Compositions and methods of use of t cells in immunotherapy

Abstract

The present disclosure relates in some aspects to methods, cells, and compositions for preparing isolated engineered immune cells comprising T cell receptors (TCRs) capable of recognizing a disease-associated antigen. In some aspects, the immune cells are T cells for use in immunotherapy. Provided In an embodiment are T cell preparation methods, including isolation, processing, incubation, and genetic engineering of cells and populations of cells. Also provided are the isolated engineered T cells and compositions produced by the methods in the present disclosure. In some aspects, the methods prepare T cells for adoptive therapy. In an embodiment, the disease-associated antigen is a cancer-associated antigen.

Description

COMPOSITIONS AND METHODS OF USE OF T CELLS IN IMMUNOTHERAPYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 595,737 filed November 2, 2023 and 63 / 682,327 filed August 12, 2024. The entire contents of the aboveidentified applications are hereby fully incorporated herein by reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (“BROD-5940WP_ST26.xml”; Size is 36,623,737 bytes and it was created on October 31, 2024) is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0003] The subject matter disclosed herein is generally directed to T cell receptors (TCRs) capable of recognizing antigens that are shared across multiple patients in a particular disease context and their use in adoptive cell therapies and vaccine compositions.BACKGROUND

[0004] Approximately 1.6 million Americans are diagnosed with neoplasia every year, and approximately 580,000 people in the United States are expected to die of the disease in 2013. Over the past few decades there been significant improvements in the detection, diagnosis, and treatment of neoplasia, which have significantly increased the survival rate for many types of neoplasia. However, only about 60% of people diagnosed with neoplasia are still alive 5 years after the onset of treatment, which makes neoplasia the second leading cause of death in the United States.

[0005] Multiple myeloma (MM), also known as plasma cell myeloma, myelomatosis, Kahler’s, is a cancer of plasma cells, a type of white blood cell normally responsible for producing antibodies in which collections of the neoplastic plasma cells accumulate in the bone marrow. It is the second most common hematologic cancer as it accounts for 10% of all hematologic malignancies and represents 1% of all cancer diagnosis and 2% of all cancer deaths. MM leads to bone lesions with 80% of patients developing osteoporosis, lytic bone lesions, or fractures during the course of the disease. MM treatments with alkylating agents, corticosteroids, proteasomeinhibitors, and immunomodulatory drugs have resulted in significant survival benefits, however relapse is inevitable and disease remains incurable with a median survival of 5 years.

[0006] Acute myeloid leukemia (AML) is a heterogeneous hematologic disorder characterized by clonal expansion of myeloid blasts in bone marrow, peripheral blood, and other tissues. Despite recent progress, current treatment of AML remains unsatisfactory with a 5-year relapse-free survival rate lower than 30%.

[0007] Various strategies are available for producing and administering engineered cells for adoptive therapy. Some available strategies include engineering immune cells expressing genetically engineered antigen receptors, such as CARs, and for suppression or repression of gene expression in the cells. Improved strategies are needed, for example, to provide a wider range of target antigens and diseases that may be treated using such cells, to improve specificity or selectivity of the cells, e.g., to avoid off-target effects, and to improve efficacy of the cells, for example, by avoiding suppression of effector functions and improving the activity and / or survival of the cells upon administration to subjects. Provided are methods, cells, compositions, kits, and systems that meet such needs.

[0008] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0009] In an embodiment, the techniques described herein relate to an isolated engineered immune cell including a T cell receptor (TCR) capable of recognizing a disease-associated antigen.

[0010] In an embodiment, the techniques described herein relate to a cell, wherein the disease- associated antigen is a virus-associated antigen. In an embodiment, the techniques described herein relate to a cell, wherein the disease-associated antigen is a cancer-associated antigen.

[0011] In an embodiment, the techniques described herein relate to a cell, wherein the cancer- associated antigens are associated with one or more hematological malignancies. In an embodiment, the techniques described herein relate to a cell, wherein the hematological malignancy is multiple myeloma (MM). In an embodiment, the techniques described herein relate to a cell, wherein the hematological malignancy is acute myeloid leukemia (AML). In an embodiment, the techniques described herein relate to a cell, wherein the hematological malignancy is chronic lymphocytic leukemia (CLL).

[0012] In an embodiment, the techniques described herein relate to a cell, wherein the disease- associated antigen is selected from SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.

[0013] In an embodiment, the techniques described herein relate to a cell, wherein the TCR includes SEQ LD NOs: 1-121, and / or a TCR alpha chain CDR3 sequence selected from SEQ ID NO: 1-62 or 41855-41902 or TCR beta chain CDR3 sequence selected from SEQ ID NO: 63-121 or 41903-41948.

[0014] In an embodiment, the techniques described herein relate to a cell, wherein the cell is a CD8 T cell. In an embodiment, the techniques described herein relate to a cell, wherein the CD8 T cell is isolated from a subject to be treated.

[0015] In an embodiment, the techniques described herein relate to a cell, wherein the cell includes one or more modifications to one or more genes that modify an immune reactivity of the cell.

[0016] In an embodiment, a method of treating cancer comprises administering the engineered immune cell to a subject in need thereof. In an embodiment, the subject suffers from a cancer that is a hematological malignancy. In an embodiment, wherein the hematological malignancy is MM, AML, or CLL.

[0017] In an embodiment, the techniques described herein relate to a vaccine including a cancer-associated antigen. In an embodiment, the techniques described herein relate to a vaccine, wherein the antigen is recognized by a TCR selected from SEQ ID NOs: 1-121 and / or a TCR alpha chain CDR3 sequence selected from SEQ ID NO: 1-62 or 41855-41902 or TCR beta chain CDR3 sequence selected from SEQ ID NO: 63-121 or 41903-41948

[0018] In an embodiment, the techniques described herein relate to a vaccine, wherein the antigen is selected from SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.

[0019] In an embodiment, the vaccine includes a polynucleotide encoding the conserved cancer antigen. In an embodiment, the polynucleotide is mRNA.

[0020] In an embodiment, wherein the vaccine includes the antigen and optionally a carrier or adjuvant.

[0021] In an embodiment, a method of treating cancer comprises administering the vaccine to a subject in need thereof. In an embodiment, the subject suffers from a hematological malignancy. In an embodiment, the hematological malignancy is multiple myeloma, acute myeloid leukemia, or chronic lymphocytic leukemia.

[0022] In an embodiment, the techniques described herein relate to a method for detecting tumor-reactive T-cell receptors (TCRs): (a) characterizing the phenotype and clonality of a population of isolated T cells to define a baseline transcriptional state; (b) segregating single isolated T cells from the population of isolated T cells into individual discrete volumes and exposing the single isolated T cells to a tumor cell; (c) identifying and retrieving single isolated T cells from the individual discrete volumes and conducting TCR alpha and beta chain sequencing; and (d) identifying antigen-reactive T cells by matching each TCR to its baseline transcriptional state using the CDR3 amino acid sequence as an endogenous barcode of each TCR.

[0023] In an embodiment, the techniques described herein relate to a method, wherein step (b) further includes capture beads to detect T cell-derived cytokines and wherein single isolated T cells are retrieved for step (c) if T cell cytokines are detected.

[0024] In an embodiment, the techniques described herein relate to a method, wherein the T cell-derived cytokines include interleukin-2 (IL-2), interferon-gamma, and tumor necrosis factor (TNF).

[0025] In an embodiment, the techniques described herein relate to a method, wherein step (b) further includes assaying for expression of surface 4-IBB as an indicator of an antigen-activated T cell.

[0026] In an embodiment, the techniques described herein relate to a method, further includes exposing a subset of the population of isolated T cells to stimulation with tumor or viral antigens and obtaining TCR sequencing TCRs using TCRV(Beta)-seq, and integrating the TCRV(beta)-seq with the baseline transcriptional state using the CDR3 amino acid sequence.

[0027] In an embodiment, the techniques described herein relate to a method further including defining an antigen-reactive TCR signature based on the identified baseline transcriptional state.

[0028] In an embodiment, the techniques described herein relate to a method, wherein characterizing the phenotype and clonality of the cells includes using high-throughput single-cell RNA sequencing (scRNA-seq), single-cell TCR sequencing (scTCR-seq) coupled with thedetection of surface proteins using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq).

[0029] In an embodiment, the techniques described herein relate to a method, wherein determining one or more epitopes on the cells to define the clonotype includes using high- throughput single-cell RNA sequencing (scRNA) and single-cell TCR sequencing.

[0030] In an embodiment, the techniques described herein relate to a method, wherein determining one or more epitopes on the cells includes using cellular indexing of the transcriptomes and epitopes by sequencing (CITE-seq).

[0031] In an embodiment, the techniques described herein relate to a method, wherein step (b) further includes optical screening to quantify T cell activation and cytokine production.

[0032] In an embodiment, the techniques described herein relate to a method, further including expanding the identified antigen-specific T cells in a cell population and delivering the cell population to a subject in need thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0034] FIGS. 1A-1H show the transcriptomic landscape of clonal T cells in the diseased bone marrow. FIG. 1A is a graphical overview of TCR discovery platform. To characterize the phenotype and clonality of bone marrow resident T cells (BMR-T), Applicants used high- throughput single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR- seq) coupled with the detection of surface proteins (that is, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), thereby defining the in vivo transcriptional state as baseline (Step 1). Functionally tumor reactive TCRs were identified in parallel using 1), a microfluidics- based forward screening approach of single BMR-T exposed to single autologous tumor cells and 2) the MANA functional expansion of specific T cells assay (MANAFEST) on BMR-T of all individuals (Step 2). Functionally screened TCRs were retrieved from their microfluidic reaction chambers and subjected to combined TCR-alpha and beta-chain sequencing (TCRA / B-seq), while MANAFEST-cultures were sequenced after 4 weeks in total with combined scRNA / TCR-seq (Step 3). The data of both assays was then used to identify and phenotypically map antigen reactiveT cells by matching each TCR to its baseline transcriptional state using the CDR3 nucleotide sequence as unique barcode of a given clone A signature of tumor reactivity was established based on the transcriptional features of tumor reactive TCRs in the establishment cohort (n=6 NDMM patients) and tested for sensitivity and specificity to prospectively identify tumor reactive TCRs in an independent validation cohort (n=6 NDMM patients) (Step 4). Lastly, the clonal dynamics and clinical relevance of tumor reactive TCRs were explored in two clinical trial cohorts with longitudinal bone marrow biopsies: newly diagnosed multiple myeloma patients undergoing induction immunochemotherapy followed by autologous stem cell transplant (ASCT; n=14 patients) and relap sed / refractory multiple patients undergoing bispecific BCMAxCD3 antibody treatment (bsAb, n=16 patients) (Step 5). FIG. IB is a UMAP of T cell subtypes with productive TCR identified in the NDMM establishment cohort (N=6 patients, n = 101,210 cells, n = 81,122 TCRs post QC). FIG. 1C is a stacked bar chart of single cell count and the respective cluster annotation per TCR-clonotype. Top 50 clonotypes per patient shown. FIG. ID is a UMAP depicting expansion ofBMR-T clonotypes (large = 0.01-1; medium = 0.001 - 0.001; small= 0.0001 - 0.001; rare = 0 - 0.0001). FIG. IE shows the relative abundance of expansion-categories within cells of each patient. FIG. IF shows the average clonotype proportion in sample as dot size by T cell subtype within each patient. FIG. 1G shows the T cell subtype composition in expanded clones (Proportion in bone marrow > 0.01) and non-expanded (Proportion in bone marrow < 0.01) clones. FIG. lH shows the gini clonality and inversed Simpson diversity indices per transcriptionally defined BMR-T cluster. N = 6 NDMM patients.

[0035] FIGS. 2A-2L shows phenotype and specificity of bone-marrow associated T cells. FIG. 2A shows representative images of microfluidics based forward TCR screening approach. Single BMR-T were co-cultured with autologous myeloma cells for 16h. Each microfluidic reaction chamber further contained capture beads to detect the T cell-derived cytokines Interleukin-2 (IL-2), Interfer on-gamma (IFN- y) and Tumor necrosis factor (TNF) and was observed for surface 4-1BB (CD137) protein expression. If one or more signals of tumor reactivity were detected, this T cell was retrieved from its reaction chamber and subjected to TCRA / B-seq (Methods). FIG. 2B is a venn diagram outlining characteristics of n = 413 TCR:Tumor recognition events. BMR-T that secreted any cytokine together with 4- IBB surface protein expression were classified as polyfunctional. FIG. 2C (Left) shows a stacked bar chart outlining distribution of TCR:Tumor recognition events in each run of the microfluidics-based forward screening assay, n= 26 assay runs. Definition of BMR-T function as in b). FIG. 2C (Right)Summary statistics of n = 1,243 TCR:Tumor recognition events. Definition of BMR-T function as in b). FIG. 2D shows summary statistics of n = 1,243 TCR:Tumor recognition events split by CD4+ or CD8+ subtype. Definition of BMR-T function as in b). Statistical significance of enrichment was determined by mixed effects analysis with Bonferroni post-hoc test for multiple hypothesis testing correction. FIG. 2E shows functional validation of cloned and mRNA transfected TCRs derived from the microfluidics-based forward screening assay. T cells were transfected with cloned TCRA / B fragments and murine TRAC / TRBC chains and co-cultured with patient-autologous multiple myeloma cells. CD69 surface expression was measured by flow cytometry. TCRs derived from N = 3 patients, n = 3 experimental replicates per patient. Statistical significance was determined by two-way ANOVA with Tukey post-hoc test for multiple hypothesis testing correction. FIG. 2F shows a UMAP of T cells in the establishment cohort colored by recognized antigen (Myeloma, SARS-CoV-2, Influenza-A, CMV, EBV and bystander (non-reactive). TCRs derived from N = 6 patients, n = 81,122 total TCRs post QC. (FIG. 2G) T cell subtype composition of BMR-T reactive to the outlined antigens. Total number of cells per antigen indicated. TCRs derived from N = 6 patients, n = 81,122 total TCRs post QC. (FIG. 2H) Scaled average transcript expression heatmap of top 10 differentially expressed genes per recognized antigen (Myeloma, SARS-CoV-2, Influenza-A, CMV, EBV and bystander (non-reactive). TCRs derived from N = 6 patients, n = 81,122 total TCRs post QC. (FIG. 21) Scaled average expression heatmap of selected marker genes per recognized antigen. (FIG. 2J) X-Y scatter of average expression of cytotoxicity and dysfunction module scores in BMR-T color-coded by antigen-recognizing TCR. Bystander T cells colored in grey. TCRs derived from N = 6 patients, n = 81, 122 total TCRs post QC. FIG. 2K is a violin plot depicting cell-wise expression of cytotoxicity signature split by antigen reactivity. Statistical significance was determined by two-way ANOVA with Tukey post-hoc test for multiple hypothesis testing correction. FIG. 2L shows a 2MHC-I blocking experiment. TCR-transgenic T cells were tested against autologous MM cells as outlined in FIG. 2E and CD137 and CD69 expression were measured by flow cytometry. MM cells were pre-incubated with either HLA- ABC blocking antibody or isotype control for 1 h before co-culture. Statistical significance was determined by two-way ANOVA with Dunnet post-hoc test for multiple hypothesis testing correction. N = 3 experimental replicates per TCR. Irrelevant TCRs were used as negative controls for MHC-I blocking (Cl, C2).

[0036] FIGS. 3A-3G show conserved transcriptional signatures of tumor reactive BMR-T. FIG. 3A shows scaled average expression heatmap of top differentially expressed genes used to define the tumor reactive TCR transcriptional signature (MM-TCR). FIG. 3B shows a ridge plot of selected marker genes per reactivity group (Myeloma, Virus (SARS-CoV-2, Influenza- A, CMV, EBV), and bystander (non-reactive) in signature establishment cohort. TCRs derived from N = 6 patients, n = 81, 122 total TCRs post QC. FIG. 3C is a UMAP of subsetted validated tumor reactive clonotypes (N=6 patients, n = 938 cells). FIG. 3D is a UMAPs overlaid with gene-weighted density of indicated genes. FIG. 3E is a violin plot indicating ITGB1 gene expression in antigenspecific BMR-T color-coded by antigen reactivity. Statistical analysis for enrichment was performed by hypergeometric testing. FIG. 3Fshows B16 gplOO-expressing and MC38 OVA- expressing tumor cells were injected into C57BL / 6J animals followed by intravenous adoptive transfer of 50:50 pmel:OT-I transgenic CD90.1 :CD45.1 T cells. Adoptively transferred T cells were detected 7 days post-injection in blood, tumors and tumor-draining lymph nodes (TDLN) by CD90.1 (pmel) or CD45.1 (OT-I) and subjected to flow cytometry analysis of phenotype and CD29 expression. FIG. 3G (Left) shows flow cytometry analysis of CD29 surface protein expression on homed TCR-transgenic T cells in TDLN from k) (Left). FIG. 3G (Right)shows flow cytometry analysis of CD44 / CD62L surface protein expression on homed TCR-transgenic T cells in TDLN from k). Statistical significance was determined by two-way ANOVA with Tukey post-hoc test for multiple hypothesis testing correction.

[0037] FIGS. 4A-4J show the clinical relevance of tumor reactive T cells in multiple myeloma. FIG. 4A shows N=6 NDMM patients (Tumor reactivity signature validation cohort) were profiled by scRNA / TCR sequencing of bone marrow and peripheral blood. TCRs were classified based on MM-TCR signature expression, followed by microfluidics tumor reactivity screening of BMR-T and autologous tumor cells. Stacked bar chart of single cell count and the respective cluster annotation per TCR-clonotype. Top 50 clonotypes per patient shown. FIG. 4B shows a UMAP of T cells in the validation cohort colored by recognized antigen (Myeloma, SARS-CoV-2, Influenza-A, CMV, EBV and bystander (non-reactive). TCRs derived from N = 6 patients, n = 61,459 total TCRs post QC. FIG. 4C shows prospective area under the curve (AUC) of receiver operator characteristic (ROC) shown (Methods). AUROC curves of MM-TCR (AUC: 0.9452), MANA Caushi5(AUC: 0.8119) NeoTCR 8 (AUC: 0.8762) and NeoTCR 4 (AUC: 0.5530)7signature scores to predict tumor reactivity in validation cohort TCRs (n = 85,268 cells,n = 3,885 validated myeloma reactive BMR-T). FIG. 4D shows a scatter plot depicting frequency of tumor reactive TCRs in the bone marrow and tumor immunogenicity metrics: tumor mutational burden (TMB in mut / MB; right y-axis) and resulting neoantigen load (total count) as per neoepitope prediction using WGS and RNA-seq of tumor cells and germline controls (Methods; left y-axis). FIG. 4E shows a graphical overview of patient cohort and procedure. N=14 NDMM patients were profiled by deep TCR sequencing (TCR-seq) of the alpha and beta TCR chain at baseline (pre-treatment) and 100 days post ASCT. N=6 NDMM patients (Tumor reactivity signature validation cohort) were profiled by scRNA / TCR sequencing of bone marrow and peripheral blood. FIG. 4F shows TCR clonality (see Methods) for each patient at each timepoint for each TCR chain. FIG. 4G shows TCR clonal dynamics over time for three donors with large increases in clonality following ASCT. Each bar represents a single beta chain TCR clone. The height of each bar at Baseline or Post-therapy represents the proportion of the total repertoire each clone occupied at that timepoint. Only clones that occupied >0.002% of the repertoire at either timepoint are shown. Bar color represents the temporal behavior of a TCR over therapy: expanded and contracted TCRs increased or decreased in size after therapy, respectively; while novel or disappeared TCRs were only observed post-therapy or at baseline, respectively. FIG. 4H shows a UMAPs depicting n = 3,829 BMR-T (initial diagnosis; top) and n = 4,188 BMR-T (dlOO post ASCT; bottom) color-coded by MM-TCR signature scored anti -tumor reactivity (predicted reactive = MM-TCR signature score > 0.42; predicted not reactive = MM-TCR signature score < 0.42). FIG. 41 show TCR clonal dynamics over time for one NDMM patient with following ASCT. Each bar represents a single TCR clone determined by scTCR-seq. Bar color represents the antitumor reactivity based on MM-TCR signature score. Log2 fold change (ASCT / initial diagnosis) shown. FIG. 4J shows a bar chart depicting average count of tumor reactive TCRs detected in the bone marrow of NDMM patients at initial diagnosis split by clinical IMWG consensus response category after induction (immuno-)chemotherapy. N=12 patients (Pt-01 to Pt-12; Table 2). Statistical significance between response groups was determined by one-way ANOVA with Tukey post-hoc test for multiple hypothesis testing correction. Statistical significance for linear trend with increasing response depth (PR - VGPR - nCR - CR MRD-) was determined by one-way ANOVA with Dunn’s post-hoc test for multiple hypothesis testing correction. PR, partial response; VGPR, very good partial response; CR, complete response; MRD, minimal residual disease.

[0038] FIGS. 5A-5I shows myeloma reactive T cells target shared cancer antigens. FIG. 5A shows total T cell counts (top) and TCR clonotype counts (bottom) retrieved from combined scRNA / TCR-seq of matching bone marrow (BM) biopsies and peripheral blood (PB) samples taken at initial diagnosis of multiple myeloma. N = 6 patients. Statistical significance was determined by a two-tailed paired t-test. FIG. 5B shows T cell antigen specificity of antigen- validated (‘deorphanized’) PB-derived (top) and BM-derived (bottom) T cells. Total number of TCRs per antigen indicated. TCR clonotypes were classified as shared between tissues if there was a fully matching CDR3 amino acid sequence across samples. TCRs derived from N = 6 patients, n = 45,777 (PB), n = 45,713 (BM) total TCRs post QC. FIG. 5C shows cells per clonotype in BM and PB averaged across all patients with matching BM and PB tissue and annotated by experimentally validated or VDJb-derived TCR antigen specificity. N = 6 biological replicates (matched patient-individual BM-PB datasets), n = 45,777 (PB), n = 45,713 (BM) total TCRs post QC FIG. 5D shows distribution of peptide amino acid (AA) length detected by immunoprecipitation of HLA class Lpeptide complexes from patient-autologous CD138+ multiple myeloma cell fraction followed by LC-MS / MS analysis (N=6 NDMM patients). FIG. 5E shows distribution of epitope calls and the non-healthy protein families they are derived from as detected by immunoprecipitation of HLA class Lpeptide complexes from patient-autologous CD 138+ multiple myeloma cell fraction followed by LC-MS / MS analysis (N=6 NDMM patients). CAAs, cancer-associated antigens; nuORFs, novel or unannotated open reading frames. FIG. 5F shows bar charts depicting tumoral MHC class Lderived antigens and the number of patients each antigen was detected in. Peptides eluted from MHC class I molecules of CD 138+ multiple myeloma cell fractions derived from N=6 NDMM patients. Only proteins with detection in >1 patient shown. CAAs, cancer-associated antigens; 5’ uORF, 5’ upstream open reading frame; 3’ dORF, 3’ downstream open reading frame; OOF, out-of-frame; ncRNA, non-coding RNA. FIG. 5G is a heatmap showing pairwise similarities of TCR TRA-TRB sequences based on scaled BLOSUM45-similarity. Only values above the 95% bootstrapping threshold as established by background distributions are displayed. TCRs are annotated by the respective patient of origin and clustered across all patients (N=12 NDMM patients). FIG. 5H (SEQ ID NO: 122-123) shows peptide-loaded MHC class I tetramer flow cytometry staining of TCR1 -expressing Pt-08 T cells (Methods). Tetramers were loaded with identified epitopes in f). Full experimental data found in Extended Data Fig. 14b. FIG. 51 (SEQ ID NO: 122-127) shows mRNA transfection and functionaltesting of transgenic TCR1 -expressing Pt-08 T cells. Tumor necrosis factor (TNF) was stained in TCR-transgenic T cells expressing the detected shared TCR in g) that were co-cultured with peptide-pulsed PBMCs (Methods). CEFT, pool of SARS-CoV-2 spike and nucleoproteins, major histocompatibility complex (MHC) class I-restricted cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza virus epitopes; MM, autologous multiple myeloma cells. N=2 independent co-cultures.

[0039] FIGS. 6A-6I show MM-TCR signature identifies TCRs responsive to bispecific antibodies. FIG. 6A provides a graphical overview of patient cohort and procedure. N=18 RRMM patients were profiled by single-cell RNA-seq and single-cell TCR-seq baseline (pre-treatment), post cycle 1 of bispecific BCMA x CD3 antibody treatment and post cycle 3 of bispecific BCMA x CD3 antibody treatment or at relapse. Patient-derived BMR-T were classified at baseline and clonal dynamics of each TCR traced over time. FIG. 6B (Left) shows a UMAP of T cell subtypes with productive TCR identified in the RRMM cohort (N=18 patients at 3 timepoints, n = 245,817 cells, n = 62,273 TCRs post QC); FIG. 6B (Right) shows a UMAP and bar chart of MM-TCR signature predictions in RRMM BMR-T at indicated timepoints (predicted reactive = MM-TCR signature score > 0.42; predicted not reactive = MM-TCR signature score < 0.42). Statistical significance between antigen specificity groups was determined by one-way ANOVA with Tukey post-hoc test for multiple hypothesis testing correction. FIG. 6C is a dot plot indicating the proportion of antigen-reactive or bystander BMR-T pre- and post-bispecific BCMAxCD3 antibody treatment, split by clinical response (N = 18 patients). Statistical significance was determined by repeated-measures ANOVA with Tukey post-hoc test for multiple hypothesis testing correction. FIG. 6D is a dot plot indicating the proportion of ITGB1+ predicted tumor reactive or non-tumor reactive BMR-T pre- and post-bispecific BCMAxCD3 antibody treatment (N = 18 patients). Statistical significance was determined by repeated-measures ANOVA with Tukey post-hoc test for multiple hypothesis testing correction. FIGs. 6E-6F show TCR clonal dynamics over time for representative RRMM patients following 3 cycles of bispecific BCMAxCD3 antibody treatment. Each bar represents a single TCR clone determined by scTCR- seq. The height of each bar at baseline or post-therapy represents the proportion of the total repertoire each clone occupied at that timepoint. Only clones that occupied >0.002% of the repertoire at either timepoint are shown. Bar color represents the anti-tumor reactivity based on MM-TCR signature score. FIGs. 6G-6H show bone marrow counts of T cells with TCRs detectedamong tumor reactive CD8+ BMR-T classified as effector-memory (EM; FIG. 6G) or progenitor- exhausted (PEX; FIG. 6H). Samples were collected from 18 patients with RRMM who experienced clinical remission (orange; n = 12) or poor clinical outcome (grey; n = 4) after 3 months of bsAb treatment. Patients with poor clinical outcome were further divided into those who did (n = 2) or did not experience (n = 4) immediate disease progression on-treatment. Single dots show values for patients with a single time point available. FIG. 61 is violin plot of MM-TCR signature expression per cell split by clinical response to bispecific BCMAxCD3 antibody treatment (nNR = 51,123 cells, nR = 64,615 cells). Statistical significance was determined by a two- tailed unpaired t-test.

