Methods and compositions comprising fusion proteins for improved immunotherapy

Abstract

Provided herein are nucleic acids, expression cassettes, modified lymphocytes comprising a sequence encoding a fusion protein comprising a domain of LTBR, a TCR or a CAR, and compositions comprising the nucleic acids, expression cassettes, modified lymphocytes. In certain embodiments, the cells are T cells. Methods of treatment using the provided compositions are also described.

Description

[0001] Government Support Statement

[0002] This invention was made with government support under 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.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic sequence listing (NYG-LIPP-158PCT.xml; size: 304,622 bytes; and creation date: March 15, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] Cellular immunotherapy using engineered autologous patient T cells redirected against selected tumor antigens has shown promising results against blood cancers, with five chimeric antigen receptors (CARs) approved by the U.S. Food and Drug Administration (FDA) to date. 6 In contrast, CAR therapy for solid tumors shows much lower overall efficacy due to suppression of T cell effector function in the tumor microenvironment. Even for hematological malignancies, most patients do not experience durable responses, with the exception of B acute lymphoblastic leukemia, where resistance is primarily due to T cell dysfunction rather than antigen loss. 7 Considerable effort has been devoted to identifying genes and pathways that contribute to T cell dysfunction 8，9 However, comprehensive, genome-wide screening for regulators of T cell function has so far been limited to loss-of-function screens. 2-4 .

[0006] Advances in CRISPR genome engineering have made it possible to easily knock out every gene in the genome in a scalable and customizable manner. Although its large size makes it challenging (though not impossible) to deliver Cas9 to primary T cells via lentivirus, 10 ), but alternative approaches have been developed that rely on the Cas9 protein 2 or mRNA 11 transient delivery of Cas9 or relying on constitutive Cas9 expression in engineered isogenic mouse strains 3However, these approaches are not suitable for gain-of-function screens in human cells, which require sustained expression of transcriptional activators that drive target gene expression.

[0007] What are needed are improved compositions and methods for more effective immunotherapy. Summary of the invention

[0008] In the first aspect, a lymphocyte is provided herein that is genetically modified to express a chimeric antigen receptor (CAR). The CAR comprises an antigen binding domain, a transmembrane domain, and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain of a gene from Table 3. In certain embodiments, the LTBR domain is an LTBR intracellular domain or a fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof.

[0009] In certain embodiments, the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

[0010] In another embodiment, the CAR comprises an antigen binding domain, a transmembrane domain, a co-stimulatory signaling domain, and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain of a gene from Table 3. In certain embodiments, the LTBR domain is an LTBR intracellular domain or a fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

[0011] On the other hand, a nucleic acid molecule is provided. The molecule comprises a sequence encoding a chimeric antigen receptor (CAR). The CAR comprises an antigen binding domain, a transmembrane domain, and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain of a gene from Table 3. In certain embodiments, the LTBR domain is an LTBR intracellular domain or a fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion in at least amino acids 393 to 435. In another embodiment, the CAR comprises an antigen binding domain, a transmembrane domain, a co-stimulatory signaling domain, and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain of a gene from Table 3. In certain embodiments, the LTBR domain is an LTBR intracellular domain or a fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

[0012] In another aspect, an expression cassette is provided, comprising a nucleic acid molecule comprising a sequence encoding a chimeric antigen receptor (CAR).

[0013] In another aspect, a method of treating cancer in a subject in need thereof is provided. The method comprises administering a composition comprising a modified lymphocyte as described herein.

[0014] In another aspect, a method of treating a viral disease in a subject in need thereof is provided. The method comprises administering a composition comprising modified lymphocytes.

[0015] In another aspect, a method of treating autoimmunity in a subject in need thereof is provided. The method comprises administering a composition comprising modified lymphocytes. In another aspect, a fusion protein is provided that comprises a LTBR domain and at least one domain from a second protein that is not a LTBR.

[0016] In another aspect, a host cell is provided, comprising a nucleic acid molecule or an expression cassette as described herein.

[0017] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A-Figure 1D A genome-scale overexpression screen to identify genes that promote proliferation of primary human T cells is shown. Figure 1A ) Overview of pooled ORF screening. CD4 + and CD8 + T cells. The barcoded genome-scale ORF library was then introduced into CD3 / CD28 stimulated T cells, and the transduced cells were subsequently selected. After 14 days in culture, the T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and restimulated to induce proliferation. By comparing the counts of specific ORF barcodes before and after cell sorting, ORFs enriched in the CFSE low population were identified. ( Figure 1B )CD4 + Normalized enrichment of individual barcodes for a given gene in the screen. ( Figure 1C ) Consistent enrichment of CFSE based on individual barcodes for each gene 低 CD4 + CFSE 低 CD8 + Robust sequencing of genes in both T cells and Figure 1D ) Enrichment of top genes (grouped by function and relevance to T cell proliferation) in individual donors and T cell populations selected for further study. Neutral genes (MHC-I complex and cell type-specific differentiation markers) are included for comparison. Gene names are colored according to differential expression in CD3 / CD28 stimulated and resting T cells (green, upregulated; red, downregulated; gray, no change; black, no expression) 41 .

[0019] Figure 2A-2F Overexpression of the top ORFs increased CD4 + and CD8 + T cell proliferation, activation and cytokine secretion. Figure 2A ) to isolate CD4+ and CD8 from screening-independent donors + T cells were then transduced with a lentivirus encoding the top-ranked ORF and a selection marker. After transduction and selection, T cells were restimulated before measuring proliferation, expression of activation markers, and cytokine secretion. Figure 2B ) The proliferation of T cells transduced with the top ranked genes was used as the relative proliferation, which was defined as the ratio of stimulated cells to the corresponding unstimulated control, normalized to tNGFR. At least two donors were tested for each overexpressed gene, in biological triplicate. Boxes show the 25th-75th percentiles, with a line at the mean; whiskers extend to the maximum and minimum values. DUPD1 is also known as DUSP29. ( Figure 2C )CD4+ and CD8 + Mean relative proliferation of ORF-transduced T cells in T cells, normalized to tNGFR. Significant genes in both or one of the T cell subsets are labeled (Student's two-sided t test P < 0.05, and false discovery rate < 0.1). Figure 2D ) Representative expression of CD25 or CD154 after restimulation. Numbers on the histograms are consistent with gated cells (CD8 + CD154 + ) or mean fluorescence intensity (MFI). The dotted line indicates the + Cells (CD8 + ) or control (tNGFR) cells. Figure 2E ) IL-2 and IFNγ secretion after restimulation, normalized to tNGFR. Only CD4 + 、CD8 + or genes that regulate T cell proliferation in two T cell subsets. At least two donors were tested in triplicate for each gene. Boxes show the 25th to 75th percentiles with a line at the mean; whiskers extend to the maximum and minimum values. ( Figure 2F )CD8 + or CD4 + A given ORF in T cells significantly (P < 0.05) improved the crossover between different T cell activation phenotypes.

[0020] Figure 3A-3E Shown that single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. Figure 3A )OverCITE-seq captures overexpressed (ORF) constructs, transcriptomes, TCR clonotypes, cell surface proteins, and therapeutic hash tags in single cells. Figure 3B ) ORF distribution ratios in resting and CD3 / CD28 stimulated T cells. Figure 3C ) Antibody-derived tag sequencing (ADT; right) yielded NGFR expression in tNGFR-transduced T cells similar to that produced by flow cytometry of T cells transduced with tNGFR (left). Untransduced cells (left) or cells assigned non-tNGFR (right) are shown in gray. ( Figure 3D)Uniform Manifold Approximation and Projection (UMAP) representation of single-cell transcriptomes after unsupervised clustering of ORF singlets captured by OverCITE-seq. The inset on the upper left identifies stimulated and resting T cells given by the treatment hash tags. 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 ) ORF prevalence in two representative clusters. Normalized residual values ​​are from chi-squared test. ORFs of interest are shown.

[0021] Figure 4A-Figure 4K It was shown that LTBR overexpression improves T cell function by activating the canonical NF-κB pathway. Figure 4A ) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are genes with a two-fold or greater change in expression and adjusted P < 0.05. Figure 4B )GO biological processes were significantly enriched in T cells overexpressing LTBR (p<0.05). Figure 4C ) CD8 + Cell viability of T cells restimulated with CD3 / CD28 for four days or left unstimulated (n = 2 donors with 3 biological replicates per donor). Figure 4D ) PD-1 expression on resting LTBR or tNGFR T cells was stimulated with a 3:1 excess of CD3 / CD28 beads every three days for up to three consecutive rounds of stimulation. Figure 4E ) ICAM-1 expression (resting) and IL-2 secretion (activated) of T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n=6 replicates from two experiments). Figure 4F ) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). Figure 4G ) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFRT cells stimulated with CD3 / CD28 antibodies for the indicated time periods. ( Figure 4H , Fig. 4I ) Phosphorylated IκBα in LTBR or tNGFR cells at rest or stimulated with CD3 / CD28 (15 min) Figure 4H ) or mature NF-κB2 ( Fig. 4I ) Quantification. Figure 4J) IFNγ secretion from stimulated LTBR or tNGFR cells following CRISPR knockout of the indicated genes (n=18, 3 sgRNAs from 2 donors in 3 biological replicates). IFNγ quantities were normalized to the corresponding non-targeting (NT) control (LTBR or tNGFR) to allow comparison of the relative effects of gene knockout on T cell activation. ( Figure 4K ) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells following CRISPR knockout of RELA or RELB (normalized to non-targeting controls in LTBR cells). Boxes show the 25th-75th percentiles with a line at the median; whiskers extend to 1.5 times the interquartile range. Unpaired two-sided t test P value ( 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 indicated.

[0022] Figure 5A-Figure 5I The top ranked genes are shown to improve antigen-specific T cell responses and tumor killing. Figure 5A-5G ) Anti-CD19 CAR and ORF were co-delivered to T cells from healthy donors. Figure 5A ) Schematic diagram of the tricistronic vector and CART cell experiment. Figure 5B , Figure 5C ) in CD8 + After T cells were co-incubated with Nalm6 cells at a 1:1 ratio overnight, IFNγ( Figure 5B ) and IL-2( Figure 5C ) (n = 3 biological replicates, representing 2 donors). Figure 5D )Nalm6 GFP + Representative images of cells co-cultured with CAR T cells or untransduced control T cells for 48 hours. Scale bar, 200 μm. ( Figure 5E ) Nalm6 GFP after co-incubation with T cells co-expressing 19-28z CAR and LTBR or tNGFR (negative control) at the indicated effector to target ratios + Cell proliferation (normalized total GFP per well). Fig. 5F ) Nalm6 GFP of T cells co-expressing 19-28z or 18-BBz CAR and top-ranked genes (n=3 biological replicates, representing 2 donors) normalized to tNGFR at an effector-to-target ratio of 0.25 and after 48 hours of co-incubation + Quantification of clearance. Figure 5G) 19-BBz CART cells co-expressing LTBR or tNGFR were co-cultured with Nalm6 cells at a 1:1 ratio every 3 days for up to 3 rounds of stimulation (n=3 biological replicates). After repeated antigen stimulation for seven days, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. ( Figure 5H ) 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 at a 1:1 ratio with CD19 + Nalm6 or CD19 - Jurkat cell lines were co-incubated (n=3 biological replicates, representing 2 patients). Secretion of IFNγ and IL2 was measured after overnight incubation. For the Nalm6 condition, the numbers in the column pairs indicated above are the fold increase in cytokine secretion by LTBR cells over tNGFR (negative control) cells. ( Fig.5I ) ORF delivery to Vγ9Vδ2 T cells. Secretion of IFNγ and IL-2 after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2 pretreated with zoledronate to promote phosphorylated antigen accumulation (n=3 biological replicates). Where appropriate, data are mean±sem.

[0023] Figure 6A-6Q The design of human ORF library screening in primary T cells is shown. Fig. 6A ) Design of barcoded vector for ORF overexpression. Figure 6B ) Distribution of the number of barcodes per ORF in the library. Figure 6C ) Vector design for quantifying 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. ( Fig.6D ) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. + After puromycin selection of T cells, rat CD2 (rCD2) expressed from EFS and CMV promoters was expressed ( Fig. 6E ) positive cell percentage and ( Fig. 6F ) Mean fluorescence intensity (MFI). Each data point indicates an individual transduction (n=3 biological replicates). Error bars are SEM. ( Figure 6G ) Distribution of ORF sizes in a genome-scale library. Fig. 6E and Fig. 6F Sizes of the TCR-rCD2 constructs tested in . Figure 6H) Titration of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated and incubated for 4 days. The gate for proliferating T cells was set to include cells that proliferated at least twice (third CFSE peak). Fig.6I ) Expansion of T cells from three healthy donors transduced with the ORF library. Figure 6J ) CD8 re-stimulated before sorting + and CD4 + Representative CFSE profiles of T cells. 低 Sorting gate. Figure 6K ) Individual barcodes or corresponding ORFs were recovered in transduced T cells and plasmids for lentiviral production. Corresponding samples from three donors were pooled together with equal read counts before counting how many barcodes or ORFs were present with at least one read. ( Figure 6L ) Distribution of reads corresponding to ORFs of different sizes. ORFs are assigned to ten quantiles according to their size, with Q1 being the smallest size and Q10 being the largest size (n=1, 161 ORFs per quantile). Boxes show 25-75 percentiles with a line at the median; whiskers extend to 1.5× interquartile range. ( Figure 6M ) CFSE calculated by folding individual barcodes into their corresponding genes 低 CD4 + and CD8 + Enrichment of genes in both T cells. Significantly enriched genes (log2 fold change above 0.5 and adjusted p-value below 0.05) are marked in red. Immune response genes of interest are marked. ( Figure 6N ) were analyzed separately for the individual screening populations (CD4 + 、CD8 + ) from Figure 6M Overlap of significantly enriched genes. Fig.6O )CD8 + Normalized enrichment of individual barcodes for a given gene in the screen. ( Figure 6P ) Figure 6M GO biological processes of genes significantly enriched in . Figure 6Q ) Overlap of significantly enriched genes and differentially expressed genes between CD3 / CD28 stimulated T cells and naive T cells 41 .

[0024] Figure 7A-7J Overexpression of ORFs selected in screening independent donors is shown. Fig. 7A ) Histogram of selected ORF expression in T cells after puromycin selection. Figure 7B ) Transduced CD4 + and CD8+ Quantification of tNGFR expression in 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 ) Correlation between ORF size and proliferation changes relative to tNGFR. The mean log2 fold change is shown. Fig.7D ) Relative to tNGFR in a single donor, restimulated CD8 + or( Fig. 7E )CD4 + Proliferation of T cells (n=3 biological replicates). Mean values ​​and SEM are shown. Figure 7F , Figure 7G ) significantly improved T cell proliferation as measured by dilution of CellTraceYellow (see Figure 2C ) ORF transduced T cells. Representative CellTraceYellow histograms and fitted distributions are shown ( Figure 7F ) and quantification of proliferation index ( Figure 7G ) (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 ) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of 4 tested) are shown (n=3 biological replicates). Fig.7I , Figure 7J ) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was performed based on isotype and fluorescence minus one control. Representative gating of S-G2-M phase cells (for stimulated T cells) is shown ( Fig.7I )as well as( Figure 7J ) Quantification (n=6 biological replicates from two 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 (from left to right). Figure 7G and Fig.7I Statistical significance of panels: One-way ANOVA with Dunnett's multiple comparisons test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM.

[0025] Figure 8A-8E Functional responses of T cells overexpressing the ORF are shown. Fig. 8A ) Quantitative expression of CD25 or CD154 after restimulation. At least two donors were tested for each gene in triplicate. Only those with significant increases in CD4 + 、CD8 + or genes involved in T cell proliferation in both T cell subsets. Mean values ​​and SEM are shown. ( Figure 8B , Figure 8C ) to antigen dose. T cells were incubated with the indicated concentrations of anti-CD3 antibodies for 24 hours and the amount of secreted IFNγ was quantified. A representative dose-response curve fit is shown ( Figure 8B ) and IC 50 Quantitative ( Figure 8C )(n=2 biological replicates). Fig.8D ) 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 are shown (n=3 biological replicates). Fig. 8E ) Multiplex quantification of selected secreted cytokines and chemokines by ORF-transduced T cells after 24 hours of CD3 / CD28 stimulation. Mean z scores of replicate measurements (from independent samples) normalized to tNGFR are shown.

[0026] Figure 9A-9J OverCITE-seq identifies ORFs and their transcriptional effects. Fig.9A ) Quality parameters for cells identified by gel bead barcoding. Negative, singlet and doublet based on cell hashing. Fig. 9B ) The proportion of stimulated and resting T cells in cells assigned to each ORF. The chi-square test p-value shows ORFs where the distribution of stimulated and resting cells is significantly skewed (uneven). ( Fig. 9C) Cell cycle corrected proportional expression of overexpressed genes in cells transduced with the corresponding ORF and negative control (tNGFR). The two-sided Wilcoxon test p-value shown above the violin plots indicates the statistical significance of gene expression levels between specific ORFs and tNGFR-transduced T cells. Boxes show 25-75 percentiles with a line at the median; 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) single cells. Fig.9D ) Expression of all ORF genes in cells assigned to each ORF. Each row is z-score normalized. ( Fig.9E ) Distribution of individual ORF frequencies in clusters. The number of ORF cells and chi-square test residuals are shown. The chi-square test p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is shown at the top of the figure. The proportion of stimulated and resting T cells in each cluster is shown below the cluster label. ( Fig.9F , Figure 9G )Rest( Fig.9F ) and stimulated ( Figure 9G )Spearman correlation between transcriptional profiles of selected ORF cells in the population. Figure 9H ) Fold change of the most 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. ( Fig.9I) 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). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is presented, normalized to the log2 fold change of tNGFR (stimulated) to tNGFR (resting). The genes of interest in each cluster are marked. ( Figure 9J ) Average TCR clonotype diversity in ORF cells.

[0027] Figure 10A-10M Functional analysis of LTBR overexpression in T cells is shown. ( Fig. 10A ) LTBR expression in indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v8 75 (n=948 donors). Boxes show 25-75 percentiles with a line at the median. Fig. 10B ) LTBR expression in peripheral blood mononuclear cells (PBMC) of 31,021 cells from 2 donors 76 The indicated cell types are derived from Harmony tSNE clustering of single-cell transcriptomes. ( Fig. 10C ) Overlap between genes significantly upregulated in LTBR cells compared to tNGFR cells identified in single cell or whole-cell RNA-seq. ( Fig. 10D , Fig.10E ) TCF1 expression in T cells transduced with LTBR or tNGFR. Fig. 10D ) shows a representative histogram of TCF1 expression and the gate of TCF1+ cells (dashed line), and ( Fig.10E )TCF1 + Quantification of cells (n = 3 biological replicates). Figure 10F-10H ) Expression of ICAM-1, CD70, CD74, and MHC-II in LTBR and tNGFRT cells. Representative histograms of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation ( Fig.10F ), n=3 donors (CD8 + ) or n = 4 donors (CD4 + )Quantitative ( Figure 10G ) and time course ( Fig. 10H )(n=3 biological replicates). Fig.10I ) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4 + and CD8 +CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45RO 阴性 CCR7 + ,CM:CD45RO + CCR7 + ,EM:CD45RO + CCR7 阴性 , effector CD45RO 阴性 CCR7 阴性 ). Fig.10J ) Representative dot plots of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. Figure 10K ) CD4 + Cell viability of T cells restimulated with CD3 / CD28 for four days or left unstimulated (n = 2 donors with 3 biological replicates per donor). Fig.10L , Figure 10M LTBR or tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every three days for up to three rounds of stimulation. After repeated stimulation, the expression of TIM-3 and LAG-3 was measured in resting cells ( Fig.10L ), and the secretion of IFNγ and IL2 was measured in restimulated cells ( Figure 10M ) (n=3 biological replicates). Fig.10E , Fig.10I Statistical significance for Figure 1 0K panels: two-sided unpaired t test; for Figure 10G Insets: two-sided paired t test. Error bars indicate SEM.

[0028] Figures 11A-11K Expression of LTBR ligands and LTBR by mRNA or deletion and point mutants is shown. Fig.11A ) IL2 secretion after 24 h of 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 were tested in triplicate. Fig. 11B , Fig. 11C ) CD4 + and CD8 + T cells were incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, and then IL2 ( Fig. 11B ) and IFNγ( Fig. 11C ). (n = 3 biological replicates). ( Fig. 10D , Fig.10E) Differentiation phenotype after restimulation of tNGFR- and LTBR-transduced T cells (n=3 biological replicates) incubated with IL2 alone or with LTA (1 ng / mL) or LIGHT (10 ng / mL) during culture ( Fig. 10D ) or proliferation ( Fig.10E ). CM: central memory. EM: effector memory. Unpaired two-sided t-test p values ​​are shown. ( Fig.11F , Fig.11I ) Transient LTBR or tNGFR expression by mRNA nucleofection ( FIG. 101F ). T cells were nucleofected with LTBR or tNGFR mRNA (n=3 biological replicates), and the expression of LTBR in LTBR cells was monitored over 21 days. Fig.11G )、tNGFR( Fig.11H ) or four up-regulated genes ( Fig.11I ) surface expression. At each time point, the expression of the target gene was normalized to the matched tNGFR control. ( Fig.11J ) Schematic representation of FLAG-tagged LTBR mutants. Figure 11K ) Expression of LTBR and FLAG in T cells transduced with LTBR mutants. Error bars indicate SEM.

[0029] Figure 12A-12I Chromatin accessibility in LTBRT cells is shown. Fig. 12A ) Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFRT cells, either resting or stimulated with CD3 / CD28 for 24 hours. Fig. 12B ) Differentially accessible chromatin regions between stimulated and resting tNGFR, stimulated and resting LTBR, ​​resting LTBR and resting tNGFR, and stimulated LTBR and stimulated tNGFR. The number of gained / lost peaks is shown (using an absolute log2 fold change of 1 and an adjusted p-value < 0.1 as cutoff). ( Fig. 12C , Fig.12D ) Chromatin accessibility of differentially expressed (adjusted p < 0.05) genes ( Fig. 12C ) or differentially accessible (adjusted p < 0.05) regions ( Fig.12D ). Two-sided t-test p values ​​are shown. Boxes show 25-75 percentiles with a line at the median; whiskers extend to 1.5× interquartile range. N=614 genes ( Fig. 12C ) or genomic region ( Fig.12D ). Fig.12E , Fig.12F ) compared with tNGFR cells at rest or stimulated for 24 h, more ( Fig.12E ) or less openings ( Fig.12F ) at the loci of chromatin accessibility profiles. 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, and 0-2350 for LAG3). ( Figure 12G ) Chromatin accessibility of resting or stimulated LTBR and tNGFR cells. Each row represents a peak significantly enriched for LTBR compared to matched tNGFR controls (log2 fold change > 1, adjusted DESeq2, p-value < 0.05). Peaks were clustered using k-means clustering, and selected genes at / near the peak from each cluster are indicated. ( Fig.12H ) Correlation of each ATAC sample (biological replicates) based on bias-corrected bias. Fig.12I ) Apical transcription factor (TF) motifs enriched in differentially accessible chromatin regions in quiescent LTBR cells compared to quiescent tNGFR cells.

[0030] Figure 13A-Figure 13P Proteomic and functional genomic assays of NF-κB activation are shown. Fig.13A ) Phospho-RELA staining of intracellular flow cytometry in LTBR and tNGFR. Gating for identification of phospho-RELA+ cells is shown. Fig. 13B , Fig. 13C ) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells that were resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel is shown ( Fig. 13B ) or quantification of band intensity relative to GAPHD ( Fig. 13C )(n=3 biological replicates). Unpaired two-sided t test p values ​​are shown. Fig.13D ) Schematic representation of the LTBR signaling pathway. Each gene is expressed based on its expression in LTBR relative to matched tNGFR cells (CD4 + and CD8 + T cells, resting or stimulated for 24 hours) were stained for differential expression ( Figure 13E-13G )Simultaneous gene knockout by CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered by nucleofection. Fig.13F ) Representative expression of target genes in LTBR cells co-expressing sgRNA targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβTCR. ( Figure 13G ) Quantification of IFNγ after restimulation (n=3 sgRNAs). Figure 13H-13O) Quantification of gene knockout efficiency based on representative protein levels. Representative histograms are shown ( Fig.13H , Fig.13J , Figure 13L ) and quantification of the relative expression levels of LTA, LIGHT and RELA ( Fig.13I , Figure 13K , Figure 13M ) (n=3 sgRNAs). Dashed lines indicate gates used to enumerate cells expressing a given protein. A representative gel is shown ( Fig.13N ) and quantification of RELB expression ( Fig.13O )(n=3 RELB sgRNAs and 2 non-targeting control sgRNAs). Figure 13P ) in CD4 transduced with LTBR + and CD8 + Identification of 274 genes identified as enriched in both T cells compared to matched tNGFR controls ("core LTBR" genes). Error bars indicate SEM.

[0031] Figure 14A-Figure 14P Co-delivery of ORF with CAR targeting CD19 is shown. ( Fig.14A ) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). Fig. 14B , Fig. 14C ) CAR expression levels as determined by staining with anti-mouse Fab F(ab′)2. Representative histograms of two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL) are shown ( Fig. 14B ) and quantification of CAR expression relative to tNGFR ( Fig. 14C ). Fig.14D ) Expansion curves of CAR+ORF transduced T cells (n=4 biological replicates). Fig.14E ) Autologous CD14 transduced with LTBR alone or CAR+LTBR + LTBR expression in monocytes and T cells. Figure 14F-14I ) ICAM-1 of T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR ( Fig.14F )、CD70( Figure 14G )、CD74( Fig.14H ) and MHC-II( Fig.14I ) expression. All data were normalized to tNGFR alone (no CAR). Unpaired two-sided t test p values ​​are shown. ( Figure 14J-Figure 14M )PD-1, an exhaustion marker in CAR+ORF T cells ( Fig.14J )、TIM-3( Figure 14K )、LAG-3( Figure 14L ) and CD39( Figure 14M ) expression. Fig.14N )Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45RO 阴性 CCR7 + ,CM:CD45RO + CCR7 + 、EM:CD45RO + CCR7 阴性 , effector CD45RO 阴性 CCR7 阴性 ). Fig.14O , Figure 14P )Activation marker CD25 in CAR+ORF T cells cultured alone or with Nalm6 cells for 24 h ( Fig.14O ) and CD69( Figure 14P ). Error bars indicate SEM. N = 3 biological replicates unless otherwise stated.

