lymphocyte according to claim 2, wherein the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof.

4. The modified lymphocyte according to claim 2 or 3, wherein the LTBR intracellular domain has a deletion of at least amino acids 393 to 435.

5. The modified lymphocyte according to any one of claims 1 to 3, comprising an expression cassette comprising an expression control sequence operably linked to the exogenous nucleic acid sequence encoding LTBR.

6. The modified lymphocyte according to any one of claims 1 to 3, further comprising an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a nucleic acid sequence encoding a T cell receptor (TCR).

7. The modified lymphocyte according to any one of claims 1 to 3, wherein the exogenous nucleic acid sequence encoding LTBR is mRNA.

8. The modified lymphocyte according to any one of claims 1 to 3, wherein the modified lymphocyte is a T cell, an NK cell, or an NK T cell.

9. The modified lymphocyte according to claim 8, wherein the modified lymphocyte is a gamma delta T cell.

10. (i) A nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid sequence encoding LTBR, (ii) A nucleic acid sequence encoding a T cell receptor and a nucleic acid sequence encoding LTBR, and / or, (iii) A nucleic acid sequence encoding a viral protein and a nucleic acid sequence encoding LTBR, An expression cassette comprising.

11. The expression cassette according to claim 10, wherein the nucleic acid sequence encoding LTBR encodes an LTBR intracellular domain, or a fragment or variant thereof.

12. A method for producing the modified lymphocyte according to any one of claims 1 to 3, comprising introducing an exogenous nucleic acid sequence encoding LTBR into a lymphocyte.

13. The method according to claim 12, wherein the exogenous nucleic acid sequence encoding LTBR encodes an LTBR intracellular domain, or a fragment or variant thereof.

14. The method according to claim 12, wherein the modified lymphocyte further comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a nucleic acid sequence encoding an engineered T cell receptor (TCR).

15. A medicament for treating cancer in a subject in need of cancer treatment, the medicament comprising the modified lymphocyte according to any one of claims 1 to 3 or the expression cassette according to claim 10 or 11.

16. A method for increasing proliferation or T cell effector function in T cells, the method comprising introducing the expression cassette according to claim 10 or 11 into the T cells.

17. A medicament for increasing the response to a vaccine composition, the medicament comprising a vaccine comprising the expression cassette according to claim 10.

18. The modified lymphocyte according to any one of claims 1 to 3, wherein the modified lymphocyte comprises an expression cassette comprising an expression control sequence and an exogenous nucleic acid sequence of the gene in Table 1.

19. (i) A nucleic acid sequence encoding a chimeric antigen receptor and a nucleic acid sequence of the gene in Table 1, (ii) A nucleic acid sequence encoding a T cell receptor and a nucleic acid sequence of the gene in Table 1, and / or, (iii) A nucleic acid sequence encoding a viral protein and a nucleic acid sequence of the gene in Table 1, An expression cassette comprising.

20. A composition comprising a modified lymphocyte comprising the expression cassette according to claim 10, 11 or 19.

21. A medicament for treating cancer in a subject in need of cancer treatment, the medicament comprising the modified lymphocyte according to claim 18.

22. A medicament for treating cancer in a subject in need of cancer treatment, the medicament comprising the expression cassette according to claim 19.

23. A medicament for treating cancer in a subject in need of cancer treatment, the medicament comprising the composition according to claim 20.

24. The modified lymphocyte according to claim 18, wherein the gene in Table 1 is LTBR, ADA, IFNλ2, IL12B, CALML3, MRPL51, DBI, GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

25. The expression cassette according to claim 19, wherein the gene in Table 1 is LTBR, ADA, IFNλ2, IL12B, CALML3, MRPL51, DBI, GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

26. The composition according to claim 20, wherein the gene in Table 1 is LTBR, ADA, IFNλ2, IL12B, CALML3, MRPL51, DBI, GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

27. The medicament according to claim 21, wherein the gene in Table 1 is LTBR, ADA, IFNλ2, IL12B, CALML3, MRPL51, DBI, GPN3, ITM2A, AHNAK, BATF, GPD1, ATF6B, AHCY, DUPD1, or AKR1C4.

28. A method for identifying a gene that modifies the therapeutic function of a modified lymphocyte when exogenously expressed in the modified lymphocyte, the method comprising: (a) obtaining a lymphocyte population; (b) transducing the lymphocyte population with a plurality of viral vectors, each viral vector of the plurality of viral vectors encoding a gene linked to one or more barcodes, to form transduced lymphocytes; (c) stimulating the transduced lymphocytes to induce activation, proliferation, and / or effector function; (d) isolating the transduced lymphocytes from the lymphocyte population of (c); (e) detecting the presence of the gene and / or the linked barcode in the isolated transduced lymphocytes, and wherein the detected gene is effective in modifying the therapeutic function of the modified lymphocyte expressing the gene.

29. The method according to claim 28, wherein the gene is linked to one or more barcodes.

30. A method for analyzing the effect of overexpression of an ORF of interest on individual cells, comprising: (a) introducing an expression cassette containing a nucleic acid encoding the ORF of interest into the individual cells to overexpress the ORF of interest; ​ (b) providing, in separate fractions, a first set of nucleic acids derived from said individual cells and first oligonucleotides having a first barcode sequence, wherein said oligonucleotides are attached to beads, and wherein said first set of nucleic acids comprises endogenous transcriptome mRNA and ORF mRNA; (c) performing RT-PCR to generate a second set of nucleic acids derived from said first set of nucleic acids, wherein the second set of nucleic acids in said fraction are attached to first oligonucleotides comprising a first nucleic acid barcode sequence, and wherein said RT-PCR is performed using RT-PCR reagents comprising primers that specifically anneal to sequences on said ORF mRNA that are not poly-A sequences, and wherein said second set of nucleic acids comprises endogenous transcriptome cDNA and ORF cDNA; (d) amplifying said second set of nucleic acids using PCR reagents comprising a second primer that specifically anneals to a sequence on said ORF cDNA that is not a poly-A sequence to generate a third set of nucleic acids; (e) detecting and / or sequencing said barcode sequence, transcriptome cDNA, and / or ORF cDNA.

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