[0040] FIGS. 7A-7L show the expansion of tumor reactive BMR-T underlies response to immune checkpoint inhibition in AML. FIG. 7A is a graphical overview of patient cohort and procedure. N=8 relapsed / refractory acute myeloid leukemia (R / R AML) patients were profiled by single-cell RNA-seq and single-cell TCR-seq baseline (pre-treatment), at response assessment / remission and at relapse. Patient-derived BMR-T were classified at baseline and clonal dynamics of each TCR traced over time. FIG. 7B is a UMAP of T cell subtypes with productive TCR identified in the R / R AML cohort (N=8 patients at 3 timepoints, n = 21,708 cells, n = 11,300 TCRs post QC). FIG. 7C is a UMAP of TCR BM classifier predictions in R / R MM BMR-T at indicated timepoints (predicted reactive = TCR BM signature score > 0.42; predicted not reactive = TCR_BM signature score < 0.42). FIG. 7D shows TCR_BM signature score in cells with a TCR exclusive pre-therapy, exclusive post-therapy or overlapping between pre- and post-therapy (n = 8 patients, 131,469 cells with productive TCR). Statistical significance was determined by oneway ANOVA with Tukey post hoc test for multiple hypothesis testing correction. FIG. 7E is a dot plot indicating the proportion of ITGB1+ antigen-reactive or bystander BMR-T pre- and post azacytidine + nivolumab (N = 8 patients). Statistical significance was determined by repeated- measures ANOVA with Turkey post-hoc test for multiple hypothesis testing correction. (FIG. 7F) UMAP depicting expansion of BMR-T clonotypes in AML patients (large = 0.01-1; medium = 0.001 - 0.001; small= 0.0001 - 0.001; rare = 0 - 0.0001). FIG. 7G shows relative abundance of expansion-categories within T cell clones of each AML patient on azacytidine + nivolumab grouped by clinical response category. FIG. 7H is a scatter plot indicating frequency among BMR- T of single T cell clones pre- and post-therapy with azacytidine + nivolumab aggregated across AML patients. The best clinical response of the patient each analyzed TCR is derived from isindicated by color. TCR classifier output for each clone indicated by shape. FIG. 7I-7K show TCR clonal dynamics over time for representative R / R AML patients on-treatment with azacytidine + nivolumab. Each bar represents a single TCR clone determined by scTCR-seq. The height of each bar at baseline or post-therapy represents the proportion of the total repertoire each clone occupied at that timepoint. Only clones that occupied >0.002% of the repertoire at either timepoint are shown. Bar color represents the anti-tumor reactivity based on TCR BM classifier score. FIG. 7L is a box plot of TCR_BM signature expression per cell split by clinical response and clinical sampling timepoint (diagnosis, remission, relapse). Statistical significance was determined by one-way ANOVA with Tukey post hoc test for multiple hypothesis testing correction.

[0041] FIG. 8A-8F show identification of antigen-specific bone-marrow associated T cells using MHC immunopeptidomes. FIG. 8A shows distribution of peptide amino acid (AA) length detected by immunoprecipitation of HLA class Lpeptide complexes from patient-autologous CD138+ multiple myeloma cell fraction followed by LC-MS / MS analysis (N=10 NDMM patients). FIG. 8B shows distribution of epitope calls and the non-healthy protein families they are derived from as detected by immunoprecipitation of HLA class Lpeptide complexes from patient-autologous CD138+ multiple myeloma cell fraction followed by LC-MS / MS analysis (N=10 NDMM patients). CAAs, cancer-associated antigens; nuORFs, novel or unannotated open reading frames. FIG. 8C shows bar charts depicting shared tumoral MHC class I-derived antigens and the number of patients each antigen was detected in. Peptides eluted from MHC class I molecules of CD 138+ multiple myeloma cell fractions derived from N=10 NDMM patients. Only proteins with detection in >2 patient shown. CAAs, cancer-associated antigens; 5’ uORF, 5’ upstream open reading frame; 3’ dORF, 3’ downstream open reading frame; OOF, out-of-frame; ncRNA, non-coding RNA. FIG. 8D shows the results MANAFEST assay in BMTC cultures of N = 9 NDMM patients exposed to the indicated antigen pools. Normalized clonal expansion for each TCR across conditions shown. Only significantly enriched TCRs as per FEST analysis are plotted. FIG. 8E shows a UMAP of identified tumor-reactive T cells in the full multiple myeloma cohort colored by screening technology. TCRs derived from N = 15 patients (Pts 01-15, n = 174,131 cells from 128,302 TCRs post QC). FIG. 8F shows stacked bar charts summarizing identified TCRs with tumor or virus specificities across all patients. Statistical significance was determined by two- way ANOVA with Tukey post-hoc test for multiple hypothesis testing correction.

[0042] FIGS. 9A-9I show myeloma reactive T cells target public or immunoglobulin-derived antigens. FIG. 9A (Left) shows a UMAP of BMTCs in the full multiple myeloma cohort colored by reactivity (Myeloma, SARS-CoV-2, Influenza-A, CMV, EBV, ambiguous (tumor / virus- reactive), and bystander (non-reactive). TCRs derived from N = 15 patients, n = 128,302 total TCRs post QC. FIG. 9A (Right) shows a UMAP of BMTCs in the full multiple myeloma cohort colored by recognized antigen. T cells responsive to shared MANA pool peptides are highlighted in yellow. TCRs derived from N = 15 patients, n = 128,302 total TCRs post QC. FIG. 9B shows T cell antigen specificity of antigen-validated (‘deorphanized’) T cells in peripheral blood (PB) and bone marrow (BM). Total number of TCRs per antigen indicated. TCR clonotypes were classified as shared between tissues if there was a fully matching CDR3 amino acid sequence across samples. TCRs derived from N = 6 patients, n = 45,777 (PB), n = 45,713 (BM) total TCRs post QC. FIG. 9C shows clonality (1 / Shannon diversity) of bone marrow TCRs split by reactivity. Statistical significance was determined by one-way ANOVA with Tukey post-hoc test for multiple hypothesis testing correction. FIG. 9D shows a X-Y scatter of average expression of cytotoxicity and dysfunction module scores in BMTC color-coded by antigen-recognizing TCR. Bystander T cells colored in grey. TCRs derived from N = 15 patients, n = 174,131 cells from n = 128,302 TCRs post QC. FIG. 9E shows a violin plot depicting cell-wise expression of cytotoxicity score split by antigen reactivity. Statistical significance was determined by two-way ANOVA with Tukey post-hoc test for multiple hypothesis testing correction. FIG. 9F shows a violin plot depicting cell-wise expression of dysfunction score split by antigen reactivity. Statistical significance was determined by two-way ANOVA with Tukey post-hoc test for multiple hypothesis testing correction. FIG. 9G shows a heatmap showing pairwise similarities of TCR TRA-TRB sequences based on scaled BLOSUM45-similarity. Only values above the 95% bootstrapping threshold as established by background distributions are displayed. TCRs are annotated by the respective patient of origin and clustered across all patients (N=15 NDMM patients). FIGS. 9H-9I show peptide-loaded MHC class I tetramer flow cytometry staining of BMTCs of various HLA-haplotypes (Methods). MHC-specific tetramers were loaded with the CTAG286-94 (RLLELHITM (SEQ ID NO: 128)) epitope for h) and 6 shared epitopes for i).

[0043] FIGS. 10A-10G show conserved transcriptional signatures of tumor-reactive BMTCs. FIG. 10A shows scaled average expression heatmap of top differentially expressed genes per reactivity category derived from N = 15 patients (Pts 01-15). FIG. 10B shows a ridge plot ofselected marker genes per reactivity group (Myeloma, Virus (SARS-CoV-2, Influenza-A, CMV, EBV), ambiguous (tumor / virus-reactive), and bystander (non-reactive) in signature establishment cohort. TCRs derived from N = 15 patients, n = 174.131 cells post QC. FIG. IOC shows a UMAP of subsetted validated tumor-reactive clonotypes (N=15 patients, n = 938 cells). FIG. 10D shows UMAPs overlaid with gene-weighted density of indicated genes. FIG. 10E shows a dot plot outlining the average expression of MM-TCR signature marker genes between bone marrow TCRs of indicated specificities in the establishment cohort. FIG. 10F shows prospective area under the curve (AUC) of receiver operator characteristic (ROC) shown (Methods). AUROC curves of MM- TCR (AUC: 0.895), MANA_Caushi5(AUC: 0.810) NeoTCR_8 (AUC: 0.812) and NeoTCR_4 (AUC: 0.563)7signature scores to predict tumor reactivity in validation cohort TCRs (N = 9 patients (Pts 07-15), n = 101,256 cells, n = 1302 validated myeloma reactive BMTCs). FIG. 10G shows a bar chart depicting average count of tumor-reactive TCRs detected in the bone marrow of NDMM patients at initial diagnosis (left) or frequency of tumor-reactive BMTCs per MM-TCR signature (right) split by remission status after induction (immuno-)chemotherapy. N=14 patients (Pt-01 to Pt-15). Induction therapy response for Pt-08 was not available. Statistical significance between response groups was determined by unpaired t-test with Welch’s correction. CR, complete response.

[0044] FIGS. 11A-11I show MM-TCR signature identifies TCRs responsive to bispecific antibodies. FIG. 11A shows a graphical overview of patient cohort and procedure. N=18 RRMM patients were profiled by single-cell RNA-seq and single-cell TCR-seq baseline (pre-treatment), post cycle 1 of bispecific BCMA x CD3 antibody treatment and post cycle 3 of bispecific BCMA x CD3 antibody treatment or at relapse. Patient-derived BMR-T were classified at baseline and clonal dynamics of each TCR traced over time. FIG. 11B (Left) shows a UMAP of T cell subtypes with productive TCR identified in the RRMM cohort (N=18 patients at 3 timepoints, n = 245,817 cells, n = 62,273 TCRs post QC). FIG. 11B (Right) shows a UMAP of MM-TCR signature predictions in RRMM BMR-T at indicated timepoints (predicted reactive = MM-TCR signature score > 0.42; predicted not reactive = MM-TCR signature score < 0.42). FIG. 11C shows a dot plot indicating the proportion of antigen-reactive or bystander BMR-T pre- and post bispecific BCMAxCD3 antibody treatment, split by clinical response (N = 18 patients). Statistical significance was determined by repeated-measures ANOVA with Tukey post-hoc test for multiple hypothesis testing correction. FIG. 11D shows TCR clonal dynamics over time for representativeRRMM patients following 3 cycles of bi specific BCMAxCD3 antibody treatment. Each bar represents a single TCR clone determined by scTCR-seq. The height of each bar at baseline or post-therapy represents the proportion of the total repertoire each clone occupied at that timepoint. Only clones that occupied >0.002% of the repertoire at either timepoint are shown. Bar color represents the anti -tumor reactivity based on MM-TCR signature score. FIG. HE shows TCR clonal dynamics over time for representative RRMM patients following 3 cycles of bispecific BCMAxCD3 antibody treatment. Each bar represents a single TCR clone determined by scTCR- seq. The height of each bar at baseline or post-therapy represents the proportion of the total repertoire each clone occupied at that timepoint. Only clones that occupied >0.002% of the repertoire at either timepoint are shown. Bar color represents the anti-tumor reactivity based on MM-TCR signature score. FIG. HF shows bone marrow counts of T cells with TCRs detected among tumor reactive CD8+ BMTC classified as effector-memory (EM) Samples were collected from 18 patients with RRMM who experienced clinical remission (orange; n = 12) or poor clinical outcome (grey; n = 4) after 3 months of bsAb treatment. Patients with poor clinical outcome were further divided into those who did (n = 2) or did not experience (n = 4) immediate disease progression on-treatment. Single dots show values for patients with a single time point available. FIG. 11G shows bone marrow counts of T cells with TCRs detected among tumor reactive CD8+ BMTC classified as progenitor-exhausted (PEX; FIG. 11H). Samples were collected from 18 patients with RRMM who experienced clinical remission (orange; n = 12) or poor clinical outcome (grey; n = 4) after 3 months of bsAb treatment. Patients with poor clinical outcome were further divided into those who did (n = 2) or did not experience (n = 4) immediate disease progression on- treatment. Single dots show values for patients with a single time point available. FIG. 11H shows a violin plot of MM-TCR signature expression per cell split by clinical response to bispecific BCMAxCD3 antibody treatment (UNR = 51,123 cells, UR = 64,615 cells). Statistical significance was determined by a two-tailed unpaired t-test. FIG. HI is shows clinical response status.

[0045] FIGS. 12A-12L show transfer of tumor-reactive TCRs by autologous stem cell transplantation. FIG. 12A shows TCR clonality (1 / Shannon diversity) for N=14 NDMM patients profiled by TRVa / p-seq at baseline (initial diagnosis) and after 100 days post ASCT for each TCR chain. FIG. 12B shows TCR clonal dynamics over time for one NDMM patient with following ASCT. Each area in the alluvial plot represents a single TCR clone determined by scTCR-seq. The height of each bar at baseline or post-therapy represents the proportion of the total repertoire eachclone occupied at that timepoint. Color shows, if the clone was found in the PBSC product. Antitumor reactivity is based on the previously defined signature, calculated on a per-clone level at diagnosis, (tumor-reactive = clone-aggregated signature score > 0.55; not reactive = clone- aggregated signature score < 0.55). FIG. 12C shows clonal dynamics of N = 9 patients with paired single-cell and VDJ data at diagnosis and post-TPL. Significant clonotype dynamics were determined by a bootstrapping approach stratified to number of cells at each time point followed by Benjamini-Hochberg correction for multiple testing. Reactivity was determined per clone with the previously established signature. FIG. 12D shows representative gating strategy of PBSC samples subjected to multiparametric flow cytometry. FIG. 12E shows frequency of CD45hi CD3+ T cells and Lin- CD34+ CD38- HSCs / MPPs in PBSC samples from N=19 multiple myeloma patients. FIG. 12F shows single-cell RNA and VDJ-sequencing data of PBSC products of 5 patients. UMAP of T cell subtypes with productive TCR. FIG. 12G shows TCR clonal dynamics over time for the patient shown in FIG. 12H after the transplantation. Each area in the alluvial plot represents a single TCR clone determined by scTCR-seq. The height of each bar at baseline or post-therapy represents the proportion of the total repertoire each clone occupied at either +100 or +360 days after stem cell transplant. Color shows if the clone was found in the PBSC product. Anti-tumor reactivity is based on the previously defined signature, calculated on a per-clone level at diagnosis. FIG. 121 shows clonal dynamics of predicted tumor-reactive clones after the autologous stem-cell transplantation. Color shows the different previously established expansion categories as described in the methods. Clone frequencies are separately compared for clones in the PBSC and predicted reactive. FIG. 12J shows differential expression analysis of clones at diagnosis based on if the clones were found in PBSC. Differential expression was assessed by DESeq2 based on patient-level pseudo-bulk aggregation of expression. Visualized as volcano plot using the R package EnhancedVolcano. FIG. 12K shows linear mixed-effects logistic regression analysis for identifying factors predicting likelihood of apheresis on a TCR-clone-level . Figure shows a forest plot of odds ratios with 95% confidence intervals, highlighting significance of tumor-reactive signature, broad cell type classification and previously established expansion characteristics. FIG. 12L shows summarized alluvial of T-cell subtype fractions of shared and unshared clones across the time course of diagnosis, PBSC and +100 and +360 days after transplantation. Figure is split into shared and unshared clonotypes, as defined by not found in the PBSC product.

[0046] Analysis of fractions of exhausted-like (PEX) T-cells throughout the time course. Comparison of fractions of shared and not-transplanted T-cells before and after the transplantation per patient (N=5). Statistical significance was determined by repeated-measures two-way ANOVA followed by Sidak’s test for multiple hypothesis testing correction.

[0047] FIGS. 13A-13D shows profiling of BMR-T in newly diagnosed multiple myeloma. FIG. 13A show a representative gating strategy used for purification of CD45+ and CD3+ cells by fluorescence- activated cell sorting (FACS). Sorted populations were then processed using the lOx Genomics 5’ single-cell sequencing strategy (methods). FIG. 13B show a Uniform Manifold Approximation and Projection (UMAP) map of T cells. Overlay highlights the average expression of indicated canonical T cell surface proteins detected by CITE-seq. EXT. FIG. 13C-13D a dot plots indicating expression of canonical marker genes across CD8+ (c) and CD4+ (d) clusters. Marker gene lists derived from Zheng et al., Science 202147, Cohen et al., Nat Cancer 202248, and Andreatta et al., Nat Commun. 202149.

[0048] FIGS. 14A-14B show bone marrow immune repertoire composition in establishment patient cohort. FIG. 14A show a Uniform Manifold Approximation and Projection (UMAP) map of reference-mapped and subsetted T cells post integration and QC split by patient and color-coded for annotated transcriptional clusters. FIG. 14B show a proportion of T cell subtypes in individual patient bone marrow samples evaluated by scRNA-seq.

[0049] FIGS. 15-16 show fluorescence imaging of BMR-T identified in establishment NDMM cohort by microfluidics-based forward tumor reactivity screening. Myeloma reactive T cells were detected among BMR-T from bone marrow biopsies of NDMM patients. Reactive T cells were identified upon detection of secreted cytokines IFN-y, IL-2, TNF (yellow) and surface expression of 4-1BB protein (CD137; blue). Per experimental run, approximately 1,400 individual CD8+ T cells were co-cultured with CD138+ autologous plasma cells after magnetic bead-based isolation from patient bone marrow samples. NEG: A reaction chamber containing a single T cell + cytokine capture beads only. POS: A reaction chamber containing a single T cell plus human aCD3 / uCD28 T cell activation beads. +, positive; (+), dim positive; (-), negative for cytokine secretion or 4- IBB expression; ND, due to a non-loaded cytokine capture bead, the respective cytokine could not be determined.

[0050] FIGS. 17A-17D show phenotypes of TCRs recovered from patient-derived tumor reactive T cells. FIG. 17A amplified V(D)J regions of TCR chains are visible at 500 to 700 bp.TCR alpha and beta chains are similar in length and therefore mostly visible as a single band. Due to alternative splicing, double bands can be generated in some cases. 5% agarose gels in TBE shown. FIG. 17B show transcriptional cluster composition of each successfully to scRNA / TCR- seq mapped CD4+ and CD8+ T cell clonotype. Relative abundance of cells in each cluster per clonotype shown. FIG. 17C show linearized DNA templates used for in vitro transcription (IVT) of retrieved and inserted TCRA / B V(D)J sequences together with murine TRAC or TRBC chains. mRNA products were then transfected into primary T cells for functional testing of transgenic TCR recognition. FIG. 17D show the representative flow cytometry gating strategy to identify T cells expressing transfected transgenic TCRs by detection of the murine TRBC chain (mTRBC). Activation state of these cells was then measured by CD69 or CD 137 surface protein expression.

[0051] FIGS. 18A-18H show functional expansion of tumor reactive T cells on BMR-T in establishment cohort. FIG. 18Ashow absolute T cell count in assay at baseline (dO) and post expansion (d28) per patient. FIG. 18B show a UMAP highlighting expanded (proportion > 0.01) clones and not expanded (proportion < 0.01) clones. Cluster phenotype annotation as in Fig. lb. FIG. 18C a UMAP of BMR-T clonal expansion categories split by patient. FIG. 18D shows a TCR clonal homeostasis per patient at baseline input of BMNC expansion culture (dO). FIG. 18E shows TCR clonal homeostasis per patient after BMNC expansion culture (d28). EXT. FIG. 18F shows Shannon diversity index of TCRs sequenced in BMR-T cultures at dO and d28 of BMNC expansion culture. Statistical significance was determined by a two-tailed paired t-test. FIG. 18G shows a bar chart of T cell subtype composition of large (proportion > 0.01 in bone marrow) clones and small (proportion < 0.01 in bone marrow) clones by patient. FIG. 18H shows a heatmap of scaled average expression of top 20 marker genes of small (proportion > 0.01) non-reactive T cell clones, large (proportion > 0.01) non-reactive T cell clones, small (proportion > 0.01) reactive T cell clones, large (proportion > 0.01) reactive T cell clones.

[0052] FIGS. 19A-19B shows retrospective and prospective TCR signature benchmarking of MM-TCR signature versus published signatures of tumor-infdtrating lymphocytes. FIG. 19A) Heatmap depicting AUROC values of published and generated signatures to predict tumor reactive TCRs in BMR-T of establishment NDMM cohort (retrospective). N=6 patients. FIG. 19B shows a heatmap depicting AUROC values of published and generated signature sig9 = MM-TCR to predict tumor reactive TCRs in BMR-T of validation NDMM cohort (prospective). N=6 patients.

[0053] FIGS. 20A-20F shows trajectory and fate mapping of tumor reactive and bystander BMR-T using RNA velocities and CellRank. FIGS. 20A-20Bshows assessment of a) average and b) patient- wise spliced versus unspliced mRNA ratio detected by 5’ scRNA-seq of primary BMR- T. FIG. 20C shows a UMAP of subclustered CD8+ T cells colored according to original cluster annotations overlaid by RNA velocities as computed by CellRank scVelo algorithm. FIG. 20Dshows density plots indicating module scores of cytotoxicity (left) and dysfunction (right) signatures overlaid on UMAP from c. Functional signatures derived from Li et al., Cell 2019a27. FIG. 20E shows module scores for T cell cytotoxicity (top) and dysfunction (bottom) for each cluster. Functional signatures derived from Li et al., Cell 2019a27. FIG. 20Fshows heatmap visualizing lineage drivers computed for tumor reactivity. Smooth gene expression for the putative tumor reactivity driver genes in latent time, using as cell-level weights the Alpha fate probabilities. Genes sorted according to their peak in latent time (proportion of cells contributing to each bin shown at the bottom), thus revealing a cascade of gene expression events.

[0054] FIGS. 21A-21E shows profiling and tumor reactivity classification of BMR-T in validation NDMM cohort. FIG. 21A shows a UMAP of T cell subtypes with productive TCR identified in the NDMM validation cohort (N=6 patients, n = 101,210 cells, n = 81,122 TCRs post QC). FIG. 21B shows a UMAP depicting expansion of BMR-T clonotypes (large = 0.01-1; medium = 0.001 - 0.001; small= 0.0001 - 0.001; rare = 0 - 0.0001). FIG. 21C shows relative abundance of expansion-categories within cells of each patient. FIG. 21D shows a UMAPs depicting BMR-T per validation cohort patient color-coded by MM-TCR signature scored antitumor reactivity (predicted reactive = MM-TCR signature score > 0.42; predicted not reactive = MM-TCR signature score < 0.42). FIG. 21E shows average clonotype proportion in sample as dot size by T cell subtype within each patient.

[0055] FIGS. 22A-22B shows tumor reactive BMR-T identified in validation NDMM cohort by microfluidics-based forward tumor reactivity screening. FIG. 22A shows myeloma reactive T cells were detected among BMR-T from bone marrow biopsies of NDMM patients. Reactive T cells were identified upon detection of secreted cytokines IFN-y, IL-2, TNF (yellow) and surface expression of 4-1BB protein (CD137; blue). Per experimental run, approximately 1,400 individual CD8+ T cells were co-cultured with CD138+ autologous plasma cells after magnetic bead-based isolation from patient bone marrow samples. NEG: A reaction chamber containing a single T cell + cytokine capture beads only. POS: A reaction chamber containing a single T cell plus humanaCD3 / aCD28 T cell activation beads. +, positive; (+), dim positive; (-), negative for cytokine secretion or 4-1BB expression; ND, due to a non-loaded cytokine capture bead, the respective cytokine could not be determined. FIG. 22B shows amplified V(D)J regions of TCR chains are visible at 500 to 700 bp. TCR alpha and beta chains are similar in length and therefore mostly visible as a single band. Due to alternative splicing, double bands can be generated in some cases. 5% agarose gels in TBE shown.

[0056] FIGS. 23A-23M shows tumor reactive T cells expand upon autologous stem cell transplantation. FIG. 23A shows the number of total TCR counts fit to log 10 for each patient’s repertoire at each timepoints for each TCR chain (ASCT cohort, N=14 patients). FIG. 23B shows TCR clonality quantified using the Renyi Entropy from order 0 to infinity for each patient, with the average of the timepoints for each treatment arm and TCR chain overlayed in bold. FIG. 23C shows the individual total TCR counts fit for each patient at each timepoint for each TCR chain. FIG. 23D (SEQ ID NO: 129-187) shows heatmap depicting longitudinal changes of TCR frequency in bone morrow between initial diagnosis and day 100 post-ASCT. TCRs classified and color-coded by MM-TCR signature score for anti-tumor reactivity (predicted reactive = MM-TCR signature score > 0.42; predicted not reactive = MM-TCR signature score < 0.42). FIG. 23E shows a Uniform Manifold Approximation and Projection (UMAP) map of reference-mapped and subsetted T cells post integration and QC split by time point (initial diagnosis (a) and day 100 post- ASCT (b)). Cluster phenotype annotation as in Fig. lb. FIG. 23F shows a T cell subtype composition in clones at initial diagnosis and post ASCT that were either classified as antimyeloma reactive (blue) or non-reactive bystander (grey). FIG. 23G (top) shows a graphical overview of patient Pt-07 and procedure. A 57-year-old male with NDMM underwent bone marrow biopsy, followed by prospective prediction of reactive BMR-T TCRs using the MM-TCR classifier. BMR-T were then tested using the microfluidics-based forward screening assay and outcomes compared on a per-clone basis between anti-tumor reactivity prediction and measured reactivity. Prospective sensitivity and reactivity of the MM-TCR classifier was then compared to published tumor reactive TCR signatures. FIG. 23G (bottom) shows a UMAPs depicting n = 9,148 BMR-T at initial diagnosis color-coded by TCR BM classifier scored anti-tumor reactivity (predicted reactive = TCR_BM signature score > 0.42; predicted not reactive = TCR_BM signature score < 0.42). N = 9148 BMR-T cells. FIG. 23H representative results of microfluidics-based forward screening assay of BMR-T isolated from Pt-07 in e). FIG. 231 UMAPs depicting n =9,148 BMR-T at initial diagnosis color-coded by validated anti-tumor reactivity. Primary transcriptional phenotype of each detected TCR annotated. FIG. 23J shows counts of tumor reactive T cells (Part of TCR1 and TCR2 clonotypes) in bone marrow and peripheral blood of Pt- 07. FIG. 23K shows prospective area under the curve (AUC) of receiver operator characteristic (ROC) shown (Methods). AUROC curves of MM-TCR (AUC: 0.9845), MANA_Caushi5 (AUC: 0.9184) NeoTCR_8 (AUC: 0.9431) and NeoTCR_4 (AUC: 0.6067)7 signature scores to predict tumor reactivity in Pt-07 TCRs (n = 9,148 cells, n = 478 validated myeloma reactive BMR-T). FIG. 23L shows summary statistics of MM-TCR classifier per detected TCR in Pt-08 (n = 9,148 cells, n = 7,548 TCRs). FIG. 23M shows blood serum IgG and M protein concentrations [g / L] in Pt-07 over time. Clinical response assessment results according to IMWG response criteria at indicated timepoints post diagnosis shown.