[0032] Figure 15A-Figure 15P The top ranked genes from the ORF screen are shown to enhance antigen-specific T cell responses. Fig.15A , Fig. 15B ) Anti-CD19 CAR and ORF were co-delivered to T cells from healthy donors. CD4 + After T cells were co-cultured with Nalm6 cells at a ratio of 1:1 overnight ( Fig.15A )IFNγ and ( Fig. 15B ) IL2 secretion (n=3 biological replicates, representing two donors). Fig. 15C , Fig.15D IFNγ of CAR+ORF or ORF T cells alone or co-incubated with Nalm6 cells for 24 h ( Fig. 15C ) or IL-2( Fig.15D )secretion.( Fig.15E ) Cytotoxicity of 19-BBz CAR T cells expressing tNGFR or LTBR ORF after co-incubation with Nalm6 GFP cells. ( Fig.15F ) Quantification of Nalm6 clearance by CAR+ORF or ORF alone T cells (relative to Nalm6 co-incubated with uninduced T cells) at different effector:target ratios. Unpaired two-sided t test p values: 0.011, 1.3x 10-4, 0.072, 0.02, 0.021, 0.52, 0.087, 1, 0.51 (from left to right). ( Figure 15G) T cells transduced with 19-28z CAR and NGFR or LTBR and CD19 + Representative images of Nalm6 GFP cells co-cultured at a 1:1 ratio for 48 h. Scale bar: 200 μm. ( Figure 15H-15J ) Repeated stimulation of CAR+ORF T cells with Nalm6 cells. After repeated stimulation with Nalm6 cells every three days for up to three rounds of stimulation, IL-2 secretion of 19-BBz CAR LTBR or tNGFR T cells re-stimulated with Nalm6 ( Fig.15I ) or Nalm6 survival rate ( Fig.15J ). Figure 15K ) 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-sided paired t test p values ​​are shown. ( Figure 15L ) Representative staining of T cells transduced with the ORF that endogenously expresses the Vγ9Vδ2 TCR. ( Figure 15M ) Quantification of T cells transduced with the ORF expressing the Vγ9Vδ2 TCR. Fig.15N , Fig.15O ) ORF-transduced Vγ9Vδ2 T cells were co-incubated with leukemia cell lines for 24 hours after IL2 ( Fig.15N ) or IFNγ( Fig.15O )secretion.( Figure 15P ) ORF-transduced Vγ9Vδ2 T cells were co-incubated with the pancreatic ductal adenocarcinoma cell line BxPC3 for 24 hours after IL2 or IFNγ secretion. Figure 15N-Figure 15P ). Error bars indicate SEM. N=3 biological replicates are shown unless otherwise stated.

[0033] Figures 16A-16F The top ranked genes that improve antigen-specific CAR T cell responses in solid tumors are shown. Fig.16A ) Anti-mesothelin CAR and ORF were co-delivered to T cells from healthy donors. Figure 16B-Figure 16D ) and the mesothelin-high cell line Capan-2 ( Fig. 16B , Fig. 16C ) or the mesothelin-low cell line BxPC3 ( Fig.16D )After overnight co-incubation, CD4+ and CD8+ T cells co-transduced with anti-mesothelin CAR and ORF secreted cytokines IFNγ and IL2. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. The dotted line indicates the level of cytokine secretion in conventional CAR T cells (i.e., co-expressing tNGFR). ( Fig.16E , Fig.16F ) After co-incubation with engineered CAR T cells at a 1:2 T cell to cancer cell ratio for 48 hours, killing GFP+mesothelin high Capan-2 or mesothelin low BxPC3. Cancer cell killing is normalized by dividing the integrated GFP signal in the well containing conventional CART cells (i.e., co-expressing tNGFR) by the integrated GFP signal in the specific sample. A ratio higher than one indicates higher killing than the matched CAR control (CAR+tNGFR), i.e., a lower GFP signal in a specific sample (and therefore a lower number of cancer cells). WT or no CAR = untransduced T cells.

[0034] Figures 17A-17D The top ranked genes are shown to improve antigen-specific TCR T cell responses in solid tumors. Fig.17A ) Anti-NY-ESO-1 TCR and ORF were co-delivered to T cells from healthy donors. Fig. 17B , Fig. 17C ) Secretion of cytokines IFNγ and IL-2 by CD8+T cells co-transduced with anti-NY-ESO-1 TCR and ORF after co-incubation overnight with melanoma cell line A375. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. The dotted line indicates the level of cytokine secretion in conventional TCR T cells (i.e., co-expressing tNGFR). (FIG. 17E, FIG. 17F) Killing of GFP+A375 cells co-incubated with engineered TCR T cells at a 1:1 ratio of T cells to cancer cells for 48 hours. Cancer cell killing was normalized by dividing the integrated GFP signal in wells containing only A375 cells but no T cells by the integrated GFP signal in a specific sample. No TCR = untransduced T cells.

[0035] Figures 18A-18G An overview of OverCITE-seq is provided.

[0036] FIG. 19 is a list of clinical trials related to chimeric antigen receptors available on clinicaltrials.gov.

[0037] FIG. 20 is a list of clinical trials related to T cell receptors available on clinicaltrials.gov.

[0038] Figure 21A-Figure 21N Insertion of the intracellular domain of LTBR into the CAR signaling domain was shown to achieve superior efficacy. Fig.21A ) Schematic representation of an anti-CD19 CAR utilizing the CD28 co-stimulatory domain. ( Fig.21B ) Schematic representation of an anti-CD19 CAR utilizing the 41-BB co-stimulatory domain. ( Fig. 21C , Fig.21D ) respectively Fig.21A and Fig.21B Surface expression of CAR in T cells transduced with the indicated constructs. No CAR indicates untransduced T cells. ( Fig.21E , Fig.21F ) respectively Fig.21A and Fig.21B Expression of LTBR-induced genes CD54 (also known as ICAM-1) and CD74 in resting T cells transduced with the indicated constructs. Surface expression was normalized to a matched control (CART cells co-expressing tNGFR). N = 2 individually transduced samples (CD4+ and CD8+ T cells). Figure 21G , Fig.21H ) respectively Fig.21A and Fig.21B Differentiation of T cells transduced with the indicated constructs (naive: CD45RO-CCR7+, central memory [CM]: CD45RO+CCR7+, effector memory [EM]: CD45RO+CCR7-, effector: CD45RO-CCR7-). Figure 21I-Figure 21L ) were co-incubated with CD19+ leukemia cell line Nalm6 overnight, and then Fig.21A and Fig.21B CD4+ and CD8+ T cells transduced with the indicated constructs secrete the cytokines IFNγ and IL2. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. The dotted line indicates the level of cytokine secretion in conventional CAR T cells (i.e., co-expressing tNGFR). ( Figure 21M , Fig.21N ) GFP+CD19+ leukemia cell line Nalm6 is killed after co-incubation with engineered fever CART cells for 48 hours or 96 hours at high (1:2 T cell to cancer cell ratio) or low (1:8 T cell to cancer cell ratio). Cancer cell killing is normalized by dividing the integrated GFP signal in the well containing conventional CAR T cells (i.e., co-expressing tNGFR) by the integrated GFP signal in the specified sample. A ratio higher than one indicates higher killing than the matched CAR control (CAR+tNGFR), i.e., a lower GFP signal in a specific sample (and therefore a lower number of Nalm6 cancer cells). WT or no CAR = untransduced T cells.

[0039] Figure 22A-22C It was shown that LTBR enhanced the activity of the first generation CAR. ( Fig.22A ) Schematic representation of anti-CD19 CAR (generation 0 - no signaling domain, generation 1 - CD3z only, generation 2 - 4-1BB and CD3z) co-expressed with tNGFR (negative control) or LTBR. ( Fig. 22B , Fig. 22C ) After overnight co-incubation with the CD19+ leukemia cell line Nalm6, Fig.22A CD4+ and CD8+ T cells transduced with the indicated constructs secrete cytokines IFNγ and IL2. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. The dotted line indicates the level of cytokine secretion in conventional second generation CAR T cells (i.e., co-expressing tNGFR). WT-untransduced T cells.

[0040] Figure 23A-23B It was shown that extracellular LTBR enhanced T cell function when fused to the handle, transmembrane and signaling domains of the second generation CAR. Fig.23A ) Schematic representation of the constructs tested. ( Fig. 23B ) After overnight incubation with CD3 and CD28 antibodies, Fig.23A CD4+ and CD8+ T cells transduced with the indicated constructs secrete the cytokines IFNγ and IL2. No specific cytokine secretion was observed in T cells incubated alone. N=2 biological replicates. Dashed lines (where applicable) show cytokine secretion in non-transduced (WT - wild type) T cells.

[0041] Figure 24A-Figure 24E , CARs with only the intracellular LTBR signaling domain enhanced T cell responses to TCR stimulation and activated the NFκB pathway. ( Fig.24A ) Schematic representation of the CAR construct, consisting of the antigen targeting moiety, the stalk (CD8 or CD28), the transmembrane domain derived from the same protein as the stalk or from the LTBR, ​​and the intracellular domain of the LTBR. ( Fig. 24B ) Schematic representation of second generation CARs co-expressed with tNGFR (negative control) or LTBR or CARs lacking any signaling domain. ( Fig.24C ) After overnight incubation with CD3 and CD28 antibodies, Fig.24A and Fig. 24B CD4+ and CD8+ T cells transduced with the indicated constructs secrete cytokines IFNγ and IL2. No specific cytokine secretion was observed in T cells incubated alone. N=3 biological replicates. Dashed lines (where applicable) show cytokine secretion in T cells expressing only the anti-CD19 targeting moiety but not the signaling domain (19-CD8+tNGFR). WT - untransduced (wild type) cells. ( Fig.24D) Expression of CD74 on resting T cells or T cells co-incubated overnight with CD19+ target cells. Dashed line shows expression of CD74 in T cells expressing only the anti-CD19 targeting moiety but not the signaling domain (19-CD8+tNGFR) - whether incubated alone or with target cells. WT - untransduced (wild type) cells.

[0042] Figure 25A-Figure 25C A schematic representation of the incorporation of LTBR into the TCR complex is shown. ( Fig.25A )αβTCR design;( Fig.25A )γδTCR design;( Fig.25A )αβT cell co-receptor design.

[0043] Figure 26A-26K It was shown that LTBR co-delivery enhanced the anti-tumor activity of B7-H3 CAR T cells. Fig.26A Differentiation of transduced and non-transduced T cells is shown (naive: CD45RO-CCR7+, central memory: CD45RO+CCR7+, effector memory: CD45RO+CCR7-, effector: CD45RO-CCR7-). Surface markers CD54 ( Fig.26B ) and CD74( Fig.26C ) expression. n = 3. The engineered T cells were co-cultured with B7-H3+ cell lines BT12, BT16 and A375 and B7-H3 阴性 After co-incubation overnight, the cell line Nalm6 secreted IFNγ ( Fig.26D ) or IL2( Fig.26E ). n = 3. After co-incubation with engineered CAR T cells at a 2:1 T cell to cancer cell ratio, GFP+B7-H3+ cell lines BT12, BT16, and A375 ( Fig.26F ) or GFP+B7-H3 阴性 Cell line Nalm6 ( Figure 26G )'s killing. Figure 26H-26K In vivo testing of B7-H3 CAR T cells in NSG mice implanted with A375 cells in the left flank is shown. The experiment was terminated at day 25 due to aggressive tumor growth in most animals. ( Fig.26H) Experimental design. Tumor volume was measured using calipers on day 13 after tumor inoculation. Based on tumor burden, mice were staged into five groups (n = 4-5 mice per group) so that there was no difference in median burden between groups. After staging, mice were injected with a 1:1 mixture of human CD4 and CD8 T cells that were either untransduced or transduced with B7-H3 CAR and control genes tNGFR or LTBR. High dose = 5 million T cells per mouse; low dose = 1 million T cells per mouse. ( Fig.26I )Weight changes after tumor transplantation. Fig.26J ) Average tumor size in each group. Once the tumor volume exceeds 200mm 3 , or at the discretion of a veterinarian blinded to the study design, mice were removed from the study and euthanized. Figure 26K ) Tumor sizes of individual mice.

[0044] Figure 27A-27G A CAR fusion with a truncated LTBR intracellular tail is shown. ( Fig.27A ) Literature-based annotation of functional elements within the LTBR signaling tail. Numbers indicate amino acid numbering according to https: / / www.uniprot.org / uniprotkb / P36941. ( Fig.27B ) Schematic diagram depicting the fusion of the LTBR intracellular tail to the C-terminus of 19-28-zCAR. For the V1-V9 variants of the intracellular tail, the dotted lines indicate which part of the LTBR protein is included in the fusion with the CAR. All sequences (amino acid and protein) are listed in the sequence listing. ( Fig.27C )Surface expression of 19-28-z CAR in transduced T cells. Fig.27D ) Differentiation of transduced and non-transduced T cells (naive: CD45RO-CCR7+, central memory: CD45RO+CCR7+, effector memory: CD45RO+CCR7-, effector: CD45RO-CCR7-). ( Fig.27E )Intracellular / nuclear expression of TCF1. Relative levels of TCF1 were normalized to the signal in 19-28-z+tNGFR CAR T cells. Fig.27F ) Surface expression of CD54 and CD74, normalized to the signal in 19-28-z+tNGFR CAR T cells. ( Figure 27G ) Secretion of IL2 and IFNγ cytokines after co-incubation of anti-CD19 CART cells with CD19+ leukemia cells Nalm6 overnight. The absolute amount of secreted cytokines was normalized to the amount secreted by 19-28-z+tNGFR CAR T cells.

[0045] Figure 28A-Figure 28NFusion of the LTBR signaling tail to a mesothelin-targeting CAR is shown. Fig.28A )Construct design. Fig.28B )CAR surface expression on transduced T cells. ( Fig.28C ) CART cell proliferation in response to antigen stimulation. T cells were isolated, transduced with lentivirus and screened. 14 days after isolation, CART cells were co-incubated with mesothelin+ PDAC cell line Capan-2 at a ratio of 1:1 (n=3). T cells were counted and split regularly to adjust to 0.5x 10 6 The density of cells / mL. Fig.28D ) Killing of GFP+mesothelin+Capan-2 cell line after co-incubation with engineered CAR T cells at a 1:4 T cell to cancer cell ratio. ( Fig.28E ) Cytokine secretion of engineered CAR T cells after 24 hours of co-incubation with mesothelin+Capan-2 cell line. ( Fig.28F ) Surface expression of CD54 and CD74 on engineered CD4 and CD8 T cells. The median fluorescence intensity of the staining for each sample was normalized by subtracting the median fluorescence intensity of the corresponding control (Meso-BB-z or Meso-28-z) and then dividing by the standard deviation of the sample group for z-score normalization. ( Figure 28G ) TCF1 is expressed intracellularly in engineered T cells. The median fluorescence intensity of the staining for each sample was normalized by subtracting the median fluorescence intensity of the corresponding control (Meso-BB-z or Meso-28-z) and then dividing by the standard deviation of the sample group for z-score normalization. ( Fig.28H ) Cytokine secretion after 24 hours of co-incubation of engineered CAR T cells with pan T cell stimulation using CD3 / CD28. ( Fig.28I ) depicts a schematic diagram of the sequential co-transduction of T cells with two lentiviral particles, one encoding a CAR targeting mesothelin and the other encoding either native full-length LTBR or a fusion of the LTBR intracellular domain with the CAR. ( Fig.28J )CAR surface expression on sequentially transduced T cells. Figure 28K ) LTBR surface expression on sequentially transduced T cells. ( Figure 28L ) Surface expression of CD54 and CD74 on sequentially transduced CD4 and CD8 T cells, normalized to corresponding non-transduced T cells. ( Figure 28M , Fig.28N ) After sequentially transduced CAR T cells were co-incubated with mesothelin+Capan-2 cell lines for 24 hours, IFNγ( Figure 28M ) or IL2( Fig.28N ) secretion.

[0046] Figure 29A-29I Fusion of the LTBR signaling tail to members of the TCR-CD3 complex is shown. ( Fig.29A )Construct design for TCR fusion. Fig.29B )TCR surface expression on transduced T cells. ( Fig.29C ) Surface expression of CD54 and CD74 on transduced T cells. ( Fig.29D ) Cytokine secretion of engineered T cells after 24 hours of incubation with NY-ESO-1+ melanoma line A375. ( Fig.29E )Construct design for CD3 fusion. Fig.29F ) Schematic depiction of sequential transduction of T cells with lentiviral particles encoding NY-ESO-1 TCR and CD3 genes, either natively or fused to the LTBR signaling tail at the C-terminus. ( Figure 29G )TCR surface expression on sequentially transduced T cells. Fig.29H ) Surface expression of CD54 and CD74 on sequentially transduced T cells. Fig.29I ) Cytokine secretion of sequentially transduced T cells after 24 h of co-incubation with the NY-ESO-1+ melanoma line A375.

[0047] Figures 30A-30G Fusion of the LTBR signaling tail to a member of the CD8 complex is shown. ( Fig. 30A )Construct design for CD8 fusion. Fig. 30B ) Schematic depiction of sequential transduction of T cells with NY-ESO-1 TCR and CD8 genes either native or fused to the LTBR intracellular tail. ( Fig. 30C , Fig.30D )CD54 on transduced T cells ( Fig. 30A ) and CD74( Fig. 30B )’s surface expression. Fig.30E )Intracellular expression of TCF1 in transduced T cells. Fig.30F ) Cytokine secretion of engineered T cells after 24 hours of co-incubation with the HLA-A2+ cell line HEK293T pulsed with the indicated concentrations of NY-ESO-1 derived peptide epitopes. ( Figure 30G ) Antigen sensitivity of engineered T cells calculated based on sigmoidal fit of the indicated cytokine secretion levels as a function of peptide concentration. MFI - median fluorescence intensity.

[0048] Fig.31ORF library vector engineering is shown.Slow virus and barcode encoding ORF library (as indicated) encoding puromycin resistance are produced in HEK293T cells, concentrated and used for transduction of activated T cells.After two days of transduction, the engineered T cells under each condition are split, and one group is selected with puromycin, and another group is treated with vehicle.After two days of selection, T cells are counted, and transduction efficiency is determined by dividing the number of viable cells under puromycin treatment conditions by the untreated control of matching.The dotted line indicates the survival rate of the untransduced T cells treated with puromycin. DETAILED DESCRIPTION

[0049] Engineering of autologous patient T cells for adoptive cell therapy has revolutionized the treatment of several types of cancer 1 . However, additional improvements are needed to increase response and cure rates. Provided herein are modified T cells comprising a nucleic acid encoding a fusion protein comprising LTBR or a domain thereof. When overexpressed in T cells, LTBR induces profound transcriptional and epigenomic remodeling, leading to increased T cell effector function and resistance to exhaustion in chronic stimulatory environments through constitutive activation of the canonical NF-κB pathway. LTBR and other higher ranked genes improve the antigen-specific response of chimeric antigen receptor T cells and γδ T cells, highlighting their potential in future cancer diagnostics and therapies 5 Provides improved CAR, TCR and related T cell therapies for the treatment of cancer and other diseases.

[0050] Unless otherwise defined in this specification, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the invention belongs and with reference to the public text, which provides a general guide for many of the terms used in this application to those skilled in the art.

[0051] As used throughout this specification and claims, the terms "comprises," "comprising," "including," and variations thereof include other components, elements, integers, steps, etc. In contrast, the term "consisting of" and variations thereof do not include other components, elements, integers, steps, etc.

[0052] It should be noted that the term "a / an" refers to one or more / one or more, for example, "T cell" should be understood to mean one or more T cells. Thus, the terms "a" (or "a kind"), "one or more / one or more" and "at least one / kind" are used interchangeably herein.

[0053] As used herein, unless otherwise indicated, the term "about" means a variability of plus or minus 10% relative to a given reference.

[0054] In addition, the use of "and / or" herein should be understood as explicitly disclosing each of the two specified features or components with or without the other. Therefore, the term "and / or" as 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" as used in phrases such as "A, B, and / or C" is intended to cover 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).

[0055] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences 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 Acids Res. 19:5081 (1991); Qhtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolim et al., Mol. Cell. Probes 8:91-98 (1994)).

[0056] The terms "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence may be single-stranded or double-stranded DNA or RNA, e.g., mRNA.

[0057] Nucleic acids described herein can be cloned using conventional molecular biology techniques, or generated de novo by DNA synthesis, which can be performed by service companies with business in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Life Technologies, Eurofins) using conventional procedures. The nucleotide sequences encoding the various aspects of the CRISPR-Cas editing system described herein are assembled and placed in any suitable genetic element, such as naked DNA, bacteriophage, transposon, cosmid, episome, etc., and the sequences carried thereon are transferred to a host cell, such as for the production of a non-viral delivery system (e.g., RNA-based systems, naked DNA, etc.) or for the production of viral vectors in packaging host cells and / or for delivery to a host cell of a subject. In one embodiment, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. The method for preparing such engineered constructs is known to nucleic acid manipulation technicians and includes 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).

[0058] "Variants" of proteins or peptides as defined in the context of the present invention can be produced, whose amino acid sequences are different from the original sequence in one or more mutations, such as one or more substituted, inserted and / or deleted amino acids. Preferably, these fragments and / or variants have the same biological function or specific activity, such as their 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 include conservative amino acid substitutions compared to their natural, i.e. non-mutated physiological sequences. Substitutions derived from the mutual exchange of amino acids of the same type are referred to as conservative substitutions. Specifically, these are amino acids with aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains, or amino acids whose side chains can enter into hydrogen bonds, such as side chains with hydroxyl functions. This means, for example, that an amino acid with a polar side chain is replaced by another amino acid with a similar polar side chain, or, for example, that an amino acid characterized by a hydrophobic side chain is replaced by another amino acid with a similar hydrophobic side chain (e.g., serine (threonine) is replaced by threonine (serine), or leucine (isoleucine) is replaced by isoleucine (leucine)). Insertions and substitutions are possible, in particular at those sequence positions which do not cause a modification of the three-dimensional structure or do not affect the binding region. Modifications of the three-dimensional structure by insertions or deletions can be easily determined, for example, using CD spectroscopy (circular dichroism spectroscopy) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, Modern Physical Methods in Biochemistry, Neuberger et al. (eds.), Elsevier, Amsterdam). Variants may also contain unnatural amino acids.

[0059] "Variants" 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 full length of the protein or peptide.

[0060] The term "gene" may refer to a DNA segment involved in producing or encoding a polypeptide chain. It may include regions before and after the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0061] As used herein, the terms "coding region" and "coding region" and grammatical variations thereof refer to an open reading frame (ORF) in a polynucleotide that produces a polypeptide or protein when expressed.

[0062] "Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. 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.

[0063] The term "domain" refers to a region of a polypeptide chain of a protein that is self-stable and folds independently of the rest of the protein. A protein domain need not be identical to the native protein from which it is derived, but may be a variant thereof, including variants with deletions, truncations, etc.

[0064] The term "coding" refers to the inherent properties of the specific sequence of nucleotides in polynucleotides such as genes, cDNAs or mRNAs, which are used to serve as templates for synthesizing other polymers and macromolecules in biological processes, and the other polymers and macromolecules have defined nucleotide sequences (i.e., rRNA, tRNA and mRNA) or defined amino acid sequences and biological properties produced therefrom. Therefore, if the transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene, cDNA or RNA encodes the protein. The coding strand (whose nucleotide sequence is consistent with the mRNA sequence and is generally provided in a sequence table) and the non-coding strand (used as a template for gene or cDNA transcription) can be referred to as encoding the protein or other products of the gene or cDNA.

[0065] Unless otherwise specified, a "nucleic acid sequence encoding an amino acid sequence" includes degenerate versions of each other and all nucleic acid sequences encoding the same amino acid sequence. Nucleic acid sequences encoding proteins or RNAs may also contain introns, to the extent that a nucleotide sequence encoding a protein may contain introns in a certain version.

[0066] The term "expression" is used herein in its broadest sense and includes the production of RNA, protein, or both RNA and protein. Expression may be transient or may be stable.

[0067] The terms "expression" and "overexpression" refer to increasing the expression of a gene or protein. The term refers to an increase in expression, such as an increase of at least 10% in the amount of mRNA or protein expressed in T cells, other lymphocytes or host cells, or an increase of at least about 20%, or at least about 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 exogenous polynucleotides encoding fusion proteins, TCRs or CARs to be expressed and / or overexpressed in cells, or inducing expression or overexpression of endogenous genes encoding proteins in cells.

[0068] The term "autologous" refers to any material derived from the same subject that is subsequently reintroduced into the subject.

[0069] The term "exogenous" refers to any material introduced or generated from outside an organism, cell, tissue or system.

[0070] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be supplied by a host cell or in an in vitro expression system. The expression vector comprises all expression vectors 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) incorporating a recombinant polynucleotide.

[0071] As used herein, "expression cassette" refers to a nucleic acid molecule encoding one or more ORFs or genes, such as effector enhancement genes, or CAR or TCR or its components. The expression cassette also contains a promoter and may contain additional regulatory elements for the expression of one or more elements of the gene editing system in the control host cell. In one embodiment, the expression cassette can be packaged into the capsid of a viral vector (e.g., a viral particle). In one embodiment, such expression cassettes for producing viral vectors as described herein are flanked by packaging signals and other expression control sequences of the viral genome, such as those described herein.

[0072] The term "regulatory element" or "regulatory sequence" refers to expression control sequences that are adjacent to a nucleic acid sequence of interest, as well as expression control sequences 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, such as the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product, when desired. See also Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include sequences that direct constitutive expression of a nucleic acid sequence in many types of target cells as well as sequences that direct expression of a nucleic acid sequence only in certain target cells (e.g., tissue-specific regulatory sequences).

[0073] "Promoter" is defined as one or more nucleic acid control sequences that guide the transcription of nucleic acids. As used herein, a promoter comprises an essential nucleic acid sequence near the transcription start site, such as a TATA element in the case of a polymerase II type promoter. A promoter may also optionally comprise a distal enhancer or repressor element, which may be positioned up to several thousand base pairs away from the transcription start site. When it comes to promoters, the term "constitutive" specifies a nucleotide sequence that, when operably connected to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. When it comes to promoters, the term "inducible" or "adjustable" specifies a nucleotide sequence that, when operably connected to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when there is an inducer corresponding to the promoter in the cell. In certain embodiments, an inducible promoter is activated in response to T cell stimulation. In certain embodiments, the promoter is a NFAT, AP1, NFκB or IRF4 promoter. When it comes to promoters, the term "tissue specificity" specifies a nucleotide sequence, and when the nucleotide sequence is operably connected to a gene encoding or specified polynucleotide, the gene product is basically produced in the cell only when the cell is a cell of the tissue type corresponding to the promoter. Other promoter elements (such as enhancers) regulate the frequency of transcription initiation. Usually, these promoter elements are located in the region of 30-110bp upstream of the start site, although it has been shown that many promoters also contain functional elements downstream of the start site. The interval between promoter elements is often flexible, so that when elements are reversed or moved relative to each other, promoter function is retained. In thymidine kinase (tk) promoters, the interval between promoter elements can be increased to 50bp before activity begins to decline. Depending on the promoter, it seems that individual elements can work in coordination or independently to activate transcription. Exemplary promoters include CMV IE genes, EF-1α., ubiquitin C or phosphoglycerol kinase (PGK) promoters.