[0057] FIGS. 24A-24E shows compartment tracing of antigen-specific patient TCRs and tumor-associated antigens detected by MHC class I immunoprecipitation. FIGS. 24A-24C show UMAPs depicting T cells in bone marrow and peripheral blood at initial diagnosis color-coded by transcriptional phenotype (a), overlap between both compartments (b), or tumor reactivity status (c). Primary transcriptional phenotype of each detected T cell annotated as in Fig. lb. FIG. 24D shows a dot plot indicating number of T cells (left) and TCR clonotypes (right) and their reactivity status in each analyzed NDMM patient with available matching bone marrow and peripheral blood. BMNCs, bone marrow mononuclear cells; PBMCs peripheral-blood mononuclear cells. FIG. 24E show bar charts depicting tumoral MHC class I-derived antigens and the number of patients each antigen was detected in. Peptides eluted from MHC class I molecules of CD 138+ multiple myeloma cell fractions derived from N=6 NDMM patients. CAAs, cancer-associated antigens; 5’ uORF, 5’ upstream open reading frame; 3’ dORF, 3’ downstream open reading frame; ncRNA, non-coding RNA; lincRNA, Long intergenic non-coding RNA.

[0058] FIG. 25 shows TCR sequence sharing in tumor and virus reactive BMR-T.

[0059] Heatmaps showing pairwise similarities of TCR TRA-TRB sequences split by tested antigen recognition and based on scaled BLOSUM45-similarity (Methods). TCRs are annotated by the respective patient of origin and clustered across all patients (N=12 NDMM patients).

[0060] FIGS. 26A-26C show epitope validation of a tumor reactive TCR shared by three NDMM patients. FIG. 26A (SEQ ID NO: 188-227) shows a network diagram of similar tumor reactive CDR3 sequences. Pairwise similarities of TCR TRA-TRB sequence are based on scaledBLOSUM45-similarity. Only events above the 95% bootstrapping threshold as established by background distributions are displayed. TCRs are annotated by the respective patient of origin and clustered across all patients (N=12 NDMM patients). Consensus TRA and TRB sequences for TCR tested in b-c) shown on the left. FIG. 26B (SEQ ID NO: 228-233) shows MHC class I- derived peptide-loaded MHC tetramer flow cytometry staining of autologous BMR-T (methods). Epitope sequences of tested tumor antigens found in Pt-08 by MHC class I immunoprecipitation indicated. FIG. 26C (SEQ ID NO: 228-233) shows fold change (FC) clonal expansion of Pt-08 BMR-T in antigen-specific T cell expansion assay from dO to d28 shown as determined by longitudinal TCR sequencing. Irradiated autologous PBMCs loaded with indicated epitopes of tumor antigens found in Pt-08 by MHC class I immunoprecipitation.

[0061] FIGS. 27A-27C show tumor reactive T cells expand upon autologous stem cell transplantation. FIG. 27A shows MM-TCR signature score in cells with a TCR exclusive pre bsAb therapy, exclusive post bsAb therapy or overlapping between pre- and post bsAb therapy (n = 16 patients, 131,469 cells with productive TCR). Statistical significance was determined by one-way ANOVA with Tukey post hoc test for multiple hypothesis testing correction. FIG. 27B shows a dot plot indicating the proportion of antigen-reactive or orphan BMR-T pre- and post bsAb treatment, split by clinical response (N = 18 patients). Statistical significance was determined by repeated-measures ANOVA with Turkey post-hoc test for multiple hypothesis testing correction. FIG. 27C shows a UMAPs and bar charts indicating the proportion of predicted tumor reactive, virus reactivy and orphan TCRs among RRMM patient BMR-T at initial diagnosis (predicted reactive = MM-TCR signature score > 0.42; predicted not reactive = MM-TCR signature score < 0.42). N=18 patients treated with bispecific BCMAxCD3 antibodies.

[0062] FIG. 28 shows fluorescence imaging of BMTCs targeting multiple myeloma by antigen-agnostic microfluidics screening.

[0063] FIGS. 29 and 30 show amplified TCRs from tumor-reactive BMTCs retrieved from antigen-agnostic microfluidics screening.

[0064] FIGS. 31A-31G show phenotype composition and cloning of TCRs targeting multiple myeloma retrieved from antigen-agnostic microfluidics screening.

[0065] FIGS 32A-32D, 33A-33D, and 34 show tumor specificity validation of TCRs targeting multiple myeloma retrieved from antigen-agnostic microfluidics screening.

[0066] FIG. 35 shows MHC class I blocking experiments of TCRs targeting multiple myeloma retrieved from antigen-agnostic microfluidics screening.

[0067] FIGS. 36A-36C shows fluorescence imaging of peripheral blood T cells targeting acute myeloid leukemia by antigen-agnostic microfluidics screening.

[0068] FIGS. 37A-37C show fluorescence imaging of peripheral blood T cells targeting chronic lymphocytic leukemia by antigen-agnostic microfluidics screening.

[0069]

[0070] FIGS. 38A-38F show compartment tracing of antigen-specific TCRs in multiple myeloma patients.

[0071] FIGS. 39A and 39B show TCR sequence similarities in tumor-reactive BMTCs in multiple myeloma patients.

[0072] FIGS. 40A and 40B show TCR sequence similarities in virus-specific and random BMTCs in multiple myeloma patients.

[0073] FIGS. 41A-41C show epitope mapping of a tumor-reactive TCR shared by three multiple myeloma patients (41A - SEQ ID NO: 234-280) (41B - SEQ ID NO: 228-233), (41C - SEQ ID NO: 228-233).

[0074] FIGS. 42A, 42B (SEQ ID NO: 281-286), and 43A-43E (43A - SEQ ID NO: 287-303) show bone marrow reactivity screening against personalized and shared antigens identified in multiple myeloma immunopeptidomes.

[0075] FIGS. 44A and 44B show retrospective and prospective TCR signature benchmarking of MM-TCR signature versus published signatures of tumor-infiltrating lymphocytes.

[0076] FIGS. 45A-45F show CD29 (JTGB1 as marker gene of tumor specific T cells.

[0077] FIGS. 46A-46F show clinical trial cohort of TCRV0 multiple myeloma patients undergoing ASCT.

[0078] FIGS. 47A and 47B show tumor-reactive T cells expand upon ASCT.

[0079] FIG. 48 shows transfer of tumor-reactive T cells with ASCT.

[0080] FIG. 49 shows persistence of tumor-reactive T cells one year after ASCT.

[0081] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTSGeneral Definitions

[0082] Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew etal. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0083] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0084] The term “optional” or “optionally” means that the subsequent described event, circumstance, or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0085] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0086] The term “about” or “approximately,” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, such as variations of + / -10% or less, +7-5% or less, + / - 1% or less, and + / -0.1% or less of and from the specified value, insofar such variations areappropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0087] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0088] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Examples of subjects / patients include humans and non-human mammals, e.g., non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. In specific embodiments, the subject is a human.

[0089] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described hereininclude some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0090] The “tumor infiltrating lymphocyte (TIL)” used herein means a lymphocyte infiltrating cancer tissue or a tumor microenvironment (TME) after moving from the bloodstream to the site of tumor tissue.

[0091] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.OVERVIEW

[0092] The present disclosure relates to a platform that may be used to identify and enrich for disease-reactive T cells in a particular disease context, for example cancer-reactive T cells. The platform enables the identification of gene expression profiles that characterize the reactive T cells allowing the disease-reactive T cells to be cloned and further characterized. These gene expression profiles may also be used as a prognostic marker to improve treatment outcomes and to select patients that would most benefit from the therapeutic modalities discussed herein. For example, as further detailed herein, the gene expressions profiles can predict a consistent response to cell based therapies, antibody based therapeutics, including bi-specific antibodies, and disease-specific vaccines. In one aspect, the embodiments disclosed herein are directed to T cell receptors from the identified cancer-reactive T cells and their use in preparing engineered cell therapy products. The present disclosure also relates to methods for identifying the specific antigens recognized by the disease-reactive T cells. As detailed further herein, the methods enable the identification of antigens that are found across multiple patients in a given disease setting leading to a convergence of shared immune responses and the potential for off-the-shelf cell therapeutics comprising T cell receptors targeting such antigens, and more effective vaccines comprising such antigens.ENGINEERED IMMUNE CELLS

[0093] In one aspect, embodiments disclosed herein are directed to engineered immune cells comprising the disease-reactive antigen receptors identified using the methods disclosed herein. The engineered immune cell may be a CD4+ T cell, a CD8+ T cell, or a natural killer (NK) T cell. The immune cell may be autologous or allogenic. The immune cell may be a chimeric antigenreceptor (CAR) T cell, wherein the CAR comprises all or an antigen-binding portion of a TCR identified using the methods disclosed herein. The engineered immune cell may be a tumorinfiltrating lymphocyte (TIL) identified as comprising or engineered to comprise TCRs identified using the methods disclosed herein and expanded ex vivo before being administered to a patient in need thereof. The ex vivo expansion may include culturing the TIL in specific culture conditions that modify a phenotype or gene expression profile of the TIL from its natural state. The TIL may also be formulated in a composition that comprises additional molecules, such as cytokines, to enhance TIL cell acceptance by a patient and / or TIL activity. The engineered immune cell may further comprise one or more modifications, for example one or more gene modifications to modify antigen processing by the cell. The one or more modifications may comprise editing to knock-out or knock-down expression of B2M, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 IB 1 (CYP1B), HER2 / neu, Wilms’ tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53 or cyclin (DI) (see W02016 / 011210). In an embodiment, the T cells are edited ex vivo by CRISPR to knock-out or knock down the expression of an antigen selected from B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI), or B-cell activating factor receptor (BAFF-R), CD38, CD138, CS-1, CD33, CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, or CD362.

[0094] In one embodiment, the engineered immune cell comprises a TCR capable of recognizing a cancer-associated antigen. In one embodiment, the cancer-associated antigen is an antigen associated with a hematological malignancy. The hematological malignancy may be a leukemia, a lymphoma, a myeloma, myelodysplastic syndrome, a myeloproliferative neoplasm, a histocytic disorder. The leukemia may be acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute promyelocytic leukemia. The lymphoma may be a Non-Hodgkin’s lymphoma or Hodgkin’s lymphoma. The Non-Hodgkin lymphoma may be diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, T-cell lymphoma, or Waldenstrom’s macroglobulinemia. The myeloma may be multiple myeloma or light chain amyloidosis myeloma. In one embodiment, the hematological malignancy is multiple myeloma. In another embodiment,the hematological malignancy is a leukemia. In one embodiment, the leukemia is acute myeloid leukemia.

[0095] In one embodiment, the engineered immune cell comprises a TCR capable of recognizing a microbial-associated antigen including virus-associated antigens, bacteria- associated antigens, fungal -associated antigens, and parasite-associated antigens.TCRs

[0096] Provided herein are TCRs or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha region and a beta chain comprising a variable beta region. The variable regions include a complementary determining region 1 (CDR-1), a complementary determining region 2 (CDR-2), and a complementary determining region 3 (CDR-3). The TCR is a heterodimer composed of two different protein chains. The highly polymorphic TCR is generated by joining of non-contiguous gene segments (VP, Dp, jp for TCRP and Va, Ja for TCRa) together with deletion / insertion of random sequences at junctions and Recombination Signal Sequences (RSS) to form the highly variable CDR3 regions. The recognition of MHC -bound peptide by the combined TCRP and TCRa proteins occurs primarily by the CDR3 regions (see e.g., Robins HS, Srivastava SK, Campregher PV, et al. Overlap and effective size of the human CD8+ T cell receptor repertoire. Sci Transl Med. 2010;2(47):47ra64). In most T cells (about 95%), these two protein chains are termed the alpha (a) and beta (P) chains. However, in a small percentage of T cells (about 5%), these two protein chains are termed the gamma and delta (y / 8) chains. The ratio of TCRs comprised of a / p chains versus y / 8 chains may change during a diseased state. When the TCR engages with antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0097] In one embodiment, the TCR or antigen-binding fragment thereof binds to or recognizes one or more peptide epitopes. In an embodiment, the TCR or antigen-binding fragment thereof, when expressed on the surface of a T cell, stimulates cytotoxic activity against a target cell. In an embodiment, the target cell is a cancer cell.

[0098] In an embodiment, the TCR is encoded by a nucleotide sequence that has been codon- optimized. In an embodiment, the alpha and / or beta chain further comprise a signal peptide. In particular embodiments, the TCR is isolated or purified or is recombinant. In an embodiment, theTCR is human. In an embodiment the TCR is monoclonal. In an embodiment, the TCR is singlechain. In an embodiment the TCR comprises two chains.

[0099] Provided herein are nucleic acid molecules encoding any of the provided TCRs, or an alpha or beta chain thereof. In one embodiment the nucleotide sequence is codon-optimized. Provided herein is a vector comprising a nucleic acid of any provided herein. In an embodiment, the vector is an expression vector. In particular embodiments, the vector is a viral vector. Provided herein is an engineered cell comprising the nucleic acid molecule of any provided herein or vector of any provided herein. Also provided herein is an engineered cell, including the TCR of any provided herein. In an embodiment the TCR is heterologous to the cell. In an embodiment, the engineered cell is a cell line. In particular embodiments, the engineered cell is a primary cell obtained from a subject. In an embodiment, the subject is a mammalian subject. In an embodiment, the subject is human. In particular embodiments, the engineered cell is a T cell. In an embodiment, the T cell is CD8+. In an embodiment, the T cell is CD4+.

[0100] In an embodiment, TCRs are identified that recognize a tumor antigen. The term “tumor antigen” as used throughout this specification refers to an antigen that is uniquely or differentially expressed by a tumor cell, whether intracellular or on the tumor cell surface (preferably on the tumor cell surface), compared to a normal or non-neoplastic cell. By means of example, a tumor antigen may be present in or on a tumor cell and not typically in or on normal cells or non-neoplastic cells (e.g., only expressed by a restricted number of normal tissues, such as testis and / or placenta), or a tumor antigen may be present in or on a tumor cell in greater amounts than in or on normal or non-neoplastic cells, or a tumor antigen may be present in or on tumor cells in a different form than that found in or on normal or non-neoplastic cells. The term thus includes tumor-specific antigens (TSA), including tumor-specific membrane antigens, tumor-associated antigens (TAA), including tumor-associated membrane antigens, embryonic antigens on tumors, growth factor receptors, growth factor ligands, etc.

[0101] In one embodiment, the engineered immune cell comprises a TCR capable of recognizing an antigen in SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.

[0102] In one embodiment, the engineered immune cell comprises a TCR comprising a TCR alpha chain CDR3 sequence selected from SEQ ID NO: 1-62, 41855-41902 or a TCR beta chainCDR3 sequence selected from SEQ ID NO: 63-121 or 41903-41948. In one embodiment, the TCR comprise recognizes CTAG2 or IGKV. In an embodiment, TCR comprises an alpha or beta chain CDR3 sequences of TCR No. 11729 or 15343 from Table 8.CARs

[0103] In some aspects, the genetically engineered antigen receptor is a T cell receptor (TCR) or a functional non-TCR antigen recognition receptor. In an embodiment, it is a chimeric antigen receptor (CAR), such as an activating or stimulatory CAR, an inhibitory CAR and / or a costimulatory CAR. Among the CARs are those with an extracellular antigen-recognition domain that specifically binds to the target antigen and an intracellular signaling domain comprising an ITAM, such as an intracellular domain of a CD3-zeta (CD3Q chain those that further comprise a costimulatory signaling region, such as a signaling domain of CD28 or 41BB. In an embodiment, the CAR comprises an extracellular antigen-recognition domain that specifically binds to the target antigen and an intracellular signaling domain that comprises a signaling portion of an immune checkpoint molecule, such as PD-1 or CTLA4.

[0104] In an embodiment, the engineered antigen receptors include chimeric antigen receptors (CARs), including activating or stimulatory CARs, costimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5 (215) (December, 2013). The CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in an embodiment, via linkers and / or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone.

[0105] In an embodiment, CAR is constructed with specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and / or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen-binding molecules, such as one or more antigenbinding fragments, domains, or portions, or one or more antibody variable domains and / or antibody molecules. In an embodiment, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0106] In an embodiment, the CAR contains an antibody or an antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell.

[0107] In an embodiment, the CAR contains a TCR-like antibody, such as an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface as an MHC-peptide complex. In an embodiment, an antibody or antigen-binding portion thereof that recognizes an MHC-peptide complex can be expressed on cells as part of a recombinant receptor, such as an antigen receptor. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MHC complexes also may be referred to as a TCR-like CAR.

[0108] In one embodiment, the engineered immune cell comprises a CAR comprising a sequence comprising a TCR alpha chain CDR3 sequence selected from SEQ ID NO: 1-62, 41855- 41902 or a TCR beta chain CDR3 sequence selected from SEQ ID NO: 63-121 or 41903-41948.Methods For Making Engineered Cells

[0109] Provided herein is a method for producing a cell of any of the provided embodiments, including introducing any of the provided vectors into a cell in vitro or ex vivo. In an embodiment, the introduction is carried out by transduction. In particular embodiments, the method further includes introducing into the cell one or more agent, wherein each of the one or more agent is independently capable of inducing genetic disruption of a T cell receptor alpha or beta chain gene. In an embodiment, the one or more agents capable of inducing a genetic disruption comprises a DNA binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the target site.

[0110] In embodiments, isolation of the cells includes one or more preparation and / or nonaffinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse, or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity, and / or resistance to particular components.

[0111] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in an embodiment, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in an embodiment contains cells other than red blood cells and platelets.

[0112] In an embodiment, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In an embodiment, the cells are washed with phosphate buffered saline (PBS). In an embodiment, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In an embodiment, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions. In an embodiment, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer’s instructions. In an embodiment, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++ / Mg++ free PBS. In an embodiment, components of a blood cell sample are removed, and the cells directly resuspended in culture media.

[0113] In an embodiment, the methods include density -based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.Methods Of Use In Adoptive Cell Therapy

[0114] In embodiments disclosed herein, the engineered cells described above may be used in novel therapeutic approaches for treating cancer. These engineered immune cells can be utilized to target hematological malignancies including MM, AML, and CLL. The engineered T cells may be autologous or allogeneic and may include modifications to further enhance their therapeutic efficacy. The engineered immune cells may comprise one or more modifications to enhance their immune reactivity, longevity, and anti-tumor effects. These modifications may include, but are not limited to, gene editing to knock out inhibitory receptors, enhance expression of co-stimulatory molecules, or secrete therapeutic cytokines. Additionally, the immune cells may include engineered receptors, such as chimeric antigen receptors (CARs) or specific T cell receptors (TCRs), to target cancer cells.

[0115] In example embodiments, identified antigen-activated T cell receptor (TCR) disclosed herein are used in constructing cells for adoptive cell transfer. In example embodiments, TCRsthat are clonal or specific to an antigen are identified. In example embodiment, the TCR CDR3 is used to generate a chimeric antigen receptor. As used herein, “ACT,” “adoptive cell therapy,” and “adoptive cell transfer” are used interchangeably. In an embodiment, adoptive cell therapy (ACT) refers to the transfer of cells to a patient with the goal of transferring the functionality and characteristics into the new host by engraftment of the cells (see, e.g., Mettananda et al., Nat Commun. 2017 Sep 4;8(1):424). As used herein, the term “engraft” or “engraftment” refers to the process of cell incorporation into a tissue of interest in vivo through contact with existing cells of the tissue. Adoptive cell therapy (ACT) can refer to the transfer of cells, most commonly immune- derived cells (e.g., T cells or NK cells), back into the same patient or into a new recipient host with the goal of transferring the immunologic functionality and characteristics into the new host. If possible, use of autologous cells helps the recipient by minimizing GVHD issues. The adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) (Zacharakis et al., (2018) Nat Med. 2018 Jun;24(6): 724-730; Besser et al., (2010) Clin. Cancer Res 16 (9) 2646-55; Dudley et al., (2002) Science 298 (5594): 850-4; and Dudley et al., (2005) Journal of Clinical Oncology 23 (10): 2346-57.) or genetically re-directed peripheral blood mononuclear cells (Johnson et al., (2009) Blood 114 (3): 535-46; and Morgan et al., (2006) Science 314(5796) 126-9) has been used to successfully treat patients with advanced solid tumors, including melanoma, metastatic breast cancer, and colorectal carcinoma, as well as patients with CD19-expressing hematologic malignancies (Kalos et al., (2011) Science Translational Medicine 3 (95): 95ra73). In an embodiment, allogenic cells immune cells are transferred (see, e.g., Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266). As described further herein, allogenic cells can be edited to reduce alloreactivity and prevent graft-versus-host disease. Thus, use of allogenic cells allows for cells to be obtained from healthy donors and prepared for use in patients as opposed to preparing autologous cells from a patient after diagnosis.

[0116] In an embodiment, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T cell therapy) of a disease (such as particularly of tumor or cancer) is a tumor-specific antigen (TSA).

[0117] In an embodiment, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T cell therapy) of a disease (such as particularly of tumor or cancer) is a neoantigen.

[0118] In an embodiment, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T cell therapy) of a disease (such as particularly of tumor or cancer) is a tumor-associated antigen (TAA) or cancer-associated antigen (CAA).

[0119] In an embodiment, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T cell therapy) of a disease (such as particularly of tumor or cancer) is a universal tumor antigen. In certain preferred embodiments, the universal tumor antigen is selected from the group consisting of: a human telomerase reverse transcriptase (hTERT), urviving, mouse double minute 2 homolog (MDM2), cytochrome P450 IB 1 (CYP1B), HER2 / neu, Wilms’ tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (DI), and any combinations thereof.

[0120] In an embodiment, an antigen (such as a tumor antigen) to be targeted in adoptive cell therapy (such as particularly CAR or TCR T cell therapy) of a disease (such as particularly of tumor or cancer) may be selected from a group consisting of: CD 19, BCMA, CD70, CLL-1, MAGE A3, MAGE A6, HPV E6, HPV E7, WT1, CD22, CD171, ROR1, MUC16, and SSX2. In certain preferred embodiments, the antigen may be CD19. For example, CD19 may be targeted in hematologic malignancies, such as in lymphomas, more particularly in B-cell lymphomas, such as without limitation in diffuse large B-cell lymphoma, primary mediastinal b-cell lymphoma, transformed follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia including adult and pediatric ALL, non-Hodgkin’s lymphoma, indolent non-Hodgkin’s lymphoma, or chronic lymphocytic leukemia. For example, BCMA may be targeted in multiple myeloma or plasma cell leukemia (see, e.g., 2018 American Association for Cancer Research (AACR) Annual meeting Poster: Allogeneic Chimeric Antigen Receptor T Cells Targeting B Cell Maturation Antigen). For example, CLL1 may be targeted in acute myeloid leukemia. For example, MAGE A3, MAGE A6, SSX2, and / or KRAS may be targeted in solid tumors. For example, HPV E6 and / or HPV E7 may be targeted in cervical cancer or head and neck cancer. For example, WT1 may be targeted in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), non-small cell lung cancer, breast, pancreatic, ovarian or colorectal cancers, or mesothelioma. For example, CD22 may be targeted in B cell malignancies, including non-Hodgkin lymphoma, diffuse large B-cell lymphoma, or acute lymphoblastic leukemia. For example, CD171 may be targeted in neuroblastoma, glioblastoma, orlung, pancreatic, or ovarian cancers. For example, R0R1 may be targeted in R0R1+ malignancies, including non-small cell lung cancer, triple negative breast cancer, pancreatic cancer, prostate cancer, ALL, chronic lymphocytic leukemia, or mantle cell lymphoma. For example, MUC 16 may be targeted in MUC16ecto+ epithelial ovarian, fallopian tube or primary peritoneal cancer. For example, CD70 may be targeted in both hematologic malignancies as well as in solid cancers such as renal cell carcinoma (RCC), gliomas (e.g., GBM), and head and neck cancers (HNSCC). CD70 is expressed in both hematologic malignancies as well as in solid cancers, while its expression in normal tissues is restricted to a subset of lymphoid cell types (see, e.g., 2018 American Association for Cancer Research (AACR) Annual meeting Poster: Allogeneic CRISPR Engineered Anti-CD70 CAR-T Cells Demonstrate Potent Preclinical Activity Against Both Solid and Hematological Cancer Cells).

[0121] Various strategies may for example be employed to genetically modify T cells by altering the specificity of the T cell receptor (TCR) for example by introducing new TCR a and 0 chains with selected peptide specificity (see U.S. Patent No. 8,697,854; PCT Patent Publications: W02003020763, W02004033685, W02004044004, W02005114215, W02006000830, W02008038002, W02008039818, W02004074322, W02005113595, WO2006125962, WO2013166321, WO2013039889, WO2014018863, WO2014083173; U.S. Patent No. 8,088,379).