[0074] The term "operably linked" refers to a functional connection between one or more regulatory sequences and a heterologous nucleic acid sequence that results 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 into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, in the case of two protein coding regions that need to be linked, be in the same reading frame.

[0075] The term "lentivirus" refers to the genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, and are therefore one of the most effective methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0076] In certain embodiments, one or more genes are encoded by a nucleic acid sequence, which is delivered to a host cell by a vector or a viral vector, many of which are known and available in the art. In one embodiment, a vector comprising an expression cassette as described herein is provided. 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, herpes simplex viruses. In one embodiment, the viral vector is replication-defective. "Replication-defective virus" refers to a viral vector, in which any viral genome sequence also packaged in a viral capsid or envelope is replication-defective, i.e., it cannot produce progeny virions, but retains the ability to infect cells.

[0077] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, and the vector particularly includes a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, for example, lentiviral vectors from Oxford BioMedica. Gene delivery technology, LENTIMAX from Lentigen TM Vector Systems, etc. Non-clinical types of lentiviral vectors are also available and will be known to those skilled in the art.

[0078] In one aspect, an engineered lentiviral vector is provided herein, comprising a sequence of SEQ ID NO: 132 or a sequence that shares at least 90% identity with SEQ ID NO: 132. In certain embodiments, the sequence shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 132. The engineered vector is particularly useful for ORF screening as described herein. In certain embodiments, an open reading frame (ORF) of a gene of interest is inserted into the vector sequence. In certain embodiments, a barcode is inserted into the vector sequence. SEQ ID NO: 133 provides an exemplary embodiment in which various primer binding sites, large nuclease recognition sites for restriction digestion, and cloning recombination sites have been inserted into the sequence at sites where ORFs and / or barcodes can be inserted. In addition to ORFs and / or barcodes, primer binding sites, restriction sites, cloning sites, etc. may also be included. In certain embodiments, the ORF and / or barcode is inserted after nucleotide 3291 of SEQ ID NO: 132.

[0079] In certain embodiments, the vector is a non-viral plasmid comprising an expression cassette as described herein, e.g., naked DNA, naked plasmid DNA, RNA, and mRNA; coupled to various compositions and nanoparticles, including, e.g., micelles, liposomes, cationic lipid-nucleic acid compositions, polysaccharide compositions and other polymers, lipid- and / or cholesterol-based nucleic acid conjugates, and other constructs as described herein. See, e.g., X. Su et al., Mol. Pharmaceutics, 2011, 8(3), pp. 774-787; Online publication: March 21, 2011; WO 2013 / 182683, WO 2010 / 053572, and WO 2012 / 170930, all of which are incorporated herein by reference.

[0080] Plasmids, other clones and expression vectors, their properties and their construction / manipulation methods that can be used according to the present invention are obvious to those skilled in the art. In one embodiment, the expression cassette as described herein is engineered into suitable genetic elements (vectors), which can be used to produce viral vectors and / or for being introduced into host cells, such as naked DNA, bacteriophages, transposons, clays, episomes, etc., and the genetic elements transfer the sequences carried thereon. The selected vector can be delivered by any suitable method, and the method comprises transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA coated pellets, viral infection and protoplast fusion. The method for preparing such constructs is known to nucleic acid manipulation technicians and comprises genetic engineering, recombinant engineering and synthetic techniques. See, for example, Sambrook et al., "Molecular Cloning: Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor, New York.

[0081] The term "transfected" refers to the process of transferring or introducing exogenous nucleic acids into host cells. A "transfected" cell is a cell transfected with exogenous nucleic acids. The cell includes the primary subject cell and its progeny.

[0082] As used herein, "transient" refers to expression of an unintegrated transgene over a period of hours, days, or weeks, wherein the 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.

[0083] RNA or DNA can be introduced into target cells using any of a variety of different methods, for example, commercially available methods include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems such as a "gene gun" (see, e.g., Nishikawa et al., Hum Gene Ther. 12(8):861-70 (2001)).

[0084] As used herein, the term "subject" means a mammal, including humans, veterinary or farm animals, livestock or pets, and animals commonly used in clinical studies. 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, cattle, sheep, non-human primates, etc. As used herein, the term "subject" can be used interchangeably with "patient".

[0085] Composition

[0086] Provided herein are compositions comprising nucleic acids, expression cassettes, and / or lymphocytes comprising coding sequences for certain genes (or fragments thereof) that have been shown to enhance T cell survival, proliferation, and / or effector function (collectively referred to herein as "effector enhancing genes"). In certain embodiments, the effector enhancing genes include any of the genes identified in Tables 1, 2, or 3.

[0087] Table 1

[0088]

[0089]

[0090]

[0091] Provided herein are expression cassettes comprising a nucleic acid sequence encoding a fusion protein comprising at least one or more fragments of an effector-enhanced gene. In certain embodiments, the gene comprises any of the genes identified in Table 1 above, or a fragment or variant thereof. In other embodiments, the gene comprises any of the genes identified in Table 2 below, or a fragment or variant thereof. In other embodiments, the gene comprises any of the genes identified in Table 3 below, or a fragment or variant thereof. The desired fragment comprises a protein domain, such as an intracellular signaling domain, a transmembrane domain, or an extracellular domain. In certain embodiments, the expression cassette comprises more than one effector-enhanced gene fragment.

[0092] As used herein, when referring to the specific gene of Table 1, Table 2 or Table 3, it is intended to use the coded sequence of the variant with full-length protein, fragment, domain or one or more substitutions in amino acid with missing or truncated. For example, in certain embodiments, nucleic acid encoding has the protein sequence of missing in N-terminus. In certain embodiments, nucleic acid encoding has the protein sequence of missing in C-terminus. In one embodiment, nucleic acid encoding has 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 acid.

[0093] In one embodiment, the effector-enhancing gene is LTBR. The receptor LTBR, ​​which is endogenously expressed by professional antigen-presenting cells rather than lymphocytes, has been identified as a strong synthetic driver of T cell proliferation and secretion of key cytokines: IL-2 and IFNγ. Using a multimodal single-cell sequencing approach, it was shown that LTBR induces profound transcriptional changes when overexpressed in T cells, activating cellular programs involved in antigen presentation and preventing apoptosis. As described herein, a platform for testing combined perturbations in T cells has been developed, by co-expressing a gene of interest (e.g., LTBR) with CRISPR sgRNAs targeting other genes, to map the signaling network in T cells. This article also demonstrates that mRNA delivery of LTBR is an alternative to constitutive lentiviral expression, highlighting the translational potential of the screening method.

[0094] In certain embodiments, the expression cassette includes a nucleic acid encoding LTBR or a fragment thereof. LTBR (lymphotoxin-β 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.

[0095] SEQ ID NO: 1

[0096]

[0097] The full-length amino acid sequence of LTBR is SEQ ID NO: 2 (Uniprot P36941):

[0098]

[0099]

[0100] The LTBR protein can be divided into three regions or domains: an extracellular domain (amino acids 31-227 of SEQ ID NO: 2); a transmembrane (or helical) domain (amino acids 228-248 of SEQ ID NO: 2); and a 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. When referring to domains herein, variants are intended to be included, including N-terminal or C-terminal truncated variants.

[0101] In certain embodiments, the expression cassette includes 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 nucleic acid encodes a protein sequence having a deletion in the N-terminus. In certain embodiments, the nucleic acid encodes a protein sequence having a deletion in 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.

[0102] In certain embodiments, the expression cassette includes a nucleic acid encoding a fragment of a domain that is a LTBR. In certain embodiments, the nucleic acid encodes the extracellular domain of the LTBR (amino acids 31-227 of SEQ ID NO: 2). In certain embodiments, the nucleic acid encodes the transmembrane domain of the LTBR (amino acids 228-248 of SEQ ID NO: 2). In certain embodiments, the nucleic acid encodes the cytoplasmic (or intracellular) domain of the 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 variants with deletions. Desirable variants of the cytoplasmic domain include variants having 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. Other desirable variants include variants having 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 having 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. In certain embodiments, the LTBR fragment includes amino acids 249-396 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment includes amino acids 249-393 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 249-387 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 249-377 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 262-435 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 297-435 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 324-435 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 345-435 of SEQ ID NO: 2. In certain embodiments, the LTBR fragment comprises amino acids 358-435 of SEQ ID NO: 2.

[0103] In other embodiments, the expression cassette includes nucleic acids encoding two or more domains of LTBR. In one embodiment, the nucleic acid encodes the cytoplasmic domain (or variant thereof) and the transmembrane domain of LTBR. In another embodiment, the nucleic acid encodes the cytoplasmic domain (or variant thereof), the transmembrane domain, and the extracellular domain of LTBR.

[0104] The fusion protein described herein also includes at least one fragment of a second protein different from the first protein, including a domain. In certain embodiments, the second protein is a protein that is a component of a T cell receptor. In other embodiments, the second protein is a protein that interacts with a T cell receptor. Such proteins include CD4, CD8A, CD8B, CD3E, CD3D, CD3G, and CD3Z.

[0105] In one embodiment, the fusion protein comprises a LTBR domain or a variant thereof and CD4. In another embodiment, the fusion protein comprises a LTBR domain or a variant thereof and CD8A and / or CD8B. In another embodiment, the fusion protein comprises a LTBR domain or a variant thereof and CD3E. In another embodiment, the fusion protein comprises a LTBR domain or a variant thereof and CD3D. In another embodiment, the fusion protein comprises a LTBR domain or a variant thereof and CD3G. In another embodiment, the fusion protein comprises a LTBR domain or a variant thereof and CD3Z. In certain embodiments, the LTBR domain is an intracellular domain. In certain embodiments, the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2 or a variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

[0106] Various isoforms of the genes identified in Table 1 are known in the art. Some are described in Table 2 below. In another embodiment, an expression cassette is provided, comprising the coding sequence of any of the alternative isoforms. One skilled in the art can identify alternative coding sequences for these genes that account for the degeneracy of the genetic code, including codon-optimized coding sequences, and use them as alternative embodiments of the compositions and methods described herein.

[0107] Table 2:

[0108]

[0109]

[0110]

[0111] Engineered T cell receptors containing LTBR domains

[0112] The present disclosure provides nucleic acid sequences encoding engineered T cell receptors, for example, T cell receptors (TCRs) incorporating LTBR domains. A chimeric antigen receptor (CAR) incorporating LTBR domains or variants thereof is also provided. Components of TCRs and CARs are further described herein. Engineered TCRs and CARs and modified T cells incorporating them are also provided.

[0113] Also provided herein is a nucleic acid sequence encoding an engineered T cell receptor, e.g., a T cell receptor (TCR) incorporating a domain or a variant thereof of one of the genes in Table 3. Also provided is a chimeric antigen receptor (CAR) incorporating a domain of one of the genes in Table 3. In certain embodiments, the domain is an intracellular domain selected from the genes in Table 3. Also provided are engineered TCRs and CARs and modified T cells incorporating them. As described below, embodiments incorporating LTBR domains are described. However, for each embodiment described for LTBR, ​​each of the genes in Table 3 has an embodiment.

[0114] Table 3

[0115]

[0116] Provided herein are nucleic acid molecules comprising the coding sequence of any of the TCRs described herein, modified to include a domain of LTBR or a gene in Table 3. Certain exemplary TCRs are provided in the sequence listings in SEQ ID NOs: 3-54. However, nucleic acids encoding all described TCRs and TCR proteins are intended to be encompassed herein.

[0117] TCR is a membrane-anchored heterodimer connected by disulfide bonds present on T cell lymphocytes, and most T cells are αβT cells with TCRs composed of α (alpha) chains and β (beta) chains. Each chain includes a variable (V) and a constant (C) domain, wherein the variable domain recognizes an antigen or a peptide presented by MHC. Using the methods described herein, TCRα and TCRβ chains with known specificity or affinity for a specific antigen, such as a tumor antigen described herein, can be introduced into T cells. Using standard molecular cloning techniques known in the art, TCRα and TCRβ chains with desired, such as enhanced specificity or affinity for a specific antigen can be isolated. Ordinary technicians can easily envision other modifications that increase the specificity, affinity or function of TCR or engineered T cells expressing TCR, for example, mutations in the antigen binding region of promoter selection, TCRα and TCRβ chains for regulating expression. Any separated or modified TCRα and TCRβ chains can be operably connected to or can be associated with one or more intracellular signaling domains described herein. Signaling can be mediated by the interaction between the antigen-bound αβ heterodimer and a CD3 chain molecule, such as CD3ζ (zeta).

[0118] A smaller subset of T cells expresses TCRs with (gamma) γ chains and δ (delta) chains. γ-δ (γδ) T cells account for 3%-10% of human circulating lymphocytes, and the Vδ2+ subpopulation 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 the major histocompatibility complex ("MHC") or human leukocyte antigen ("HLA"). Most Vδ2+ T cells also express the Vγ9 chain and are stimulated by exposure to 5-carbon pyrophosphate compounds, which are intermediates of the mevalonate and non-mevalonate sterol / isoprenoid synthesis pathways. Responses to isopentenyl pyrophosphate (5-carbon) are common in healthy humans. Another subset of γδ T cells, Vδ1+, accounts for a much smaller percentage of T cells in the blood circulation, but is commonly found in epithelial mucosa and skin. γδ T cells have a variety of functions, including killing tumor cells and pathogen-infected cells. Stimulation by γδ TCR improves the ability of cytotoxicity, cytokine secretion and other effector functions. The TCR of γδT cells has unique specificity, and the cells themselves appear at a high clonal frequency, thus allowing rapid innate-like responses to tumors and pathogens. See, for example, Park and Lee, Experimental and Molecular Medicine (Exp Mol Med). March 2021; 53 (3): 318-327., which is incorporated herein by reference.

[0119] In certain embodiments, the T cell comprises a nucleic acid sequence encoding a TCR (e.g., a TCR targeting a tumor antigen) comprising a LTBR domain. In certain embodiments, the TCR comprises an LTBR intracellular domain or a variant thereof as described herein. In one embodiment, the variant has a deletion in at least amino acids 393 to 435 of SEQ ID NO: 2. In one embodiment, a variant of the intracellular domain comprises amino acids 249-392 of SEQ ID NO: 2. However, additional residues may be deleted. Thus, desirable variants of the intracellular domain include variants having a sequence 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. Other desirable variants include variants having the sequence of 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 variants 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.

[0120] In certain embodiments, the T cell comprises a nucleic acid sequence encoding a TCR, e.g., a modified TCR targeting a tumor antigen described herein, comprising a domain of a gene in Table 3. In certain embodiments, the TCR comprises an intracellular domain from a gene in Table 3 or a variant thereof, as described herein.

[0121] In one embodiment, the TCR comprises the LTBR intracellular domain fused to the C-terminus of the TCR alpha chain. In another embodiment, the TCR comprises the LTBR intracellular domain fused to the C-terminus of the TCR beta chain. Fig.25A , Fig.29A. In one embodiment, the TCR comprises a LTBR intracellular domain fused to the C-terminus of the TCRδ chain. In another embodiment, the TCR comprises a LTBR intracellular domain fused to the C-terminus of the TCRγ chain. In another embodiment, the TCR comprises a LTBR intracellular domain fused to the C-terminus of CD3γ. In another embodiment, the TCR comprises a LTBR intracellular domain fused to the C-terminus of CD3δ. In another embodiment, the TCR comprises a LTBR intracellular domain fused to the C-terminus of one or two CD3ε. In another embodiment, the TCR comprises a LTBR intracellular domain fused to the C-terminus of one or two CD3ζ. See Fig.25A and 25B .

[0122] In one embodiment, the TCR comprises an intracellular domain of a gene in Table 3 fused to the C-terminus of a TCR alpha chain. In another embodiment, the TCR comprises an intracellular domain of a gene in Table 3 fused to the C-terminus of a TCR beta chain. Fig.25A . In one embodiment, the TCR comprises an intracellular domain of a gene in Table 3 fused to the C-terminus of a TCRδ chain. In another embodiment, the TCR comprises an intracellular domain of a gene in Table 3 fused to the C-terminus of a TCRγ chain. In another embodiment, the TCR comprises an intracellular domain of a gene in Table 3 fused to the C-terminus of CD3γ. In another embodiment, the TCR comprises an intracellular domain of a gene in Table 3 fused to the C-terminus of CD3δ. In another embodiment, the TCR comprises an intracellular domain of a gene in Table 3 fused to the C-terminus of one or two CD3ε. In another embodiment, the TCR comprises an intracellular domain of a gene in Table 3 fused to the C-terminus of one or two CD3ζ.

[0123] In another embodiment, the LTBR intracellular domain is fused to the C-terminal intracellular tail of CD4. In another embodiment, the LTBR intracellular domain is fused to the C-terminal intracellular tail of CD8α and / or CD8β. Fig.25C .

[0124] In another embodiment, the intracellular domain of the gene in Table 3 is fused to the C-terminal intracellular tail of CD4. In another embodiment, the intracellular domain of the gene in Table 3 is fused to the C-terminal intracellular tail of CD8α and CD8β.

[0125] In other embodiments, at least one domain of the LTBR is delivered to lymphocytes by direct modification of the endogenous genome. Various techniques for modification of the endogenous genome, including CRISPR, zinc finger nucleases, TALEN, etc., are known in the art. See, e.g., Azangou-Khyavy et al., CRISPR / Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer, Front. Immunol., September 29, 2020 https: / / doi.org / 10.3389 / fimmu.2020.02062 , which is incorporated herein by reference. In one embodiment, the LTBR intracellular domain or a fragment thereof is inserted into the genome of a lymphocyte. In other embodiments, at least one domain of a gene in Table 3 is delivered to a lymphocyte by direct modification of the endogenous genome.

[0126] In certain embodiments, the TCR is a known TCR, as identified in Figure 20, modified as described herein to include a domain of LTBR or a gene (or variant thereof) in Table 3. 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. Clinical Cancer Res. May 1, 2014; 20 (9): 2457-65. doi: 10.1158 / 1078-0432. CCR-13-3017. Electronic version March 14, 2014. PMID: 24634374; PMCID: PMC4070853. In other embodiments, 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; Abstract 102). In other embodiments, 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 other embodiments, the TCR targets MAGE A3. In other embodiments, the TCR targets GD-2. In other embodiments, the TCR targets P53. In other embodiments, the TCR targets LAGE-A1. In other embodiments, the TCR targets GP100.

[0127] In any of the embodiments described herein, the modified TCR can replace the CAR described herein to generate T cells. The engineered TCR described herein can replace the CAR in any of the embodiments described herein.

[0128] Chimeric Antigen Receptor (CAR) Containing LTBR Domain

[0129] The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a handle / hinge, a transmembrane domain, and a cytoplasmic signaling domain (also referred to as an intracellular signaling domain), wherein the cytoplasmic signaling domain comprises a functional signaling domain derived from a stimulatory molecule as defined below. See Jayaraman et al., CAR-T design: Elements and their synergistic function, "eBioMedicine", 58: 102931 (August 2020), which is incorporated herein by reference. In some embodiments, the stimulatory molecule is TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, 4-1BB or CD3-ζ. In a specific embodiment, the stimulatory molecule is a ζ chain associated with a T cell receptor complex. As used herein, for ease of reference, the term CD3ζ may be used when referring to a stimulatory molecule. However, the present invention is intended to provide similar embodiments in which CD3ζ is replaced by another suitable stimulatory molecule. In one embodiment, the stimulatory molecule is 4-1BB. In one embodiment, the stimulatory molecule is CD28. In another embodiment, the stimulatory molecule is LTBR, ​​and the stimulatory signaling domain comprises an LTBR intracellular domain or a variant thereof. In another embodiment, the stimulatory molecule is a gene in Table 3, and the stimulatory signaling domain comprises an intracellular domain of a gene in Table 3.

[0130] In one embodiment, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule as defined below (also referred to as "costimulatory signaling domain"). In one embodiment, the costimulatory molecule is selected from the costimulatory molecules described herein, such as OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand bound to CD83, ICAM-1, LFA-1 (CD11a / CD18), ICOS and 4-1BB (CD137), or any combination thereof. In certain embodiments, the costimulatory molecule is LTBR, ​​and the costimulatory signaling domain comprises an LTBR intracellular domain or a variant thereof. In another embodiment, the costimulatory molecule is a gene in Table 3, and the costimulatory signaling domain comprises an intracellular domain of a gene in Table 3.

[0131] As described herein, CAR itself includes one or more LTBR domains. In some embodiments, the LTBR domain can replace the domain from the existing CAR construct, such as those described in Figure 19. In other embodiments, in addition to the domain from the existing CAR construct, the LTBR domain is also included, such as those described in Figure 19.

[0132] As described herein, the CAR itself includes one or more domains from the genes in Table 3. In some embodiments, the domains from the genes in Table 3 can replace the domains in existing CAR constructs, such as those described in Figure 19. In other embodiments, in addition to the domains from the existing CAR constructs, domains from the genes in Table 3 are also included, such as those described in Figure 19.

[0133] In certain embodiments, the handle and transmembrane are from the same molecule, e.g., LTBR, ​​CD8, or CD28. In other embodiments, the handle and transmembrane are from different molecules, e.g., CD8 handle and LTBR TM, CD28 handle and LTBR TM, etc.

[0134] In one embodiment, CAR includes an optional leader sequence at the amino terminus (N terminus) of CAR fusion protein. In one embodiment, CAR further includes a leader sequence at the N terminus of an extracellular antigen binding domain, wherein during cell processing and CAR localization to cell membrane, the leader sequence is optionally cut from the scFv domain.

[0135] In one embodiment, 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 derived from LTBR or a variant thereof. Without wishing to be bound by theory, T cells engineered with CARs lacking the CD3ζ chain do not induce specific cytokine secretion in the presence of CD19+ leukemic cells. However, these cells induce NFκB-induced genes (as shown by CD74 expression (see Fig.24E )), and induced IFNγ and IL2 secretion upon stimulation with CD3 and CD28 antibodies ( Fig.24C and 24D ).

[0136] In certain embodiments, in addition to the LTBR signaling domain, the transmembrane domain is derived from LTBR. In certain embodiments, in addition to the LTBR signaling domain and (optionally) the transmembrane domain, the stalk is derived from LTBR.

[0137] In one embodiment, 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-derived LTBR or a variant thereof.

[0138] In one embodiment, CAR includes a chimeric fusion protein, the chimeric fusion protein includes an extracellular antigen recognition domain, a transmembrane domain, an intracellular signaling domain and a functional signaling domain, the intracellular signaling domain includes a functional co-signaling domain derived from LTBR or a variant thereof, the functional signaling domain is derived from a stimulatory molecule (e.g., CD3 ζ chain). The placement of the LTBR co-signaling domain can vary according to the desired function of the CAR. In certain embodiments, the LTBR co-signaling domain or its variant is placed between the transmembrane domain and the signaling domain (e.g., CD3 ζ). In other embodiments, CD3 ζ is placed between the transmembrane domain and the LTBR co-signaling domain or its variant.

[0139] In certain embodiments, in addition to the signaling domain, the transmembrane domain is derived from a gene in Table 3. In certain embodiments, in addition to the signaling domain and (optionally) the transmembrane domain, the stalk is derived from a gene in Table 3.

[0140] In one embodiment, 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 derived from a gene in Table 3 or a variant thereof.

[0141] In one embodiment, CAR includes a chimeric fusion protein, the chimeric fusion protein includes an extracellular antigen recognition domain, a transmembrane domain, an intracellular signaling domain and a functional signaling domain, the intracellular signaling domain includes a functional co-signaling domain derived from LTBR or a variant thereof, at least one additional functional co-signaling domain, the functional signaling domain is derived from a stimulatory molecule (e.g., CD3ζ chain). The placement of the LTBR co-signaling domain can vary depending on the desired function of the CAR. In certain embodiments, the LTBR co-signaling domain or its variant is placed between the transmembrane domain and other co-signaling domains (e.g., CD28 or 4-1BB). In certain embodiments, the LTBR co-signaling domain or its variant is placed between other co-signaling domains and the signaling domain (e.g., CD3ζ). In other embodiments, the LTBR co-signaling domain or its variant is placed downstream of the signaling domain (e.g., CD3ζ). See, e.g., Fig.21A and Fig.21B.

[0142] In certain embodiments, in addition to the LTBR signaling domain, the transmembrane domain is derived from LTBR. In certain embodiments, in addition to the LTBR signaling domain and (optionally) the transmembrane domain, the stalk is derived from LTBR.

[0143] In one embodiment, CAR includes a chimeric fusion protein, the chimeric fusion protein includes an extracellular antigen recognition domain, a transmembrane domain, an intracellular signaling domain and a functional signaling domain, the intracellular signaling domain includes a functional co-signaling domain or a variant thereof derived from a gene in Table 3, at least one additional functional co-signaling domain, and the functional signaling domain is derived from a stimulatory molecule (e.g., CD3 ζ chain). The placement of the co-signaling domain can vary according to the desired function of CAR. In certain embodiments, the co-signaling domain or a variant thereof of the gene in Table 3 is placed between the transmembrane domain and other co-signaling domains (e.g., CD28 or 4-1BB). In certain embodiments, the co-signaling domain or a variant thereof from a gene in Table 3 is placed between other co-signaling domains and a signaling domain (e.g., CD3 ζ). In other embodiments, the co-signaling domain or a variant thereof from a gene in Table 3 is placed downstream of a signaling domain (e.g., CD3 ζ).

[0144] The present disclosure provides a nucleic acid sequence encoding any one of the CARs described herein, e.g., a DNA or RNA construct. This also refers to a nucleic acid sequence encoding a known CAR, such as one of those shown in Figure 19, but modified to include a LTBR domain or a variant thereof, as described herein.

[0145] The present disclosure provides a nucleic acid sequence encoding any one of the CARs described herein, e.g., a DNA or RNA construct. This also refers to a nucleic acid sequence encoding a known CAR, such as one of those shown in Figure 19, but modified to include a domain or a variant thereof of the gene in Table 3, as described herein.

[0146] In certain embodiments, CAR targets CD19. In certain embodiments, CAR is a known CAR, such as one of those shown in Figure 19, but modified to include an LTBR domain (or a gene in Table 3) or a variant thereof, as described herein. In one embodiment, CAR is a modified axicabtagene ciloleucel. In another embodiment, CAR is a modified Brexucabtagene autoleucel. In another embodiment, CAR is a modified Tisagenlecleucel. In another embodiment, CAR is a modified Lisocabtagenemaraleucel. In certain embodiments, CAR targets B cell maturation antigen (BCMA). In another embodiment, CAR is a modified Idecabtagene vicleucel. In one embodiment, CAR is a modified ciltacabtagene autoleucel. In certain embodiments, CAR targets mesothelin. In certain embodiments, CAR targets ROR1. In certain embodiments, CAR targets B7-H3. In certain embodiments, CAR targets CD33. In certain embodiments, CAR targets EGFR806. In certain embodiments, CAR targets IL13Rα2. In certain embodiments, CAR targets GD2. In certain embodiments, CAR targets HER2. In certain embodiments, CAR targets glycan 3. In certain embodiments, CAR targets CD7. In certain embodiments, CAR targets NY-ESO-1. In certain embodiments, CAR targets CD30. In certain embodiments, CAR targets MAGE-A1. In certain embodiments, CAR targets LMP2. In certain embodiments, CAR targets PD1. In certain embodiments, CAR targets mutant KRAS G12V. In certain embodiments, CAR targets CD20. In certain embodiments, CAR targets CD22. In certain embodiments, CAR targets CD171. In certain embodiments, CAR targets CD123. In certain embodiments, CAR targets CD38. In certain embodiments, CAR targets CD10. In certain embodiments, CAR targets BAFFR. In certain embodiments, CAR targets PSMA. In certain embodiments, 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. See Srinivasan et al., 1972 Investigation of ALLO-316: A Fratricide-Resistant Allogeneic CAR T Targeting CD70 As a Potential Therapy for the Treatment of AML, 62nd ASH Annual Meeting and Exposition (62. nd ASH Annual Meeting and Exposition, December 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. Nature Medicine. 2017 Dec;23(12):1416-1423. doi:10.1038 / nm.4444. Epub 2017 Nov 13. PMID: 29131157.

[0147] Exemplary sequences of CARs and TCRs used in the Examples are provided in the sequence listings of SEQ ID NOs: 3-54. Other exemplary antibody sequences that can be used for the antigen recognition domain are provided in the sequence listings of SEQ ID NOs: 63-83.

[0148]

[0149] Other chimeric antigen receptors as modified herein include those used to treat 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 a skilled artisan.

[0150] The present disclosure provides a nucleic acid sequence encoding any of the TCRs described herein, e.g., a DNA or RNA construct. This also refers to a nucleic acid sequence encoding a known TCR, such as one of those shown in Figure 20, but modified to include an LTBR domain (or a gene in Table 3) or a fragment thereof, as described herein. Various other engineered T cell receptors are known in the art or can be designed by a skilled artisan. Such TCRs include those currently undergoing clinical testing, such as those identified in Figure 20.

[0151] The expression cassette referred to herein comprises a nucleic acid molecule encoding one or more biologically useful nucleic acid sequences (e.g., gene cDNA encoding fusion protein, CAR, TCR, mRNA, etc.) and a regulatory sequence operably connected thereto, the regulatory sequence instructs or regulates the transcription, translation and / or expression of the nucleic acid sequence and its gene product. The operably connected sequence comprises a regulatory sequence adjacent to the nucleic acid sequence and a regulatory sequence that acts in trans or at a distance to control the sequence. Such regulatory sequences generally include one or more of, for example, promoters, enhancers, introns, Kozak sequences, polyadenylation sequences, and TATA signals. The expression cassette may contain a regulatory sequence upstream (5'), for example, one or more of a promoter, enhancer, intron, etc., and one or more of an enhancer, or a regulatory sequence downstream (3'), for example, including a 3' untranslated region of a polyadenylation site, and other elements. Therefore, in addition to the coding sequence of a fusion protein, CAR or TCR, the expression cassette may also include an expression control sequence.

[0152] The expression control sequence comprises a promoter. In some embodiments, it is desirable to utilize a promoter with high transcriptional activity. Certain strong constitutive promoters are known in the art, including but not limited to CMV promoter, EF-1α promoter, CBG promoter, CB7 promoter, etc. Alternatively, other promoters may be utilized, such as adjustable (inducible) promoters [see, for example, WO2011 / 126808 and WO 2013 / 049493, which are incorporated herein by reference], or promoters that respond to physiological cues. In certain embodiments, an inducible promoter is activated in response to T cell stimulation. In certain embodiments, the promoter is a NFAT, AP1, NFκB or IRF4 promoter.

[0153] In certain embodiments, the expression cassette may also contain one or more IRES or 2A sequences to allow expression of multiple coding sequences from the same expression cassette. As exemplified herein, in one embodiment, a TCR directed to NY-ESO-1 is provided in which the LTBR intracellular domain is expressed in series with the TCRα (or TCRβ) chain. Figure 5A , in a lentiviral vector comprising a 2A sequence. The construction of such boxes and vectors is known in the art and is described in the examples herein. See, for example, Sack et al. Profound tissue specificity in proliferation control is the basis of cancer drivers and aneuploidy patterns. Cell. April 5, 2018; 173(2): 499-514.e2 and Yang et al. A public genome-scale lentiviral expression library of human ORFs. Nat Methods. August 2011; 8(8): 659-661, which are incorporated herein by reference.

[0154] In certain aspects, provided herein are compositions comprising modified lymphocytes, the modified lymphocytes comprising nucleic acids and / or expression cassettes described herein. In one embodiment, host lymphocytes are T cells. In another embodiment, host lymphocytes are natural killer (NK) cells. In certain embodiments, compositions include cell colonies, the cell colonies comprising mixed colonies of lymphocytes (e.g., αβT cells and NKT cells). In other embodiments, compositions include cells, the cells comprising colonies enriched for specific lymphocyte colonies.

[0155] As used herein, the phrase "T cell" refers to a lymphocyte that expresses a T cell receptor molecule. T cells include human αβ (alpha beta) T cells and human γδ (gamma delta) T cells. T cells include but are not limited to naive T cells, stimulated T cells, primary T cells (e.g., uncultured), 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 may be CD4+, CD8+, or CD4+ and CD8+. T cells may also be CD4-, CD8-, or CD4- and CD8-. T cells may be helper cells, such as T H 1. TH 2. T H 3. T H 9. T H 17 or T FH T cells can be cytotoxic T cells. T cells can also be regulatory T cells. Regulatory T cells (Tregs) can be FOXP3+ or FOXP3-. T cells can be α / β T cells or γ / δ T cells. In some cases, T cells are CD4+CD25 hi CD127 lo Regulatory T cells. In some cases, the T cells are regulatory T cells selected from the group consisting of type 1 regulatory (Tr1), T H 3. CD8+CD28-, Treg17 and Qa-1 restricted T cells or combinations or subpopulations thereof. In some cases, the T cells are FOXP3 + T cells. In some cases, the T cells are CD4 + CD25 lo CD127 hi Effector T cells. In some cases, the T cells are CD4 + CD25 lo CD127 hi CD45RA hi CD45RO- naive T cells. In some cases, the T cells are Vγ9Vδ2 T cells. In some embodiments, the T cells express viral antigens. In other embodiments, the T cells express cancer antigens. The T cells can be genetically manipulated recombinant T cells.

[0156] As used herein, the phrase "primary" in the context of primary cells is not transformed or immortalized cells. Such primary cells can be cultivated, subcultured or passed down a limited number of times (for example, cultivate 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 adapt to in vitro culture conditions. In some cases, primary cells are separated from organisms, systems, organs or tissues, optionally sorted, and directly utilized without cultivating or subculture. In some cases, primary cells are stimulated, activated or differentiated. For example, primary T cells can be activated by contacting (for example, cultivating in the presence of) with CD3, CD28 agonists, IL-2, IFN-γ or their combinations.

[0157] method

[0158] Methods for preparing modified host cells

[0159] Also provided herein are methods for preparing modified cells and compositions containing modified cells as described herein. Methods for modifying cells, such as lymphocytes, to introduce exogenous sequences are known in the art, such as including fusion proteins, CAR or TCR coding sequences or more than one expression cassette or expression vector of these sequences. For example, see, for example, WO 2016 / 109410A2, which is incorporated herein by reference. In certain embodiments, more than one exogenous sequence is introduced.

[0160] As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and / or functionally. Cells may be modified by the introduction of nucleic acids. Modification may refer to altering the expression of a gene in a lymphocyte, for example, by the introduction of an exogenous nucleic acid encoding a gene.

[0161] The lymphocytes provided herein can be genetically modified by, for example, transfection, transduction, or electroporation to express nucleic acid sequences encoding fusion proteins, TCRs, or CARs, as described herein. Depending on the clinical situation, such as the patient's condition or the condition to be treated, extended or permanent expression of the gene and / or, for example, for robust and lasting CAR activity, such as anti-tumor activity, may be desirable. In such embodiments, lymphocytes are genetically modified using a vector comprising a nucleic acid sequence encoding a gene disclosed herein, such as transduction, such as viral transduction, to confer desired effector functions. In other embodiments, transient expression of the gene is desired. In such embodiments, effector enhancement genes may be expressed using, for example, mRNA or an adjustable promoter.

[0162] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, for example, mammalian, bacterial, yeast or insect cells, by any of the techniques of the art. For example, expression vectors can be transferred to host cells by physical, chemical or biological means.

[0163] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Vols. 1-4, Cold Spring Harbor Press, New York). A suitable method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0164] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used methods for inserting genes into mammals, e.g., human cells. Viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0165] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used 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 acids are available, such as delivering polynucleotides with targeted nanoparticles or other suitable submicron-sized delivery systems. In the case of utilizing a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that nucleic acids are introduced into host cells (in vitro, in vitro, or in vivo) using lipid formulations. On the other hand, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, connected to liposomes via a linking molecule associated with liposomes and oligonucleotides, captured in liposomes, compounded with liposomes, dispersed in a solution containing lipids, mixed with lipids, merged with lipids, contained as a suspension in lipids, containing micelles or compounded with micelles, or otherwise associated with lipids. The composition associated with lipid, lipid / DNA or lipid / expression vector is not limited to any specific structure in solution. For example, the composition can exist as a bilayer structure, micelle, or "collapsed" structure. The composition can also be simply dispersed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids. For example, lipids include naturally occurring fat droplets in the cytoplasm, and compounds containing long-chain aliphatic hydrocarbons and their derivatives (e.g., fatty acids, alcohols, amines, amino alcohols, and aldehydes). Lipofectamine-nucleic acid complexes are also considered.

[0166] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose cells to the inhibitors of the invention, a variety of assays can be performed to confirm that the recombinant nucleic acid sequence is present in the host cell. Such assays include, for example, Southern and Northern blotting, RT-PCR and PCR, biochemical assays, such as, for example, by immunological methods (ELISA and Western blotting) to detect the presence or absence of specific peptides.

[0167] In certain embodiments, an expression vector is provided, which comprises a coding sequence of a fusion protein, the fusion protein comprising a LTBR domain and a domain of a non-LTBR protein. In other embodiments, the expression vector comprises a coding sequence of one or more components of a CAR or TCR. In other embodiments, the expression vector comprises a coding sequence of one or more components of a CAR or TCR comprising one or more LTBR domains. In other embodiments, the expression vector comprises a coding sequence of one or more components of a CAR or TCR comprising one or more domains of a gene in Table 1. The expression vector comprises all expression vectors known in the art, including clays, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., slow viruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporated with recombinant polynucleotides. In one embodiment, the expression vector is a slow virus. If more than one expression vector is used, each expression vector can be selected individually from vectors known in the art.

[0168] Provided herein is a method for preparing a population of immune effector cells (e.g., T cells, NK cells) modified to express fusion proteins, TCRs or CARs described herein. The method for preparing such immune cells includes introducing exogenous nucleic acids encoding LTBR domain fusion proteins into cells. Also provided herein is a method for preparing a population of immune effector cells (e.g., T cells, NK cells) modified to express CARs or TCRs comprising one or more LTBR domains. The method for preparing such immune cells includes introducing exogenous nucleic acids encoding CARs or TCRs into cells. In certain embodiments, immune effector cells including fusion proteins described herein have a long life span and / or are anti-apoptotic.

[0169] In the examples described below, for convenience, methods of preparing modified T cells are described. However, alternative embodiments using other types of immune cells, such as NK T cells or NK cells are conceivable. Suitable methods are known in the art.

[0170] In short, exemplary methods include providing an immune effector cell (e.g., T cell) colony, and optionally, removing T regulatory cells, e.g., CD25+T cells, from the colony. In one embodiment, the immune effector cell colony is autologous to the subject to which the cell therapy will be administered. In one embodiment, the immune effector cell colony is allogeneic to the subject to which the cell therapy will be administered. In one embodiment, anti-CD25 antibodies or fragments thereof or CD25 binding partners, such as IL-2 are used to remove T regulatory cells, e.g., CD25+T cells, from the colony. In one embodiment, anti-CD25 antibodies or fragments thereof or CD25 binding partners are conjugated with substrates, e.g., beads, or otherwise coated on substrates, e.g., beads. In one embodiment, anti-CD25 antibodies or fragments thereof are conjugated with substrates as described herein. In one embodiment, anti-CD25 antibody molecules or fragments thereof are used to remove T regulatory cells, e.g., CD25+T cells, from the colony. In another embodiment, CD25+ cells are not removed.

[0171] Another exemplary method comprises providing immune effector cell (for example, T cell) colony, and enrichment CD8+ cell colony and subsequently enrichment CD4+ cell colony.In one embodiment, anti-CD8 and anti-CD4 antibody or its fragment or CD8 binding ligand or CD4 binding ligand are used to enrich CD8+ and CD4+T cell colony.In one embodiment, anti-CD4 or anti-CD8 antibody or its fragment or CD4 or anti-CD8 binding ligand is conjugated with substrate (for example, bead), or is otherwise coated on substrate (for example, bead).In one embodiment, anti-CD4 antibody or anti-CD8 or its fragment is conjugated with substrate as described herein.

[0172] In one embodiment, the method further includes transducing cells from a population with one or more vectors, the vector including nucleic acids encoding fusion proteins, CARs or TCRs as described herein. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors or retroviral vectors. In one embodiment, cells from a T cell population are transduced with a vector once, for example, within one day after obtaining an immune effector cell population from a blood sample of a subject, the blood sample is obtained, for example, by a single blood collection technique. In one embodiment, the method further includes generating an RNA engineered cell population transiently expressing exogenous RNA from a T cell population. The method includes introducing in vitro transcribed RNA or synthetic RNA into cells from a population, wherein RNA includes nucleic acids encoding fusion proteins, TCRs or CARs containing LTBR domains.

[0173] In another embodiment, the method further includes transducing cells from a population with one or more vectors, the vector including a nucleic acid encoding a CAR or TCR comprising one or more LTBR domains as described herein. In one embodiment, the vector is selected from the group consisting of: DNA, RNA, plasmid, lentiviral vector, adenoviral vector or retroviral vector. In one embodiment, cells from a T cell population are transduced once with a vector, for example, within one day after obtaining an immune effector cell population from a blood sample of a subject, the blood sample being obtained, for example, by a single blood collection technique. In one embodiment, the method further includes generating an RNA engineered cell population transiently expressing exogenous RNA from a T cell population. The method includes introducing in vitro transcribed RNA or synthetic RNA into a cell from a population, wherein RNA includes a nucleic acid encoding a CAR or TCR comprising one or more LTBR domains as described herein.

[0174] 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 for 3 or 4 days in culture, and the resulting cells are more potent than the same cells expanded for 9 days in culture under the same culture conditions. Effectiveness can be defined by, for example, various T cell functions, such as proliferation, target cell killing, cytokine production, activation, migration or a combination thereof.

[0175] Treatment

[0176] In certain aspects, the present invention also provides a method for treating cancer in a subject. In certain embodiments, the method comprises administering to a subject a cell expressing a fusion protein as described herein, so as to treat cancer in the subject. In certain embodiments, the cell further expresses CAR. In other embodiments, the cell further expresses TCR. As used herein in describing the method of treatment, for convenience, LTBR is used as an exemplary effector enhancing gene. However, in alternative embodiments, other genes in Table 3 are also used in the method for modified cells containing CAR or TCR. In one embodiment, the method comprises obtaining cells from a patient, modifying the cells as described herein, and administering the cells to the patient.

[0177] In certain embodiments, the method comprises administering to a subject a cell expressing a CAR or TCR comprising one or more LTBR domains as described herein, such that the subject's cancer is treated. In one embodiment, the method comprises obtaining cells from a patient, modifying the cells as described herein, and administering the cells to a patient.

[0178] In certain embodiments, the method comprises administering to a subject a cell expressing a CAR or TCR comprising one or more domains from a gene in Table 3 as described herein, such that the subject's cancer is treated. In one embodiment, the method comprises obtaining a cell from a patient, modifying the cell as described herein, and administering the cell to a patient.

[0179] As used herein, the term "cancer" refers to any disease, condition, trait, genotype, or phenotype characterized by unregulated cell growth or replication. In certain embodiments, the compositions disclosed herein are administered, e.g., according to the methods disclosed herein, to treat cancer. In certain embodiments, the cancer is selected from the group consisting of adrenocortical carcinoma, advanced cancer, anal cancer, aplastic anemia, biliary tract cancer, bladder cancer, bone cancer, bone metastases, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary cause, Castleman disease, disease), cervical cancer, colon / rectum cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, 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 myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer, small cell lung cancer, lung carcinoid tumors, skin lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal and Sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, adult soft tissue sarcoma, basal and squamous cell skin cancer, melanoma, small intestine cancer, gastric cancer, testicular cancer, laryngeal cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms' tumor, secondary cancers caused by cancer treatment, and any combination thereof. Examples of cancers that can be treated by modified cells (e.g., LTBR CART or LTBR TCR-T cells, or LTBR-containing CART or TCR-T cells) are hematological cancers. On the one hand, blood cancer includes but is not limited to leukemia (such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and myelodysplastic syndrome) and malignant lymphoproliferative conditions, including lymphoma (such as multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma and small cell and large cell follicular lymphoma). In other embodiments, blood cancer can include, for example, leukemia, such as minimal residual disease (MRD) of AML or MDS. Other cancers include breast cancer, lung cancer, prostate cancer, colorectal cancer, esophageal cancer, gastric cancer, bladder cancer, pancreatic cancer, kidney cancer, cervical cancer, liver cancer, ovarian cancer and testicular cancer. In other embodiments, cancer is a solid tumor cancer. In certain embodiments, cancer is one of those cancers listed in Figure 19 or Figure 20.

[0180] In certain embodiments, the CAR is selected from Akilenbine Tecartus TM ), Abecma TM ), Leakey Myron Tisaron

[0181] Autoimmune diseases are conditions caused by abnormal immune attacks on the body, and they greatly increase morbidity, mortality, and medical expenses worldwide. Because T cells play a key role in the process of autoimmune diseases, engineered T cell therapy has emerged, and the therapy is also considered to be a potential method to overcome the obstacles in the current treatment of autoimmune diseases. Autoreactions or autoantibodies play a key role in the process of autoimmune diseases. Therefore, engineering T cells to express chimeric autoantibody receptors (CAARs) is a strategy for treating autoimmune diseases. In one embodiment, CAR includes CAAR. See, for example, Zhang et al., Chimeric antigen receptor T-cell therapy beyond cancer: current practice and future prospects, Immunotherapy, September 2020; 12 (13): 1021-1034. doi: 10.2217 / imt-2020-0009. Electronic version July 30, 2020, the document 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, for example, Chen et al., Immunotherapy Deriving from CAR-T Cell Treatment in Autoimmune Diseases, Journal of Immunology Research, Vol. 2019, December 31, 2019, the document is incorporated herein by reference. Other conditions that can be treated with the compositions described herein include graft-versus-host disease (GVHD) and transplant rejection.

[0182] In another embodiment, the subject has a virus-driven cancer. In certain embodiments, the virus-driven cancer is selected from the following:

[0183]

[0184]

[0185] On the one hand, the method includes applying an effective amount of cells expressing effector-enhanced genes described herein (e.g., LTBR CART or LTBR TCR-T cells) or cells expressing CAR / TCR containing LTBR (CART or TCR-T cells containing LTBR) in combination with an effective amount of another therapy to a subject in need. As used herein, "combination" administration means delivering two (or more) different treatments to a subject during the process of the subject suffering from the disease, for example, after the subject has been diagnosed with the disease and before the disease has been cured or eliminated or the treatment has stopped for other reasons. The two or more treatments are delivered. In some embodiments, the delivery of a treatment still occurs at the beginning of the delivery of the second treatment, so that there is overlap in the administration. This is sometimes referred to as "simultaneous" or "simultaneous delivery" in this article. In other embodiments, the delivery of a treatment ends before the delivery of another treatment begins. In some embodiments of any case, treatment is more effective due to combined administration. For example, the second treatment is more effective, e.g., a comparable effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation is seen with the first treatment. In some embodiments, delivery results in a reduction in symptoms or other parameters associated with the condition that is greater than the reduction that would be observed with one treatment delivered in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive. Delivery may be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0186] Expression effector enhancing gene cells described herein (e.g., LTBR CART or LTBR TCR-T cells) or expressing CAR / TCR containing LTBR cells (LTBR containing CART or TCR-T cells) and at least one other therapeutic agent can be administered simultaneously or sequentially in the same or separate compositions. For sequential administration, expression effector enhancing gene cells described herein (e.g., LTBR CART or LTBRTCR-T cells) cells can be first administered, and other agents can be administered secondly, or the order of administration can be reversed.

[0187] Cells expressing effector-enhanced genes (e.g., LTBR CART or LTBR TCR-T cells) or expressing CAR / TCR containing LTBR (CART or TCR-T cells containing LTBR) and / or other therapeutic agents, surgeries or modalities can be administered during a time period when the disease is active, or during a time period when the disease is in remission or the disease is less active. Cells expressing effector-enhanced genes (e.g., LTBR CART or LTBR TCR-T cells) or expressing CAR / TCR containing LTBR (CART or TCR-T cells containing LTBR) can be administered before other treatments, simultaneously with treatment, after treatment, or during remission of the disease.

[0188] When administered in combination, cells expressing effector enhancement genes (e.g., LTBRCART or LTBRTCR-T cells) or cells expressing CAR / TCR containing LTBR (CART or TCR-T cells containing LTBR) and additional agents (e.g., a second agent or a third agent) or all can be administered in an amount or dosage higher, lower or the same as each agent used alone, for example, as a monotherapy or dosage. In certain embodiments, cells expressing effector enhancement genes (e.g., LTBR CART or LTBR TCR-T cells) or cells expressing CAR / TCR containing LTBR (CART or TCR-T cells containing LTBR), additional agents (e.g., a second agent or a third agent) or all are administered in an amount or dosage lower than each agent used alone, for example, as a monotherapy or dosage (e.g., at least 20%, at least 30%, at least 40% or at least 50%). In other embodiments, the amount or dose of cells expressing an effector enhancer gene (e.g., LTBRCART or LTBRTCR-T cells) or cells expressing a CAR / TCR containing LTBR (LTBR-containing CART or TCR-T cells), an additional agent (e.g., a second agent or a third agent), or all is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dose of each agent used alone, e.g., as a monotherapy, required to achieve the same therapeutic effect.

[0189] In other aspects, cells expressing effector enhancer genes described herein (e.g., LTBR CART or LTBR TCR-T cells) or cells expressing CAR / TCR containing LTBR (LTBR containing CART or TCR-T cells) can be used in combination with surgery, chemotherapy, radiation, immunosuppressants such as cyclosporin, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunosuppressants such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation or peptide vaccines as described in Izumoto et al. 2008 J Neurosurg 108:963-971 in a treatment regimen.

[0190] In some cases, cells expressing effector enhancer genes as described herein (e.g., LTBRCART or LTBR TCR-T cells) or cells expressing CAR / TCR containing LTBR (LTBR-containing CART or TCR-T cells) are combined with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, antiemetics (or antiemetics), analgesics, cytoprotective agents, and combinations thereof.

[0191] In one embodiment, cells expressing effector-enhancing genes as described herein (e.g., LTBRCART or LTBR TCR-T cells) or cells expressing CAR / TCR containing LTBR (CART or TCR-T cells containing LTBR) can be used in combination with chemotherapeutic agents. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (doxorubicin) (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine (vinblastine), vincristine (vincristine), vindesine (vindesine), vinorelbine (vinorelbine)), alkylating agents (e.g., cyclophosphamide, decarbazine (decarbazine), melphalan (melphalan), ifosfamide (ifosfamide) , temozolomide), immune cell antibodies (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), antimetabolites (including, for example, folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-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 checkpoint inhibitors (e.g., PD-1 or PD-L1 inhibitors, e.g., pembrolizumab) brolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi).

[0192] Common chemotherapeutic agents considered for combination therapy include anastrozole Bicalutamide Bleomycin sulfate busulfan Busulfan Injection Capecitabine N4-pentyloxycarbonyl-5-deoxy-5-fluorocytosine, carboplatin Carmustine Chlorambucil Cisplatin Cladribine Cyclophosphamide Cytosine arabinoside, cytosine arabinoside Cytarabine liposome injection Dacarbazine Dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride Daunorubicin citrate liposome injection Dexamethasone, docetaxel Doxorubicin Hydrochloride Etoposide Fludarabine phosphate 5-Fluorouracil Flutamide Tezacitibine, Gemcitabine (difluorodeoxycetidine), Hydroxyurea Idarubicin Ifosfamide Irinotecan L-Asparaginase Calcium folinate, melphalan 6-Mercaptopurine Methotrexate Mitoxantrone Mylotarg, paclitaxel Phoenix (yttrium 90 / MX-DTPA), pentostatin, polyphenylpropanone 20 with carmustine implant Tamoxifen citrate Teniposide 6-thioguanine, thiotepa, tirapazamine Topotecan hydrochloride for injection Vinblastine Vincristine Vinorelbine

[0193] Treatment with a combination of chemotherapeutic agents and cells expressing effector-enhanced genes (e.g., LTBR CART or LTBR TCR-T cells) or cells expressing CAR / TCR containing LTBR (CART or TCR-T cells containing LTBR) described herein can be used to treat hematological cancers described herein, such as AML. In an embodiment, a combination of chemotherapeutic agents and cells expressing effector-enhanced genes (e.g., LTBR CART or LTBR TCR-T cells) or cells expressing CAR / TCR containing LTBR (CART or TCR-T cells containing LTBR) can be used for targeting, such as killing cancer stem cells, such as leukemia stem cells, such as targeting in subjects with AML. In an embodiment, a combination of chemotherapeutic agents and cells expressing effector-enhanced genes (e.g., LTBRCART or LTBR TCR-T cells) or cells expressing CAR / TCR containing LTBR (CART or TCR-T cells containing LTBR) can be used to treat minimal residual disease (MRD). MRD refers to a small amount of cancer cells remaining in a subject during treatment, such as during chemotherapy or after treatment. MRD is usually the main cause of recurrence. The present invention provides a method for treating cancer, such as MRD, comprising administering a chemotherapeutic agent in combination with cells expressing effector-enhancing genes (e.g., LTBRCART or LTBRTCR-T cells) or cells expressing CAR / TCR containing LTBR (LTBR-containing CART or TCR-T cells), e.g., as described herein.

[0194] In one embodiment, a chemotherapeutic agent is administered before administering a cell expressing an effector-enhancing gene (e.g., LTBR CART or LTBR TCR-T cell) or expressing a CAR / TCR containing LTBR (CART or TCR-T cell containing LTBR). In a chemotherapy regimen in which more than one administration of a chemotherapeutic agent is desired, the chemotherapy regimen is started or completed before administering a cell expressing an effector-enhancing gene (e.g., LTBR CART or LTBR TCR-T cell) or expressing a CAR / TCR containing LTBR (CART or TCR-T cell containing LTBR). In embodiments, the chemotherapeutic agent is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days or 30 days prior to administration of cells expressing effector enhancer genes (e.g., LTBRCART or LTBRTCR-T cells) or cells expressing CAR / TCR containing LTBR (LTBR-containing CART or TCR-T cells). In embodiments, the chemotherapy regimen is started or completed at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days or 30 days prior to administration of cells expressing effector enhancer genes (e.g., LTBR CART or LTBR TCR-T cells) or cells expressing CAR / TCR containing LTBR (LTBR-containing CART or TCR-T cells).