[0122] As an alternative to, or addition to, TCR modifications, chimeric antigen receptors (CARs) may be used in order to generate immunoresponsive cells, such as T cells or natural killer cells (NK), specific for selected targets, such as malignant cells, with a wide variety of receptor chimera constructs having been described (see U.S. Patent Nos. 5,843,728; 5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014; 6,753,162; 8,211,422; and, PCT Publication WO92 15322).

[0123] In general, CARs are comprised of an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen-binding domain that is specific for a predetermined target (see, e.g., Gong Y, Klein Wolterink RGJ, Wang J, Bos GMJ, Germeraad WTV. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. J Hematol Oncol. 2021;14(l):73; Guedan S, Calderon H, Posey AD Jr, Maus MV. Engineering and Design of Chimeric Antigen Receptors. Mol Ther Methods Clin Dev. 2018;12: 145-156; Petersen CT, Krenciute G. Next Generation CAR T Cells for theImmunotherapy of High-Grade Glioma. Front Oncol. 2019;9:69; and Lu H, Zhao X, Li Z, Hu Y, Wang H. From CAR-T Cells to CAR-NK Cells: A Developing Immunotherapy Method for Hematological Malignancies. Front Oncol. 2021). While the antigen-binding domain of a CAR is often an antibody or antibody fragment (e.g., a single chain variable fragment, scFv), the binding domain is not particularly limited so long as it results in specific recognition of a target. For example, In an embodiment, the antigen-binding domain may comprise a receptor, such that the CAR is capable of binding to the ligand of the receptor. Alternatively, the antigen-binding domain may comprise a ligand, such that the CAR is capable of binding the endogenous receptor of that ligand.

[0124] The antigen-binding domain of a CAR is generally separated from the transmembrane domain by a hinge or spacer. The spacer is also not particularly limited, and it is designed to provide the CAR with flexibility. For example, a spacer domain may comprise a portion of a human Fc domain, including a portion of the CH3 domain, or the hinge region of any immunoglobulin, such as IgA, IgD, IgE, IgG, or IgM, or variants thereof. Furthermore, the hinge region may be modified to prevent off-target binding by FcRs or other potential interfering objects. For example, the hinge may comprise an IgG4 Fc domain with or without a S228P, L235E, and / or N297Q mutation (according to Kabat numbering) to decrease binding to FcRs. Additional spacers / hinges include, but are not limited to, CD4, CD8, and CD28 hinge regions.

[0125] The transmembrane domain of a CAR may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane bound or transmembrane protein. Transmembrane regions of particular use in this disclosure may be derived from CD8, CD28, CD3, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, TCR. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0126] Alternative CAR constructs may be characterized as belonging to successive generations. First-generation CARs typically consist of a single-chain variable fragment of anantibody specific for an antigen, for example comprising a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD8a hinge domain and a CD8a transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3^ or FcRy (scFv-CD3(^ or scFv-FcRy; see U.S. Patent No. 7,741,465; U.S. Patent No. 5,912,172; U.S. Patent No. 5,906,936). Second-generation CARs incorporate the intracellular domains of one or more costimulatory molecules, such as CD28, 0X40 (CD134), or 4-1BB (CD137) within the endodomain (for example scFv-CD28 / OX40 / 4-lBB-CD3(^; see U.S. Patent Nos. 8,911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761). Third-generation CARs include a combination of costimulatory endodomains, such a CD3^-chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, 0X40, 4-1BB, CD2, CD7, LIGHT, LFA-1, NKG2C, B7-H3, CD30, CD40, PD-1, or CD28 signaling domains (for example scFv-CD28-4-lBB-CD3(^ or scFv-CD28- OX40-CD3(^; see U.S. Patent No. 8,906,682; U.S. Patent No. 8,399,645; U.S. Pat. No. 5,686,281; PCT Publication No. WO2014134165; PCT Publication No. W02012079000). In an embodiment, the primary signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma Rlla, DAP10, and DAP12. In certain preferred embodiments, the primary signaling domain comprises a functional signaling domain of CD3(^ or FcRy. In an embodiment, the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the group consisting of CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CD 11 a, LFA-1, ITGAM, CD l ib, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D. In an embodiment, the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the groupconsisting of: 4-1BB, CD27, and CD28. In an embodiment, a chimeric antigen receptor may have the design as described in U.S. Patent No. 7,446,190, comprising an intracellular domain of CD3^ chain (such as amino acid residues 52-163 of the human CD3 zeta chain, as shown in SEQ ID NO: 14 of US 7,446,190), a signaling region from CD28 and an antigen-binding element (or portion or domain; such as scFv). The CD28 portion, when between the zeta chain portion and the antigenbinding element, include the transmembrane and signaling domains of CD28 (such as amino acid residues 114-220 of SEQ ID NO: 10, full sequence shown in SEQ ID NO: 6 of US 7,446,190; these include the following portion of CD28 as set forth in Genbank identifier NM 006139 (sequence version 1, 2 or 3): lEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS)) (SEQ. ID NO: 304). Alternatively, when the zeta sequence lies between the CD28 sequence and the antigenbinding element, intracellular domain of CD28 is used alone (such as amino sequence set forth in SEQ ID NO: 9 of US 7,446,190). Hence, certain embodiments employ a CAR comprising (a) a zeta chain portion comprising the intracellular domain of human CD3(^ chain, (b) a costimulatory signaling region, and (c) an antigen-binding element (or portion or domain), wherein the costimulatory signaling region comprises the amino acid sequence encoded by SEQ ID NO: 6 of US 7,446,190.

[0127] Alternatively, costimulation may be orchestrated by expressing CARs in antigenspecific T cells, chosen to be activated and expanded following engagement of their native a0TCR, for example by antigen on professional antigen-presenting cells, with attendant costimulation. In addition, additional engineered receptors may be provided on the immunoresponsive cells, for example to improve targeting of a T cell attack and / or minimize side effects

[0128] By means of an example and without limitation, Kochenderfer et al., (2009) J Immunother. 32 (7): 689-702 described anti-CD19 chimeric antigen receptors (CAR). FMC63- 28Z CAR contained a single chain variable region moiety (scFv) recognizing CD 19 derived from the FMC63 mouse hybridoma (described in Nicholson et al., (1997) Molecular Immunology 34: 1157-1165), a portion of the human CD28 molecule, and the intracellular component of the human TCR-^ molecule. FMC63-CD828BBZ CAR contained the FMC63 scFv, the hinge and transmembrane regions of the CD8 molecule, the cytoplasmic portions of CD28 and 4-1BB, and the cytoplasmic component of the TCR-(^ molecule. The exact sequence of the CD28 moleculeincluded in the FMC63-28Z CAR corresponded to Genbank identifier NM_006139; the sequence included all amino acids starting with the amino acid sequence IEVMYPPPY (SEQ. ID NO: 305) and continuing all the way to the carboxy-terminus of the protein. To encode the anti-CD19 scFv component of the vector, the authors designed a DNA sequence which was based on a portion of a previously published CAR (Cooper et al., (2003) Blood 101 : 1637-1644). This sequence encoded the following components in frame from the 5’ end to the 3’ end: an Xhol site, the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor a-chain signal sequence, the FMC63 light chain variable region (as in Nicholson et al., supra), a linker peptide (as in Cooper et al., supra), the FMC63 heavy chain variable region (as in Nicholson et al., supra), and a Notl site. A plasmid encoding this sequence was digested with Xhol and Notl. To form the MSGV- FMC63-28Z retroviral vector, the Xhol and Notl-digested fragment encoding the FMC63 scFv was ligated into a second Xhol and Notl-digested fragment that encoded the MSGV retroviral backbone (as in Hughes et al., (2005) Human Gene Therapy 16: 457-472) as well as part of the extracellular portion of human CD28, the entire transmembrane and cytoplasmic portion of human CD28, and the cytoplasmic portion of the human TCR-^ molecule (as in Maher et al., 2002) Nature Biotechnology 20: 70-75). The FMC63-28Z CAR is included in the KTE-C19 (axicabtagene ciloleucel) anti-CD19 CAR-T therapy product in development by Kite Pharma, Inc. for the treatment of inter alia patients with relapsed / refractory aggressive B-cell non-Hodgkin lymphoma (NHL). Accordingly, In an embodiment, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may express the FMC63-28Z CAR as described by Kochenderfer et al. (supra). Hence, In an embodiment, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may comprise a CAR comprising an extracellular antigen-binding element (or portion or domain; such as scFv) that specifically binds to an antigen, an intracellular signaling domain comprising an intracellular domain of a CD3(^ chain, and a costimulatory signaling region comprising a signaling domain of CD28. Preferably, the CD28 amino acid sequence is as set forth in Genbank identifier NM_006139 (sequence version 1, 2 or 3) starting with the amino acid sequence IEVMYPPPY (SEQ ID NO: 305) and continuing all the way to the carboxy-terminus of the protein. The sequence is reproduced herein: lEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 304).Preferably, the antigen is CD19, more preferably the antigen-binding element is an anti-CD19 scFv, even more preferably the anti-CD19 scFv as described by Kochenderfer et al. (supra).

[0129] Additional anti-CD19 CARs are further described in WO2015187528. More particularly Example 1 and Table 1 of WO2015187528, incorporated by reference herein, demonstrate the generation of anti-CD19 CARs based on a fully human anti-CD19 monoclonal antibody (47G4, as described in US20100104509) and murine anti-CD19 monoclonal antibody (as described in Nicholson et al. and explained above). Various combinations of a signal sequence (human CD8-alpha or GM-CSF receptor), extracellular and transmembrane regions (human CD8- alpha) and intracellular T cell signaling domains (CD28-CD3^; 4-lBB-CD3(^; CD27-CD3(^; CD28- CD27-CD3L) 4-lBB-CD27-CD3(^; CD27-4-1BB-CD3 CD28-CD27-FceRI gamma chain; or CD28-FceRI gamma chain) were disclosed. Hence, In an embodiment, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may comprise a CAR comprising an extracellular antigen-binding element that specifically binds to an antigen, an extracellular and transmembrane region as set forth in Table 1 of WO2015187528 and an intracellular T cell signaling domain as set forth in Table 1 of WO2015187528. Preferably, the antigen is CD19, more preferably the antigen-binding element is an anti-CD19 scFv, even more preferably the mouse or human anti-CD19 scFv as described in Example 1 of WO2015187528. In an embodiment, the CAR comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 as set forth in Table 1 of WO2015187528.

[0130] By means of an example and without limitation, chimeric antigen receptor that recognizes the CD70 antigen is described in W02012058460A2 (see also, Park et al., Oral Oncol. 2018 Mar;78: 145-150; and Jin et al., Neuro Oncol. 2018 Jan 10;20(l):55-65). CD70 is expressed by diffuse large B-cell and follicular lymphoma and also by the malignant cells of Hodgkin’s lymphoma, Waldenstrom’s macroglobulinemia and multiple myeloma, and by HTLV-1- and EBV-associated malignancies. (Agathanggelou et al. Am.J.Pathol. 1995; 147: 1152-1160; Hunter et al., Blood 2004; 104:4881. 26; Lens et al., J Immunol. 2005;174:6212-6219; Baba et al., J Virol. 2008;82:3843-3852.) In addition, CD70 is expressed by non-hematological malignancies such as renal cell carcinoma and glioblastoma. (Junker et al., J Urol. 2005;173:2150-2153; Chahlavi et al.,Cancer Res 2005;65:5428-5438) Physiologically, CD70 expression is transient and restricted to a subset of highly activated T, B, and dendritic cells.

[0131] By means of an example and without limitation, chimeric antigen receptor that recognizes BCMA has been described (see, e.g., US20160046724A1; WO2016014789A2; W02017211900AI; WO2015158671A1; US20180085444A1; WO2018028647A1;US20170283504A1; and WO2013154760A1).

[0132] In an embodiment, the immune cell may, in addition to a CAR or exogenous TCR as described herein, further comprise a chimeric inhibitory receptor (inhibitory CAR) that specifically binds to a second target antigen and is capable of inducing an inhibitory or immunosuppressive or repressive signal to the cell upon recognition of the second target antigen. In an embodiment, the chimeric inhibitory receptor comprises an extracellular antigen-binding element (or portion or domain) configured to specifically bind to a target antigen, a transmembrane domain, and an intracellular immunosuppressive or repressive signaling domain. In an embodiment, the second target antigen is an antigen that is not expressed on the surface of a cancer cell or infected cell or the expression of which is downregulated on a cancer cell or an infected cell. In an embodiment, the second target antigen is an MHC-class I molecule. In an embodiment, the intracellular signaling domain comprises a functional signaling portion of an immune checkpoint molecule, such as for example PD-1 or CTLA4. Advantageously, the inclusion of such inhibitory CAR reduces the chance of the engineered immune cells attacking non-target (e.g., non-cancer) tissues.

[0133] Alternatively, T cells expressing CARs may be further modified to reduce or eliminate expression of endogenous TCRs to reduce off-target effects. Reduction or elimination of endogenous TCRs can reduce off-target effects and increase the effectiveness of the T cells (U.S. 9, 181 ,527). T cells stably lacking expression of a functional TCR may be produced using a variety of approaches. T cells internalize, sort, and degrade the entire T cell receptor as a complex, with a half-life of about 10 hours in resting T cells and 3 hours in stimulated T cells (von Essen, M. et al. 2004. J. Immunol. 173:384-393). Proper functioning of the TCR complex requires the proper stoichiometric ratio of the proteins that compose the TCR complex. TCR function also requires two functioning TCR zeta proteins with ITAM motifs. The activation of the TCR upon engagement of its MHC-peptide ligand requires the engagement of several TCRs on the same T cell, which all must signal properly. Thus, if a TCR complex is destabilized with proteins that do not associateproperly or cannot signal optimally, the T cell will not become activated sufficiently to begin a cellular response.

[0134] Accordingly, In an embodiment, TCR expression may eliminated using RNA interference (e.g., shRNA, siRNA, miRNA, etc.), CRISPR, or other methods that target the nucleic acids encoding specific TCRs (e.g., TCR-a and TCR-P) and / or CD3 chains in primary T cells. By blocking expression of one or more of these proteins, the T cell will no longer produce one or more of the key components of the TCR complex, thereby destabilizing the TCR complex and preventing cell surface expression of a functional TCR.

[0135] In some instances, CAR also may comprise a switch mechanism for controlling expression and / or activation of the CAR. For example, a CAR may comprise an extracellular, transmembrane, and intracellular domain, in which the extracellular domain comprises a targetspecific binding element that comprises a label, binding domain, or tag that is specific for a molecule other than the target antigen that is expressed on or by a target cell. In such embodiments, the specificity of the CAR is provided by a second construct that comprises a target antigen binding domain (e.g., an scFv or a bispecific antibody that is specific for both the target antigen and the label or tag on the CAR) and a domain that is recognized by or binds to the label, binding domain, or tag on the CAR. See, e.g., WO 2013 / 044225, WO 2016 / 000304, WO 2015 / 057834, WO 2015 / 057852, WO 2016 / 070061, US 9,233,125, US 2016 / 0129109. In this way, a T cell that expresses the CAR can be administered to a subject, but the CAR cannot bind its target antigen until the second composition comprising an antigen-specific binding domain is administered.

[0136] Alternative switch mechanisms include CARs that require multimerization to activate their signaling function (see, e.g., US 2015 / 0368342, US 2016 / 0175359, US 2015 / 0368360) and / or an exogenous signal, such as a small molecule drug (US 2016 / 0166613, Yung et al., Science, 2015), to elicit a T cell response. Some CARs may also comprise a “suicide switch” to induce cell death of the CAR T cells following treatment (Buddee et al., PLoS One, 2013) or to downregulate expression of the CAR following binding to the target antigen (WO 2016 / 011210).

[0137] Alternative techniques may be used to transform target immunoresponsive cells, such as protoplast fusion, lipofection, transfection or electroporation. A wide variety of vectors may be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids, or transposons, such as a Sleeping Beauty transposon (see U.S. Patent Nos. 6,489,458; 7,148,203; 7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, forexample using 2nd generation antigen-specific CARs signaling through CD3(j and either CD28 or CD137. Viral vectors may for example include vectors based on HIV, SV40, EBV, HSV or BPV. In an embodiment, inducible gene switches are used to regulate expression of a CAR or TCR (see, e.g., Chakravarti, Deboki et al. “Inducible Gene Switches with Memory in Human T Cells for Cellular Immunotherapy.” ACS synthetic biology vol. 8,8 (2019): 1744-1754).

[0138] Cells that are targeted for transformation may for example include T cells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, human embryonic stem cells, tumor-infdtrating lymphocytes (TIL) or a pluripotent stem cell from which lymphoid cells may be differentiated. T cells expressing a desired CAR may for example be selected through co-culture with y-irradiated activating and propagating cells (AaPC), which co-express the cancer antigen and co-stimulatory molecules. The engineered CAR T cells may be expanded, for example by coculture on AaPC in presence of soluble factors, such as IL-2 and IL-21. This expansion may for example be carried out to provide memory CAR+ T cells (which may for example be assayed by non-enzymatic digital array and / or multi-panel flow cytometry). In this way, CAR T cells may be provided that have specific cytotoxic activity against antigen-bearing tumors (optionally in conjunction with production of desired chemokines such as interferon-y). CAR T cells of this kind may for example be used in animal models, for example to treat tumor xenografts.

[0139] In an embodiment, ACT includes co-transferring CD4+ Thl cells and CD8+ CTLs to induce a synergistic antitumour response (see, e.g., Li et al., Clin Transl Immunology. 2017 Oct; 6(10): el60).

[0140] In an embodiment, antigen specificity can be conferred to Tregs by engineering the expression of transgenic T cell receptor (TCR) or chimeric antigen receptor (CAR), such as to modulate immune responses in organ transplant and autoimmune diseases (see, e.g., Arjomandnejad M, Kopec AL, Keeler AM. Biomedicines. 2022;10(2):287). Regulatory T cells (Tregs) are a T cell subset known for their immunomodulatory function. Expression of CD4, CD25, and the master transcription factor, forkhead box P3 (FOXP3), are the main characteristic markers of conventional Tregs. However, other regulatory immune cells with different properties such as CD8+ Tregs, or type 1 regulatory T cells (Tri) have been described. Id. Tregs are divided into “natural” Tregs that develop in the thymus or “induced” Tregs that are generated in the periphery. Id. Regulatory T cells suppress immune responses through multiple mechanisms including direct interaction with other immune cells or by producing immunosuppressivecytokines such as interleukin- 10 (IL-10) and Transforming growth factor beta (TGF-0). Id. Directing Tregs towards a desired antigen may boost the overall response and lower the risk of broad and systemic immunosuppression or generation of an inflammatory response. Id.

[0141] In an embodiment, Thl7 cells are transferred to a subject in need thereof. Thl7 cells have been reported to directly eradicate melanoma tumors in mice to a greater extent than Thl cells (Muranski P, et al., Blood. 2008 Jul 15; 112(2):362-73; and Martin-Orozco N, et al., Immunity. 2009 Nov 20; 31 (5):787-98). Those studies involved an adoptive T cell transfer (ACT) therapy approach, which takes advantage of CD4+ T cells that express a TCR recognizing tyrosinase tumor antigen. Exploitation of the TCR leads to rapid expansion of Thl 7 populations to large numbers ex vivo for reinfusion into the autologous tumor-bearing hosts.

[0142] In an embodiment, ACT may include autologous iPSC-based vaccines, such as irradiated iPSCs in autologous anti-tumor vaccines (see e.g., Kooreman, Nigel G. et al., Cell Stem Cell 22, 1-13, 2018).

[0143] Unlike T cell receptors (TCRs) that are MHC restricted, CARs can potentially bind any cell surface-expressed antigen and can thus be more universally used to treat patients (see Irving et al., Front. Immunol., 03 April 2017). In an embodiment, in the absence of endogenous T cell infiltrate (e.g., due to aberrant antigen processing and presentation), which precludes the use of TIL therapy and immune checkpoint blockade, the transfer of CAR T cells may be used to treat patients (see, e g., Hinrichs CS, Rosenberg SA. Immunol Rev (2014) 257(1):56— 71).

[0144] Approaches such as the foregoing may be adapted to provide methods of treating and / or increasing survival of a subject having a disease, such as a neoplasia, for example by administering an effective amount of an immunoresponsive cell comprising an antigen recognizing receptor that binds a selected antigen, wherein the binding activates the immunoresponsive cell, thereby treating or preventing the disease (such as a neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant reaction).

[0145] In an embodiment, the treatment is administered after lymphodepleting pretreatment in the form of chemotherapy (typically a combination of cyclophosphamide and fludarabine) or radiation therapy. Initial studies in ACT had short lived responses and the transferred cells did not persist in vivo for very long (Houot et al., Cancer Immunol Res (2015) 3(10): 1115-22; and Kamta et al., Front. Oncol. (2017) 7:64). Immune suppressor cells like Tregs and MDSCs may attenuate the activity of transferred cells by outcompeting them for the necessary cytokines. Not being boundby a theory lymphodepl eting pretreatment may eliminate the suppressor cells allowing the TILs to persist.

[0146] In one embodiment, the treatment is administrated into patients undergoing an immunosuppressive treatment (e.g., glucocorticoid treatment). The cells, or population of cells, may be made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent. In an embodiment, the immunosuppressive treatment provides for the selection and expansion of the immunoresponsive T cells within the patient.

[0147] In an embodiment, the treatment is administered before primary treatment (e.g., surgery or radiation therapy) to shrink a tumor before the primary treatment. In another embodiment, the treatment is administered after primary treatment to remove any remaining cancer cells.

[0148] In an embodiment, immunometabolic barriers are targeted therapeutically prior to and / or during ACT to enhance responses to ACT or CAR T cell therapy and to support endogenous immunity (see, e.g., Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel, Front. Immunol., 03 April 2017, doi.org / 10.3389 / fimmu.2017.00267).

[0149] The administration of cells or population of cells, such as immune system cells or cell populations, such as more particularly immunoresponsive cells or cell populations, as disclosed herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The cells or population of cells can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intrathecally, by intravenous or intralymphatic injection, or intraperitoneally. In an embodiment, the disclosed CARs are delivered or administered into a cavity formed by the resection of tumor tissue (i.e. intracavity delivery) or directly into a tumor prior to resection (i.e. intratumoral delivery). In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0150] The administration of the cells or population of cells comprises administering 104- 109 cells per kg body weight, preferably 105 to 106 cells / kg body weight including all integer values of cell numbers within those ranges. Dosing in CAR T cell therapies may for example involve administration of from 106 to 109 cells / kg, with or without a course of lymphodepletion,for example with cyclophosphamide. The cells or population of cells can be administrated in one or more doses. In another embodiment, the effective amount of cells are administrated as a single dose. In another embodiment, the effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions are within the ordinary skill of one in the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.

[0151] In an example embodiment, the effective amount of cells can be any amount ranging from about 1 or 2 cells to 1x101 cells / mL, 1x1020 cells / mL or more, such as about 1x101 cells / mL, 1x102 cells / mL, 1x103 cells / mL, 1x104 cells / mL, 1x105 cells / mL, 1x106 cells / mL, 1x107 cells / mL, 1x108 cells / mL, 1x109 cells / mL, 1x1010 cells / mL, 1x1011 cells / mL, 1x1012 cells / mL, 1x1013 cells / mL, 1x1014 cells / mL, 1x1015 cells / mL, 1x1016 cells / mL, 1x1017 cells / mL, 1x1018 cells / mL, 1x1019 cells / mL, to / or about 1x1020 / cells / mL or any numerical value or subrange within any of these ranges.

[0152] In another embodiment, the effective amount of cells or composition comprising those cells are administrated parenterally. The administration can be an intravenous administration. The administration can be directly done by injection within a tumor.

[0153] To guard against possible adverse reactions, engineered immunoresponsive cells may be equipped with a transgenic safety switch, in the form of a transgene that renders the cells vulnerable to exposure to a specific signal. For example, the herpes simplex viral thymidine kinase (TK) gene may be used in this way, for example by introduction into allogeneic T lymphocytes used as donor lymphocyte infusions following stem cell transplantation (Greco, et al., Front. Pharmacol. 2015; 6: 95). In such cells, administration of a nucleoside prodrug such as ganciclovir or acyclovir causes cell death. Alternative safety switch constructs include inducible caspase 9, for example triggered by administration of a small-molecule dimerizer that brings together two nonfunctional icasp9 molecules to form the active enzyme. A wide variety of alternative approaches to implementing cellular proliferation controls have been described (see U.S. PatentPublication No. 20130071414; PCT Patent Publication WO2011146862; PCT Patent Publication W02014011987; PCT Patent Publication W02013040371; Zhou et al. BLOOD, 2014, 123 / 25:3895 - 3905; Di Stasi et al., The New England Journal of Medicine 2011; 365: 1673-1683; Sadelain M, The New England Journal of Medicine 2011; 365:1735-173; Ramos et al., Stem Cells 28(6): 1107-15 (2010)).

[0154] In a further refinement of adoptive therapies, genome editing may be used to tailor immunoresponsive cells to alternative implementations, for example providing edited CAR T cells (see Poirot et al., 2015, Multiplex genome edited T-cell manufacturing platform for “off-the-shelf ’ adoptive T-cell immunotherapies, Cancer Res 75 (18): 3853; Ren et al., 2017, Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition, Clin Cancer Res. 2017 May l;23(9):2255-2266. doi: 10.1158 / 1078-0432.CCR-16-1300. Epub 2016 Nov 4; Qasim et al., 2017, Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells, Sci Transl Med. 2017 Jan 25;9(374); Legut, et al., 2018, CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells. Blood, 131(3), 311-322; Georgiadis et al., Mol Ther. 2018 May 2;26(5): 1215-1227; and Roth, T.L. Curr Hematol MaligRep 15, 235-240 (2020)). Cells may be edited using any CRISPR system and method of use thereof as described herein. CRISPR systems may be delivered to an immune cell by any method described herein. In preferred embodiments, cells are edited ex vivo and transferred to a subject in need thereof. Immunoresponsive cells, CAR T cells or any cells used for adoptive cell transfer may be edited. Editing may be performed for example to insert or knock-in an exogenous gene, such as an exogenous gene encoding a CAR or a TCR, at a preselected locus in a cell (e.g. TRAC locus); to eliminate potential alloreactive T cell receptors (TCR) or to prevent inappropriate pairing between endogenous and exogenous TCR chains, such as to knock-out or knock-down expression of an endogenous TCR in a cell; to disrupt the target of a chemotherapeutic agent in a cell; to block an immune checkpoint, such as to knock-out or knock-down expression of an immune checkpoint protein or receptor in a cell; to knock-out or knock-down expression of other gene or genes in a cell, the reduced expression or lack of expression of which can enhance the efficacy of adoptive therapies using the cell; to knock-out or knock-down expression of an endogenous gene in a cell, said endogenous gene encoding an antigen targeted by an exogenous CAR or TCR; to knock-out or knock-down expression of one or more MHC constituent proteins in a cell; to activate a T cell; to modulate cells such that the cells are resistant to exhaustion or dysfunction; and / or increase thedifferentiation and / or proliferation of functionally exhausted or dysfunctional CD8+ T cells (see PCT Patent Publications: WO2013176915, WO2014059173, WO2014172606, WO2014184744, and WO2014191128).

[0155] In an embodiment, editing may result in inactivation of a gene. By inactivating a gene, it is intended that the gene of interest is not expressed in a functional protein form. In a particular embodiment, the CRISPR system specifically catalyzes cleavage in one targeted gene thereby inactivating said targeted gene. The nucleic acid strand breaks caused are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). However, NHEI is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions (Indel) and can be used for the creation of specific gene knockouts. Cells in which a cleavage induced mutagenesis event has occurred can be identified and / or selected by well-known methods in the art. In an embodiment, homology directed repair (HDR) is used to concurrently inactivate a gene (e.g., TRAC) and insert an endogenous TCR or CAR into the inactivated locus.

[0156] Hence, In an embodiment, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to insert or knock-in an exogenous gene, such as an exogenous gene encoding a CAR or a TCR, at a preselected locus in a cell. Conventionally, nucleic acid molecules encoding CARs or TCRs are transfected or transduced to cells using randomly integrating vectors, which, depending on the site of integration, may lead to clonal expansion, oncogenic transformation, variegated transgene expression and / or transcriptional silencing of the transgene. Directing of transgene(s) to a specific locus in a cell can minimize or avoid such risks and advantageously provide for uniform expression of the transgene(s) by the cells. Without limitation, suitable ‘safe harbor’ loci for directed transgene integration include CCR5 or AAVS 1. Homology-directed repair (HDR) strategies are known and described elsewhere in this specification allowing to insert transgenes into desired loci (e.g., TRAC locus).

[0157] Further suitable loci for insertion of transgenes, in particular CAR or exogenous TCR transgenes, include without limitation loci comprising genes coding for constituents of endogenous T cell receptor, such as T cell receptor alpha locus (TRA) or T cell receptor beta locus (TRB), for example T cell receptor alpha constant (TRAC) locus, T cell receptor beta constant 1 (TRBC1)locus or T cell receptor beta constant 2 (TRBC1) locus. Advantageously, insertion of a transgene into such locus can simultaneously achieve expression of the transgene, potentially controlled by the endogenous promoter, and knock-out expression of the endogenous TCR. This approach has been exemplified in Eyquem et al., (2017) Nature 543: 113-117, wherein the authors used CRISPR / Cas9 gene editing to knock-in a DNA molecule encoding a CD19-specific CAR into the TRAC locus downstream of the endogenous promoter; the CAR-T cells obtained by CRISPR were significantly superior in terms of reduced tonic CAR signaling and exhaustion.

[0158] T cell receptors (TCR) are cell surface receptors that participate in the activation of T cells in response to the presentation of antigen. The TCR is generally made from two chains, a and P, which assemble to form a heterodimer and associates with the CD3 -transducing subunits to form the T cell receptor complex present on the cell surface. Each a and P chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. As for immunoglobulin molecules, the variable region of the a and P chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T cells. However, in contrast to immunoglobulins that recognize intact antigen, T cells are activated by processed peptide fragments in association with an MHC molecule, introducing an extra dimension to antigen recognition by T cells, known as MHC restriction. Recognition of MHC disparities between the donor and recipient through the T cell receptor leads to T cell proliferation and the potential development of graft versus host disease (GVHD). The inactivation of TCRa or TCRp can result in the elimination of the TCR from the surface of T cells preventing recognition of alloantigen and thus GVHD. However, TCR disruption generally results in the elimination of the CD3 signaling component and alters the means of further T cell expansion.

[0159] Hence, In an embodiment, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, is performed to knock-out or knock-down expression of an endogenous TCR in a cell. For example, NHEJ-based or HDR-based gene editing approaches are employed to disrupt the endogenous TCR alpha and / or beta chain genes. For example, gene editing system or systems, such as CRISPR / Cas system or systems, can be designed to target a sequence found within the TCR beta chain conserved between the beta 1 and beta 2 constant region genes (TRBC1 and TRBC2) and / or to target the constant region of the TCR alpha chain (TRAC) gene.

[0160] Allogeneic cells are rapidly rejected by the host immune system. It has been demonstrated that, allogeneic leukocytes present in non-irradiated blood products will persist for no more than 5 to 6 days (Boni, Muranski et al. 2008 Blood 1;112(12):4746-54). Thus, to prevent rejection of allogeneic cells, the host’s immune system usually has to be suppressed to some extent. However, in the case of adoptive cell transfer the use of immunosuppressive drugs also have a detrimental effect on the introduced therapeutic T cells. Therefore, to effectively use an adoptive immunotherapy approach in these conditions, the introduced cells would need to be resistant to the immunosuppressive treatment. Thus, in a particular embodiment, the present invention further comprises a step of modifying T cells to make them resistant to an immunosuppressive agent, preferably by inactivating at least one gene encoding a target for an immunosuppressive agent. An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. An immunosuppressive agent can be, but is not limited to a calcineurin inhibitor, a target of rapamycin, an interleukin-2 receptor a-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid, or an immunosuppressive antimetabolite. The present invention allows conferring immunosuppressive resistance to T cells for immunotherapy by inactivating the target of the immunosuppressive agent in T cells. As non-limiting examples, targets for an immunosuppressive agent can be a receptor for an immunosuppressive agent such as: CD52, glucocorticoid receptor (GR), a FKBP family gene member and a cyclophilin family gene member.

[0161] In an embodiment, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, is performed to block an immune checkpoint, such as to knock-out or knock-down expression of an immune checkpoint protein or receptor in a cell. Immune checkpoints are inhibitory pathways that slow down or stop immune reactions and prevent excessive tissue damage from uncontrolled activity of immune cells. In an embodiment, the immune checkpoint targeted is the programmed death-1 (PD-1 or CD279) gene (PDCD1) (see, e.g., Rupp LJ, Schumann K, Roybal KT, et al. CRISPR / Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep. 2017;7(1 ):737). In other embodiments, the immune checkpoint targeted is cytotoxic T lymphocyte-associated antigen (CTLA-4). In additional embodiments, the immune checkpoint targeted is another member of the CD28 and CTLA4 Ig superfamily such as BTLA,LAG3, ICOS, PDL1 or KIR. In further additional embodiments, the immune checkpoint targeted is a member of the TNFR superfamily such as CD40, 0X40, CD 137, GITR, CD27 or TIM-3.

[0162] Additional immune checkpoints include Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) (Watson HA, et al., Biochem Soc Trans. 2016 Apr 15;44(2):356- 62). SHP-1 is a widely expressed inhibitory protein tyrosine phosphatase (PTP). In T cells, it is a negative regulator of antigen-dependent activation and proliferation. It is a cytosolic protein, and therefore not amenable to antibody-mediated therapies, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adoptive transfer strategies, such as chimeric antigen receptor (CAR) T cells. Immune checkpoints may also include T cell immunoreceptor with Ig and ITIM domains (TIGIT / Vstm3 / WUCAM / VSIG9) and VISTA (Le Mercier I, et al., (2015) Front. Immunol. 6:418).

[0163] WO2014172606 relates to the use of MT1 and / or MT2 inhibitors to increase proliferation and / or activity of exhausted CD8+ T cells and to decrease CD8+ T cell exhaustion (e.g., decrease functionally exhausted or unresponsive CD8+ immune cells). In an embodiment, metallothioneins are targeted by gene editing in adoptively transferred T cells.

[0164] In an embodiment, targets of gene editing may be at least one targeted locus involved in the expression of an immune checkpoint protein. Such targets may include, but are not limited to CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2, BTLA, CD 160, TIGIT, CD96, CRT AM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4), TNFRSF10B, TNFRSF10A, CASP8, C ASP 10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40, 0X40, CD137, GITR, CD27, SHP-1, TIM-3, CEACAM-1, CEACAM-3, or CEACAM-5. In preferred embodiments, the gene locus involved in the expression of PD-1 or CTLA-4 genes is targeted. In other preferred embodiments, combinations of genes are targeted, such as but not limited to PD-1 and TIGIT.

[0165] By means of an example and without limitation, WO2016196388 concerns an engineered T cell comprising (a) a genetically engineered antigen receptor that specifically binds to an antigen, which receptor may be a CAR; and (b) a disrupted gene encoding a PD-L1, an agent for disruption of a gene encoding a PD- LI, and / or disruption of a gene encoding PD-L1, wherein the disruption of the gene may be mediated by a gene editing nuclease, a zinc finger nuclease(ZFN), CRISPR / Cas9 and / or TALEN. WO2015142675 relates to immune effector cells comprising a CAR in combination with an agent (such as CRISPR, TALEN or ZFN) that increases the efficacy of the immune effector cells in the treatment of cancer, wherein the agent may inhibit an immune inhibitory molecule, such as PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, or CEACAM-5. Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR and electrotransfer of Cas9 mRNA and gRNAs targeting endogenous TCR, P-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.

[0166] In an embodiment, cells are engineered to express a CAR, wherein expression and / or function of methylcytosine dioxygenase genes (TET1, TET2 and / or TET3) in the cells has been reduced or eliminated, such as by CRISPR, ZNF or TALEN (for example, as described in WO20 1704916).

[0167] In an embodiment, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, is performed to knock-out or knock-down expression of an endogenous gene in a cell, said endogenous gene encoding an antigen targeted by an exogenous CAR or TCR, thereby reducing the likelihood of targeting of the engineered cells. In an embodiment, the targeted antigen is one or more antigen selected from the group consisting of CD38, CD138, CS-1, CD33, CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, CD362, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, Wilms’ tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (DI), B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI), and B-cell activating factor receptor (BAFF-R) (for example, as described in WO2016011210 and WO2017011804).

[0168] In an embodiment, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to knock-out or knock-down expression of one or more MHC constituent proteins, such as one or more HLA proteins and / or beta-2 microglobulin (B2M), in a cell, whereby rejection of non-autologous (e.g., allogeneic) cells by the recipient’s immune system can bereduced or avoided. In preferred embodiments, one or more HLA class I proteins, such as HLA- A, B and / or C, and / or B2M are knocked-out or knocked-down. Preferably, B2M is knocked-out or knocked-down. By means of an example, Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, P-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.

[0169] In other embodiments, at least two genes are edited. Pairs of genes include, but are not limited to PD1 and TCRa, PD1 and TCR , CTLA-4 and TCRa, CTLA-4 and TCR , LAG3 and TCRa, LAG3 and TCR , Tim3 and TCRa, Tim3 and TCRp, BTLA and TCRa, BTLA and TCRp, BY55 and TCRa, BY55 and TCRp, TIGIT and TCRa, TIGIT and TCRp, B7H5 and TCRa, B7H5 and TCRP, LAIR1 and TCRa, LAIR1 and TCRP, SIGLEC10 and TCRa, SIGLEC10 and TCRP, 2B4 and TCRa, 2B4 and TCRp, B2M and TCRa, B2M and TCRp.

[0170] In an embodiment, a cell may be multiply edited (multiplex genome editing) as taught herein to (1) knock-out or knock-down expression of an endogenous TCR (for example, TRBC1, TRBC2 and / or TRAC), (2) knock-out or knock-down expression of an immune checkpoint protein or receptor (for example PD1, PD-L1 and / or CTLA4); and (3) knock-out or knock-down expression of one or more MHC constituent proteins (for example, HLA-A, B and / or C, and / or B2M, preferably B2M).

[0171] Whether prior to or after genetic modification of the T cells, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. T cells can be expanded in vitro or in vivo.

[0172] Immune cells may be obtained using any method known in the art. In one embodiment, allogenic T cells may be obtained from healthy subjects. In one embodiment T cells that have infiltrated a tumor are isolated. T cells may be removed during surgery. T cells may be isolated after removal of tumor tissue by biopsy. T cells may be isolated by any means known in the art. In one embodiment, T cells are obtained by apheresis. In one embodiment, the method comprises obtaining a bulk population of T cells from a tumor sample by any suitable method known in the art. For example, a bulk population of T cells can be obtained from a tumor sample by dissociating the tumor sample into a cell suspension from which specific cell populations can be selected.Suitable methods of obtaining a bulk population of T cells may include, but are not limited to, any one or more of mechanically dissociating (e.g., mincing) the tumor, enzymatically dissociating (e.g., digesting) the tumor, and aspiration (e.g., as with a needle).

[0173] The bulk population of T cells obtained from a tumor sample may comprise any suitable type of T cell. Preferably, the bulk population of T cells obtained from a tumor sample comprises tumor infiltrating lymphocytes (TLLs).

[0174] The tumor sample may be obtained from any mammal. In a preferred embodiment, the tumor sample is obtained from a human. In an embodiment, the tumor sample is obtained from a subject to be treated.

[0175] T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleen tissue, and tumors. In an embodiment of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis are washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or many, if not all, divalent cations. Initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow- through” centrifuge (for example, the Cobe 2991 cell processor) according to the manufacturer’s instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample can be removed, and the cells can be directly resuspended in culture media.

[0176] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28+, CD4+, CDC, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selectiontechniques. For example, in one preferred embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3><28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, or XCYTE DYNABEADS™ for an incubation time sufficient for positive selection of the desired T cells. In one embodiment, the period is about 30 minutes. In a further embodiment, the incubation time ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the incubation time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the incubation time is from 10 hours to 24 hours. In one preferred embodiment, the incubation time is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells.

[0177] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA- DR, and CD 8.

[0178] Further, monocyte populations (i.e., CD14+ cells) may be depleted from blood preparations by a variety of methodologies, including anti-CD14 coated beads or columns, or utilization of the phagocytotic activity of these cells to facilitate removal. Accordingly, in one embodiment, the invention uses paramagnetic particles of a size sufficient to be engulfed by phagocytotic monocytes. In an embodiment, the paramagnetic particles are commercially available beads, for example, those produced by Life Technologies under the trade name Dynabeads™. In one embodiment, other non-specific cells are removed by coating the paramagnetic particles with “irrelevant” proteins (e g., serum proteins or antibodies). Irrelevant proteins and antibodies include those proteins and antibodies or fragments thereof that do not specifically target the T cells to be isolated. In an embodiment, the irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin.

[0179] In brief, such depletion of monocytes is performed by preincubating T cells isolated from whole blood, apheresed peripheral blood, or tumors with one or more varieties of irrelevant or non-antibody coupled paramagnetic particles at any amount that allows for removal of monocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to 2 hours at 22 to 37 degrees C., followed by magnetic removal of cells which have attached to or engulfed the paramagnetic particles. Such separation can be performed using standard methods available in the art. For example, any magnetic separation methodology may be used including a variety of which are commercially available, (e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)). Assurance of requisite depletion can be monitored by a variety of methodologies known to those of ordinary skill in the art, including flow cytometric analysis of CD14 positive cells, before and after depletion.

[0180] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In an embodiment, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml are used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[0181] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels ofCD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5x 106 / mL. In other embodiments, the concentration used is from about 1 x 105 / ml to 1 * 106 / mL, and any integer value in between.

[0182] T cells can also be frozen. Without wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media, the cells then are frozen to -80° C at a rate of 1° C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used, such as uncontrolled freezing immediately at -20° C or in liquid nitrogen.

[0183] T cells for use in the present invention also may be antigen-specific T cells. For example, tumor-specific T cells can be used. In an embodiment, antigen-specific T cells are isolated from a patient of interest, such as a patient afflicted with a cancer or an infectious disease. In one embodiment, neoepitopes are determined for a subject, and T cells specific to these antigens are isolated. Antigen-specific cells for use in expansion may also be generated in vitro using any number of methods known in the art (e.g., as described in U.S. Patent Publication No. US 20040224402 and U.S. Pat. Nos. 6,040,177). Antigen-specific cells for use in the present invention also may be generated using any number of methods known in the art (e.g., as described in Current Protocols in Immunology and Current Protocols in Cell Biology, both published by John Wiley & Sons, Inc., Boston, Mass).

[0184] In a related embodiment, it may be desirable to sort or otherwise positively select (e.g. via magnetic selection) the antigen specific cells prior to or following one or two rounds of expansion. Sorting or positively selecting antigen-specific cells can be carried out using peptide- MHC tetramers (Altman, et al., Science. 1996 Oct. 4; 274(5284):94-6). In another embodiment, the adaptable tetramer technology approach is used (Andersen et al., 2012 Nat Protoc. 7:891-902). Tetramers are limited by the need to utilize predicted binding peptides based on prior hypotheses, and the restriction to specific HLAs. Peptide-MHC tetramers can be generated using techniques known in the art and can be made with any MHC molecule of interest and any antigen of interest as described herein. Specific epitopes to be used in this context can be identified using numerousassays known in the art. For example, the ability of a polypeptide to bind to MHC class I may be evaluated indirectly by monitoring the ability to promote incorporation of 1251 labeled P2- microglobulin (P2m) into MHC class I / p2m / peptide heterotri meric complexes (see Parker et al., J. Immunol. 152: 163, 1994).

[0185] In one embodiment, cells are directly labeled with an epitope-specific reagent for isolation by flow cytometry followed by characterization of phenotype and TCRs. In one embodiment, T cells are isolated by contacting with T cell specific antibodies. Sorting of antigenspecific T cells, or generally any cells of the present invention, can be carried out using any of a variety of commercially available cell sorters, including, but not limited to, MoFlo sorter (DakoCytomation, Fort Collins, Colo.), FACSAria™, FACSArray™, FACSVantage™, BD™ LSR II, and FACSCalibur™ (BD Biosciences, San Jose, Calif).

[0186] In a preferred embodiment, the method comprises selecting cells that also express CD3. The method may comprise specifically selecting the cells in any suitable manner. Preferably, the selecting is carried out using flow cytometry. The flow cytometry may be carried out using any suitable method known in the art. The flow cytometry may employ any suitable antibodies and stains. Preferably, the antibody is chosen such that it specifically recognizes and binds to the particular biomarker being selected. For example, the specific selection of CD3, CD8, TIM-3, LAG-3, 4-1BB, or PD-1 may be carried out using anti-CD3, anti-CD8, anti-TIM-3, anti-LAG-3, anti-4-lBB, or anti-PD-1 antibodies, respectively. The antibody or antibodies may be conjugated to a bead (e.g., a magnetic bead) or to a fluorochrome. Preferably, the flow cytometry is fluorescence-activated cell sorting (FACS). TCRs expressed on T cells can be selected based on reactivity to autologous tumors. Additionally, T cells that are reactive to tumors can be selected for based on markers using the methods described in patent publication Nos. WO2014133567 and WO2014133568, herein incorporated by reference in their entirety. Additionally, activated T cells can be selected for based on surface expression of CD 107a.

[0187] In one embodiment of the invention, the method further comprises expanding the numbers of T cells in the enriched cell population. Such methods are described in U.S. Patent No. 8,637,307 and is herein incorporated by reference in its entirety. The numbers of T cells may be increased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), more preferably at least about 10- fold (or at least about 20-, at least about 30-, at least about 40-, at least about 50-, at least about 60-, at least about 70-, at least about 80-, or at least about 90-fold), more preferably at least about100-fold, more preferably at least about 1,000 fold, or most preferably at least about 100,000-fold. The numbers of T cells may be expanded using any suitable method known in the art. Exemplary methods of expanding the numbers of cells are described in patent publication No. WO 2003057171, U.S. Patent No. 8,034,334, and U.S. Patent Application Publication No. 2012 / 0244133, each of which is incorporated herein by reference.

[0188] In one embodiment, ex vivo T cell expansion can be performed by isolation of T cells and subsequent stimulation or activation followed by further expansion. In one embodiment of the invention, the T cells may be stimulated or activated by a single agent. In another embodiment, T cells are stimulated or activated with two agents, one that induces a primary signal and a second that is a co-stimulatory signal. Ligands useful for stimulating a single signal or stimulating a primary signal and an accessory molecule that stimulates a second signal may be used in soluble form. Ligands may be attached to the surface of a cell, to an Engineered Multivalent Signaling Platform (EMSP), or immobilized on a surface. In a preferred embodiment, both primary and secondary agents are co-immobilized on a surface, for example a bead or a cell. In one embodiment, the molecule providing the primary activation signal may be a CD3 ligand, and the co-stimulatory molecule may be a CD28 ligand or 4- IBB ligand.

[0189] In an embodiment, T cells comprising a CAR or an exogenous TCR, may be manufactured as described in WO2015120096, by a method comprising: enriching a population of lymphocytes obtained from a donor subject; stimulating the population of lymphocytes with one or more T cell stimulating agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule which encodes the CAR or TCR, using a single cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the population of transduced T cells for a predetermined time to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium. In an embodiment, T cells comprising a CAR or an exogenous TCR, may be manufactured as described in W02015120096, by a method comprising: obtaining a population of lymphocytes; stimulating the population of lymphocytes with one or more stimulating agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; transducing the population ofactivated T cells with a viral vector comprising a nucleic acid molecule which encodes the CAR or TCR, using at least one cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the population of transduced T cells to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium. The predetermined time for expanding the population of transduced T cells may be 3 days. The time from enriching the population of lymphocytes to producing the engineered T cells may be 6 days. The closed system may be a closed bag system. Further provided is population of T cells comprising a CAR or an exogenous TCR obtainable or obtained by said method, and a pharmaceutical composition comprising such cells.

[0190] In an embodiment, T cell maturation or differentiation in vitro may be delayed or inhibited by the method as described in W02017070395, comprising contacting one or more T cells from a subject in need of a T cell therapy with an AKT inhibitor (such as, e.g., one or a combination of two or more AKT inhibitors disclosed in claim 8 of W02017070395) and at least one of exogenous Interleukin-7 (IL-7) and exogenous Interleukin- 15 (IL- 15), wherein the resulting T cells exhibit delayed maturation or differentiation, and / or wherein the resulting T cells exhibit improved T cell function (such as, e.g., increased T cell proliferation; increased cytokine production; and / or increased cytolytic activity) relative to a T cell function of a T cell cultured in the absence of an AKT inhibitor.

[0191] In an embodiment, a patient in need of a T cell therapy may be conditioned by a method as described in WO2016191756 comprising administering to the patient a dose of cyclophosphamide between 200 mg / m2 / day and 2000 mg / m2 / day and a dose of fludarabine between 20 mg / m2 / day and 900 mg / m2 / day.

[0192] In an embodiment, a patient in need of adoptive cell transfer may be administered a TLR agonist to enhance anti-tumor immunity (see, e.g., Urban-Wojciuk, et al., Front Immunol. 2019; 10: 2388; and Kaczanowska et al., J Leukoc Biol. 2013 Jun; 93(6): 847-863). In an embodiment, TLR agonists are delivered in a nanoparticle system (see, e.g., Buss and Bhatia, Proc Natl Acad Sci.Methods Of Use In Stem Cell Transplant

[0193] Autologous stem cell transplantation (ASCT) represents a therapeutic approach for treating hematological malignancies such as multiple myeloma (MM), acute myeloid leukemia(AML), and chronic lymphocytic leukemia (CLL). This method involves the collection and reinfusion of hematopoietic stem cells (HSCs) into patients following high-dose chemotherapy or radiation therapy designed to eradicate malignant cells. The reinfused HSCs subsequently repopulate the bone marrow, facilitating the recovery of the patient's hematopoietic system. As disclosed herein, the engineered cells may be included in compositions used for ASCT. The antigen-activated TCRs disclosed herein may be engineered into patient derived T cells and included in compositions used for ASCT. As discussed in further detail in the Examples section below, the antigen-activated TCRs may enhance patient response to ASCT therapy and help sustain cancer remission. The identification, characterization, and utilization of these tumor- reactive TCRs represent a targeted therapeutic strategy that holds promise for improving patient prognosis and achieving durable remissions.IMMUNOGENIC COMPOSITIONS

[0194] Described In an embodiment herein are immunogenic compositions that can contain one or more disease associated antigens, e.g. cancer associated antigens (CAAs) and / or one or more polynucleotides encoding the one or more CAAs. In an embodiment, the cancer associated antigen is a conserved cancer antigen. In this context herein, “conserved cancer antigen” refers to a cancer associated antigen of a cancer cell that is recognized by a TCR comprising a conserved cancer gene signature. In an embodiment, the CAA (including but not limited to, a conserved cancer antigen) is a peptide or polypeptide antigen found in SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC. In an embodiment, the CAA (including but not limited to, a conserved cancer antigen) is a polynucleotide. In an embodiment, the CAA (including but not limited to, a conserved cancer antigen) is recognized by a TCR. In an embodiment, the CAA (including but not limited to, a conserved cancer antigen) is capable of presentation in an MHC I (HLA I) or MCH II (HLA II) molecule on a cancer cell. In an embodiment, the CAA (including but not limited to, a conserved cancer antigen) is capable of presentation in an MHC I (HLA I) or MCH II (HLA II) molecule as identified in SEQ ID NO: 29988-41854.

[0195] In an embodiment the CAA is selected from SEQ ID NOs: 325-4747, SEQ ID NOs:4748-4778, SEQ ID NO:s 4779-4902, SEQ ID NO:s 4903-4927, SEQ ID NOs: 4928-26232, SEQ ID NO: 26233-26364, SEQ ID NO: 26365-26738, SEQ ID NO: 26739-28624, SEQ ID NOs:26825-28633, SEQ ID NOs: 28634-28675, SEQ ID NOs: 28676-29125, SEQ ID NOs: 29126- 29987, or SEQ ID NO: 29988-41854.