[0195] When an "immunologically effective amount", "anti-tumor effective amount", "tumor suppressive 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 age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). Generally speaking, the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 cells / kg body weight, in some instances 10 5 Up to 10 6Cells / kg body weight, including all integer values ​​within those ranges. T cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New England Journal of Medicine (NewEng.J.of Med.) 319:1676, 1988). As used herein, the "effective amount" of the term medicament is an amount sufficient to produce a beneficial or desired result such as a clinical result, and therefore, the "effective amount" depends on the environment in which it is applied. For example, in the case of administering a medicament for treating a tumor, the effective amount of the medicament is, for example, sufficient to reduce or reduce tumor size or inhibit tumor growth compared to the response obtained without administering the medicament. The term "effective amount" can be used interchangeably with "effective dose", "therapeutically effective dose" or "therapeutically effective dose".

[0196] Also provided herein is a method for vaccinating a subject with a combination vaccine, the combination vaccine comprising at least two nucleic acid sequences encoding at least one effector enhancing gene and at least one viral protein. In one embodiment, the effector enhancing gene is LTBR. In another embodiment, the viral protein is a coronavirus spike protein. Some embodiments provide a vaccine, the vaccine comprising an RNA polynucleotide with an open reading frame encoding an effector enhancing gene, an RNA polynucleotide with an open reading frame encoding an effector enhancing gene, a viral protein, and a pharmaceutically acceptable carrier or excipient deployed in a cationic lipid nanoparticle (LNP). The vaccine described herein (e.g., an LNP-encapsulated mRNA vaccine) produces an effective level, concentration, and / or titer of antigen-specific antibodies in the blood or serum of a vaccinated subject. See, for example, US2018 / 0311336A1, which is incorporated herein by reference in its entirety.

[0197] As used herein, the term "treatment" and variants thereof, such as "treat" or "treating" refer to clinical interventions that attempt to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing or reducing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In certain embodiments, the compositions described herein are used to delay the development of the disease or slow the progression of the disease.

[0198] Likewise, 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, prevention of metastasis or reduction in the extent of metastasis, and / or prolongation of survival of an animal with cancer compared to a control. In certain embodiments, treatment results in a reduced risk of distant recurrence or metastasis.

[0199] With respect to the description of the invention provided herein, in another embodiment, it is contemplated that each of the compositions described herein is useful in the methods of the invention. In addition, it is also contemplated that in another embodiment, each of the compositions described herein that can be used in the methods is itself an embodiment of the invention.

[0200] The following examples are provided for illustrative purposes only, and the invention should in no way be construed as limited to such examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0201] Examples

[0202] Example 1: Materials and Methods

[0203] Isolation and culture of primary human T cells

[0204] Standard buffy coats containing peripheral blood from unidentified healthy donors were collected and purchased from the New York Blood Center under an IRB exemption protocol. All donors provided informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using Lymphoprep (Stemcell) gradient centrifugation. For most assays, CD8 + and CD4 + First, CD8 was isolated by magnetic positive selection using the EasySep Human CD8 Positive Selection Kit II (Stem Cells, Inc.). + Then, using EasySep human CD4 + T cell isolation kit (Stem Cell Corporation) was used to separate CD4 + T cells. γδ T cells were isolated by magnetic negative selection using the EasySep Human γ / δ T Cell Isolation Kit (Stem Cell Corporation). Immediately after isolation, T cells were resuspended in T cell culture medium supplemented with 10 ng ml -1 Immunocult-XF T cell expansion medium (Stem Cell Corporation) was composed of recombinant human IL-2 (Stem Cell Corporation).

[0205] T cells were activated with Immunocult human CD3 / CD28 T cell activator (Stem Cell Corporation) at 25 μl / 10 6 cells / ml. Typically, T cells were transduced with concentrated lentivirus 24 hours after isolation. For 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, 2 μg ml -1 Puromycin was used to select lentiviral-transduced T cells.

[0206] Every 2-3 days, split T cells or replace the culture medium to maintain 1 × 10 6 -2×10 6 During the culture period, the lentiviral-transduced T cells were maintained in a medium containing 2 μg ml -1 Puromycin-containing medium. 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 using anti-Vγ9 PE antibody (Biolegend) and anti-PE microbeads (Miltenyi Biotec) in the presence of the protein kinase inhibitor dasatinib according to the manufacturer's recommendations before functional assays to prevent activation-induced cell death due to TCR cross-linking 42 PBMCs from a patient with diffuse large B-cell lymphoma (S14-02164) were obtained from the Perlmutter Cancer Center under a protocol approved by the Perlmutter Cancer Center Institutional Review Board.

[0207] Vector design and molecular cloning

[0208] All vectors used were cloned using Gibson assembly (NEB). Figures 1A to 1D The experiments shown used a lentiviral backbone from a pHAGE plasmid. 14 For all other experiments, the backbone from lentiCRISPRv2 (Addgene 52961) was used. ORFs were PCR amplified for cloning from genome-scale libraries for screening.

[0209] 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 gBlock (IDT). For CAR-ORF cloning, CAR-P2A-puro-T2A (partial) was first inserted into XbaI- and EcoRI-cut lentiCRISPRv2. For subsequent ORF insertions, the plasmid was cut with HpaI located within partial T2A and EcoRI. The following vectors were deposited to 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).

[0210] Nuclease and CRISPR guide RNA design

[0211] All sgRNAs were designed using the GUIDES web tool 43 Guides targeting the initial protein coding exon were selected (preference for targeting protein family domains was enabled in the guide (GUIDES)) as well as minimizing off-target scores and maximizing on-target scores. For Cas9 nuclease nucleofection, purified sNLS-SpCas9-sNLS nuclease (Aldevron) was used.

[0212] Preparation of ORF library plasmids for paired-end sequencing

[0213] The previously described genome-scale ORF library was re-amplified using Endura electrocompetent cells (Lucigen). 14The identity of the ORF and the matching 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 labeled using TnY transposase. 44 . The fragmented plasmid was then amplified in a PCR reaction using a forward primer that binds to the handle introduced by TnY and a reverse primer that binds to a sequence downstream of the barcode. All transposon and PCR primer oligonucleotides were synthesized by IDT. The resulting amplicons were sequenced on a NextSeq 500. Forward reads (containing ORFs) were mapped to the GRCh38.101 CDS transcriptome annotation using STARv.2.7.3a (mapping quality ≥ 10) 45 . Using paired-end reads, a 24-nucleotide barcode downstream of the constant plasmid sequence was also captured. The ORF-barcode combinations were tabulated and this table was further organized by excluding any spurious pairings that could have been caused by sequencing or PCR errors. Specifically, a permutation test was performed to identify the maximum number of ORF-barcode combinations expected by random chance, after which only ORF-barcode combinations with counts exceeding this maximum number were retained. All non-coding elements were excluded from the reference, and barcodes with a Levenshtein distance less than 2 were then collapsed.

[0214] Cell culture

[0215] 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 hours. MycoAlert PLUS (Lonza) was used to test cell lines for mycoplasma routinely and the results were negative. No cell lines were identified in this study.

[0216] Lentivirus production

[0217] The third generation lentiviral transfer plasmid was co-transfected into HEK293FT cells with packaging plasmid psPAX2 (Addgene 12260) and envelope plasmid pMD2.G (Addgene 12259) using polyethyleneimine linear MW25000 (Polysciences) to produce lentivirus. After 72 hours, the supernatant was collected, filtered through a 0.45-μm Steriflip-HV filter (Millipore), and the virus was concentrated using a lentiviral precipitation solution (Alstem). The concentrated lentivirus was resuspended in T cell culture medium containing IL-2 and stored at -80°C.

[0218] Pooled ORF library screening

[0219] For pooled ORF library screening, CD4 + and CD8 + T cells were collected from at least 500 × 10 6 The amount of lentivirus used for transduction was titrated to produce a transduction efficiency of 20%-30% to minimize the probability 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 maintain 1 × 10 6 -2×10 6 On day 14 after isolation, T cells were collected, counted, labeled with 5 μM CFSE (Bio-Bio), and lysed with CD3 / CD28 activator (Stem Cell) at 1.56 μl / 1×10 6 4 cells were stimulated. In this step, an aliquot of cells representing 1,000-fold library coverage was frozen for use as a pre-stimulation control. After 4 days of stimulation, cells were collected and an aliquot of cells representing 1,000-fold library coverage was frozen for use as a pre-sorting control. The remaining cells were stained with LIVE / DEAD Violet cell viability dye (Thermo Fisher Scientific) and sorted for CFSE using a Sony SH800S cell sorter. 低 Cells (corresponding to the bottom 15% of the distribution). Genomic DNA was isolated and two rounds of PCR were performed to amplify the ORF barcodes and add Illumina adapters 46 .

[0220] Pooled ORF screening analysis

[0221] For most analyses, an equal number of reads from all three donors were merged into each bin before trimming and alignment. Barcodes were mapped to the reference library after adapter trimming with Cutadapt v.1.13 (-m 24 -e 0.1 -discard-untrimmed) using Bowtie v.1.1.2 (-v1 -m 1 -best-tier). 47 , 48. All subsequent analyses were performed in RStudio v.1.1.419 with R 4.0.0.2. To calculate individual barcode enrichment, barcode counts were normalized to the total number of reads per sample (with pseudocounts added) and log2 transformed. To calculate ORF enrichment, raw barcode counts were first collapsed by gene before normalization and log2 transformation.

[0222] Enrichment analysis was performed at both the barcode and gene levels. 49 For statistical analysis of barcode enrichment, CFSE 低 Samples were compared with the corresponding input (pre-stimulation) using CD4 + and CD8 + As a replica. Using DESeq2 50 Statistical analysis of ORF enrichment was performed. Raw gene counts were obtained by collapsing barcodes into corresponding genes. 低 Samples were compared to corresponding input (both pre-stimulated and pre-sorted) using CD4 + and CD8 + As a replica. Using the topGO package 51 The DESeq2 standard (log2-transformed fold change > 0.5, P adj <0.05) were used for GO enrichment (biological process). 低 Genes enriched in the screen (DESeq2 analysis) were overlapped with differentially expressed genes after CD3 / CD28 stimulation using data from the Database of Immune Cell eQTL, Expression, and Epigenome (DICE; https: / / dice-database.org / ) 41 For differentially expressed genes, the following DICE datasets were used: 'T cells, CD4, naive' versus 'T cells, CD4, naive [activated]', 'T cells, CD8, naive' versus 'T cells, CD8, naive [activated]'. Significant differential expression (P adj <0.05).

[0223] Proliferation assay

[0224] Transduced T cells were collected on day 14 after isolation, counted, and expressed as 2.5 × 10 4 Cells / well were plated in a round-bottom 96-well plate and two sets of triplicate wells were transduced each time. One set of triplicate wells was transduced in a 5% flask supplemented with 10 ng ml -1 The cells were cultured in Immunocult-XF T cell expansion medium supplemented with IL-2, and another set of triplicate wells was further supplemented with 1.56 μl CD3 / CD28 activator / 1 ml of medium. The cells were cultured for 4 days and then collected and stained with LIVE / DEAD Violet cell viability dye. Before flow cytometry acquisition, the cells were resuspended in D-PBS containing 10% v / v precision counting beads (Bio-Bio). For quantification, the number of live cell events was normalized to the number of bead events for each sample. Then, for each ORF, the normalized number of live cells in the wells supplemented with CD3 / CD28 activators was divided by the average number of live cells in the control wells to quantify T cell proliferation. In order to be able to compare donors with CD4 + / CD8 + Comparisons between T cells were made, and proliferation of T cells transduced with a given ORF was finally normalized to that of a matched tNGFR control.

[0225] In addition to the counting bead assay, a dye dilution assay was also used to measure proliferation. For this assay, transduced T cells were collected on day 14 after isolation, washed with D-PBS, and then labeled with 5 μM CellTrace Yellow (CTY) in D-PBS for 20 minutes at room temperature. Excess dye was removed by washing with a five-fold excess of RPMI-1640 supplemented with 10% Serum Plus-II. The labeled cells were then plated at 2.5×10 4 10 cells / well were plated in a round-bottom 96-well plate. One set of triplicate wells was cultured in supplemented Immunocult-XF T cell expansion medium (i.e., without IL-2), and another set of triplicate wells was supplemented with 10 ng ml -1 IL-2 and 1.56 μl CD3 / CD28 activator / 1 ml of culture medium. Cells were cultured for 4 days and then collected and stained with LIVE / DEAD Violet cell viability dye. To quantify the proliferation index, events were first gated on live T cells in FlowJo (Treestar) and exported for further analysis in R / RStudio using flowFit and flowCore packages. 52. Unstimulated cells were used to determine parental population size and localization to account for differences in staining intensity between samples. These fitted parental population parameters were then used to fit the CTY profile of the matched stimulated sample, modeled as a Gaussian distribution, assuming log2 distance peaks due to cell division and dye dilution. The fitted CTY profile was visually inspected for consistency with the original CTY profile and used to calculate the proliferation index. The proliferation index was defined as the sum of cells in all generations divided by the calculated number of parental cells present at the start of the assay.

[0226] Flow cytometry of cell surface and intracellular markers

[0227] For CD25 (IL2RA) and CD154 (CD40L) quantification, T cells were stained with CD3 / CD28 activator (6.25 μl / 10 6 cells) for 6 hours (CD8 + CD154 staining in 24 hours) or 24 hours (CD4 + and CD8 + CD25 staining in both, and CD4 + For Ki-67 and 7-amino-actinomycin D (7-AAD) staining, T cells were kept overnight in Immunocult-XFT cell expansion medium without IL-2 and then treated with CD3 / CD28 activator (25 μl / 10 6 In other cases, T cells were stained without stimulation. To detect secreted proteins, CD3 / CD28 activator (25 μl / 10 6 T cells were stimulated with IL-12B (5 μg ml) for 24 h (LTA, LIGHT) and the protein transport inhibitor brefeldin A (5 μg ml -1 ) and monensin (2 μM).

[0228] First, cells were collected, washed with D-PBS and stained with LIVE / DEAD Violet cell viability dye in the dark at room temperature for 5 minutes, followed by surface antibody staining on ice for 20 minutes. After surface antibody staining (where applicable), cells were washed with PBS and collected or intracellular staining was performed on a Sony SH800S cell sorter. For intracellular staining, cells were resuspended in appropriate fixation buffer. The following fixation buffers were used for specific protein detection: fixation buffer for IL12B and MS4A3 staining (Baijin Biotechnology); eukaryotic transcription factor fixation for BATF, TCF1 and FLAG staining (Baijin Biotechnology); and FoxP3 / transcription factor fixation reagent for Ki-67 (eBioscience (eBioscience)). After being resuspended in the fixation buffer, cells were incubated in the dark at room temperature for 1 hour. After incubation, cells were washed twice in appropriate permeabilization buffer. The following permeabilization buffers were used: intracellular staining permeabilization wash buffer (Bio-Bio) for IL12B and MS4A3 staining; eukaryotic Perm buffer (Bio-Bio) 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 in the appropriate permeabilization buffer and acquired on a Sony SH800S flow cytometer. For cell cycle analysis, cells were immediately treated with 0.5 μg ml -1 7-AAD was further stained for 5 minutes. Appropriate isotype, fluorescence minus one, and biological controls were used for gating. Typically, 5,000-10,000 real-time events were recorded per sample.

[0229] Flow cytometric detection of phosphorylated proteins

[0230] Prior to detection of phosphorylated proteins, T cells were rested in Immunocult-XF T cell expansion medium without IL-2 for 24 h. CD3 / CD28 activator (25 μl / 10 6) were stimulated to resting cells, and the stimulation time is indicated in the corresponding figure. After the stimulation period, the cells were immediately fixed with a 1:1 volume ratio of pre-warmed fixation buffer (Baijin Bio) at 37°C for 15 minutes and washed twice with cell staining buffer (D-PBS + 2% FBS). Following the manufacturer's protocol, the cells were resuspended in the remaining volume and permeabilized in 1 ml of pre-cooled True-Phos Perm buffer (Baijin Bio) while vortexing. The cells were incubated in True-Phos Perm buffer at -20°C for 60 minutes. 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) at room temperature for 30 minutes. After staining, the cells were washed twice in cell staining buffer and collected on a Sony SH800S cell sorter. For CD4 + or CD8 + Cells were gated and levels of RELA and phospho-RELA were determined using appropriate isotype and biological controls.

[0231] Western blot detection of proteins and phosphorylated proteins

[0232] T cells expressing tNGFR or LTBR were collected and induced with CD3 / CD28 activator (25 μl / 10 6Cells were rested or stimulated for 15 minutes, washed with 1x D-PBS, and lysed on ice for 1 hour with TNE buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) in the presence of a protease inhibitor cocktail (Bimake B14001) and a phosphatase inhibitor cocktail (Cell Signaling Technologies 5872S). Cell lysates were centrifuged at 10,000 g for 10 minutes, and protein concentrations were determined using the 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). PageRuler prestained protein ladder (Thermo Fisher Scientific) was used to determine protein size. The gel was run in 1x Tris-glycine-SDS buffer (IBI Scientific) at 120 V for approximately 120 min. Proteins were transferred to a nitrocellulose membrane (Bio-Rad) at 100 V for 100 min in the presence of pre-chilled 1x Tris-glycine transfer buffer (Thermo Fisher Scientific) supplemented with 20% methanol.

[0233] Immunoblots were blocked with 5% skim milk dissolved in 1x PBS containing 1% Tween-20 (PBST) and incubated overnight at 4°C 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-IκBαSer32 (1:1,000 dilution, Cell Signaling, 14D4), rabbit anti-NF-κB p100 / p52 (1:1,000 dilution, Cell Signaling, 4882) and rabbit anti-RELB (1:1,000 dilution, Cell Signaling, C1E4). After primary antibodies, blots were incubated 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-COR926-32212). Blots were imaged using Odyssey CLx (LI-COR) and quantified using ImageJv.1.52.

[0234] Quantification of cytokine secretion

[0235] To measure secreted IFNγ and IL-2, T cells were first collected and rested in medium without IL-2 for 24 h. They were then counted and counted at 2.5 × 10 4 Cells / well were plated in a round-bottom 96-well plate and incubated in the absence of IL-2, with or without CD3 / CD28 activator (25 μl / 10 6 Cells) were incubated in the culture medium for 24 hours. Then, the cell supernatant was collected and diluted, and the plate reader Infinite F200 Pro (Tecan company (Tecan)) was used for cytokine quantification by enzyme-linked immunosorbent assay (human IL-2 or IFNγDuoSet, R&D system company (R&D Systems)). Multiple quantification of cytokines and chemokines secreted in resting or stimulated T cells was performed using human cytokine / chemokine 48-Plex discovery assay array (Eve Technology Company (Eve Technologies)).

[0236] T cell killing assay

[0237] CD19 was first transduced with a lentiviral vector encoding EGFPd2PEST-NLS and a puromycin resistance gene. + Nalm6 cells 53 Throughout the culture, the transduced cells were kept under puromycin selection to maintain stable EGFP expression, and puromycin was removed from the culture medium only prior to the killing assay. T cells were transduced with vectors encoding CARs specific for CD19 using 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, the transduced T cells were inoculated with 5×10 4 Nalm6 GFP +Cells were combined in triplicate at the indicated effector: target ratios in flat 96-well plates pre-coated with 0.01% poly-l-ornithine (EMD Millipore) in Immunocult medium without IL-2. The wells were then imaged using an Incucyte SX1, using 20x magnification and collecting 4 images every 2 hours for 120 hours. For each well, the integrated GFP intensity was normalized relative to the 2 hour time point to allow cells to fully settle after plating.

[0238] In vitro mRNA preparation

[0239] Templates for in vitro transcription were generated by PCR from plasmids encoding LTBR or tNGFR, and the resulting amplicon contained a T7 promoter upstream of the ORF. The purified templates were then used for in vitro transcription with capping and poly-A tailing using the HiScribe T7ARC AmRNA kit (NEB) with capping.

[0240] Nucleofection of primary T cells

[0241] Activated T cells were nucleofected with in vitro transcribed mRNA 24 hours after activation or with Cas9 protein 48 hours after activation. Cells were collected, washed twice in PBS, and cultured at 5 × 10 5 10 cells / 20 μl were resuspended in P3 primary cell Nucleofector solution (Lonza). Immediately after resuspension, 1 μg mRNA or 10 μg Cas9 (Aldevron) was added (not exceeding 10% v / v of the reaction) and the cells were nuclear transfected using the E0-11 5 program on the 4D-Nucleofector (Lonza). After nuclear transfection, the cells were resuspended in preheated Immunocult medium containing IL-2 and recovered at 37°C with 5% CO2 for 20 minutes. After recovery, the cells were plated at 1×10 6 Cells / ml were plated for downstream assays.

[0242] OverCITE-seq sample preparation and sequencing

[0243] For single-cell sequencing, CD8 + T cells were transduced with ORFs individually and maintained under puromycin selection for 14 days. The transduced cells were then combined and split into two conditions: one cultured for 24 hours in the presence of IL-2 alone; the other further supplemented with 6.25 μl CD3 / CD28 activator / 10 6 After stimulation, cells were collected, counted, and analyzed at 2 × 10 710 cells / ml were resuspended in staining buffer (PBS containing 2% BSA + 0.01% Tween-20). Then, 10% (v / v) human TruStain FcX Fc receptor blocking solution (Bio-Bio) was added and the cells were incubated at 4°C for 10 minutes. After Fc receptor blocking, the cell concentration was adjusted to 5×10 6 cells / ml and split the stimulated and unstimulated cells into 4 conditions each. Each condition received a different oligonucleotide-conjugated (barcoded) CytoHash antibody to allow pooling of different conditions in the same 10x Genomics Chromium lane 23 After 20 minutes of co-incubation on ice, cells were washed three times with staining buffer and counted using Trypan blue exclusion. Cell viability was typically about 95%.

[0244] Then, cells stained with different hash antibodies were 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, Biogen). The cells were stained on ice for 30 minutes, washed three times with staining buffer, resuspended in PBS, and filtered through a 40-μm cell strainer. The cells were then counted and the concentration was adjusted to 1×10 6 ml -1 To load into 10x Genomics Chromium, 3×10 4 The cells were combined with Chromium Next GEM Single Cell 5′ v2 Master Mix (10x Genomics) supplemented with a custom reverse primer combined with a puromycin resistance cassette for enhanced ORF transcript capture during the reverse transcription stage. The custom reverse primer was added at a 1:3 ratio to the poly-dT primer included in the master mix.

[0245] For cDNA amplification, additional primers for amplifying sample hash and surface antigen barcodes are included 23, and a nested reverse primer that binds to the puromycin resistance cassette downstream of the ORF. After cDNA amplification, SPRI beads are used for size selection of the resulting PCR products: small-sized (less than 300 bp) sample hashes and surface antigen barcodes are physically separated from larger cDNA and ORF amplicons for downstream processing. Sample hashes and surface antigen barcodes are also processed 22 . The amplified cDNA was then divided into three conditions for the construction of a gene expression library, an αβTCR library, and an ORF library. 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 resulting library was verified on a bioanalyzer using a high sensitivity DNA kit (Agilent). The libraries were sequenced on a NextSeq 500. For the gene expression library, more than 25,000 reads were generated per cell. For the other libraries, more than 5,000 reads were generated per cell.

[0246] OverCITE-seq data analysis

[0247] Gene expression unique molecular identifier (UMI) count matrices and TCR clonotypes were obtained using 10x Genomics Cell Ranger 3.1.0. Kallisto v.0.46.0 was used 55 and bustools v.0.39.3 56 Generate hash tag oligonucleotide (HTO) and antibody UMI count matrices. First use Bowtie2 v.2.2.8 57 ORF reads were aligned to plasmid references and indexed to relevant ORFs, after which UMI count matrices were generated using kallisto and bustools. All modalities were normalized using the centered log ratio (CLR) transformation. 5 Cell doublets and negatives were identified using the 8 functions and then excluded from downstream analysis. The UMI cutoff quantile of HTODemux was optimized to maximize singlet recovery using a grid search with values ​​between 0 and 1. ORF singlets were identified using MULTIseqDemux 5 9. Then, cells with low-quality gene expression metrics were excluded, and cells with fewer than 200 unique RNA features or more than 5% of reads mapping to the mitochondrial transcriptome were removed.

[0248] The count matrix was then loaded into Seurat v.4.0.1 60Cell cycle correction and scaling of gene expression data were performed using the CellCycleScoring function with default genes, followed by scaling of the data using the ScaleData function. 6 1 Principal component (PC) optimization was performed on the scaled and corrected data, where all PCs were selected until the first insignificant PC was used for clustering. Clustering of cells was performed using a shared nearest neighbor (SNN) based clustering algorithm and UMAP dimensionality reduction was used. 62 Cluster PCs were projected into 2D space for visualization. Cluster marker analysis was performed using the FindAllMarkers function, with the hypothesis set defined as the presence of positive and negative markers in at least 25% of cluster cells and a log2-transformed fold change threshold of 0.25 compared to non-cluster cells. 50 Differential expression analysis of ORFs was performed to identify up- and down-regulated genes in cells expressing the ORFs compared to NGFR (control) cells, where differential expression was defined as using the Storey method 63 Differential expression was calculated with q < 0.1.

[0249] Global RNA-seq and analysis

[0250] CD3 / CD28 activator (25 μl / 10 6 cells) stimulate CD4 + and CD8 + T cells were transduced with LTBR or tNGFR for 24 hours or were left unstimulated (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 described. 64 Briefly, RNA was reverse transcribed using SMARTScribe reverse transcriptase (Takara Bio) and a poly(dT) oligonucleotide containing a portion of the Nextera handle. The resulting cDNA was then PCR amplified for 3 cycles using OneTaq polymerase (NEB) and transposed using the homemade transposase TnY 44Labeling was performed at 55°C for 5 minutes. Immediately thereafter, labeled DNA was purified on a MinElute column (Qiagen) and PCR amplification was performed for 12 cycles using OneTaq polymerase and barcoded primers. PCR products were purified using double (0.5x-0.8x) SPRI cleanup (Agencourt) and size distribution was determined using Tapestation (Agilent). Samples were sequenced on a NextSeq 500 (Illumina) using the v2.575 cycle kit (paired end). Kallisto v.0.46.0 was used. 55 Paired-end reads were aligned to the transcriptome (Human Ensembl v.96 reference 65 ) for comparison and use the tximport package 66 Loaded into RStudio1.1.419 with R4.0.0.2. Using DESeq2 50 Differential gene expression analysis was performed using the topGO package 51 GO enrichment (biological process) was performed on genes that passed the DESeq2 criteria (log2 transformed fold change > 1, Padj < 0.05).

[0251] ATAC-seq library preparation

[0252] CD3 / CD28 activator (25 μl / 10 6 cells) stimulate CD8 + LTBR and tNGFR T cells were either stimulated for 24 h or left unstimulated (n = 2 biological replicates). Whole-mount ATAC-seq was performed as previously described. 44 Briefly, cell membranes were lysed in RSB buffer (10 mM Tris-HCL pH 7.4, 3 mM MgCl2, 10 mM NaCl) freshly supplemented with 0.1% IGEPAL. After pipetting up and down, nuclei were isolated by centrifugation at 500 g for 5 min at 4°C. After discarding the supernatant, the nuclei were resuspended in 5% PBS containing the homemade transposase TnY protein. 44 Labeled DNA (TD) buffer 44 After purification on a MinElute column (Qiagen), the DNA polymerase Pfu X7 was used to 44The labeled DNA was amplified by PCR for 12 cycles with the barcoded primers. The PCR products were purified by 1.5x SPRI cleanup (Agencourt) and checked for characteristic nucleosome banding patterns using TapeStation (Agilent). The samples were sequenced on NextSeq500 (Illumina) using the v2.575 cycle kit (single end).

[0253] ATAC-seq analysis

[0254] Using Bowtie2 v.2.4.4 57 Assemble single-end reads with Gencode hg38 master 67 Then, SAMtools v.1.9 was used to compare the 68 Alignments with low mapping quality (MAPQ < 30) were filtered out, and the filtered BAM files were subsequently processed. 68 Sorting and indexing were performed using Picard v.4.1.8.1 69 Removed read duplicates. Use MACS3v.3.0.0 70 Peaks were called with default parameters (-g 2.7e9 -q 0.05).

[0255] To construct the joint feature space (‘joint peaks’) used for most downstream analyses, pairs of biological replicate narrow peak files were intersected using BEDTools v.2.29.0 (using bedtools intersect) to retain only peaks found in both replicates. 71. After marking shared peaks between replicates, biological replicates at each shared peak (with at least 1 bp overlap) were merged using bedtools merge. In this new peak BED file, each shared peak contained all sequences found under the peak in any one biological replicate. Next, the union of each of these peak files (LTBR resting, LTBR stimulated, tNGFR resting, tNGFR stimulated) was taken; any peaks with at least 1 bp overlap were merged. Using the unioned peaks, a peak read count matrix (union_peaks × ATAC_samples) was generated, where each entry in the matrix corresponds to the number of reads that overlapped with this peak in a given sample, referred to as the per-peak ATAC_matrix. Overlapping reads were taken directly from the BAM files (converted to BED) that provided the alignments for each sample. Therefore, the matrix contains a column for each biological replicate. Although the samples differed minimally in the aligned reads, each entry in the matrix was normalized by the number of reads that overlapped with the TSS region in each sample. In this way, any differences in reads or alignment depth between samples would be properly normalized. In addition to the per-peak ATAC matrix, a per-gene ATAC matrix was constructed as follows: the total ATAC reads for a gene were assigned as the sum of the normalized reads of the per-peak ATAC matrix for all peaks within 3 kb of the start or end coordinate of the gene.

[0256] The two ATAC matrices (per peak and per gene) were imported into Rv.4.1.1 for gene and peak enrichment analysis using DESeq2v.1.32.0. For comparison between ATAC-seq and RNA-seq, an adjusted P value of <0.05 statistical threshold was used, and the log2-transformed change fold was >0 (for an increase in ATAC or RNA) or the log2-transformed change fold was <0 (for a decrease in ATAC or RNA). For transcription factor motif analysis, Chrom-VARv.1.14.072 was used as follows: For each test and control condition, the columns and sample subsets of the per-peak matrix and the joint feature space were used to construct a summarized experimental object. The matchMotifs function was used to annotate transcription factor motifs. The enrichment deviation between the test and control conditions was calculated using the computeDeviations function.

[0257] To generate read accumulation tracks at specific genomic loci, dereplicated reads (BAMs) from biological replicates were pooled using samtools merge. These pooled replicate BAM files were converted to bigWig files using the bamCoverage function from deeptools v.3.4.2 and setting the scaleFactor to the relative number of TSSs found in the pooled biological replicates compared to all other sample aggregates 73. Using the bigWig files, the pyGenomeTracks v.3.6 74 Plot read accumulation.

[0258] Finally, k-means clustering was performed on ATAC peaks near genes with increased chromatin accessibility. First, using DEseq2 on the ATAC per-gene matrix, genes with a log2-transformed fold change >1 and an adjusted P value <0.05 in either of the two comparisons (that is, genes with increased chromatin accessibility) were identified: (1) stimulated LTBR relative to stimulated tNGFR; (2) LTBR quiescent relative to tNGFR quiescent. After identifying these genes, all accessibility peaks in the per-peak ATAC matrix within a 3kb gene body were isolated; this subset of peaks from the per-peak ATAC matrix was used as input for clustering. This subset of ATAC peaks was then k-means clustered using deeptools (computeMatrix and plotHeatmap functions), with k=4 clusters and a read window of 6kb.

[0259] Statistical analysis

[0260] According to the data type (depending on the normality of the distribution), the data between the two groups were compared using a two-tailed unpaired Student's t-test or Mann-Whitney test. Unless otherwise stated, P values ​​less than or equal to 0.05 were considered statistically significant in all analyses and were not corrected in multiple comparisons. In the case of multiple comparison corrections, the P value was adjusted using the Benjamini-Hochberg method. Unless otherwise stated, the results of all groups are expressed as mean ± sem. Statistical analysis was performed in Prism (GraphPad) and RStudio (Rstudio PBC). Flow cytometry data were analyzed using FlowJo v.10.7.1 (Treestar Company (Treestar)).

[0261] Example 2: Genome-scale screen for synthetic drivers of T cell proliferation

[0262] Using a lentiviral library of barcoded human ORFs, we expressed cells in primary human CD4 + and CD8 + A genome-scale gain-of-function screen was performed in T cells. It was shown that T cells with the strongest proliferative phenotype were enriched in both known and unknown immune response regulators, many of which are not normally expressed by peripheral T cells. The top-ranked ORFs in cells from screening-independent donors were validated, and it was further demonstrated that these ORFs not only drove T cell proliferation, but also increased the expression of activation markers and the secretion of key proinflammatory cytokines. In order to obtain a more comprehensive understanding of the mechanism of action of these genes, a single-cell sequencing method coupled with direct ORF capture was developed. LTBR, ​​one of the top-ranked ORFs not expressed by lymphocytes, was identified as a key driver of profound transcriptional and epigenetic remodeling by increasing NF-κB signaling, which led to a significant increase in the secretion of proinflammatory cytokines and resistance to apoptosis. Finally, it was shown that the top-ranked ORFs enhanced antigen-specific T cell function in the context of CD19-guided CAR T cells and a wide range of tumor-reactive γδ T cells from healthy donors and patients with blood cancers.

[0263] Genome-scale ORF screening in T cells

[0264] To avoid reliance on constitutive expression of large bacterial proteins or chromatin accessibility near target genes 13 , decided to use a lentiviral library of human ORFs; this library contains nearly 12,000 full-length genes, each with about 6 barcodes 14 ( Figure 1A , Figure 6A-6G ). Previously, genome-scale loss-of-function screens in human T cells focused on CD4 + or CD8 + However, CD4 + and CD8 + Both T cells are required for durable tumor control in adoptive therapy 15 , 16, such as FDA approval of CD4 + and CD8 + This was further demonstrated by anti-CD19 CART cells at a 1:1 ratio 1 Therefore, it was decided to use the ORF library to identify genes that promote CD4 + and CD8+ T cell proliferation genes ( Figure 1A , Figure 6H-6J ).

[0265] Transduction of the lentiviral ORF library into CD4 + and CD8 +T cells, and after a brief culture period (14 days), the cells were restimulated to identify proliferation drivers in response to TCR stimulation. The majority of individual ORF barcodes were captured, as well as nearly all ORFs, including the largest ORF ( Figure 6K , Figure 6L Comparing the relative frequencies of genes in the most highly proliferating cells with those in the unsorted cells revealed that genes known to be involved in immune processes were enriched in the top-ranked ORFs ( Figure 6M , Figure 6N ). Identification of MAPK3 (encoding ERK1), a key mediator of T cell function 17 , co-stimulatory 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 have shown that overexpression of IL12B and BATF promotes proliferation, cytotoxicity, and cytokine secretion of CAR T cells. 19 , 20.

[0266] Each ORF in the library was linked to an average of six DNA barcodes ( Figure 6B To increase confidence in the top ranked ORFs in the pooled screen, proliferating CD4 + and CD8 + Enrichment of individual barcodes corresponding to a given ORF in cells ( Figure 6B , Figure 6C , Fig.6O ). For most ORFs, multiple individual barcodes per gene were enriched in the highly proliferative population, thus indicating that the observed enrichment was not due to pseudoclonal growth or PCR bias. Surprisingly, the most significantly enriched gene was the lymphotoxin-β receptor (LTBR), a gene widely expressed in stromal and myeloid cells but completely absent in lymphocytes.

[0267] Overall, the enriched ORFs spanned a range of different biological processes. The top enriched gene ontology (GO) biological processes included lymphocyte proliferation, interferon-γ (IFNγ) production, and NF-κB signaling ( Figure 6P ). It was observed that upon stimulation with CD3 and CD28 (CD3 / CD28), the enriched ORFs showed only a slight preference for endogenously upregulated genes of T cells and were in fact expressed in all categories of differential expression ( Figure 6QThis 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, it was decided to test a broad range of ORFs that function in multiple pathways associated with T cell fitness and display different endogenous regulatory patterns (Figure 1E).

[0268] The forward ORF enhances T cell function

[0269] 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-linked puromycin resistance gene from the same promoter. Truncated nerve growth factor receptor (tNGFR), lacking its intracellular domain, was selected as a control with no effect on T cell phenotype. 21 .CD4 + and CD8 + Populations were isolated from several selection-independent healthy donors and transduced with individual ORFs ( Figure 2A Flow cytometry was used to analyze the representative ORFs and confirmed their stable and uniform expression in both subsets of T cells during the experiment ( Fig. 7A , Figure 7B ).

[0270] Fourteen days after isolation, cells were restimulated and the relative increase in cell number was measured. It was found that 16 tested ORFs significantly improved cell proliferation compared to tNGFR, and proliferation was significantly higher in CD4 + With CD8 + There was a good correlation between the cells (Spearman's r = 0.61, P = 0.002) ( Figure 2B , Figure 2C , Figure 7C-7H Having determined that the top ORFs promote T cell proliferation, we next tested whether there were also changes in other T cell phenotypes and functions, such as increased cell cycle entry, expression of the activation markers IL2RA (CD25) and CD40L (CD154), and cytokine secretion. Most of the ORFs tested showed no differences in circulation (Figure 2I, Figure 2J), but showed higher expression of both CD25 and CD154 in T cells after stimulation (Figure 2I, Figure 2J). Figure 2D , Fig. 8A ), further confirming its effect in improving the magnitude of T cell responses.

[0271] Finally, the secretion of cytokines interleukin-2 (IL-2) and IFNγ was measured after CD3 / CD28 restimulation ( Figure 2E , Figure 8B-Figure 8EAlthough the screen was not designed to identify genes that regulate cytokine secretion, several 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, ​​which increased CD4 + and CD8 + The secretion of both cytokines in T cells increased more than fivefold.

[0272] Single-cell analysis of ORF phenotypes

[0273] Based on the quantification of how each ORF affects proliferation, activation, and cytokine release, we next sought to better understand the underlying mechanisms driving these changes in cell state. To gain a more comprehensive understanding of the mechanisms of action of individual ORFs and provide a multidimensional characterization of the phenotypic changes they induce, a single-cell sequencing strategy with direct ORF capture was developed. This approach, OverCITE-seq (Overexpression-compatible Cell Indexing of Transcriptomes and Epitopes by Sequencing), expands on previously developed methods for quantifying surface antigens 22 and CRISPR perturbations 23 The method is based on the results of a novel lentiviral template-switching approach and allows high-throughput single-cell analysis of T cell pools with different ORFs. Briefly, during template switching, mRNA from the lentivirally integrated ORF is specifically reverse transcribed by primers that bind to constant sequences of transcripts downstream of the ORF and barcoded along with the cellular transcriptome. The resulting cDNA pool is then separated to construct gene expression and ORF expression profiles, respectively. Figure 3A , Figure 3B , Fig.9A ).

[0274] OverCITE-seq was optimized and applied to CD8 + Pools of ~30 ORFs in T cells. Pools of cells were either unstimulated ('resting') or stimulated with CD3 / CD28 antibodies to mimic TCR activation. To gain confidence in how ORFs were assigned to each single cell, we exploited the fact that the protein produced by the control gene tNGFR is expressed on the cell surface and can therefore be captured by the DNA barcoded antibodies. 23 The proportion of cells designated 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 a given ORF had overall the strongest expression of the corresponding gene ( Figure 9B-9D ), indicating that the ORF capture strategy reliably assigns genetic perturbations to each single cell.

[0275] Unsupervised clustering shows a clear separation of stimulated and resting T cells. Within these activation-driven superclusters, individual clusters associated with specific cell states or functions can be observed, such as cell cycle (clusters 1 and 9), macromolecular biosynthesis (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 contribute to a given cluster phenotype ( Fig.9E ), but a significant enrichment of two ORFs, CDK1 and CLIC1, was observed in cluster 1, which is characterized by increased expression of genes responsible for chromosome condensation in cell cycle preparation ( Figure 3E ). An even stronger enrichment was observed for cluster 10, which consisted almost entirely of cells expressing LTBR.

[0276] To investigate the mechanisms of gene perturbations with the strongest transcriptional changes, we examined the transcriptional profiles of CD3 / CD28-stimulated ORFT cells compared with unstimulated control T cells ( Figure 9F-Figure 9I ). This approach allows the identification of gene modules that are shared between different perturbations, or that are specific to a specific perturbation. For example, LTBR and CDK1 showed the strongest enrichment for genes involved in RNA metabolism and the cell cycle (CDK4, HSPA8, and BTG3), as well as the tumor necrosis factor (TNF) signaling pathway (TNFAIP3, TRAF1, and CD70). FOSB appears to drive a program opposite to LTBR in terms of genes involved in TCR signaling (CD3D, CD3E, LAPTM5, and LAT), cytokine response (GATA3 and TNFRSF4), and the NF-κB pathway (NFKB2, NFKBIA, and UBE2N). Finally, it was determined that the observed phenotype was the result of the genetic perturbation and not of a single clone, since in reality each single cell expresses a unique TCR clonotype ( Figure 9J This result highlights the utility of OverCITE-seq’s multimodal capture approach, yielding each T cell’s transcriptome, clonotype, cell surface proteome, cell hash (for treatment or stimulation condition), and lentiviral ORF identity.

[0277] LTBR improves multiple T cell functions

[0278] LTBR has been identified as a pro-inflammatory cytokine secretion Figure 2E ) and profound transcriptional remodeling ( Figure 3D , Figure 3E ) and decided to investigate its mechanism of action in more detail. LTBR belongs to the tumor necrosis factor receptor superfamily (TNFRSF) and is expressed on a variety of non-immune cell types and bone marrow-derived immune cells, but is absent in lymphocytes ( Fig. 10A , Fig. 10B Using global RNA sequencing (RNA-seq), we compared global gene expression between LTBR- and tNGFR-transduced cells with or without TCR stimulation ( Figure 4A , Figure 4B , Fig. 10C LTBR cells also express the MHC-II invariant chain (encoded by CD74), in addition to upregulation of MHC-I and II genes (HLA-C, HLA-B, HLA-DPB1, HLA-DPA1, and HLA-DRB6) and transcription factors essential for MHC-II expression (RFX5 and CIITA). Notably, CD74 has been shown to activate the pro-survival NF-κB pathway in B cells, specifically by upregulating the anti-apoptotic genes TRAF1 and BIRC3 (both of which are also upregulated in cells overexpressing LTBR) 24. Similarly, strong upregulation of LTBR cells has been shown to promote CD8 + T cells 25 BATF3 involved in the survival of IL-2 was also observed 26 The transcription factor JUNB is responsible for T cell self-renewal 27 The RNA-seq results were confirmed at the protein level ( Figure 10D-10I LTBR cells are also more resistant to activation-induced cell death and retain greater functionality after repeated stimulation ( Figure 4C , Figure 4D , Figure 10J-Figure 10M ).

[0279] LTBR signaling is triggered in its endogenous setting (in myeloid cells) by the heterotrimer of lymphotoxin-α (LTA) and lymphotoxin-β (LTB) or by LIGHT (encoded by the TNFSF14 gene). Since LTA, LTB, and LIGHT are expressed by activated T cells, attempts were made to elucidate whether the addition of exogenous LTA or LIGHT could modulate cytokine secretion, differentiation, or proliferation of T cells overexpressing LTBR stimulated by CD3 / CD28; however, no effect of exogenous ligands on LTBRT cell function was found ( Figures 11A-11E ). Thus, although LTBR can enhance TCR-driven T cell responses, it does not drive activation itself, which would be a potential safety issue and result in loss of antigen specificity of the engineered T cell response. It was also determined that constitutive expression of LTBR is necessary to maintain its phenotype, but there is a considerable lag time between loss of detectable LTBR expression and loss of phenotype ( Figure 11F-11I ), indicating that transient expression of LTBR may be a safe approach for therapeutic applications.

[0280] Finally, to identify the key domains that drive LTBR protein activity in T cells, a series of point or deletion mutations of LTBR were designed ( Figure 4E , Fig.11J ). In general, the N-terminus of LTBR was found to be less sensitive to deletions than the C-terminus. Similarly, by binding to LTA and LTB 28 Partial reduction of the LTBR phenotype was achieved by introducing three alanine point mutations into key residues of the LTBR or by removing the signal peptide. Using C-terminal deletions, it was found that a mutant version of the LTBR lacking residues 393–435 was indistinguishable from the full-length LTBR, ​​whereas deletion of residues 377–435 completely abolished the LTBR phenotype despite comparable, if not higher, expression levels (Fig. I1K), which could be due to the expression of TRAF2, TRAF3, or TRAF5. 29 In addition, deletion of the self-association domain 30 (324-377) also completely abolished the phenotype.

[0281] LTBR acts through canonical NF-κB in T cells

[0282] LTBR overexpression was shown to induce widespread transcriptomic changes in T cells, accompanied by changes in T cell function ( Figure 4A , Figure 4B Therefore, we sought to determine whether perturbations in gene expression in LTBR cells were accompanied by epigenetic changes using assays for transposase-accessible chromatin by sequencing (ATAC-seq). Figure 12A-12G Comparison of the enrichment of specific transcription factor motifs in differentially accessible chromatin regions identified NF-κB p65 (RELA) as the most enriched transcription factor in LTBR cells ( Fig.12H , Fig.12I Notably, NF-κB p65 and NFAT-AP-1 were the two transcription factors most enriched in open chromatin in resting T cells (both LTBR and tNGFR) upon stimulation, which is consistent with their roles in T cell activation. 31 , only NF-κB p65 showed strong enrichment in LTBR cells, regardless of stimulation ( Figure 4F ). This result suggests that LTBR induces a partial T cell activation state but still requires signal 1 (TCR stimulation) for full activation.

[0283] It was then decided to investigate changes in protein expression and / or phosphorylation of members of the NF-κB signaling pathway. A more rapid phosphorylation of p65 (RELA) and a strong increase in the phosphorylation of the NF-κB inhibitor IκBα, which was targeted for degradation, were observed; both effects enhance the activation or transcription of NF-κB ( Figure 4G , Figure 4H , Figure 13A-13C In addition to changes in the canonical NF-κB pathway, upregulation of RELB and NF-κB p52, key mediators of the non-canonical NF-κB pathway, was also detected ( Fig. 4I , Fig. 13B , Fig. 13C ).

[0284] Having established that LTBR activates both canonical and noncanonical NF-κB pathways, we sought to determine the molecular basis of this phenomenon by interfering with key genes in the LTBR and NF-κB pathways. This was achieved by co-delivering LTBR or tNGFR and CRISPR constructs targeting 11 genes involved in the LTBR signaling pathway. 32 ( Figure 4J , Figure 13D-Figure 13O ). Knockdown 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 perturbations of LIGHT (also known as TNFSF14), ASK1 (also known as MAP3K5), and RELA had stronger effects on control cells than on 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, the observation that loss of either TRAF2 or TRAF3 promoted IFNγ secretion only in tNGFR cells is consistent with previous findings that T cells from dominant-negative mice expressing TRAF2 overreact to TCR stimulation. 33 .

[0285] To investigate the potential role of canonical vs non-canonical NF-κB signaling in LTBR T cells, we decided to analyze the global effect of loss of RELA or RELB on the LTBR-driven gene expression profile. Using global RNA-seq on T cells overexpressing either 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 13P ).

[0286] ORF enhances antigen-specific responses

[0287] So far, we have shown that the top ranked genes from the ORF screen improve T cell function using non-specific pan-TCR stimulation. Next, we sought to determine whether similar improvements could be observed using antigen-specific stimulation ( Figure 5A To this end, several top-ranked genes were co-expressed using two FDA-approved CARs targeting the B-cell marker CD19 ( Figures 14A-14D Using LTBR as an example, it was demonstrated that ORF expression can be achieved using this tricistronic vector ( Figure 14E-14I ).

[0288] Since the two CARs use different costimulatory domains, either CD28 or 4-1BB, we wanted to determine if the top genes selected using CD28 costimulation would also work in the presence of 4-1BB costimulation. Almost all of the top genes tested improved upregulation of CD25 and antigen-specific cytokine secretion with no significant differences in differentiation or exhaustion phenotypes, except for AKR1C4 ( Figure 5B , Figure 5C , Figure 14J-Figure 14P , Figures 15A-15D ).

[0289] Although IL-2 and IFNγ production is critical 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 on CD28 CAR T cells ( Figure 5D-5F , Fig.15E , Fig.15F ). Notably, it was observed that CAR T cells co-expressing LTBR tended to form large cell clusters; these clusters were generally absent in wells with control cells but were consistent with the overall high expression of adhesion molecules such as ICAM-1 in cells expressing LTBR ( Figure 15G Another important feature of effective antitumor T cells is the ability to remain functional despite prolonged exposure to antigen. This is consistent with previous findings in the setting of LTBR alone ( Figure 4D ), after repeated stimulation with target cells, LTBR-expressing CART cells showed better functionality than matched tNGFR-expressing CAR T cells ( Figure 5G , Figure 15H-15J ).

[0290] T cells from healthy donors are relatively easy to engineer and rarely show signs of dysfunction in culture, whereas autologous T cells from patients with cancer are often dysfunctional, showing limited proliferation and effector function. 34To investigate whether the top-ranked genes could improve CAR T-cell responses in healthy T cells and potentially dysfunctional T cells derived from patients, 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, increased secretion of IL-2 and IFNγ from LTBR CAR T cells was observed to be similar to that from healthy donors, indicating that the identified ORFs can be successfully used to engineer T cells from patients with lymphoma ex vivo ( Figure 5H , Figure 15K ). Notably, there was no secretion of cytokines in response to CD19- cells, indicating that overexpression of LTBR did not induce spurious, antigen-independent responses.

[0291] Screening and subsequent validation were performed in αβ T cells, a major subset of T cells in human peripheral blood. While αβ T cell-based immunotherapy has shown considerable potential in the clinic, γδ T cells present an attractive alternative due to their lack of MHC restriction, ability to target widely expressed stress markers in a cancer type-agnostic manner and more innate-like properties. 5 Therefore, we sought to determine whether the top genes validated in αβ T cells translated into γδ T cells. After co-incubation with leukemia or pancreatic ductal adenocarcinoma cells, increased IL-2 and IFNγ secretion from γδ T cells transduced with the top-ranked genes was observed ( Fig.5I , Figure 15L-Figure 15P ). Thus, the top genes selected may act on signaling pathways that are conserved even among highly differentiated T cell subsets, highlighting their broad applicability in cancer immunotherapy.

[0292] discuss

[0293] In summary, a genome-scale gain-of-function screen was developed in primary human T cells, in which nearly 12,000 full-length genes were tested in a massively parallel manner for their effects on TCR-driven proliferation. To our knowledge, the largest previously published gain-of-function study in primary T cells involved 36 constructs, including full-length genes and synthetic receptors. 35. This approach relies on construct delivery via donor DNA and Cas9-mediated targeting inserts. While the use of donor DNA for target gene delivery allows for greater flexibility in construct design, specifically for engineered synthetic receptors, the approach is more difficult to scale and acquire in terms of cost and complexity than the lentiviral libraries used here. Therefore, ORF-based gain-of-function screening is easily applicable to a plethora of T cell phenotypes and environments, and it provides opportunities for clinical translation. In fact, all FDA-approved CAR therapies already rely on lentiviral or retroviral integration of the CAR transgene, and therefore the addition of an ORF in this system does not pose significant challenges to manufacturing or regulation. The use of mRNA encoding an ORF delivered to CAR T cells prior to infusion is another translational approach, specifically if there are safety concerns about the mode of action of a particular ORF.

[0294] Gain-of-function screening has the potential to reveal regulators that are tightly controlled, restricted to specific developmental stages, or expressed only under certain circumstances. As shown herein, LTBR is not normally present in cells of lymphoid origin, but, due to an intact signaling pathway, can play a synthetic role when introduced into T cells. While constitutive activation of other TNFRSF members may result in similar phenotypes, one of the features that distinguishes LTBR (and appears to result in its enrichment, but not that of other TNFRSF members in the screen) is the formation of an autocrine loop, whereby the receptor and its ligand are present in the same cell. Of particular note, expression of LTBR promotes IL-2 secretion, as this cytokine is only produced by T cells and not by cell types that endogenously express LTBR. In addition to promoting cytokine secretion, overexpression of LTBR promotes stemness (expression of TCF1) and reduces activation-induced apoptosis, as well as providing a degree of protection against phenotypic and functional hallmarks of T cell exhaustion, all of which are features that cannot be recapitulated by cell types that endogenously express LTBR. Previous work overexpressing LTBR in cell lines has shown that LTBR has a pro-apoptotic effect 36 , in direct contrast to the phenotypes observed in primary T cells. Analysis at the transcriptional and protein levels revealed that LTBR drives constitutive activation of both the canonical and non-canonical NF-κB pathways. However, using epigenomic profiling and CRISPR-based functional perturbations, we showed that the phenotypic and functional changes resulting from LTBR expression are primarily mediated through activation of the canonical NF-κB pathway, whereas changes in the non-canonical pathway may not be necessary for the observed phenotypes, in contrast to the well-established role of non-canonical NF-κB activation in cells endogenously expressing LTBR37.