[0196] Reference to “Major histocompatibility complex” (MHC) refers to a protein, generally a glycoprotein, that contains a polymorphic peptide binding site or binding groove that can, in some cases, complex with peptide antigens of polypeptides, including peptide antigens processed by the cell machinery. In some cases, MHC molecules can be displayed or expressed on the cell surface, including as a complex with peptide, i.e. MHC-peptide complex, for presentation of an antigen in a conformation recognizable by an antigen receptor on T cells, such as a TCRs or TCR- like antibody. Generally, MHC class I molecules are heterodimers having a membrane spanning a chain, in some cases with three a domains, and a non-covalently associated P2 microglobulin. Generally, MHC class II molecules are composed of two transmembrane glycoproteins, a and P, both of which typically span the membrane. An MHC molecule can include an effective portion of an MHC that contains an antigen binding site or sites for binding a peptide and the sequences necessary for recognition by the appropriate antigen receptor. In an embodiment, MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a MHC-peptide complex is recognized by T cells, such as generally CD8+ T cells, but in some cases CD4+ T cells. In an embodiment, MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are typically recognized by CD4+ T cells. Generally, MHC molecules are encoded by a group of linked loci, which are collectively termed H-2 in the mouse and human leukocyte antigen (HLA) in humans. Hence, typically human MHC can also be referred to as human leukocyte antigen (HLA).

[0197] The term “MHC-peptide complex” or “peptide-MHC complex” or variations thereof, refers to a complex or association of a peptide antigen and an MHC molecule, such as, generally, by non-covalent interactions of the peptide in the binding groove or cleft of the MHC molecule. In an embodiment, the MHC-peptide complex is present or displayed on the surface of cells. In an embodiment, the MHC-peptide complex can be specifically recognized by an antigen receptor, such as a TCR, TCR-like CAR or antigen-binding portions thereof.

[0198] In an embodiment, the CAA(s) (including but not limited to, a conserved cancer antigen(s)) are selected from a peptide selected from or are encoded by a polynucleotide selected from SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA,AGTACTTAT, GCTGCGTCC, GAGGTCACC.. In an embodiment, the conserved cancer antigens are or are encoded by a polynucleotide selected from a target sequence of SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC. In an embodiment, the conserved cancer antigens are selected from a peptide selected from or are encoded by a polynucleotide selected from SEQ ID NO: 325- 41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC. In an embodiment, the conserved cancer antigens are or are encoded by a polynucleotide selected from atarget sequence of SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.. In an embodiment, the one or more polynucleotides encoding the one or more CAAs (including but not limited to, conserved cancer antigens) is DNA. In an embodiment, the one or more polynucleotides encoding the one or more CAAs (including but not limited to, conserved cancer antigens) is RNA. In an embodiment, the one or more polynucleotides encoding the one or more CAAs (including but not limited to, conserved cancer antigens) is mRNA.

[0199] In an embodiment, the immunogenic composition can stimulate an immune response in a subject to which it is administered. In an embodiment, the immune response is a cell-mediated immune response. In an embodiment, the immune response is a humoral immune response. In an embodiment, the immune response includes B-cell, plasma cell, and / or antibody production (collectively referred to as a B-cell response). In an embodiment, the immune response includes a T-cell production (also referred to as a T-cell response). In an embodiment, the T-cell response includes CD 4+ T-cell production, CD8+ T cell production, or both. In an embodiment, the immune response includes both a B-cell and T-cell response.

[0200] In an embodiment, the immunogenic composition is formulated as a vaccine. In an embodiment, the immunogenic composition is formulated as a protein or peptide vaccine. In an embodiment, the immunogenic composition is formulated as a DNA vaccine. In an embodiment, the immunogenic composition is formulated as an RNA, such as an mRNA, vaccine. In an embodiment, the immunogenic composition or formulation thereof is a cancer vaccine. In other words, In an embodiment, the immunogenic composition can stimulate an immune responseagainst a cancer. In an embodiment, the immune response stimulated by the cancer vaccine is effective to reduce or eliminate the cancer in subject. In an embodiment, the cancer is a blood cancer. In an embodiment, the cancer is a white blood cell cancer. In an embodiment, the cancer is multiple myeloma.

[0201] In an embodiment, the immunogenic compositions may be combined with one or more antigenic components and / or anti-viral therapeutics, anti-proliferative therapeutics, anti -neoplastic therapeutics, and / or chemotherapeutics. In some examples, such combination may elicit cellular and / or antibody-mediated immune response, e.g., production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma-delta T cells.

[0202] These and other embodiments are described in greater detail elsewhere herein.Cancer Associated Antigen Polynucleotides and Polypeptides

[0203] In an embodiment, the CAA (including but not limited to, conserved cancer antigen) is a peptide or a polypeptide or a polynucleotide encoding said peptide or polypeptide. In an embodiment the CAA (including but not limited to, conserved cancer antigen) is recognized by a TCR or component thereof. In an embodiment, the TCR that recognizes a CAA (including but not limited to, conserved cancer antigen) described comprises a TCR alpha CDR3 sequence selected from SEQ ID NOs: 1-62, 41855-41902, or a TCR beta CDR3 sequence selected from SEQ ID NO: 63-121[ or 41903-41948.

[0204] In an embodiment the CAA is peptide selected from SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC, or a combination thereof. In an embodiment, the polynucleotides are codon optimized for expression in humans or non-human animals. In an embodiment, the one or more CAA polynucleotides or encoding polynucleotides (including, but not limited to, one or more conserved antigen polynucleotides or conserved antigen encoding polynucleotides) has a sequence corresponding to a (a) an annotated region of a genome; (b) an unannotated region of a genome; (c) a mutation; (d) a 5’UTR; (e) a 3’UTR; (f) an open reading frame; (g) a non-canonical open reading frames (nuORFs), or (h) any combination thereof. In an embodiment, the one or more CAA polynucleotides or encoding polynucleotides (including, but not limited to, one or more conserved antigen polynucleotides or conserved antigen encoding polynucleotides) has a sequencecorresponding to a (a) an annotated region of a cancer cell genome; (b) an unannotated region of a cancer cell genome; (c) a mutation; (d) a 5’UTR of a cancer cell; (e) a 3’UTR or a cancer cell; (f) an open reading frame of a cancer cell; (g) a non-canonical open reading frames (nuORFs) of a cancer cell, or (h) any combination thereof. In an embodiment, the cancer cell is a blood cancer cell. In an embodiment, the cancer cell is a white blood cell cancer cell. In an embodiment the cancer cell is a plasma cell. In an embodiment, the cancer is multiple myeloma, and the cancer cell is a multiple myeloma cell.

[0205] In an embodiment, a CAA antigen or antigen encoding polynucleotide (including, but not limited to, a conserved cancer antigen or conserved antigen encoding polynucleotide) of the present invention has 50-100% identity a peptide of SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC. In an embodiment, a CAA antigen or antigen encoding polynucleotide (including, but not limited to, a conserved cancer antigen or conserved antigen encoding polynucleotide) of the present invention of the present invention has 50%, to / or 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to a peptide of SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.

[0206] The terms “percent (%) sequence identity”, and the like, generally refer to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of polypeptides that may or may not share a common evolutionary origin. Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.), etc.Sizes of polynucleotides and polypeptides

[0207] In an embodiment, the polynucleotides may be any length reasonable to encode an epitope. In an embodiment, the polynucleotides range in length from about 10 to about 200 or more polynucleotides. In an embodiment, the polynucleotides in length from 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, to / or 200 nucleotides in length.

[0208] In an embodiment, the polypeptides may be any length that is reasonable for an epitope. For example, the polypeptides may have a size of from 5 to 30 or more, e.g., from 5 to 25, from 5 to 20, from 5 to 15, from 5 to 10, from 6 to 10, from 7 to 9, or from 8 to 9 amino acids. For example, the polypeptides may have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In an embodiment, the optimal length of a polypeptide may be determined based the immunogenicity of the polypeptides of different lengths when introduced to a cell or subject.Modifications on polypeptides

[0209] In an embodiment, polypeptides of the present invention herein may comprise one or more modifications (e.g., post-translational modifications). In some cases, the polypeptides may comprise cysteinylated Cysteine. Other examples of modifications include ubiquitination, phosphorylation, sulfonation, glycosylation, acetylation, methylation, ADP-ribosylation, methionine oxidation, cysteine oxidation, cysteine lipidation, farnesylation, geranylation, pyroglutamation, and deamidation. In an embodiment, the polypeptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acids that are each independently modified with an ubiquitination, phosphorylation, sulfonation, glycosylation, acetylation, methylation, ADP- ribosylation, methionine oxidation, cysteine oxidation, cysteine lipidation, farnesylation, geranylation, pyroglutamation, or deamidation.Synthetic mRNA

[0210] In an embodiment, the CAA polynucleotide of the present invention is mRNA, e.g., synthetic mRNA. In an embodiment, the synthetic mRNA may comprise coding sequence(s) for one or more CAA polypeptides herein. In an embodiment, the synthetic mRNA is or is encoded by a CAA encoding polynucleotide described elsewhere herein.

[0211] A synthetic mRNA may be an mRNA produced through an in vitro transcription reaction or through artificial (non-natural) chemical synthesis or through a combination thereof. In an embodiment, the synthetic mRNA further comprises a poly A tail, a Kozak sequence, a 3’ untranslated region, a 5’ untranslated region, or any combination thereof. Poly A tails in particular can be added to a synthetic RNA using a variety of art-recognized techniques, e.g., using poly A polymerase, using transcription directly from PCR products, or by ligating to the 3’ end of a synthetic RNA with RNA ligase.

[0212] The synthetic mRNA may comprise one or more stabilizing elements that maintain or enhance the stabilities of mRNA, e.g., reducing or preventing degradation of the mRNA. Examples of stabilizing elements include untranslated regions (UTR) at their 5 '-end (5'UTR) and / or at their 3 '-end (3 'UTR), in addition to other structural features, such as a 5 '-cap structure or a 3'-poly(A) tail. The stabilizing elements may be a histone stem-loop, e.g., a histone stem loop added by a stem-loop binding protein (SLBP).Delivery Vehicles

[0213] The CAA polynucleotides and / or peptides of the present invention can be incorporated into a delivery vehicle. The delivery vehicles can be used to deliver a CAA polynucleotide and / or peptide of the present invention to a cell. Thus, also described in certain example embodiments herein delivery vehicles, including but not limited to, vectors and virus particles that can deliver a CAA polynucleotide and / or polypeptide of the present invention, which is also generally referred to as “cargo” in this context. It will be appreciated that other molecules can also be included as cargo. The delivery vehicles may deliver the cargo into cells, tissues, organs, or organisms (e.g., animals or plants). The cargos may be packaged, carried, or otherwise associated with the delivery vehicles. The delivery vehicles may be selected based on the types of cargo to be delivered, and / or the delivery is in vitro and / or in vivo. Examples of delivery vehicles include vectors, viruses (e.g., virus particles), non-viral vehicles, and other delivery reagents described herein.

[0214] The delivery vehicles described herein can have a greatest dimension or greatest average dimension (e.g., diameter or greatest average diameter) of less than 100 microns (pm). In an embodiment, the delivery vehicles have a greatest dimension or greatest average dimension of less than 10 pm. In an embodiment, the delivery vehicles may have a greatest dimension or greatest average dimension of less than 2000 nanometers (nm). In an embodiment, the delivery vehicles may have a greatest dimension or greatest average dimension of less than 1000 nanometers (nm).In an embodiment, the delivery vehicles may have a greatest dimension or greatest average dimension (e.g., diameter or average diameter) of less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 150nm, or less than lOOnm, less than 50nm. In an embodiment, the delivery vehicles may have a greatest dimension or greatest average dimension ranging between 25 nm and 200 nm.

[0215] In an embodiment, the delivery vehicles may be or comprise particles. For example, the delivery vehicle may be or comprise nanoparticles (e.g., particles with a greatest dimension or greatest average dimension (e.g., diameter or greatest average diameter) no greater than 1000 nm. The particles may be provided in different forms, e.g., as solid particles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of particles, or combinations thereof. Metal, dielectric, and semiconductor particles may be prepared, as well as hybrid structures (e.g., core-shell particles).

[0216] In an embodiment, the delivery vehicles are nanoparticles. Exemplary nanoparticles are described in WO 2008042156, US 20130185823, and WO2015089419. In general, a “nanoparticle” refers to any particle having a diameter of less than 1000 nm. In an embodiment, nanoparticles of the invention have a greatest dimension or greatest average dimension (e.g., diameter or average diameter) of 500 nm or less. In other embodiments, nanoparticles of the invention have a greatest dimension or greatest average dimension ranging between 25 nm and 200 nm. In other embodiments, nanoparticles of the invention have a greatest dimension or greatest average dimension of 100 nm or less. In other embodiments, nanoparticles of the invention have a greatest dimension or greatest average dimensions ranging between 35 nm and 60 nm. It will be appreciated that reference made herein to particles or nanoparticles can be interchangeable, where appropriate. Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically sub 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present invention. Semi-solid and soft nanoparticles have been manufactured and are within the scope of the present invention. Nanoparticles with one half hydrophilic and the other half hydrophobic are termed Janus particles and are particularly effective for stabilizing emulsions. They can self-assemble at water / oil interfaces and act as solid surfactants.

[0217] Particle characterization (including e.g., characterizing morphology, dimension, etc.) is done using a variety of different techniques. Common techniques are electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALD1-TOF), ultraviolet-visible spectroscopy, dual polarization interferometry and nuclear magnetic resonance (NMR). Characterization (dimension measurements) may be made as to native particles (i.e., preloading) or after loading of the cargo (herein cargo refers to e.g., one or more components of CRISPR-Cas system e.g., CRISPR enzyme or mRNA or guide RNA, or any combination thereof, and may include additional carriers and / or excipients) to provide particles of an optimal size for delivery for any in vitro, ex vivo and / or in vivo application of the present invention. In certain preferred embodiments, particle dimension (e.g., diameter) characterization is based on measurements using dynamic laser scattering (DLS). Mention is made of US Patent No. 8,709,843; US Patent No. 6,007,845; US Patent No. 5,855,913; US Patent No. 5,985,309; US. Patent No. 5,543,158; and the publication by James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) published online 11 May 2014, doi: 10.1038 / nnano.2014.84, describing particles, methods of making and using them and measurements thereof.Vectors and Vector systems

[0218] Also provided herein are vectors that can contain one or more of the CAA polynucleotides of the present invention described elsewhere herein. In an embodiment, the vector can contain one or more polynucleotides encoding one or more polypeptides, such as a CAA polypeptide, of the present invention described elsewhere herein. The vectors can be useful in producing bacterial, fungal, yeast, plant cells, animal cells, and transgenic animals that can express one or more CAA polynucleotides and / or polypeptides of the present invention described elsewhere herein. Within the scope of this disclosure are vectors containing one or more of the polynucleotide sequences described herein. The vectors and / or vector systems can be used, for example, to express one or more of the polynucleotides in a cell, such as a producer cell, to produce virus particles containing one or more polynucleotide(s) of the present invention described elsewhere herein. Other uses for the vectors and vector systems described herein are also within the scope of this disclosure. In general, and throughout this specification, the term “vector” refers to a tool that allows or facilitates the transfer of an entity from one environment to another. Insome contexts which will be appreciated by those of ordinary skill in the art, “vector” can be a term of art to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements.

[0219] Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively -linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0220] Recombinant expression vectors can be composed of a nucleic acid (e.g., a polynucleotide) of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which can be selected on the basis of the host cells to be used for expression, that is operatively- linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” and “operatively-linked” are used interchangeably herein and further defined elsewhere herein. In the context of a vector, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or ina host cell when the vector is introduced into the host cell). Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells. These and other embodiments of the vectors and vector systems are described elsewhere herein.

[0221] In an embodiment, the vector can be a viral vector. In an embodiment, the viral vector is an is an adeno-associated virus (AAV), adenovirus vector, a retroviral vector, or lentiviral vector.

[0222] These and others are further detailed and described elsewhere herein.Cell-based Vector Amplification and Expression

[0223] Vectors may be introduced and propagated in a prokaryote or prokaryotic cell. In an embodiment, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e g., amplifying a plasmid as part of a viral vector packaging system). The vectors can be viral-based or non-viral based. In an embodiment, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism.

[0224] Vectors can be designed for expression of the polynucleotides and / or polypeptides of the present invention described herein (e.g., nucleic acid transcripts, proteins, enzymes, and combinations thereof) in a suitable host cell. In an embodiment, the suitable host cell is a prokaryotic cell. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. In an embodiment, the suitable host cell is a eukaryotic cell.

[0225] In an embodiment, the suitable host cell is a suitable bacterial cell. Suitable bacterial cells include but are not limited to bacterial cells from the bacteria of the species Escherichia coli. Many suitable strains of E. coli are known in the art for expression of vectors. These include, but are not limited to Pirl, Stbl2, Stbl3, Stbl4, TOP 10, XL1 Blue, and XL 10 Gold. In an embodiment, the host cell is a suitable insect cell. Suitable insect cells include those from Spodoptera frugiperda. Suitable strains of S. frugiperda cells include, but are not limited to, Sf9 and Sf21. In an embodiment, the host cell is a suitable yeast cell. In an embodiment, the yeast cell can be from Saccharomyces cerevisiae. In an embodiment, the host cell is a suitable mammalian cell. Many types of mammalian cells have been developed to express vectors. Suitable mammalian cells include, but are not limited to, HEK293, Chinese Hamster Ovary Cells (CHOs), mouse myeloma cells, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-Rb50,HepG G2, DIKX-X11, J558L, Baby hamster kidney cells (BHK), and chicken embryo fibroblasts (CEFs). Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

[0226] In an embodiment, the vector can be a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa(Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif). As used herein, a “yeast expression vector” refers to a nucleic acid that contains one or more sequences encoding an RNA and / or polypeptide and may further contain any desired elements that control the expression of the nucleic acid(s), as well as any elements that enable the replication and maintenance of the expression vector inside the yeast cell. Many suitable yeast expression vectors and features thereof are known in the art; for example, various vectors and techniques are illustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (Humana Press, New York, 2007) andBuckholz, R.G. and Gleeson, M.A. (1991) Biotechnology (NY) 9(11): 1067- 72. Yeast vectors can contain, without limitation, a centromeric (CEN) sequence, an autonomous replication sequence (ARS), a promoter, such as an RNA Polymerase III promoter, operably linked to a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., auxotrophic, antibiotic, or other selectable markers). Examples of expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2p plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and episomal plasmids.

[0227] In an embodiment, the vector is a baculovirus vector or expression vector and can be suitable for expression of polynucleotides and / or proteins in insect cells. In an embodiment, the suitable host cell is an insect cell. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39). rAAV (recombinant Adeno-associated viral) vectors are preferably produced in insect cells, e.g., Spodoptera frugiperda Sf9 insect cells, grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial vendors, e.g., Sigma Aldrich (EX-CELL 405).

[0228] In an embodiment, the vector is a mammalian expression vector. In an embodiment, the mammalian expression vector is capable of expressing one or more polynucleotides and / or polypeptides in a mammalian cell. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). The mammalian expression vector can include one or more suitable regulatory elements capable of controlling expression of the one or more polynucleotides and / or proteins in the mammalian cell. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. More detail on suitable regulatory elements are described elsewhere herein.

[0229] For other suitable expression vectors and vector systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0230] In an embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). With regards to these prokaryotic and eukaryotic vectors, mention is made of U.S. Patent 6,750,059, the contents of which are incorporated by reference herein in their entirety. Other embodiments can utilize viral vectors, with regards to which mention is made of U.S. Patent application 13 / 092,085, the contents of which are incorporated by reference herein in their entirety. Tissue-specific regulatory elementsare known in the art and in this regard, mention is made of U.S. Patent 7,776,321, the contents of which are incorporated by reference herein in their entirety. In an embodiment, a regulatory element can be operably linked to one or more polynucleotides of the present invention so as to drive expression of the one or more polynucleotides of the present invention described herein.

[0231] In an embodiment, the vector can be a fusion vector or fusion expression vector. In an embodiment, fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus, carboxy terminus, or both of a recombinant protein. Such fusion vectors can serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. In an embodiment, expression of polynucleotides (such as non-coding polynucleotides) and proteins in prokaryotes can be carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polynucleotides and / or proteins. In an embodiment, the fusion expression vector can include a proteolytic cleavage site, which can be introduced at the junction of the fusion vector backbone or other fusion moiety and the recombinant polynucleotide or protein to enable separation of the recombinant polynucleotide or protein from the fusion vector backbone or other fusion moiety subsequent to purification of the fusion polynucleotide or protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301- 315) and pET l id (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0232] In an embodiment, one or more vectors driving expression of one or more polynucleotides of the present invention described herein are introduced into a cell, such as a host cell for viral particle production and / or a target cell to which a polypeptide of the present invention is to be expressed.Cell-Free Vector and Polynucleotide Expression

[0233] In an embodiment, the polynucleotide encoding one or more CAA polynucleotides or polypeptides of the present invention can be expressed from a vector or suitable polynucleotide in a cell-free in vitro system. In other words, the polynucleotide can be transcribed and optionally translated in vitro. In vitro transcription / translation systems and appropriate vectors are generally known in the art and commercially available. Generally, in vitro transcription and in vitro translation systems replicate the processes of RNA and protein synthesis, respectively, outside of the cellular environment. Vectors and suitable polynucleotides for in vitro transcription can include T7, SP6, T3, promoter regulatory sequences that can be recognized and acted upon by an appropriate polymerase to transcribe the polynucleotide or vector.

[0234] In vitro translation can be stand-alone (e.g., translation of a purified polyribonucleotide) or linked / coupled to transcription. In an embodiment, the cell-free (or in vitro) translation system can include extracts from rabbit reticulocytes, wheat germ, and / or E. coli. The extracts can include various macromolecular components that are needed for translation of exogenous RNA (e.g., 70S or 80S ribosomes, tRNAs, aminoacyl-tRNA, synthetases, initiation, elongation factors, termination factors, etc.). Other components can be included or added during the translation reaction, including but not limited to, amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase (eukaryotic systems)) (phosphoenol pyruvate and pyruvate kinase for bacterial systems), and other co-factors (Mg2+, K+, etc.). As previously mentioned, in vitro translation can be based on RNA or DNA starting material. Some translation systems can utilize an RNA template as starting material (e.g., reticulocyte lysates and wheat germ extracts). Some translation systems can utilize a DNA template as a starting material (e.g., E coli-based systems). In these systems transcription and translation are coupled and DNA is first transcribed into RNA, which is subsequently translated. Suitable standard and coupled cell-free translation systems are generally known in the art and are commercially available.Vector Features

[0235] The vectors can include additional features that can confer one or more functionalities to the vector, the polynucleotide to be delivered, a virus particle produced there from, or polypeptide expressed thereof. Such features include, but are not limited to, regulatory elements, selectable markers, molecular identifiers (e.g., molecular barcodes), stabilizing elements, and thelike. It will be appreciated by those skilled in the art that the design of the expression vector and additional features included can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc.Regulatory Elements

[0236] In an embodiment, the polynucleotides and / or vectors thereof described herein (such as the polynucleotides of the present invention, such as a viral polynucleotides of the present invention) can include one or more regulatory elements that can be operatively linked to the polynucleotide. The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences) and cellular localization signals (e.g., nuclear localization signals). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissuespecific regulatory sequences). A tissue-specific promoter can direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In an embodiment, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41 :521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I (Mol.Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit P-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).

[0237] In an embodiment, the regulatory sequence can be a regulatory sequence described in U.S. Pat. No. 7,776,321, U.S. Pat. Pub. No. 2011 / 0027239, and International Patent Publication No. WO 2011 / 028929, the contents of which are incorporated by reference herein in their entirety. In an embodiment, the vector can contain a minimal promoter. In an embodiment, the minimal promoter is the Mecp2 promoter, tRNA promoter, or U6. In a further embodiment, the minimal promoter is tissue specific. In an embodiment, the length of the vector polynucleotide the minimal promoters and polynucleotide sequences is less than 4.4Kb.

[0238] To express a polynucleotide, the vector can include one or more transcriptional and / or translational initiation regulatory sequences, e.g., promoters, that direct the transcription of the gene and / or translation of the encoded protein in a cell. In an embodiment a constitutive promoter may be employed. Suitable constitutive promoters for mammalian cells are generally known in the art and include, but are not limited to SV40, CAG, CMV, EF-la, -actin, RSV, and PGK. Suitable constitutive promoters for bacterial cells, yeast cells, and fungal cells are generally known in the art, such as a T-7 promoter for bacterial expression and an alcohol dehydrogenase promoter for expression in yeast.

[0239] In an embodiment, the regulatory element can be a regulated promoter. “Regulated promoter” refers to promoters that direct gene expression not constitutively, but in a temporally- and / or spatially-regulated manner, and includes tissue-specific, tissue-preferred and inducible promoters. Regulated promoters include conditional promoters and inducible promoters. In an embodiment, conditional promoters can be employed to direct expression of a polynucleotide in a specific cell type, under certain environmental conditions, and / or during a specific state of development. Suitable tissue specific promoters can include, but are not limited to, liver specific promoters (e.g. APOA2, SERPIN Al (hAAT), CYP3A4, and MIR122), pancreatic cell promoters (e.g. INS, IRS2, Pdxl, Alx3, Ppy), cardiac specific promoters (e.g. Myh6 (alpha MHC), MYL2 (MLC-2v), TNI3 (cTnl), NPPA (ANF), Slc8al (Next)), central nervous system cell promoters (SYN1, GFAP, INA, NES, MOBP, MBP, TH, FOXA2 (HNF3 beta)), skin cell specific promoters (e.g. FLG, K14, TGM3), immune cell specific promoters, (e.g. ITGAM, CD43 promoter, CD14 promoter, CD45 promoter, CD68 promoter), urogenital cell specific promoters (e.g. Pbsn, Upk2, Sbp, Ferll4), endothelial cell specific promoters (e.g. ENG), pluripotent and embryonic germ layercell specific promoters (e.g. Oct4, NANOG, Synthetic Oct4, T brachyury, NES, SOX17, FOXA2, MIR122), and muscle cell specific promoter (e.g. Desmin). Other tissue and / or cell specific promoters are generally known in the art and are within the scope of this disclosure.