[0295] In many studies, gene overexpression has been used for preclinical enhancement of CAR T cell therapy. For example, arming CAR T cells with cytokines such as IL-12 or IL-18, which are not normally produced by T cells but are known to improve T cell function when secreted by other types of cells, has been shown to improve their anti-tumor activity. 38 , 39. It is noteworthy that previous studies have found that CART cell exhaustion can be identified by RNA-seq as exhausted cells 40 Future studies adapting genome-wide gain-of-function screens to relevant immunotherapy models will lead to advanced target selection for engineering synthetic cell therapies that can overcome the immunosuppressive tumor microenvironment and eradicate established cancers.

[0296] Example 3: Improving CAR solid tumor response

[0297] It has been shown that in the context of B cell leukemia, LTBR and several other top-ranked genes (ORFs, open reading frames) identified in the screen enhance the anti-tumor response of anti-CD19 CARs. In the case of pancreatic cancer, it was tested herein whether similar activity improvements could be seen by combining two clinically tested anti-mesothelin CARs (using 4-1BB or CD28 costimulatory domains). T cells co-expressing CAR and ORF were tested against Capan-2 and BxPC3, which are pancreatic cancer cell lines expressing high levels of mesothelin CAR targets and BxPC3, which are pancreatic cancer cell lines expressing low levels of mesothelin ( Fig.16A ).

[0298] 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 either CAR (41BB or CD28) against the anti-mesothelin high cell line Capan-2 ( Fig. 16B In terms of promoting IL-2 secretion, significant improvements were observed when LTBR and, to a lesser extent, AHNAK were used compared to negative (tNGFR) controls ( Fig. 16C In terms of reactivity against the mesothelin-low lineage BxPC3, improvements relative to negative controls were observed primarily in T cells overexpressing IL12B or LTBR ( Fig.16D ).

[0299] Cytokine secretion is one aspect of producing an anti-tumor response, and another aspect is direct cytotoxicity. Therefore, the ability of CART cells co-expressing the top genes to kill GFP+Capan2 or BxPC3 cells was tested ( Fig.16E , Fig.16F ). In view of the improvement in cytokine secretion, the increased cytotoxicity against mesothelin high Capan-2 exhibited by CAR T cells overexpressing any of the top six genes tested (including GPD1) was also observed against BxPC3 cells. Therefore, it is concluded that the top genes identified in the screen (including but not limited to AHNAK, BATF, GPD1, IFNL2, IL12B and LTBR) can enhance the responsiveness of different CARs (anti-CD19 shown previously, anti-mesothelin shown here) in different cancer types (including liquid tumors such as B cell leukemia and solid tumors such as pancreatic cancer) and under different target antigen densities (mesothelin high and mesothelin low cell lines) using different costimulatory domains (CD28 or 4-1BB).

[0300] Example 4: Improving TCR activity in solid tumors

[0301] T cell therapy can rely on the use of CAR or TCR to redirect cells to a given tumor target. The advantage of the former is that it can target tumors of different patients regardless of their HLA haplotypes, while the latter can also target intracellular antigens (because epitopes from all cell proteins are sampled and displayed on them by HLA molecules). Clinically tested TCRs for epitopes from NY-ESO-1 are used herein, which are typically expressed in many cancer histologies, including but not limited to melanoma, multiple myeloma, sarcoma, and lung cancer. Due to size limitations, TCR and genes (ORF, open reading frame) are delivered to two separate lentiviruses for co-transduction of T cells ( Fig.17A The dual-transduced T cells were then selected using puromycin (only T cells transduced with the ORF lentivirus will survive) and using antibody-based selection for NY-ESO-1 TCR-positive cells (in the presence of dasatinib to prevent T cell activation and thus activation-induced cell death during selection).

[0302] The engineered CD8+ T cells were then tested against the HLA-A2+NY-ESO-1+ melanoma cell line A375. Most of the genes tested increased the secretion of one or both of the cytokines IFNγ and IL2 ( Fig. 17B , Fig. 17CDirect cytotoxicity against A375 cells was also measured and demonstrated that all genes tested exhibited cytotoxicity superior to that exhibited by TCR-transduced T cells co-expressing the negative control gene tNGFR ( Fig.17D ). Therefore, it was concluded that the top genes identified in the screen (including but not limited to AHNAK, BATF, GPD1, IFNL2, IL12B and LTBR) can enhance the responsiveness of T cells engineered with cancer-specific TCRs.

[0303] Example 5: Intracellular signaling domains from LTBR enhance CAR T cell activity

[0304] It has been demonstrated that LTBR overexpression in T cells, in combination with anti-CD19 CARs that utilize CD28 or 4-1BB co-stimulatory domains in addition to the CD3z signaling domain, increases cytokine (IFNγ and IL-2) secretion and target cell killing (B-cell leukemia cell line Nalm6). It has also been demonstrated that CAR T cells co-expressing LTBR have increased expression of core genes activated by LTBR, ​​such as adhesion molecules ICAM-1 (also known as CD54) and CD74, which are an invariant part of the MHC-II complex and have a proposed anti-apoptotic effect. Finally, it has been demonstrated that T cells overexpressing LTBR, ​​with or without CAR, exhibit a less differentiated, “younger” phenotype (predominantly central memory [CM], with a reduced frequency of terminally differentiated effector cells), which could be beneficial in the context of cell therapy.

[0305] Here, the intracellular portion of LTBR (https: / / www.uniprot.org / uniprot / P36941, amino acids 249-435) was inserted into the intracellular domain of the most commonly used CAR construct to see if: 1) this insertion would not disrupt the CAR; 2) this insertion conferred similar phenotypic and functional benefits as expressing the full-length LTBR as a separate gene in a polycistronic construct; and 3) this insertion increased CAR activity above that of CAR and LTBR delivered together but as separate proteins. To this end, three constructs were first engineered whereby the intracellular domain of LTBR was inserted into different locations in a CAR construct identical to the CAR construct used in the FDA-approved Akirame (denoted herein as 19-28-z), specifically: 1) downstream of the CD28 stalk and transmembrane domain, but upstream of the CD28 signaling and CD3z signaling domains; 2) downstream of the CD28 signaling domain but upstream of the CD3z signaling domain; and 3) downstream of both the CD28 and CD3z signaling domains ( Fig.21A). These three constructs were then transduced into CD4+ and CD8+ T cells from healthy donors and compared with full-length CARs expressed with the control genes tNGFR (19-28-z+tNGFR) or LTBR (19-28-z+LTBR), respectively. Two additional constructs were also engineered whereby the intracellular domain of LTBR was inserted into a different position in the CAR construct, which was identical to the CAR construct used in the FDA-approved tesalencyclase (represented here as 19-BB-z), specifically: 1) downstream of the 4-1BB signaling domain but upstream of the CD3z signaling domain; and 2) downstream of both the 4-1BB and CD3z signaling domains ( Fig.21B ). These two constructs were then transduced into CD4+ and CD8+ T cells from healthy donors and compared with full-length CAR expressed with the control gene tNGFR (19-BB-z+tNGFR) or LTBR (19-BB-z+LTBR), respectively.

[0306]

[0307]

[0308] We first verified that surface CAR expression was similar between these constructs, thus indicating that insertion of the LTBR intracellular domain had no significant effect on CAR expression or protein stability and that the observed functional effects were not due to CAR expression levels ( Fig. 21C , Fig.21D ). Since the full-length LTBR induced higher expression of several genes that are beneficial for cell therapy, including CD54 and CD74, it was tested whether the inclusion of an intracellular LTBR domain in the CAR phenocopies the effects of the full-length LTBR. Indeed, it was observed that the upregulation of both markers was higher in cells expressing LTBR as the second or third component in the intracellular CAR domain, i.e., 19-28-LTBR-z, 19-28-z-LTBR, ​​19-BB-LTBR-z, and 19-BB-z-LTBR ( Fig.21E , Fig.21F This suggests that including only the signaling domain of LTBR in the CAR may have a more profound effect on T cell status than expressing the entire protein. It was also observed that there was an increase in central memory (CM) and a decrease in effector phenotype in cells expressing LTBR as a second or third component in the intracellular CAR domain compared to cells expressing full-length LTBR ( Figure 21G , Fig.21H ).

[0309] After determining that some of the tested constructs not only phenotypically replicated the effects of full-length LTBR, ​​but were in fact superior, attempts were made to determine whether they also improved the antigen-specific response of CAR T cells to CD19+ cancer cells. In terms of cytokine secretion, it was observed that the 19-LTBR-28-z construct generally performed worse than the control (“conventional” CART cells, i.e., 19-28-z+tNGFR), while 19-28-LTBR-z performed similarly to the control. Most importantly, 19-28-zLTBR was superior to T cells expressing CAR and full-length LTBR (respectively) in IFNg secretion, and superior to control CART cells ( Fig.21I , Fig.21J In the case of CARs utilizing the 4-1BB domain, constructs containing the LTBR in the second position (19-BBLTBR-z) were generally improved over control CAR T cells and performed similarly to CAR T cells co-expressing the full-length LTBR, ​​while constructs containing the LTBR in the third position even outperformed the CAR + full-length LTBR in all cases tested ( Figure 21K , Figure 21K ).

[0310] Although cytokine secretion (IFNg, IL2) is critical for T cells to reshape the tumor microenvironment and stimulate T cell proliferation, another key aspect of anticancer activity is direct cytotoxicity, which is primarily performed by CD8+ T cells. Therefore, the ability of engineered T cells to kill CD19+ cancer cells was tested. Interestingly, all CD28 LTBR constructs showed improvements over control CARs at high T cell doses. More importantly, the 19-28-z-LTBR construct was also able to effectively kill tumor cells at low T cell doses, with T cells expressing 19-28-z CAR and full-length LTBR providing only a slight benefit over conventional CARs ( Figure 21M ). This was also true in the case of the 4-1BB CAR: both constructs tested showed improvements over the conventional CAR at high T cell doses, but only the 19-BB-z-LTBR construct was more cytotoxic at low T cell doses ( Fig.21NThis is particularly important for three reasons: 1) clinical efficacy: it is clinically impossible to control the number of CAR T cells that encounter tumor cells except by infusing more T cells, so they should be able to destroy cancer cells with the greatest possible potency; 2) safety: if large portions of the disease are rapidly eliminated, the incidence of serious adverse events, especially cytokine release syndrome, may be reduced, especially if this disease can be alleviated at lower T cell numbers; and 3) manufacturing: some patients' T cells fail to expand sufficiently in vitro to reach the recommended cell dose prior to re-infusion due to fatigue / dysfunction - if the recommended cell dose can be reduced due to superior potency, this may make more patients eligible for this treatment.

[0311] Example 6: Intracellular LTBR as a replacement for CD28 or 4-1BB in second-generation CARs

[0312] The first generation CAR construct contains only the CD3z signaling domain connected to the target recognition modality. Due to the lack of costimulatory signals, these first generation receptors are not efficient in vitro and especially in vivo, so it is necessary to provide costimulation by including CD28 or 4-1BB signaling domains in the second generation CAR (currently approved by the FDA). It is hoped that the presence of LTBR can improve the response of the first generation CAR.

[0313] Therefore, constructs with only the target recognition domain and the transmembrane portion of the CAR (generation 0) and constructs that also contained the intracellular CD3z signaling domain (generation 1) were designed and compared with a second generation CAR utilizing a 4-1BB co-stimulatory domain ( Fig.22A Each generation of CAR was co-expressed with tNGFR (negative control) or full-length LTBR.

[0314] SEQ ID NO name type 21 19+tNGFR AA 22 19+tNGFR DNA 23 19+LTBR AA 24 19+LTBR DNA 25 19-z+tNGFR AA 26 19-z+tNGFR DNA 27 19-z+LTBR AA 28 19-z+LTBR DNA

[0315] When co-incubated with the CD19+ leukemia cell line Nalm6, the generation 0 CAR showed no response in terms of cytokine (IFNγ, IL2) secretion ( Fig. 22B , Fig. 22C). As expected, T cells expressing the first generation CAR and tNGFR showed a weaker response than the second generation CAR. Interestingly, when the first generation CAR was co-expressed with LTBR, ​​the response was stronger in all cases compared to the conventional second generation CAR (19-BB-z+tNGFR), and in some cases, similar to the co-expression of the second generation CAR and LTBR. Therefore, CARs containing CD3z and LTBR signaling domains can provide an attractive alternative to conventional second generation CARs (i.e., costimulatory domains containing CD28 or 4-1BB sources). The results described in Example 5 further support this.

[0316] SEQ ID NO name type 29 LTBR-28-z AA 30 LTBR-28-z DNA 31 NGFR-28-z AA 32 NGFR-28-z DNA 33 LTBR-28TM-28-z AA 34 LTBR-28TM-28-z DNA 35 NGFR-28TM-28-z AA 36 NGFR-28TM-28-z DNA

[0317] Example 7: Use of the extracellular domain of LTBR

[0318] It has been previously shown that LTBR overexpression improves T cell phenotype and function through ligand-dependent and -independent mechanisms. It was desirable to assess whether additional T cell activation could be provided through the same mechanism (ligand binding or tonic signaling) when the extracellular domain of LTBR was linked to a signaling domain derived from second generation CAR T cells.

[0319] To this end, constructs expressing the full-length extracellular domain of LTBR (including the transmembrane portion) were generated (LTBR: https: / / www.uniprot.org / uniprot / P36941, amino acids 1-248), or constructs utilizing the handle and transmembrane portion from an FDA-approved CAR, i.e., CD28 handle and transmembrane or CD8 handle and transmembrane (LTBR: https: / / www.uniprot.org / uniprot / P36941, amino acids 1-227). In some cases, the extracellular domain of LTBR was linked to the CD28 and CD3z or 4-1BB and CD3z signaling domains, as in FDA-approved CARs. In all iterations of the constructs, a matching NGFR fragment was used as a negative control ( Fig.23A ).

[0320] SEQ ID NO name type 37 LTBR-CD8-BB-z AA 38 LTBR-CD8-BB-z DNA 39 NGFR-CD8-BB-z AA 40 NGFR-CD8-BB-z DNA 41 19-CD8-LTBR AA 42 19-CD8-LTBR DNA 43 19-CD8-LTBR(TM) AA 44 19-CD8-LTBR(TM) DNA 45 19-28-LTBR AA 46 19-28-LTBR DNA 47 19-28-LTBR(TM) AA 48 19-28-LTBR(TM) DNA

[0321] When T cells were incubated alone, no cytokine (IFNγ, IL2) secretion was observed, indicating that ligand availability or tonic signaling mediated by LTBR was insufficient to activate the cells. However, when T cells were activated through their T cell receptors (CD3 and CD28 antibodies), a strong increase in the secretion of both IFNg and IL2 was observed by T cells expressing a construct containing the extracellular portion of LTBR fused to the CD8 stalk and transmembrane domain and the 4-1BB and CD3z signaling domains (LTBR-CD8-BB-z) Fig. 23B , Fig.23C ). This result indicates that fusion of the extracellular domain of LTBR to CAR instead of or in addition to a targeting moiety (usually derived from a scFv antibody fragment) may be a useful approach to enhance T cell function.

[0322] Example 8: Use of the extracellular domain of LTBR

[0323] It has been previously shown that LTBR overexpression improves T cell phenotype and function through ligand-dependent and -independent mechanisms. It was desirable to assess whether additional T cell activation could be provided through the same mechanism (ligand binding or tonic signaling) when the extracellular domain of LTBR was linked to a signaling domain derived from second generation CAR T cells.

[0324] To this end, constructs expressing the full-length extracellular domain of LTBR (including the transmembrane portion) were generated (LTBR: https: / / www.uniprot.org / uniprot / P36941, amino acids 1-248), or constructs utilizing the handle and transmembrane portion from an FDA-approved CAR, i.e., CD28 handle and transmembrane or CD8 handle and transmembrane (LTBR: https: / / www.uniprot.org / uniprot / P36941, amino acids 1-227). In some cases, the extracellular domain of LTBR was linked to the CD28 and CD3z or 4-1BB and CD3z signaling domains, as in FDA-approved CARs. In all iterations of the constructs, a matching NGFR fragment was used as a negative control ( Fig.24A ).

[0325] When T cells were incubated alone, no cytokine (IFNg, IL2) secretion was observed, indicating that ligand availability or tonic signaling mediated by LTBR was insufficient to activate the cells. However, when T cells were activated through their T cell receptors (CD3 and CD28 antibodies), a strong increase in the secretion of both IFNg and IL2 was observed by T cells expressing a construct containing the extracellular portion of LTBR fused to the CD8 stalk and transmembrane domain and the 4-1BB and CD3z signaling domains (LTBR-CD8-BB-z) Fig. 24B , Fig.24C ). This result indicates that fusion of the extracellular domain of LTBR to CAR instead of or in addition to a targeting moiety (usually derived from a scFv antibody fragment) may be a useful approach to enhance T cell function.

[0326] Example 9: Modified TCR Complex

[0327] The intracellular signaling domain of LTBR (https: / / www.uniprot.org / uniprot / P36941, amino acids 249-435*) is fused to different components of the TCR complex. Fig.25A Existing designs are shown that improve T cell function (i.e., NY-ESO-1 TCR and LTBR are expressed separately, see Example 4), and the LTBR signaling domain is fused to different components of the TCR-CD3 complex, particularly at the C-terminus of the intracellular domains of CD3ε (https: / / www.uniprot.org / uniprot / P07766), CD3γ (https: / / www.uniprot.org / uniprot / P09693), CD3δ (https: / / www.uniprot.org / uniprot / P04234), and CD247 (also known as CD3ζ, https: / / www.uniprot.org / uniprot / P20963). The LTBR signaling domain can also be fused to the C-terminus of the TCR-α or TCR-β chain.

[0328] Similar to the way the LTBR intracellular domain is used to modify the αβTCR complex, the γδTCR complex can also be modified ( Fig.25B ).like Fig.5I As shown, expression of full-length LTBR enhanced γδ T cell function.

[0329] αβT cells use CD4 to co-receive antigens presented by MHC-II (HLA-DR, HLA-DP, HLA-DQ), or use CD8 (composed of CD8a and CD8b chains) to co-receive antigens presented by MHC-I (HLA-A, HLA-B, HLA-C). Therefore, intracellular LTBR can be fused to the C-terminal intracellular tail of CD4 or the C-terminal intracellular tail of CD8a or CD8b to improve T cell function ( Fig.25C ).

[0330] Example 10: LTBR co-delivery enhances the anti-tumor function of CAR targeting B7-H3

[0331] It has been previously shown that co-delivery of LTBR with anti-CD19 CARs into primary T cells can improve their activity against hematological cancers. See, e.g., Legut et al., Nature, (2022). Similar results have been shown in the case of co-delivery of LTBR with anti-mesothelin CARs to improve the activity of T cells against a range of solid tumors. The application of LTBR co-delivery has now been extended to anti-B7-H3 CARs that are being clinically tested against a range of solid tumors, including but not limited to pediatric cancers, brain tumors, sarcomas, and melanomas. See, e.g., Theruvath et al., Nature Medicine (2020).

[0332] When LTBR was co-delivered with an anti-B7-H3 CAR (MGA271-28-z) to primary human T cells, it was observed (as was previously the case with CD19 and mesothelin CARs) that LTBR had a positive effect on the CAR T cell phenotype. Specifically, compared with CAR T cells co-expressing the control gene tNGFR, LTBR CAR T cells displayed a predominantly central memory phenotype and had few terminally differentiated effector cells ( Fig.26A LTBR CART cells also strongly upregulated CD54 and CD74, markers of LTBR activity ( Fig.26B , Fig.26C ).

[0333] In terms of antitumor efficacy, LTBR CAR T cells, both CD4 and CD8, showed stronger responses than control CAR T cells against cancer cells expressing B7-H3 (including two atypical teratoid rhabdoid tumor lines BT12 and BT16, and melanoma line A375), while remaining inactive against the B7-H3-negative leukemia line Nalm6 ( Figure 26D-26G Improvements in antitumor responses were observed in all T cell functions tested, including secretion of the key cytokines IFNγ and IL2, as well as direct cell killing.

[0334] Finally, the efficacy and safety of LTBR CAR T cells were evaluated in a mouse xenograft model. A375 tumors were implanted into the flank of NSG mice and allowed to grow for two weeks to reminiscent of advanced human solid tumors ( Fig.26H When body weight was monitored, no significant weight loss was observed in mice treated with CART cells (whether containing LTBR or not), indicating that LTBR did not cause any significant toxicity ( Fig.26I When tumor size was measured, it was observed that at both doses tested, the overall tumor burden caused by LTBR CAR T cells was lower than that of mice treated with control CAR T cells or untransduced T cells, indicating that LTBR improved CAR T cell efficacy in this model ( Fig.26J , Figure 26K Due to the rapid progression of this advanced solid tumor model, this study was terminated 12 days after T cell injection, precluding monitoring of the long-term effects of LTBR T cells on tumor regression and survival.

[0335] Example 11: Identification of the minimal intracellular domain of LTBR that drives its function in T cells

[0336] It has been previously shown that the intracellular tail of LTBR was fused to the C-terminus of a CAR targeting CD19 using a CD28 or 4-1BB design (FMC6.3-28-z-LTBR or FMC6.3-BB-z-LTBR) to produce CAR T cells that exhibit superior anti-tumor efficacy and LTBR-driven phenotypes when the full-length CAR and full-length LTBR were co-expressed as separate molecules. Here, it was hoped to determine whether truncations (N-terminal or C-terminal) of the LTBR signaling tail would result in smaller but equally effective or more effective CAR constructs than CAR constructs incorporating the full-length LTBR signaling tail.

[0337] Constructs LTBR amino acids (Uniprot: P36941) The SEQ ID NOs contained in full length 249：435 84、85 V1 249：396 86、87 V2 249：393 88、89 V3 249：387 90、91 V4 249：377 92、93 V5 262：435 94、95 V6 297：435 96、97 V7 324：435 98、99 V8 345：435 100、101 V9 358：435 102、103

[0338] First, the LTBR intracellular tail was annotated based on the literature to identify key functional domains ( Fig.27A Then, a series of 4 N-terminal truncations and 5 C-terminal truncations of the LTBR tail were designed to gradually remove the unannotated parts (which were believed to have no biological role) and expand into the previously annotated key signaling domains ( Fig.27B). The resulting truncated mutants were then fused to the C-terminus of CD28-based CD19 CARs and compared to CD28-based CARs incorporating a full-length LTBR signaling tail (19-28-z-LTBR) or conventional CARs co-expressed with LTBR or control gene tNGFR (19-28-z). The sequences are shown in SEQ ID NOs: 84-103. Each sequence comprises 19-28-z (SEQ ID NOs: 134, 135), LTBR variants as indicated above, P2A, and puromycin.

[0339] All CAR-LTBR fusions elicited similar levels of CAR surface expression ( Fig.27C ).

[0340] In terms of T cell differentiation state, LTBR truncated fusions display a range of phenotypes, with some variants (e.g., V7) closely resembling full-length LTBR, ​​while others (e.g., V4) are more reminiscent of conventional CAR T cells that do not express LTBR ( Fig.27D Similarly, full-length LTBR fusions and most truncation mutants except V3 and V4 resulted in enhanced expression of TCF1, a key transcription factor controlling T cell stemness and self-renewal ( Fig.27E Another indicator of successful induction of the LTBR program, which involves differential expression of hundreds of genes critical for immune responses (see, e.g., Legut et al., Nature (2022)), is surface expression of CD54 and CD74 markers. Although some truncated variants, such as V3, V4, and V9, failed to upregulate CD54 and CD74, the remaining variants showed similar activity to full-length LTBR fusions ( Fig.27F ).

[0341] In addition to phenotypic measurements, antigen-specific antitumor responses were determined by co-incubating T cells expressing anti-CD19 CAR (with or without LTBR fusion) with CD19+ leukemia cells. To quantify this response, secretion of key antitumor cytokines IFNγ and IL2 was measured ( Figure 27G). Consistent with CD54 and CD74 quantification, similar increases were observed for the "conventional" CAR (19-28-z+tNGFR) and V5-V7 variants as for full-length LTBR fusions. Despite elevated CD54 and CD74 levels, cytokine secretion was not improved for V1 and V2, while V3 and V4 showed almost no cytokine secretion, suggesting that these designs interfered with the CAR itself. Interestingly, the V8 variant caused the greatest upregulation of CD54 and CD74, but poor cytokine secretion, while V9 showed mild upregulation of CD54 and CD74, but significantly improved anti-tumor activity. This suggests that the phenotypic effects of LTBR (CD54, CD74) can be decoupled from the functional effects (IL2, IFNγ).

[0342] Example 12: Fusion of LTBR signaling tail with mesothelin-targeting CAR

[0343] It has been previously shown that co-expression of full-length LTBR with a mesothelin-targeting CAR using both 4-1BB and CD28 designs improves the anti-tumor reactivity of CAR T cells, and that fusion of the intracellular tail of LTBR to a CD19-targeting CAR using both 4-1BB and CD28 designs achieves better phenotype and function than co-expression of a conventional CD19-targeting CAR and LTBR as two separate molecules.

[0344] To extend this observation, the intracellular tail of LTBR was fused to a mesothelin-targeting CAR (see for a schematic representation of the CAR-LTBR fusion design). Fig.28A ). Specifically, based on the optimal LTBR positioning within the CAR construct determined in the CD19 system, the C-terminus of the meso-28-zCAR was selected for LTBR fusion. Replacing the CD28 costimulatory domain with LTBR was also investigated, placing it upstream or downstream of CD3z to test whether LTBR could provide costimulation in place of CD28.

[0345] SEQ ID NO name type 104 Meso-28-z-LTBR-P2A-puroR DNA 105 Meso-28-z-LTBR-P2A-puroR AA 106 Meso-BB-z-LTBR-P2A-puroR DNA 107 Meso-BB-z-LTBR-P2A-puroR AA 108 Meso-LTBR-z-P2A-puroR(CD28 handle) DNA 109 Meso-LTBR-z-P2A-puroR(CD28 handle) AA 110 Meso-LTBR-z-P2A-puroR (CD8 handle) DNA 111 Meso-LTBR-z-P2A-puroR (CD8 handle) AA 112 Meso-z-LTBR-P2A-puroR(CD28 handle) DNA 113 Meso-z-LTBR-P2A-puroR(CD28 handle) AA 114 Meso-z-LTBR-P2A-puroR (CD8 handle) DNA 115 Meso-z-LTBR-P2A-puroR (CD8 handle) AA

[0346] When looking at the surface expression of the CAR, in contrast to the data in the CD19 system, it was observed that fusing the LTBR tail to any position within the mesothelin CAR resulted in a significant reduction in the surface expression of the CAR ( Fig.28B ).

[0347] To assess the functional impact of reduced CAR expression in fusion with LTBR, ​​key functional outputs of CAR-driven responses to mesothelin-positive cancer cells were measured: T cell proliferation, direct cancer cell killing, and secretion of key cytokines. In terms of antigen-driven proliferation, co-expression of the full-length LTBR caused the strongest expansion, but the CAR-LTBR fusion was still superior to conventional unmodified CAR ( Fig.28C In terms of cancer cell killing, a CAR design lacking CD28 but containing the LTBR tail located downstream of CD3z (Meso-z-LTBR) showed similar killing to conventional CAR co-expressed with the full-length LTBR ( Fig.28D Surprisingly, although the mesothelin CAR-LTBR fusions had better or comparable functional activity in terms of proliferation and killing, two of the three constructs tested (Meso-28-z-LTBR and Meso-LTBR-z) showed little to no activity when cytokine secretion was measured ( Fig.28E ). Meso-z-LTBRT cells were able to produce cytokines when co-incubated with mesothelin+ cancer cells, but to a lesser extent than conventional Meso-28-z CAR T cells.

[0348] Given that CAR-specific function is reduced after fusion to the LTBR tail, it was desirable to understand the LTBR-specific function of these constructs. When looking at the surface expression of key markers of LTBR activity, namely CD54 and CD74 ( Fig.28F ), and the intracellular / nuclear expression of the transcription factor TCF1 ( Figure 28G ), it was observed that all three CAR-LTBR fusion constructs drove a stronger LTBR program than co-expression of CAR and full-length LTBR. This phenomenon was observed in both the CD28 and 4-1BB designs. Therefore, it was hypothesized that the mesothelin CAR-LTBR fusion may not be the best choice as a CAR, but is a superior non-natural version of the LTBR. To test this further, the engineered T cells were stimulated by the endogenous T cell receptor as an alternative to providing them with signal 1 instead of using CAR-mesothelin binding. Supporting the hypothesis, it was observed that in cases where the CAR-LTBR fusion only required the provision of LTBR-like signal 2 co-stimulation but not CAR-like signal 1 stimulation, the CAR-LTBR fusion T cell functional response was superior to the response in T cells expressing CAR and LTBR as two independent molecules ( Fig.28H ).

[0349] To demonstrate the utility of using CAR-LTBR fusions as superior LTBR-like molecules for use in conjunction with independent antigen receptors, native LTBR or Meso-z-LTBR was co-delivered with Meso-28-zCART to T cells ( Fig.28I All tested constructs showed the same expression of CAR on the surface ( Fig.28J); similarly, co-delivery of CAR and LTBR on separate viruses resulted in strong surface expression of LTBR, ​​similar to expression in T cells transduced with only the LTBR virus but not the CAR virus, highlighting the potential of delivery of LTBR on separate lentiviruses as an alternative to co-expression of antigen receptors from the same lentiviral cassette ( Figure 28K ).

[0350] In this co-delivery setting, CAR+Meso-z-LTBR T cells upregulated LTBR marker genes CD54 and CD74 to a similar extent as CAR+LTBR T cells ( Figure 28L CAR+Meso-z-LTBR T cells showed that the antigen-specific response to mesothelin+ cancer cells was also better than that of CAR T cells alone, and even outperformed CAR+natural LTBRT cells in IFNγ secretion ( Figure 28M , Fig.28N ). In summary, the utility of the Meso-z-LTBR synthetic fusion receptor as a replacement for the naturally occurring full-length LTBR has been demonstrated.

[0351] Example 13: Fusion of the LTBR signaling tail to members of the TCR-CD3 complex.

[0352] It has been previously shown that co-expression of full-length LTBR with NY-ESO-1 targeting the αβ TCR improves the anti-tumor reactivity of engineered T cells, and fusion of the intracellular tail of LTBR to a CD19-targeting CAR utilizing both 4-1BB and CD28 designs achieved better phenotypes and functions than co-expression of conventional CD19-targeting CAR and LTBR as two separate molecules.

[0353] To extend this observation, the intracellular tail of LTBR was fused to the α or β chain of the TCR heterodimer or the δ, ε, γ or ζ chain of the CD3 complex. CD3 constitutively associates with the TCR and is required for its surface expression and downstream signaling (https: / / www.pnas.org / doi / 10.1073 / pnas.1420936111).

[0354] To first test whether the LTBR signaling tail could be used as a direct fusion to enhance TCR activity, constructs were generated containing the LTBR intracellular tail linked to the C-terminus of the short intracellular tail of either TCRα or TCRβ ( Fig.29A ) and tested them together with unmodified TCR co-expressed with full-length LTBR or the irrelevant gene tNGFR.

[0355] SEQ ID NO name type 116 TCRa:LTBR-P2A-TCRb DNA 117 TCRa:LTBR-P2A-TCRb AA 118 TCRa-P2A-TCRb:LTBR DNA 119 TCRa-P2A-TCRb:LTBR AA 120 CD3D:LTBR-P2A-puroR DNA 121 CD3D:LTBR-P2A-puroR AA 122 CD3E:LTBR-P2A-puroR DNA 123 CD3E:LTBR-P2A-puroR AA 124 CD3G:LTBR-P2A-puroR DNA 125 CD3G:LTBR-P2A-puroR AA 126 CD3Z:LTBR-P2A-puroR DNA 127 CD3Z:LTBR-P2A-puroR AA

[0356] We first investigated whether LTBR fusion affects the surface expression of TCR. We noted that fusion of LTBR to TCR-β, but not TCR-α, significantly reduced its surface expression ( Fig.29B ). We then examined the surface expression of CD54 and CD74, key phenotypic markers of LTBR activity. Only LTBR-TCR-β caused an increase in the expression of CD54 and CD74, but LTBR-TCR-α did not, albeit to a lesser extent than full-length LTBR ( Fig.29C In terms of antigen-specific activity, both LTBR-TCR-α and LTBR-TCR-β fusions proved to be ineffective, eliciting cytokine secretion levels similar to those of untransduced cells after co-incubation with the NY-ESO-1+ melanoma cell line A375 ( Fig.29D ). We therefore concluded that direct fusion of the LTBR tail to the TCR chain causes disruption of TCR expression and / or function.

[0357] The CD3 molecule is essential for mediating TCR signaling. Therefore, fusions of all four CD3 polypeptides to the LTBR signaling tail were engineered ( Fig.29E ), and CD3-LTBR fusion or unmodified CD3 gene was sequentially transduced into T cells together with NY-ESO-1TCR ( Fig.29F In terms of TCR surface expression, there were no major differences between the different conditions, as expected, since T cells already express all CD3 genes ( Figure 29G In terms of phenotypic markers of LTBR activity, it was observed that CD3-LTBR fusions elicited higher overall expression of CD54 and CD74 than the matched unmodified CD3 construct ( Fig.29H CD3-LTBR fusion failed to increase the activity of TCR-engineered T cells above control levels in terms of antigen-specific responses after co-incubation with the NY-ESO-1+ melanoma line A375 ( Fig.29I Interestingly, overexpression of unmodified CD3ε or CD3ζ elicited a small but significant increase in antigen-specific responses, albeit to a lesser extent than overexpression of full-length LTBR. Overall, LTBR-CD3 fusions failed to outperform native full-length LTBR in phenotypic and functional assays, likely due to competition with endogenous, highly expressed components of the CD3 complex.

[0358] Example 14: Fusion of the LTBR signaling tail to members of the CD8 complex.

[0359] It has been shown that co-expression of full-length LTBR with NY-ESO-1 targeting αβTCR improves the anti-tumor reactivity of engineered T cells (WO 2023 / 279049), and fusion of the intracellular tail of LTBR to a CD19-targeting CAR using both 4-1BB and CD28 designs achieves better phenotype and function than co-expression of conventional CD19-targeting CAR and LTBR as two independent molecules.

[0360] To extend this observation, the intracellular tail of LTBR was fused to CD8α or CD8β molecules ( Fig. 30A The CD8α-CD8β heterodimer is essential for efficient engagement of a TCR (such as the NY-ESO-1 targeting αβ TCR used here) with a peptide antigen (here, the NY-ESO-1 derived peptide SLLMWITQC) presented by an MHC-I molecule (here, HLA-A2).

[0361] SEQ ID NN name type 128 CD8a:LTBR-P2A-puroR DNA 129 CD8a:LTBR-P2A-puroR AA 130 CD8b:LTBR-P2A-puroR DNA 131 CD8b:LTBR-P2A-puroR AA

[0362] In this experiment, CD8 T cells expressing endogenous CD8α-CD8β heterodimers were sequentially transduced with NY-ESO-1 targeting the TCR and the modified gene to be overexpressed: control gene tNGFR, full-length LTBR, ​​CD8α (wild type or fused to the LTBR signaling tail), or CD8β (wild type or fused to the LTBR signaling tail) ( Fig. 30B ).

[0363] First, we measured the surface expression of CD54 and CD74, markers of LTBR activity, and the intracellular expression of the key transcription factor TCF1. As expected, T cells transduced with NY-ESO-1 TCR co-expressing LTBR showed significantly increased expression of both surface markers and transcription factors compared to the control gene tNGFR ( Figure 30C-Figure 30E ). T cells transduced with the CD8-LTBR fusion showed a slight increase in expression of CD54 and CD74, but not TCF1, compared with their wild-type counterparts, indicating that LTBR signaling is at least partially active in these constructs.

[0364] Finally, the effect of CD8-LTBR fusion on the functional response of engineered T cells was tested. The NY-ESO-1 antigen is expressed on various tumor types (Thomas et al., NY-ESO-1 Based Immunotherapy of Cancer: Current Perspectives, Front. Immunol., May 1, 2018, Section Cancer Immunity and Immunotherapy, Vol. 9-2018). To test the sensitivity and magnitude of response of LTBR engineered T cells to this antigen, HLA-A2+ cell lines were pulsed with different concentrations of the immunodominant peptide epitope SLLMWITQC derived from the NY-ESO-1 protein. After 24 hours of co-incubation of T cells with the peptide presenting cell line, T cell responses were measured based on the secretion of two key cytokines, IFNγ and IL2 ( Fig.30F , Figure 30G As expected, co-expression of the full-length LTBR induced much stronger T cell responses to antigen than did the matched control tNGFR, even at concentrations as low as 10 -7 The same was true at peptide concentrations of 1.4 M. LTBR-transduced T cells also showed a >3-fold increase in sensitivity to peptide concentration (IC of IFNγ secretion by LTBR 50 For 6x 10 -7 M, tNGFR is 2x10 -6 M). Importantly, CD8-LTBR fusions also increased the magnitude of responses to both cytokines measured compared to matched controls. Thus, the functionality and utility of synthetic CD8-LTBR fusions as alternatives to full-length native LTBRs were demonstrated.

[0365] Example 15: ORF library vector engineering to improve transduction efficiency

[0366] It has been previously demonstrated that pooled barcoded lentiviral libraries containing >12,000 human genes can be used to discover new modulators of key T cell functions (see, e.g., Legut et al., Nature 2022). Here, it is shown that engineering of the lentiviral vector backbone greatly increases the functional titer of the library virus, improving the scalability of this approach. In terms of transduction efficiency, the improved ORF library vector (SEQ ID NO: 132) showed a >5-fold increase relative to the original vector, making it possible to achieve a transduction rate of approximately 30% with as little as 50ul of lentivirus (which is ideal for pooled screening) ( Fig.31). As expected, due to its smaller size, the engineered vector backbone without the ORF library showed higher transduction efficiency.

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[0411] All publications cited in this specification are incorporated herein by reference. U.S. Provisional Patent Application No. 63 / 320,100, filed on March 15, 2022, is incorporated herein by reference. Although the present invention has been described with reference to specific embodiments, it should be understood that modifications may be made without departing from the spirit of the present invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A lymphocyte genetically modified to express a chimeric antigen receptor (CAR), wherein the CAR comprises: a) antigen binding domain; b) a transmembrane domain; and c) signal transduction domain; At least one of the domains comprises a LTBR domain.

2. The modified lymphocyte of claim 1, wherein the LTBR domain is the LTBR intracellular domain or a fragment or variant thereof.

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

5. The modified lymphocyte according to any one of claims 1 to 4, wherein the transmembrane domain is a LTBR domain.

6. The modified lymphocyte according to any one of claims 1 to 5, wherein the extracellular domain is a LTBR domain.

7. The modified lymphocyte according to any one of claims 1 to 6, wherein the lymphocyte is a T cell.

8. The modified lymphocyte according to any one of claims 1 to 7, wherein the lymphocyte is an αβ T cell or a γδ T cell, optionally a Vγ9Vδ2 T cell.

9. The modified lymphocyte according to any one of claims 1 to 6, wherein the lymphocyte is a NK cell or a NK T cell.

10. The modified lymphocyte of any one of claims 1 to 9, wherein the LTBR intracellular domain is positioned between a co-stimulatory signaling domain and the signaling domain of (c).

11. The modified lymphocyte of any one of claims 1 to 9, wherein the LTBR domain is positioned after the signaling domain of (c).

12. A lymphocyte genetically modified to express a chimeric antigen receptor (CAR), wherein the CAR comprises: a) antigen binding domain; b) transmembrane domain; c) a co-stimulatory signaling domain; and d) signal transduction domain; At least one of the domains comprises a LTBR domain.

13. The modified lymphocyte of claim 12, wherein the LTBR domain is the LTBR intracellular domain or a fragment or variant thereof.

14. The modified lymphocyte of claim 13, wherein the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion or variant thereof.

15. The modified lymphocyte of claim 13 or 14, wherein the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

16. The modified lymphocyte of any one of claims 12 to 15, wherein the transmembrane domain is a LTBR domain.

17. The modified lymphocyte according to any one of claims 12 to 16, wherein the extracellular domain is a LTBR domain.

18. The modified lymphocyte according to any one of claims 12 to 17, wherein the lymphocyte is a T cell.

19. The modified lymphocyte according to any one of claims 12 to 18, wherein the lymphocyte is an αβT or γδT cell, optionally a Vγ9Vδ2 T cell.

20. The modified lymphocyte according to any one of claims 12 to 18, wherein the lymphocyte is a NK cell or a NK T cell.

21. The modified lymphocyte of any one of claims 12 to 20, wherein the LTBR intracellular domain is positioned between the transmembrane domain of (b) and the co-stimulatory signaling domain of (c).

22. The modified lymphocyte of any one of claims 1 to 20, wherein the LTBR intracellular domain is positioned between the co-stimulatory signaling domain of (c) and the signaling domain of (d).

23. The modified lymphocyte of any one of claims 1 to 20, wherein the LTBR domain is positioned after the signaling domain of (c).

24. The modified lymphocyte according to any one of claims 1 to 23, wherein the CAR is selected from Axicabtagene ciloleucel Brexucabtageneautoleucel (Tecartus TM )、Idecabtagene vicleucel (Abecma TM ), Lisocabtagene maraleucel Tisagenlecleucel Or one of the CARs found in Figure 19, which is modified to contain a LTBR domain.

25. A nucleic acid molecule comprising a sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: a) antigen binding domain; b) a transmembrane domain; and c) signal transduction domain; At least one of the domains comprises a LTBR domain.

26. The nucleic acid molecule of claim 25, wherein the LTBR domain is the LTBR intracellular domain or a fragment or variant thereof.

27. The nucleic acid molecule of claim 26, wherein the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion or variant thereof.

28. The nucleic acid molecule of claim 26 or 27, wherein the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

29. The nucleic acid molecule of any one of claims 25 to 28, wherein the transmembrane domain is a LTBR domain.

30. The nucleic acid molecule according to any one of claims 25 to 29, wherein the extracellular domain is a LTBR domain.

31. The nucleic acid molecule of any one of claims 25 to 30, wherein the LTBR intracellular domain is positioned between a co-stimulatory signaling domain and the signaling domain of (c).

32. The nucleic acid molecule of any one of claims 25 to 30, wherein the LTBR domain is positioned after the signaling domain of (c).

33. A nucleic acid molecule comprising a sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: a. Antigen binding domain; b. transmembrane domain; c. a co-stimulatory signaling domain; and d. Signal transduction domain; At least one of the domains comprises a LTBR domain.

34. The nucleic acid molecule of claim 33, wherein the LTBR domain is the LTBR intracellular domain or a fragment or variant thereof.

35. The nucleic acid molecule of claim 34, wherein the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion or variant thereof.

36. The nucleic acid molecule of claim 34 or 35, wherein the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

37. The nucleic acid molecule of any one of claims 33 to 36, wherein the transmembrane domain is a LTBR domain.

38. The nucleic acid molecule of any one of claims 33 to 37, wherein the extracellular domain is a LTBR domain.

39. The nucleic acid molecule of any one of claims 33 to 38, wherein the LTBR intracellular domain is positioned between the transmembrane domain of (b) and the co-stimulatory signaling domain of (c).

40. The nucleic acid molecule of any one of claims 33 to 39, wherein the LTBR intracellular domain is positioned between the co-stimulatory signaling domain of (c) and the signaling domain of (d).

41. The nucleic acid molecule of any one of claims 33 to 40, wherein the LTBR domain is positioned after the signaling domain of (c).

42. The nucleic acid molecule according to any one of claims 25 to 41, wherein the CAR is selected from Akiramecetib. Tecartus TM ), Abecma TM ), Leakey Myron Tisaron Or one of the CARs found in Figure 19, which is modified to contain a LTBR domain.

43. An expression cassette comprising the nucleic acid molecule according to any one of claims 25 or 42.

44. The expression cassette of claim 43, further comprising an expression control sequence.

45. The expression cassette of claim 44, wherein the expression control sequence comprises a promoter.

46. ​​The expression cassette of claim 45, wherein the promoter is a constitutive promoter or an inducible promoter.

47. A modified lymphocyte comprising a nucleic acid molecule according to any one of claims 25 to 42 or an expression cassette according to any one of claims 43 to 46.

48. A method of producing a modified lymphocyte, the method comprising introducing into the lymphocyte a nucleic acid molecule according to any one of claims 25 to 42 or an expression cassette according to any one of claims 43 to 46.

49. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising the modified lymphocytes of any one of claims 1 to 24 or 47.

50. The method of claim 49, wherein the subject has a lymphoma, optionally B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma.

51. The method of claim 49, wherein the subject has a solid tumor cancer.

52. The method of claim 49, wherein the subject has leukemia.

53. The method of claim 49, wherein the subject has multiple myeloma.

54. The method of claim 49, wherein the subject has a virally driven cancer.

55. The method of claim 54, wherein the subject has HPV.

56. The method of claim 54, wherein the subject has a cancer selected from the group consisting of Burkitt's lymphoma, liver cancer, Kaposi's sarcoma, cervical cancer, head cancer, neck cancer, anal cancer, pancreatic cancer, melanoma, oral cancer, pharyngeal cancer, penile cancer, adult T-cell lymphoma, or merkel cell carcinoma.

57. A method of treating a viral disease in a subject in need thereof, the method comprising administering to the subject a composition comprising the modified lymphocytes of any one of claims 1 to 24 or 47.

58. The method of claim 57, wherein the disease is HIV or HPV.

59. A method of treating autoimmunity in a subject in need thereof, the method comprising administering to the subject a composition comprising the modified lymphocytes of any one of claims 1 to 24 or 47.

60. The method of claim 29, wherein the disease is an autoimmune disorder.

61. A method of increasing proliferation or T cell effector function including cytokine production and / or secretion, the method comprising administering to a subject a composition comprising a modified lymphocyte according to any one of claims 1 to 24 or 47.

62. The method of claim 61, wherein the T cells are obtained from a human prior to treating the T cells to overexpress LTBR, ​​and the treated T cells are reintroduced into the human.

63. A fusion protein comprising a LTBR domain and at least one domain from a second protein that is not a LTBR.

64. The fusion protein of claim 63, wherein the second protein is CD4.

65. The fusion protein of claim 63, wherein the second protein is CD8A or CD8B.

66. The fusion protein of claim 63, wherein the second protein is CD3E.

67. The fusion protein of claim 63, wherein the second protein is CD3D.

68. The fusion protein of claim 63, wherein the second protein is CD3zeta.

69. The fusion protein of claim 63, wherein the second protein is CD3Z.

70. The fusion protein of any one of claims 63 to 69, wherein the LTBR domain is the LTBR intracellular domain or a fragment or variant thereof.

71. The fusion protein of claim 70, wherein the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion or variant thereof.

72. The fusion protein of claim 70 or 71, wherein the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

73. A nucleic acid molecule comprising a sequence encoding a fusion protein according to any one of claims 63 to 72.

74. A host cell comprising the nucleic acid molecule according to claim 73.

75. The host cell of claim 74 which is a lymphocyte.

76. The host cell of claim 75, which is a T cell.

77. A lymphocyte genetically modified to express a T cell receptor (TCR), wherein the TCR comprises an alpha chain fused to a LTBR intracellular domain.

78. A lymphocyte genetically modified to express a T cell receptor (TCR), wherein the TCR comprises a beta chain fused to a LTBR intracellular domain.

79. A lymphocyte genetically modified to express a T cell receptor (TCR), wherein the TCR comprises a gamma chain fused to a LTBR intracellular domain.

80. A lymphocyte genetically modified to express a T cell receptor (TCR), wherein the TCR comprises a delta chain fused to a LTBR intracellular domain.

81. The modified lymphocyte of any one of claims 77 to 80, wherein the LTBR domain is the LTBR intracellular domain or a fragment or variant thereof.

82. The modified lymphocyte of claim 81, wherein the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion or variant thereof.

83. The modified lymphocyte of claim 81 or 82, wherein the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

84. The modified lymphocyte of any one of claims 77 to 83, wherein the lymphocyte is a T cell.

85. The modified lymphocyte according to any one of claims 77 to 84, wherein the lymphocyte is an αβ T cell or a γδ T cell, a Vγ9Vδ2 T cell.

86. The modified lymphocyte of any one of claims 77 to 83, wherein the lymphocyte is a NK cell or a NK T cell.

87. The modified lymphocyte of any one of claims 77 to 86, wherein the TCR is tebentafusp-tebn Or one of the TCRs found in Figure 20, which has been modified to contain a LTBR domain.

88. A nucleic acid molecule comprising a sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: a. Antigen binding domain; b. transmembrane domain; and c. Signal transduction domain; At least one of the domains comprises a LTBR domain.

89. The nucleic acid molecule of claim 88, wherein the LTBR domain is the LTBR intracellular domain or a fragment or variant thereof.

90. The nucleic acid molecule of claim 89, wherein the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion or variant thereof.

91. The nucleic acid molecule of claim 88 or 89, wherein the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

92. An expression cassette comprising the nucleic acid molecule according to any one of claims 88 or 91.

93. The expression cassette of claim 92, further comprising an expression control sequence.

94. The expression cassette of claim 93, wherein the expression control sequence comprises a promoter.

95. The expression cassette of claim 94, wherein the promoter is a constitutive promoter or an inducible promoter.

96. A modified lymphocyte comprising a nucleic acid molecule according to any one of claims 88 to 91 or an expression cassette according to any one of claims 92 to 95.

97. A method of producing a modified lymphocyte, the method comprising introducing into the lymphocyte a nucleic acid molecule according to any one of claims 88 to 91 or an expression cassette according to any one of claims 92 to 95.

98. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising the modified lymphocytes of any one of claims 77 to 87 or 96.

99. The method of claim 98, wherein the subject has a lymphoma, optionally B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma.

100. The method of claim 99, wherein the subject has a solid tumor cancer.

101. The method of claim 98, wherein the subject has leukemia.

102. The method of claim 98, wherein the subject has multiple myeloma.

103. The method of claim 98, wherein the subject has multiple uveal melanomas.

104. The method of claim 98, wherein the subject has a virally driven cancer.

105. The method of claim 98, wherein the subject has HPV.

106. The method of claim 104, wherein the subject has a cancer selected from the group consisting of 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.

107. A method of increasing proliferation or T cell effector function including cytokine production and / or secretion, the method comprising administering to a subject a composition comprising a modified lymphocyte according to any one of claims 77 to 87 or 96.

108. The method of claim 107, wherein the T cells are obtained from a human prior to modifying the lymphocytes, and the modified lymphocytes are reintroduced into the human.

109. A nucleic acid molecule according to XX, wherein the CAR targets CD19, mesothelin, ROR1, B7-H3, IL13Rα2, GD2, Her2, phosphatidylinositol glycan 3, CD7, NY-ESO-1, CD30, MAGE-A1, LMP2, PD1, KRAS G12V, CD20, CD22, CD171, CD123, CD38, CD10, BAFFR, PSMA or mucin.

110. A fusion protein comprising: a. Antigen binding domain; b.LTBR; c. and at least one domain from a second protein that is not a LTBR.

111. The fusion protein of claim 110, wherein the second protein is CD4.

112. The fusion protein of claim 110, wherein the second protein is CD8A or CD8B.

113. The fusion protein of claim 110, wherein the second protein is CD3E.

114. The fusion protein of claim 110, wherein the second protein is CD3D.

115. The fusion protein of claim 110, wherein the second protein is CD3zeta.

116. The fusion protein of claim 110, wherein the second protein is CD3Z.

117. The fusion protein of any one of claims 110 to 116, wherein the LTBR domain is the LTBR intracellular domain or a fragment or variant thereof.

118. A nucleic acid molecule comprising a sequence encoding a fusion protein according to any one of claims 110 to 117.

119. A modified lymphocyte comprising the nucleic acid molecule according to claim 118.

120. A modified lymphocyte comprising the nucleic acid molecule according to claim 118 and further comprising a nucleic acid molecule encoding CAR.

121. The modified lymphocyte of claim 120, wherein both the CAR and the antigen binding domain target mesothelin.

122. A lymphocyte genetically modified to express a T cell receptor (TCR) and a CD8 alpha chain fused to the LTBR intracellular domain.

123. A lymphocyte genetically modified to express a T cell receptor (TCR) and a CD8 beta chain fused to the LTBR intracellular domain.

124. The modified lymphocyte of claim 122 or 123, wherein the LTBR domain is the LTBR intracellular domain or a fragment or variant thereof.

125. The modified lymphocyte of claim 124, wherein the LTBR intracellular domain comprises amino acids 249 to 435 of SEQ ID NO: 2, or a fragment, deletion or variant thereof.

126. The modified lymphocyte of claim 124 or 125, wherein the LTBR intracellular domain has a deletion in at least amino acids 393 to 435.

127. The modified lymphocyte of any one of claims 122 to 126, wherein the lymphocyte is a T cell.

128. The modified lymphocyte according to any one of claims 122 to 127, wherein the lymphocyte is an αβ T cell or a γδ T cell, a Vγ9Vδ2 T cell.

129. The modified lymphocyte of any one of claims 122 to 126, wherein the lymphocyte is a NK cell or a NK T cell.

130. The modified lymphocyte according to any one of claims 122 to 129, wherein the TCR is Tibenfossi or one of the TCRs found in Figure 20.

131. An engineered lentiviral vector comprising the sequence of SEQ ID NO: 132, or a sequence sharing at least 90% identity with SEQ ID NO: 132, optionally with an open reading frame (ORF) of a gene of interest and / or a barcode inserted within the sequence.

132. The engineered vector of claim 131, wherein the ORF and / or the barcode is inserted after nucleotide 3291 of SEQ ID NO: 132.

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