[0240] Inducible / conditional promoters can be positively inducible / conditional promoters (e g. a promoter that activates transcription of the polynucleotide upon appropriate interaction with an activated activator, or an inducer (compound, environmental condition, or other stimulus) or a negative / conditional inducible promoter (e.g. a promoter that is repressed (e.g. bound by a repressor) until the repressor condition of the promotor is removed (e g. inducer binds a repressor bound to the promoter stimulating release of the promoter by the repressor or removal of a chemical repressor from the promoter environment). The inducer can be a compound, environmental condition, or other stimulus. Thus, inducible / conditional promoters can be responsive to any suitable stimuli such as chemical, biological, or other molecular agents, temperature, light, and / or pH. Suitable inducible / conditional promoters include, but are not limited to, Tet-On, Tet-Off, Lac promoter, pBad, AlcA, LexA, Hsp70 promoter, Hsp90 promoter, pDawn, XVE / OlexA, GVG, and pOp / LhGR.

[0241] Where expression in a plant cell is desired, the components of the CRISPR-Cas system described herein are typically placed under control of a plant promoter, i.e., a promoter operable in plant cells. The use of different types of promoters is envisaged.

[0242] A constitutive plant promoter is a promoter that can express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant (referred to as “constitutive expression”). One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. In particular embodiments, one or more of the polynucleotides of the present invention are expressed under the control of a constitutive promoter, such as the cauliflower mosaic virus 35S promoter issue-preferred promoters can be utilized to target enhanced expression in certain cell types within a particular plant tissue, for instance vascular cells in leaves or roots or in specific cells of the seed. Examples of particular promoters for expression of one or more polynucleotides of the present invention in plants can be found in e.g., Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Yamamoto etal., (1997) Plant J 12:255-65; Hire et al, (1992) Plant Mol Biol 20:207-18, Kuster et al, (1995) Plant Mol Biol 29:759-72, and Capana et al., (1994) Plant Mol Biol 25:681 -91.

[0243] Examples of promoters that are inducible and that can allow for spatiotemporal control of gene editing or gene expression may use a form of energy. The form of energy may include but is not limited to sound energy, electromagnetic radiation, chemical energy and / or thermal energy. Examples of inducible systems include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc.), or light inducible systems (Phytochrome, LOV domains, or cryptochrome), such as a Light Inducible Transcriptional Effector (LITE) that direct changes in transcriptional activity in a sequence-specific manner. The components of a light inducible system may include one or more polynucleotides of the present invention described herein, a light-responsive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcriptional activation / repression domain. In an embodiment, the vector can include one or more of the inducible DNA binding proteins provided in International Patent Publication No. WO 2014 / 018423 and US Patent Publication Nos., 2015 / 0291966, 2017 / 0166903, 2019 / 0203212, which describe e.g., embodiments of inducible DNA binding proteins and methods of use and can be adapted for use with the present invention.

[0244] In an embodiment, transient or inducible expression can be achieved by including, for example, chemical-regulated promotors, i.e., whereby the application of an exogenous chemical induces gene expression. Modulation of gene expression can also be obtained by including a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters include, but are not limited to, the maize ln2-2 promoter, activated by benzene sulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568- 77), the maize GST promoter (GST-11-27, WO93 / 01294), activated by hydrophobic electrophilic compounds used as pre-emergent herbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7) activated by salicylic acid. Promoters which are regulated by antibiotics, such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991 ) Mol Gen Genet 227:229-37; U.S. Patent Nos. 5,814,618 and 5,789,156) can also be used herein.

[0245] In an embodiment, the polynucleotide, vector, or system thereof can include one or more elements capable of translocating and / or expressing one or more polynucleotides of the present invention to / in a specific cell component or organelle. Such organelles can include, but arenot limited to, nucleus, ribosome, endoplasmic reticulum, Golgi apparatus, chloroplast, mitochondria, vacuole, lysosome, cytoskeleton, plasma membrane, cell wall, peroxisome, centrioles, etc. Such regulatory elements can include, but are not limited to, nuclear localization signals (examples of which are described in greater detail elsewhere herein), any such as those that are annotated in the LocSigDB database (see e.g., genome.unmc.edu / LocSigDB / and Negi et al., 2015. Database. 2015: bav003; doi: 10.1093 / database / bav003), nuclear export signals (e.g., LXXXLXXLXL and others described elsewhere herein), endoplasmic reticulum localization / retention signals (e.g., KDEL (SEQ ID NO: 306), KDXX, KKXX, KXX, and others described elsewhere herein; and see e.g. Liu et al. 2007 Mol. Biol. Cell. 18(3): 1073-1082 and Gorleku et al., 2011. J. Biol. Chem. 286:39573-39584), mitochondria (see e.g., Cell Reports. 22:2818-2826, particularly at Fig. 2; Doyle et al. 2013. PLoS ONE 8, e67938; Funes et al. 2002. J. Biol. Chem. 277:6051-6058; Matouschek et al. 1997. PNAS USA 85:2091-2095; Oca-Cossio et al., 2003. 165:707-720; Waltner et al., 1996. J. Biol. Chem. 271 :21226-21230; Wilcox et al., 2005. PNAS USA 102: 15435-15440; Galanis et al., 1991. FEBS Lett 282:425-430, peroxisome (e.g. (S / A / C)-(K / R / H)-(L / A), SLK, (R / K)-(L / V / I)-XXXXX-(H / Q)-(L / A / F). Suitable protein targeting motifs can also be designed or identified using any suitable database or prediction tool, including but not limited to Minimotif Miner (http:minimotifminer.org, http: / / mitominer.mrc- mbu.cam.ac.uk / release-4.0 / embodiment.do?name=Protein%20MTS), LocDB (see above), PTSs predictor (), TargetP-2.0 (http: / / www.cbs.dtu.dk / services / TargetP / ), ChloroP (http: / / www.cbs.dtu.dk / services / ChloroP / ); NetNES (www.cbs.dtu.dk / services / NetNES / ), Predotar (https: / / urgi.versailles.inra.fr / predotar / ), and SignalP(http: / / www.cbs.dtu.dk / services / SignalP / ).Selectable Markers and Tags

[0246] One or more of the polynucleotides of the present invention can be operably linked, fused to, or otherwise modified to include a polynucleotide that encodes or is a selectable marker or tag, which can be a polynucleotide or polypeptide. In an embodiment, the polynucleotide encoding a polypeptide selectable marker can be incorporated with the polynucleotide of the present invention, such as a viral polynucleotide, such that the selectable marker polypeptide, when translated, is inserted between two amino acids between the N- and C- terminus of the polypeptide of the present invention or is present at the N- and / or C-terminus of the polypeptide of the presentinvention. In an embodiment, the selectable marker or tag is a polynucleotide barcode or unique molecular identifier (UMI).

[0247] It will be appreciated that the polynucleotide encoding such selectable markers or tags can be incorporated into a polynucleotide encoding one or more polypeptides of the present invention, such as a viral polypeptide, described herein in an appropriate manner to allow expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein, and will be instantly appreciated by one of ordinary skill in the art in view of this disclosure. Many such selectable markers and tags are generally known in the art and are intended to be within the scope of this disclosure.

[0248] Suitable selectable markers and tags include, but are not limited to, affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag; solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP, and GST; chromatography tags such as those consisting of polyanionic amino acids, such as FLAG-tag; epitope tags such as V5-tag, Myc-tag, HA-tag and NE-tag; protein tags that can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging), DNA and / or RNA segments that contain restriction enzyme or other enzyme cleavage sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT)) and the like; DNA and / or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and / or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as P-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (REP), luciferase, and cell surface proteins); polynucleotides that can generate one or more new primer sites for PCR (e.g., the juxtaposition of two DNA sequences not previously juxtaposed), DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g. GFP, FLAG- and His-tags), and, DNA sequences that make a molecular barcode or unique molecular identifier (UMI), DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art.

[0249] Selectable markers and tags can be operably linked to one or more polypeptides of the present invention herein via suitable linker, such as a glycine or glycine serine linkers as short as GS or GG up to (GGGGG)3 (SEQ ID NO: 307) or (GGGGS)3 (SEQ ID NO: 308). Other suitable linkers are described elsewhere herein.

[0250] The vector or vector system can include one or more polynucleotides encoding one or more targeting moieties. In an embodiment, the targeting moiety encoding polynucleotides can be included in the vector or vector system, such as a viral vector system, such that they are expressed within and / or on the virus particle(s) produced such that the virus particles can be targeted to specific cells, tissues, organs, etc. In an embodiment, the targeting moiety encoding polynucleotides can be included in the vector or vector system such that the polynucleotide(s) and / or products expressed therefrom (e.g., polypeptides) include the targeting moiety and can be targeted to specific cells, tissues, organs, etc. In an embodiment, such as non-viral carriers, the targeting moiety can be attached to the carrier (e g., polymer, lipid, inorganic molecule etc.) and can be capable of targeting the carrier and any attached or associated polynucleotide(s) and / or polypeptides of the present invention to specific cells, tissues, organs, etc.Codon Optimization of Vector Polynucleotides

[0251] As described elsewhere herein, the polynucleotide encoding one or more polypeptides of the present invention described herein can be codon optimized. In an embodiment, one or more polynucleotides contained in a vector (“vector polynucleotides”) described herein that are in addition to an optionally codon optimized polynucleotide encoding one or more polypeptides of the present invention, such as viral polypeptides, described herein can be codon optimized. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimalgene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp / codon / and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In an embodiment, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a DNA / RNA-targeting Cas protein corresponds to the most frequently used codon for a particular amino acid. As to codon usage in yeast, reference is made to the online Yeast Genome database available at http: / / www.yeastgenome.org / community / codon usage. shtml, or B[ED1] ennetzen and Hall, J Biol Chem. 1982 Mar 25;257(6):3026-31. As to codon usage in plants including algae, reference is made to Campbell and Gowri, Plant Physiol. 1990 Jan; 92(1): 1—11.; as well as Murray et al, Nucleic Acids Res. 1989 Jan 25;17(2):477-98; or Morton BR, J Mol Evol. 1998 Apr;46(4):449- 59.

[0252] The vector polynucleotide can be codon optimized for expression in a specific celltype, tissue type, organ type, and / or subject type. In an embodiment, a codon optimized sequence is a sequence optimized for expression in a eukaryote, e.g., humans (i.e., being optimized for expression in a human or human cell), or for another eukaryote, such as another animal (e.g., a mammal or avian) as is described elsewhere herein. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, the polynucleotide is codon optimized for a specific cell type. Such cell types can include, but are not limited to, epithelial cells (including skin cells, cells lining the gastrointestinal tract, cells lining other hollow organs), nerve cells (nerves, brain cells, spinal column cells, nerve support cells (e.g. astrocytes, glial cells, Schwann cells etc.) , muscle cells (e.g., cardiac muscle, smooth muscle cells, and skeletal muscle cells), connective tissue cells (fat and other soft tissue padding cells, bone cells, tendon cells, cartilage cells), blood cells, stem cells and other progenitor cells, immune system cells, germ cells, and combinations thereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, the polynucleotide is codon optimized for a specific tissue type. Such tissue types can include, but are not limited to, muscle tissue, connective tissue, connective tissue, nervous tissue, and epithelialtissue. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, the polynucleotide is codon optimized for a specific organ. Such organs include, but are not limited to, muscles, skin, intestines, liver, spleen, brain, lungs, stomach, heart, kidneys, gallbladder, pancreas, bladder, thyroid, bone, blood vessels, blood, and combinations thereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein.

[0253] [In an embodiment, a vector polynucleotide is codon optimized for expression in particular cells, such as prokaryotic or eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as discussed herein, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.Vector Construction

[0254] The vectors described herein can be constructed using any suitable process or technique. In an embodiment, one or more suitable recombination and / or cloning methods or techniques can be used to the vector(s) described herein. Suitable recombination and / or cloning techniques and / or methods can include, but not limited to, those described in U.S. Patent Publication No. US 2004 / 0171156 Al. Other suitable methods and techniques are described elsewhere herein.

[0255] Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81 :6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). Any of the techniques and / or methods can be used and / or adapted for constructing an AAV or other vector described herein. nAAV vectors are discussed elsewhere herein.

[0256] In an embodiment, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In an embodiment, one or more insertion sites (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide polynucleotides are used, a single expression construct may be used to target nucleic acid-targeting activity to multiple different, corresponding target sequences within a cell. For example, a single vector may compriseabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, or more guide polynucleotides. In an embodiment, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more such guide-polynucleotide-containing vectors may be provided, and optionally delivered to a cell.

[0257] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more polynucleotides and / or polypeptides of the present invention, such as one or more viral polynucleotides and / or polypeptides, described herein are as used in the foregoing documents, such as International Patent Publication No. WO 2014 / 093622 (PCT / US2013 / 074667) and are discussed in greater detail herein.Viral Vectors

[0258] In an embodiment, the vector is a viral vector. The term of art “viral vector” and as used herein in this context refers to polynucleotide based vectors that contain one or more elements from or based upon one or more elements of a virus that can be capable of expressing and packaging a polynucleotide, such as a viral polynucleotide of the present invention, into a virus particle and producing said virus particle when used alone or with one or more other viral vectors (such as in a viral vector system). Viral vectors and systems thereof can be used for producing viral particles for delivery of and / or expression of one or more polynucleotides and / or polypeptides of the present invention described herein. The viral vector can be part of a viral vector system involving multiple vectors. In an embodiment, systems incorporating multiple viral vectors can increase the safety of these systems. Suitable viral vectors can include retroviral -based vectors, lentiviral-based vectors, adenoviral-based vectors, adeno associated vectors, helper-dependent adenoviral (HdAd) vectors, hybrid adenoviral vectors, herpes simplex virus-based vectors, poxvirus-based vectors, and Epstein-Barr virus-based vectors. Other embodiments of viral vectors and viral particles produce therefrom are described elsewhere herein. In an embodiment, the viral vectors are configured to produce replication incompetent viral particles for improved safety of these systems.

[0259] In an embodiment, the virus structural component, which can be encoded by one or more polynucleotides in a viral vector or vector system, comprises one or more capsid proteins including an entire capsid. In an embodiment, such as wherein a viral capsid comprises multiple copies of different proteins, the delivery system can provide one or more of the same protein or a mixture of such proteins. For example, AAV comprises 3 capsid proteins, VP1, VP2, and VP3,thus delivery systems of the invention can comprise one or more of VP1, and / or one or more of VP2, and / or one or more of VP3. Accordingly, the present invention is applicable to a virus within the family Adenoviridae, such as Atadenovirus, e.g., Ovine atadenovirus D, Aviadenovirus, e.g., Fowl aviadenovirus A, Ichtadenovirus, e.g., Sturgeon ichtadenovirus A, Mastadenovirus (which includes adenoviruses such as all human adenoviruses), e.g., Human mastadenovirus C, and Siadenovirus, e.g., Frog siadenovirus A. Thus, a virus of within the family Adenoviridae is contemplated as within the invention with discussion herein as to adenovirus applicable to other family members. Target-specific AAV capsid variants can be used or selected. Non-limiting examples include capsid variants selected to bind to chronic myelogenous leukemia cells, human CD34 PBPC cells, breast cancer cells, cells of lung, heart, dermal fibroblasts, melanoma cells, stem cell, glioblastoma cells, coronary artery endothelial cells and keratinocytes. See, e.g., Buning et al, 2015, Current Opinion in Pharmacology 24, 94-104. From teachings herein and knowledge in the art as to modifications of adenovirus (see, e g., US Patents 9,410,129, 7,344,872, 7,256,036, 6,911,199, 6,740,525; Matthews, “Capsid-Incorporation of Antigens into Adenovirus Capsid Proteins for a Vaccine Approach,” Mol Pharm, 8(1): 3-11 (2011)), as well as regarding modifications of AAV, the skilled person can readily obtain a modified adenovirus that has a large payload protein, despite that heretofore it was not expected that such a large protein could be provided on an adenovirus. And as to the viruses related to adenovirus mentioned herein, as well as to the viruses related to AAV mentioned elsewhere herein, the teachings herein as to modifying adenovirus and AAV, respectively, can be applied to those viruses without undue experimentation from this disclosure and the knowledge in the art.Retroviral and Lentiviral Vectors

[0260] Retroviral vectors can be composed of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Suitable retroviral vectors for the CRISPR-Cas systems can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66: 1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J.Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Selection of a retroviral gene transfer system may therefore depend on the target tissue.

[0261] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and are described in greater detail elsewhere herein. A retrovirus can also be engineered to allow for conditional expression of the inserted transgene, such that only certain cell types are infected by the lentivirus.

[0262] Lentiviruses are complex retroviruses that can infect and express their genes in both mitotic and post-mitotic cells. Advantages of using a lentiviral approach can include the ability to transduce or infect non-dividing cells and their ability to typically produce high viral titers, which can increase efficiency or efficacy of production and delivery. Suitable lentiviral vectors include, but are not limited to, human immunodeficiency virus (HlV)-based lentiviral vectors, feline immunodeficiency virus (FlV)-based lentiviral vectors, simian immunodeficiency virus (SIV)- based lentiviral vectors, Moloney Murine Leukaemia Virus (Mo-MLV), Visna.maedi virus (VMV)-based lentiviral vector, carpine arthritis-encephalitis virus (CAEV)-based lentiviral vector, bovine immune deficiency virus (BlV)-based lentiviral vector, and Equine infectious anemia (EIAV)-based lentiviral vector. In an embodiment, an HIV-based lentiviral vector system can be used. In an embodiment, a FIV-based lentiviral vector system can be used.

[0263] In an embodiment, the lentiviral vector is an EIAV-based lentiviral vector or vector system. EIAV vectors have been used to mediate expression, packaging, and / or delivery in other contexts, such as for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8: 275 - 285). In another embodiment, RetinoStat®, (see, e.g., Binley et al., HUMAN GENE THERAPY 23:980- 991 (September 2012)), which describes RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. Any of these vectors described in these publications can be modified for polynucleotides and / or polypeptides of the present invention described herein.

[0264] In an embodiment, the lentiviral vector or vector system thereof can be a first- generation lentiviral vector or vector system thereof. First-generation lentiviral vectors can contain a large portion of the lentivirus genome, including the gag and pol genes, other additional viral proteins (e g., VSV-G) and other accessory genes (e.g., vif, vprm vpu, nef, and combinationsthereof), regulatory genes (e.g., tat and / or rev) as well as the gene of interest between the LTRs. First generation lentiviral vectors can result in the production of virus particles that can be capable of replication in vivo, which may not be appropriate for some instances or applications.

[0265] In an embodiment, the lentiviral vector or vector system thereof can be a second- generation lentiviral vector or vector system thereof. Second-generation lentiviral vectors do not contain one or more accessory virulence factors and do not contain all components necessary for virus particle production on the same lentiviral vector. This can result in the production of a replication-incompetent virus particle and thus increase the safety of these systems over first- generation lentiviral vectors. In an embodiment, the second-generation vector lacks one or more accessory virulence factors (e.g., vif, vprm, vpu, nef, and combinations thereof). Unlike the first- generation lentiviral vectors, no single second-generation lentiviral vector includes all features necessary to express and package a polynucleotide into a virus particle. In an embodiment, the envelope and packaging components are split between two different vectors with the gag, pol, rev, and tat genes being contained on one vector and the envelope protein (e.g., VSV-G) are contained on a second vector. The gene of interest, its promoter, and LTRs can be included on a third vector that can be used in conjunction with the other two vectors (packaging and envelope vectors) to generate a replication-incompetent virus particle.

[0266] In an embodiment, the lentiviral vector or vector system thereof can be a third- generation lentiviral vector or vector system thereof. Third-generation lentiviral vectors and vector systems thereof have increased safety over first- and second-generation lentiviral vectors and systems thereof because, for example, the various components of the viral genome are split between two or more different vectors but used together in vitro to make virus particles, they can lack the tat gene (when a constitutively active promoter is included up-stream of the LTRs), and they can include one or more deletions in the 3’LTR to create self-inactivating (SIN) vectors having disrupted promoter / enhancer activity of the LTR. In an embodiment, a third-generation lentiviral vector system can include (i) a vector plasmid that contains the polynucleotide of interest and upstream promoter that are flanked by the 5’ and 3’ LTRs, which can optionally include one or more deletions present in one or both of the LTRs to render the vector self-inactivating; (ii) a “packaging vector(s)” that can contain one or more genes involved in packaging a polynucleotide into a virus particle that is produced by the system (e.g., gag, pol, and rev) and upstream regulatory sequences (e.g., promoter(s)) to drive expression of the features present on the packaging vector,and (iii) an “envelope vector” that contains one or more envelope protein genes and upstream promoters. In an embodiment, the third-generation lentiviral vector system can include at least two packaging vectors, with the gag-pol being present on a different vector than the rev gene.

[0267] In an embodiment, self-inactivating lentiviral vectors with an siRNA targeting a common exon shared by HIV tat / rev, a nucleolar-localizing TAR decoy, and an anti-CCR5- specific hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) can be used / and or adapted to the polypeptides and / or polynucleotides of the present invention described elsewhere herein.

[0268] In an embodiment, the pseudotype and infectivity or tropisim of a lentivirus particle can be tuned by altering the type of envelope protein(s) included in the lentiviral vector or system thereof. As used herein, an “envelope protein” or “outer protein” means a protein exposed at the surface of a viral particle that is not a capsid protein. For example, envelope or outer proteins typically comprise proteins embedded in the envelope of the virus. In an embodiment, a lentiviral vector or vector system thereof can include a VSV-G envelope protein. VSV-G mediates viral attachment to an LDL receptor (LDLR) or an LDLR family member present on a host cell, which triggers endocytosis of the viral particle by the host cell. Since LDLR is expressed by a wide variety of cells, viral particles expressing the VSV-G envelope protein can infect or transduce a wide variety of cell types. Other suitable envelope proteins can be incorporated based on the host cell that a user desires to be infected by a virus particle produced from a lentiviral vector or system thereof described herein and can include, but are not limited to, feline endogenous virus envelope protein (RD114) (see, e.g., Hanawa et al. Molec. Ther. 2002 5(3) 242-251), modified Sindbis virus envelope proteins (see, e.g., Morizono et al. 2010. J. Virol. 84(14) 6923-6934; Morizono et al. 2001. J. Virol. 75:8016-8020; Morizono et al. 2009. J. Gene Med. 11 :549-558; Morizono et al. 2006 Virology 355:71-81; Morizono et al J. Gene Med. 11:655-663, Morizono et al. 2005 Nat. Med. 11 :346-352), baboon retroviral envelope protein (see e.g., Girard-Gagnepain et al. 2014. Blood. 124: 1221-1231); Tupaia paramyxovirus glycoproteins (see e.g., Enkirch T. et al., 2013. Gene Ther. 20: 16-23); measles virus glycoproteins (see e.g., Funke et al. 2008. Molec. Ther. 16(8): 1427-1436), rabies virus envelope proteins, MLV envelope proteins, Ebola envelope proteins, baculovirus envelope proteins, filovirus envelope proteins, hepatitis El and E2 envelope proteins, gp41 and gpl20 of HIV, hemagglutinin, neuraminidase, M2 proteins of influenza virus, and combinations thereof.

[0269] In an embodiment, the tropism of the resulting lentiviral particle can be tuned by incorporating cell targeting peptides into a lentiviral vector such that the cell targeting peptides are expressed on the surface of the resulting lentiviral particle. In an embodiment, a lentiviral vector can contain an envelope protein that is fused to a cell targeting protein (see, e.g., Buchholz et al. 2015. Trends Biotechnol. 33:777-790; Bender et al. 2016. PLoS Pathog. 12(el005461); and Friedrich et al. 2013. Mol. Ther. 2013. 21 : 849-859.

[0270] In an embodiment, a split-intein-mediated approach to target lentiviral particles to a specific cell type can be used (see, e g., Chamoun-Emaneulli et al. 2015. Biotechnol. Bioeng. 112:2611-2617, Ramirez et al. 2013. Protein. Eng. Des. Sei. 26:215-233. In these embodiments, a lentiviral vector can contain one half of a splicing-deficient variant of the naturally split intein from Nostoc punctiforme fused to a cell targeting peptide and the same or different lentiviral vector can contain the other half of the split intein fused to an envelope protein, such as a bindingdeficient, fusion-competent virus envelope protein. This can result in production of a virus particle from the lentiviral vector or vector system that includes a split intein that can function as a molecular Velcro linker to link the cell-binding protein to the pseudotyped lentivirus particle. This approach can be advantageous for use where surface-incompatibilities can restrict the use of, e.g., cell targeting peptides.

[0271] In an embodiment, a covalent-bond-forming protein-peptide pair can be incorporated into one or more of the lentiviral vectors described herein to conjugate a cell targeting peptide to the virus particle (see, e.g., Kasaraneni et al. 2018. Sci. Reports (8) No. 10990). In an embodiment, a lentiviral vector can include an N-terminal PDZ domain of InaD protein (PDZ1) and its pentapeptide ligand (TEFCA (SEQ ID NO: 309)) from NorpA, which can conjugate the cell targeting peptide to the virus particle via a covalent bond (e.g., a disulfide bond). In an embodiment, the PDZ1 protein can be fused to an envelope protein, which can optionally be binding deficient and / or fusion competent virus envelope protein and included in a lentiviral vector. In an embodiment, the TEFCA (SEQ ID NO: 309) can be fused to a cell targeting peptide and the TEFCA-CPT (SEQ ID NO: 309) fusion construct can be incorporated into the same or a different lentiviral vector as the PDZl-envenlope protein construct. During virus production, specific interaction between the PDZ1 and TEFCA (SEQ ID NO: 309) facilitates producing virus particles covalently functionalized with the cell targeting peptide and thus capable of targeting a specific cell-type based upon a specific interaction between the cell targeting peptide and cellsexpressing its binding partner. This approach can be advantageous for use where surfaceincompatibilities can restrict the use of, e.g., cell targeting peptides.

[0272] Lentiviral vectors have been disclosed as in the treatment for Parkinson’ s Disease, see, e.g., US Patent Publication No. 20120295960 and US Patent Nos. 7303910 and 7351585. Lentiviral vectors have also been disclosed for the treatment of ocular diseases, see, e g., US Patent Publication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also been disclosed for delivery to the brain, see, e.g., US Patent Publication Nos. US20110293571; US20110293571, US20040013648, US20070025970, US20090111106 and US Patent No. US7259015. Any of these systems or a variant thereof can be used to deliver a polynucleotide of the present invention described herein to a cell.

[0273] In an embodiment, a lentiviral vector system can include one or more transfer plasmids. Transfer plasmids can be generated from various other vector backbones and can include one or more features that can work with other retroviral and / or lentiviral vectors in the system that can, for example, improve safety of the vector and / or vector system, increase virial titers, and / or increase or otherwise enhance expression of the desired insert to be expressed and / or packaged into the viral particle. Suitable features that can be included in a transfer plasmid can include, but are not limited to, 5’LTR, 3’LTR, SIN / LTR, origin of replication (Ori), selectable marker genes (e.g., antibiotic resistance genes), Psi (T), RRE (rev response element), cPPT (central polypurine tract), promoters, WPRE (woodchuck hepatitis post-transcriptional regulatory element), SV40 polyadenylation signal, pUC origin, SV40 origin, Fl origin, and combinations thereof.

[0274] In another embodiment, Cocal vesiculovirus envelope pseudotyped retroviral or lentiviral vector particles are contemplated (see, e.g., US Patent Publication No. 20120164118 assigned to the Fred Hutchinson Cancer Research Center). Cocal virus is in the Vesiculovirus genus, and is a causative agent of vesicular stomatitis in mammals. Cocal virus was originally isolated from mites in Trinidad (Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964)), and infections have been identified in Trinidad, Brazil, and Argentina from insects, cattle, and horses. Many of the vesiculoviruses that infect mammals have been isolated from naturally infected arthropods, suggesting that they are vector-borne. Antibodies to vesiculoviruses are common among people living in rural areas where the viruses are endemic and laboratory-acquired; infections in humans usually result in influenza-like symptoms. The Cocal virus envelope glycoprotein shares 71.5%identity at the amino acid level with VSV-G Indiana, and phylogenetic comparison of the envelope gene of vesiculoviruses shows that Cocal virus is serologically distinct from, but most closely related to, VSV-G Indiana strains among the vesiculoviruses. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) and Travassos da Rosa et al., Am. J. Tropical Med. & Hygiene 33:999-1006 (1984). The Cocal vesiculovirus envelope pseudotyped retroviral vector particles may include for example, lentiviral, alpharetroviral, betaretroviral, gammaretroviral, deltaretroviral, and epsilonretroviral vector particles that may comprise retroviral Gag, Pol, and / or one or more accessory protein(s) and a Cocal vesiculovirus envelope protein. In an embodiment of these embodiments, the Gag, Pol, and accessory proteins are lentiviral and / or gammaretroviral. In an embodiment, a retroviral vector can contain encoding polypeptides for one or more Cocal vesiculovirus envelope proteins such that the resulting viral or pseudoviral particles are Cocal vesiculovirus envelope pseudotyped.Adenoviral vectors, Helper-dependent Adenoviral vectors, and Hybrid Adenoviral Vectors

[0275] In an embodiment, the vector can be an adenoviral vector. In an embodiment, the adenoviral vector can include elements such that the virus particle produced using the vector or system thereof can be serotype 2 or serotype 5. In an embodiment, the polynucleotide to be delivered via the adenoviral particle can be up to about 8 kb. Thus, In an embodiment, an adenoviral vector can include a DNA polynucleotide to be delivered that can range in size from about 0.001 kb to about 8 kb. Adenoviral vectors have been used successfully in several contexts (see, e.g., Teramato et al. 2000. Lancet. 355: 1911-1912; Lai et al. 2002. DNA Cell. Biol. 21 :895- 913; Flotte et al., 1996. Hum. Gene. Ther. 7: 1145-1159; and Kay et al. 2000. Nat. Genet. 24:257- 261.

[0276] In an embodiment the vector can be a helper-dependent adenoviral vector or system thereof. These are also referred to in the art as “gutless” or “gutted” vectors and are a modified generation of adenoviral vectors (see e.g., Thrasher et al. 2006. Nature. 443:E5-7). In an embodiment of the helper-dependent adenoviral vector system one vector (the helper) can contain all the viral genes required for replication but contains a conditional gene defect in the packaging domain. The second vector of the system can contain only the ends of the viral genome, one or more polynucleotides of the present invention described elsewhere herein, and the native packaging recognition signal, which can allow selective packaged release from the cells (see e.g., Cideciyan et al. 2009. N Engl J Med. 361 :725-727). Helper-dependent adenoviral vector systemshave been successful for gene delivery in several contexts (see, e.g., Simonelli et al. 2010. J Am Soc Gene Ther. 18:643-650; Cideciyan et al. 2009. N Engl J Med. 361:725-727; Crane et al. 2012. Gene Ther. 19(4):443-452; Alba et al. 2005. Gene Ther. 12: 18-S27; Croyle et al. 2005. Gene Ther. 12:579-587; Amalfitano et al. 1998. J. Virol. 72:926-933; and Morral et al. 1999. PNAS. 96: 12816- 12821). The techniques and vectors described in these publications can be adapted for inclusion and delivery of the polynucleotides of the present invention described herein. In an embodiment, the polynucleotide to be delivered via the viral particle produced from a helper-dependent adenoviral vector or system thereof can be up to about 37 kb. Thus, In an embodiment, an adenoviral vector can include a DNA polynucleotide to be delivered that can range in size from about 0.001 kb to about 37 kb (see e.g. Rosewell et al. 2011. J. Genet. Syndr. Gene Ther. Suppl. 5:001).

[0277] In an embodiment, the vector is a hybrid-adenoviral vector or system thereof. Hybrid adenoviral vectors are composed of the high transduction efficiency of a gene-deleted adenoviral vector and the long-term genome-integrating potential of adeno-associated, retroviruses, lentivirus, and transposon based-gene transfer. In an embodiment, such hybrid vector systems can result in stable transduction and limited integration site. See e.g., Balague et al. 2000. Blood. 95:820-828; Morral et al. 1998. Hum. Gene Ther. 9:2709-2716; Kubo and Mitani. 2003. J. Virol. 77(5): 2964-2971; Zhang et al. 2013. PloS One. 8(10) e76771; and Cooney et al. 2015. Mol. Ther. 23(4):667-674), whose techniques and vectors described therein can be modified and adapted for use for delivering the polynucleotide of the present invention described elsewhere herein. In an embodiment, a hybrid-adenoviral vector can include one or more features of a retrovirus and / or an adeno-associated virus. In an embodiment the hybrid-adenoviral vector can include one or more features of a spuma retrovirus or foamy virus (FV). See e.g., Ehrhardt et al. 2007. Mol. Ther. 15: 146-156 and Liu et al. 2007. Mol. Ther. 15:1834-1841, whose techniques and vectors described therein can be modified and adapted for use for delivering one or more polynucleotides of the present invention described herein. Advantages of using one or more features from the FVs in the hybrid-adenoviral vector or system thereof can include the ability of the viral particles produced therefrom to infect a broad range of cells, a large packaging capacity as compared to other retroviruses, and the ability to persist in quiescent (non-dividing) cells. See also e.g., Ehrhardt et al. 2007. Mol. Ther. 156: 146-156 and Shuji et al. 2011. Mol. Ther. 19:76-82, whose techniquesand vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention.Adeno Associated Viral (AAV) Vectors

[0278] In an embodiment, the vector can be an adeno-associated virus (AAV) vector. See, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94: 1351 (1994). Although similar to adenoviral vectors in some of their features, AAVs have some deficiency in their replication and / or pathogenicity and thus can be safer than adenoviral vectors. In an embodiment the AAV can integrate into a specific site on chromosome 19 of a human cell with no observable side effects. In an embodiment, the capacity of the AAV vector, system thereof, and / or AAV particles can be up to about 4.7 kb. The AAV vector or system thereof can include one or more regulatory molecules. In an embodiment the regulatory molecules can be promoters, enhancers, repressors and the like, which are described in greater detail elsewhere herein. In an embodiment, the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory proteins. In an embodiment, the one or more regulatory proteins can be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof.

[0279] The AAV vector or system thereof can include one or more polynucleotides that can encode one or more capsid proteins. The capsid proteins can be selected from VP1, VP2, VP3, and combinations thereof. The capsid proteins can be capable of assembling into a protein shell of the AAV virus particle. In an embodiment, the AAV capsid can contain 60 capsid proteins. In an embodiment, the ratio of VP1 :VP2:VP3 in a capsid can be about 1 : 1 : 10.

[0280] In an embodiment, the AAV vector or system thereof can include one or more adenovirus helper factors or polynucleotides that can encode one or more adenovirus helper factors. Such adenovirus helper factors can include, but are not limited, El A, E1B, E2A, E4ORF6, and VA RNAs. In an embodiment, a producing host cell line expresses one or more of the adenovirus helper factors.

[0281] The AAV vector or system thereof can be configured to produce AAV particles having a specific serotype. In an embodiment, the serotype can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9 or any combinations thereof. In an embodiment, the AAV can be AAV1, AAV-2, AAV-5 or any combination thereof. One can select the AAV of the AAV with regard to the cells to be targeted; e g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV-1, AAV-2, AAV-5 or any combination thereof for targeting brain and / or neuronal cells; and one can select AAV-4 for targeting cardiac tissue; and one can select AAV8 for delivery to the liver. Thus, In an embodiment, an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and / or neuronal cells can be configured to generate AAV particles having serotypes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof. In an embodiment, an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue can be configured to generate an AAV particle having an AAV- 4 serotype. In an embodiment, an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver can be configured to generate an AAV having an AAV-8 serotype. In an embodiment, the AAV vector is a hybrid AAV vector or system thereof. Hybrid AAVs are AAVs that include genomes with elements from one serotype that are packaged into a capsid derived from at least one different serotype. For example, if it is the rAAV2 / 5 that is to be produced, and if the production method is based on the helper-free, transient transfection method discussed above, the 1st plasmid and the 3rd plasmid (the adeno helper plasmid) will be the same as discussed for rAAV2 production. However, the second plasmid, the pRepCap will be different. In this plasmid, called pRep2 / Cap5, the Rep gene is still derived from AAV2, while the Cap gene is derived from AAV5. The production scheme is the same as the above-mentioned approach for AAV2 production. The resulting rAAV is called rAAV2 / 5, in which the genome is based on recombinant AAV2, while the capsid is based on AAV5. It is assumed the cell or tissue-tropism displayed by this AAV2 / 5 hybrid virus should be the same as that of AAV5.

[0282] A tabulation of certain AAV serotypes as to these cells can be found in Grimm, D. et al, J. Virol. 82: 5887-5911 (2008). The AAV can be any one of the serotypes.

[0283] In an embodiment, the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector. In an embodiment, the “gutless” AAV vector or system thereof can have the cis-acting viral DNA elements involved in genome amplification and packaging in linkage with the heterologous sequences of interest (e.g., the CRISPR-Cas system polynucleotide(s)).

[0284] In an embodiment, the AAV vectors are produced in in insect cells, e g., Spodoptera frugiperda Sf9 insect cells, grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial vendors, e.g., Sigma Aldrich (EX-CELL 405).

[0285] In an embodiment, an AAV vector or vector system can contain or consists essentially of one or more polynucleotides encoding one or more polynucleotides of the present invention, such as one or more viral polynucleotides.

[0286] In another embodiment, the invention provides a polypeptide of the present invention operatively coupled with Adeno Associated Virus (AAV), e.g., an AAV comprising a polypeptide of the present invention as a fusion, with or without a linker, to or with an AAV capsid protein such as VP1, VP2, and / or VP3. More particularly, modifying the knowledge in the art, e.g., Rybniker et al., [ED2] J Virol. Dec 2012; 86(24): 13800-13804; Lux K, et al. 2005. J. Virol.79: 11776-11787; Munch RC, et al. 2013[ED3] . Mol. Ther. 21 : 109-118 ; and Warrington KH, Jr, et al. 2004. J. Virol. 78:6595-6609, each incorporated herein by reference, one can obtain a modified AAV capsid of the invention. It will be understood by those skilled in the art that the modifications described herein if inserted into the AAV cap gene may result in modifications in the VP1, VP2 and / or VP3 capsid subunits. Alternatively, the capsid subunits can be expressed independently to achieve modification in only one or two of the capsid subunits (VP1, VP2, VP3, VP1+VP2, VP1+VP3, or VP2+VP3). One can modify the cap gene to have expressed at a desired location a non-capsid protein advantageously a large payload protein, such as a polypeptide of the present invention. Likewise, these can be fusions, with the protein, e.g., large payload protein such as a polypeptide of the present invention fused in a manner analogous to prior art fusions. See, e.g., US Patent Publication 20090215879; Nance et al., Hum Gene Ther. 26(12):786-800 (2015) and documents cited therein, incorporated herein by reference. One of ordinary skill in the art, from this disclosure, can make and use modified AAV or AAV capsid as in the herein invention, and through this disclosure one knows now that payload proteins, such as large payload proteins, can be fused to the AAV capsid. Accordingly, this approach is also applicable to a virus in the genus Dependoparvovirus or in the family Parvoviridae, for instance, AAV, or a virus of Amdoparvovirus, e.g., Carnivore amdoparvovirus 1, a virus of Aveparvovirus, e.g., Galliform aveparvovirus 1, a virus of Bocaparvovirus, e.g., Ungulate bocaparvovirus 1, a virus of Copiparvovirus, e g., Ungulate copiparvovirus 1, a virus of Dependoparvovirus, e g., Adeno- associated dependoparvovirus A, a virus of Erythroparvovirus, e.g., Primate erythroparvovirus 1, a virus of Protoparvovirus, e.g., Rodent protoparvovirus 1, a virus of Tetraparvovirus, e.g., Primate tetraparvovirus 1. Thus, a virus of within the family Parvoviridae or the genus Dependoparvovirusor any of the other foregoing genera within Parvoviridae is contemplated as within the invention with discussion herein as to AAV applicable to such other viruses.

[0287] In one embodiment, the invention provides a non-naturally occurring or engineered composition comprising a polypeptide of the present invention which is part of or tethered to an AAV capsid domain, i.e., VP1, VP2, or VP3 domain of Adeno- Associated Virus (AAV) capsid. In an embodiment, part of or tethered to an AAV capsid domain includes associated with associated with a AAV capsid domain. In an embodiment, the polypeptide of the present invention may be fused to the AAV capsid domain. In an embodiment, the fusion may be to the N-terminal end of the AAV capsid domain. As such, In an embodiment, the C- terminal end of the polypeptide of the present invention is fused to the N- terminal end of the AAV capsid domain. In an embodiment, an NLS and / or a linker (such as a GlySer linker) may be positioned between the C- terminal end of the polypeptide of the present invention and the N- terminal end of the AAV capsid domain. In an embodiment, the fusion may be to the C-terminal end of the AAV capsid domain. In an embodiment, this is not preferred due to the fact that the VP1, VP2 and VP3 domains of AAV are alternative splices of the same RNA and so a C- terminal fusion may affect all three domains. In an embodiment, the AAV capsid domain is truncated. In an embodiment, some or all of the AAV capsid domain is removed. In an embodiment, some of the AAV capsid domain is removed and replaced with a linker (such as a GlySer linker), typically leaving the N- terminal and C- terminal ends of the AAV capsid domain intact, such as the first 2, 5 or 10 amino acids. In this way, the internal (non-terminal) portion of the VP3 domain may be replaced with a linker. It is particularly preferred that the linker is fused to the polypeptide of the present invention. A branched linker may be used, with the polypeptide of the present invention fused to the end of one of the branches. This allows for some degree of spatial separation between the capsid and the polypeptide of the present invention. In this way, the polypeptide of the present invention is part of (or fused to) the AAV capsid domain.

[0288] In other embodiments, the polypeptide of the present invention may be fused in frame within, i.e., internal to, the AAV capsid domain. Thus, In an embodiment, the AAV capsid domain again preferably retains its N- terminal and C- terminal ends. In this case, a linker is preferred, In an embodiment, either at one or both ends of the polypeptide of the present invention. In this way, the polypeptide of the present invention is again part of (or fused to) the AAV capsid domain. In an embodiment, the positioning of the polypeptide of the present invention is such that thepolypeptide of the present invention is at the external surface of the viral capsid once formed. In one embodiment, the invention provides a non-naturally occurring or engineered composition comprising a polypeptide of the present invention associated with a AAV capsid domain of Adeno- Associated Virus (AAV) capsid. As used herein, the term “associated” means fused, bound to, or tethered to. In an embodiment, the polypeptide of the present invention is tethered to the VP1, VP2, or VP3 domain. This is via a connector protein or tethering system such as the biotinstreptavidin system. In one example, a biotinylation sequence (15 amino acids) therefore is fused to the polypeptide of the present invention.

[0289] When the AAV capsid domain is fused with streptavidin, they form a highly stable association due to their very strong affinity for each other. This is especially true if the fusion is located at the N-terminus of the AVV capsid domain. Accordingly, In an embodiment, provided herein is a composition or system comprising a polypeptide of the present inventi on-biotin fused to a streptavidin- AAV capsid domain. The polypeptide of the present invention-biotin and streptavidin- AAV capsid domain form a single complex when the two parts are fused together. NLSs also may be incorporated between the polypeptide of the present invention and the biotin; and / or between the streptavidin and the AAV capsid domain.

[0290] In an embodiment, provided herein is a fusion of a polypeptide of the present invention with a connector protein specific for a high affinity ligand for that connector, and the AAV VP2 domain is bound to said high affinity ligand. For example, streptavidin can be the connector fused to the polypeptide of the present invention, while biotin is bound to the AAV VP2 domain. Upon co-localization, the streptavidin will bind to the biotin, thus connecting the polypeptide of the present invention to the AAV VP2 domain. The reverse arrangement also is possible. In an embodiment, a biotinylation sequence (15 amino acids) is fused to the AAV VP2 domain, in particular the N- terminus of the AAV VP2 domain. [ED4]

[0291] In an embodiment, the biotinylated AAV capsids with streptavidin-polypeptide of the present invention are assembled in vitro. This way the AAV capsids assemble in a straightforward manner and the polypeptide of the present invention-streptavidin fusion may be added after assembly of the capsid.

[0292] [In other embodiments, a biotinylation sequence (15 amino acids) is fused to the polypeptide of the present invention, which is fused with the AAV VP2 domain fused withstreptavidin, wherein, in preferred embodiments, the fusion is located at the N-terminus of the AVV capsid domain.

[0293] In an embodiment, the polypeptide of the present invention and the AAV VP2 domain are fused. In an embodiment, the fusion is to the N- terminal end of the polypeptide of the present invention. In an embodiment, the AAV and polypeptide of the present invention are associated via fusion. In an embodiment, the AAV and polypeptide of the present invention are associated via fusion including a linker. Suitable linkers are discussed herein but include Gly Ser linkers. Fusion to the N- terminus of AAV VP2 domain is preferred. In an embodiment, the polypeptide of the present invention comprises at least one Nuclear Localization Signal (NLS). In a further embodiment, the present invention provides compositions comprising the polypeptide of the present invention and associated AAV VP2 domain or the polynucleotides or vectors described herein. Such compositions and formulations are discussed elsewhere herein.

[0294] Alternatively, a tether may be to fuse or otherwise associate the AAV capsid domain to an adaptor protein that binds to or recognizes a corresponding RNA sequence or motif. In an embodiment, the adaptor comprises a binding protein which recognizes and binds (or is bound by) an RNA sequence specific for said binding protein. In preferred embodiments, the MS2 binding protein recognizes and binds (or is bound by) an RNA sequence specific for the MS2 protein (see Konermann et al. Dec 2014, cited infra, incorporated herein by reference).

[0295] In an embodiment, the AAV capsid domain is associated with the adaptor protein, and the polypeptide of the present invention is tethered to the adaptor protein of the AAV capsid domain. In an embodiment, the polypeptide of the present invention is tethered to the adaptor protein of the AAV capsid domain via the polypeptide of the present invention being in a complex with a modified guide, see Konermann et al. In an embodiment, the modified guide is an sgRNA. In an embodiment, the modified guide comprises a distinct RNA sequence; see, e.g., International Patent Application No. PCT7US14 / 70175, incorporated herein by reference. In an embodiment, distinct RNA sequence is an aptamer.

[0296] In an embodiment, the positioning of the polypeptide of the present invention is such that the polypeptide of the present invention is at the internal surface of the viral capsid once formed. In one embodiment, the invention provides a non-naturally occurring or engineered composition comprising a polypeptide of the present invention associated with (i.e., fused, bound to, tethered to) an internal surface of an AAV capsid domain. In an embodiment, the polypeptideof the present invention is tethered to the VP1, VP2, or VP3 domain such that it locates to the internal surface of the viral capsid once formed, wherein the polypeptide of the present invention is tethered to the VP 1, VP2, or VP3 domain via a connector protein or a tethering system such as the biotin-streptavidin system as described above and / or elsewhere herein.Herpes Simplex Viral Vectors

[0297] In an embodiment, the vector can be a Herpes Simplex Viral (HSV)-based vector or system thereof. HSV systems can include the disabled infections single copy (DISC) viruses, which are composed of a glycoprotein H defective mutant HSV genome. When the defective HSV is propagated in complementing cells, virus particles can be generated that are capable of infecting subsequent cells permanently replicating their own genome but are not capable of producing more infectious particles. See e g., 2009. Trobridge. Exp. Opin. Biol. Ther. 9: 1427-1436, whose techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention. In an embodiment where an HSV vector or system thereof is utilized, the host cell can be a complementing cell. In an embodiment, HSV vector or system thereof can be capable of producing virus particles capable of delivering a polynucleotide cargo of up to 150 kb. Thus, In an embodiment, the polynucleotide(s) of the present invention included in the HSV-based viral vector or system thereof can sum from about 0.001 to about 150 kb. HSV- based vectors and systems thereof have been successfully used in several contexts including various models of neurologic disorders. See, e.g., Cockrell et al. 2007. Mol. Biotechnol. 36: 184- 204; Kafri T. 2004. Mol. Biol. 246:367-390; Balaggan and Ali. 2012. Gene Ther. 19: 145-153; Wong et al. 2006. Hum. Gen. Ther. 2002. 17: 1-9; Azzouz et al. J. Neruosci. 22L10302-10312; and Betchen and Kaplitt. 2003. Curr. Opin. Neurol. 16:487-493, whose techniques and vectors described therein can be modified and adapted for use to delivery one or more polynucleotides and / or polypeptides of the present invention of the present invention.Poxyirus Vectors

[0298] In an embodiment, the vector is a poxvirus vector or system thereof. In an embodiment, the poxvirus vector results in cytoplasmic expression of one or more polynucleotides and / or polypeptides of the present invention. In an embodiment the capacity of the poxvirus vector or system thereof is about 25 kb or more. In an embodiment, the poxvirus vector or system thereof includes one or more polynucleotides of the present invention described herein.Viral Vectors for delivery to plants

[0299] The systems and compositions can be delivered to plant cells using viral vehicles. In particular embodiments, the compositions and systems can be introduced in the plant cells using a plant viral vector (e.g., as described in Scholthof et al. 1996, Annu Rev Phytopathol. 1996;34:299- 323). Such viral vectors can be a vector from a DNA virus, e.g., geminivirus (e.g., cabbage leaf curl virus, bean yellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, maize streak virus, tobacco leaf curl virus, or tomato golden mosaic virus) or nanovirus (e.g., Faba bean necrotic yellow virus). The viral vector can be a vector from an RNA virus, e g., tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus), potexvirus (e.g., potato virus X), or hordeivirus (e.g., barley stripe mosaic virus). The replicating genomes of plant viruses can be non-integrative vectors.Virus Particle Production from Viral VectorsRetroviral Production

[0300] In an embodiment, one or more viral vectors and / or system thereof are delivered to a suitable cell line for production of virus particles containing the polynucleotide or other payload to be delivered to a host cell. Suitable host cells for virus production from viral vectors and systems thereof described herein are known in the art and are commercially available. For example, suitable host cells include HEK 293 cells and its variants (HEK 293T and HEK 293TN cells). In an embodiment, the suitable host cell for virus production from viral vectors and systems thereof described herein can stably express one or more genes involved in packaging (e.g., pol, gag, and / or VSV-G) and / or other supporting genes.

[0301] In an embodiment, after delivery of one or more viral vectors to the suitable host cells for or virus production from viral vectors and systems thereof, the cells are incubated for an appropriate length of time to allow for viral gene expression from the vectors, packaging of the polynucleotide to be delivered (e.g., a viral polynucleotide of the present invention), and virus particle assembly, and secretion of mature virus particles into the culture media. Various other methods and techniques are generally known to those of ordinary skill in the art.

[0302] Mature virus particles can be collected from the culture media by a suitable method. In an embodiment, this can involve centrifugation to concentrate the virus. The titer of the composition containing the collected virus particles can be obtained using a suitable method. Such methods can include transducing a suitable cell line (e.g. NIH 3T3 cells) and determining transduction efficiency, infectivity in that cell line by a suitable method. Suitable methods includePCR-based methods, flow cytometry, and antibiotic selection-based methods. Various other methods and techniques are generally known to those of ordinary skill in the art. The concentration of virus particle can be adjusted as needed. In an embodiment, the resulting composition containing virus particles contains 1 X101 -1 X 1020 particles / mL.

[0303] Lentiviruses can be prepared from any lentiviral vector or vector system described herein. In one example embodiment, after cloning pCasESlO (which contains a lentiviral transfer plasmid backbone), HEK293FT at low passage (p=5) can be seeded in a T-75 flask to 50% confluence the day before transfection in DMEM with 10% fetal bovine serum and without antibiotics. After 20 hours, the media can be changed to OptiMEM (serum-free) media and transfection of the lentiviral vectors can done 4 hours later. Cells can be transfected with 10 pg of lentiviral transfer plasmid (pCasESlO) and the appropriate packaging plasmids (e.g., 5 pg of pMD2.G (VSV-g pseudotype), and 7.5ug of psPAX2 (gag / pol / rev / tat)). Transfection can be carried out in 4mL OptiMEM with a cationic lipid delivery agent (50uL Lipofectamine 2000 and lOOul Plus reagent). After 6 hours, the media can be changed to antibiotic-free DMEM with 10% fetal bovine serum. These methods can use serum during cell culture, but serum-free methods are preferred.

[0304] Following transfection and allowing the producing cells (also referred to as packaging cells) to package and produce virus particles with packaged cargo, the lentiviral particles can be purified. In an exemplary embodiment, virus-containing supernatants can be harvested after 48 hours. Collected virus-containing supernatants can first be cleared of debris and filtered through a 0.45um low protein binding (PVDF) filter. They can then be spun in an ultracentrifuge for 2 hours at 24,000 rpm. The resulting virus-containing pellets can be resuspended in 50ul of DMEM overnight at 4 degrees C. They can be then aliquoted and used immediately or immediately frozen at -80 degrees C for storage.AAV Particle Production

[0305] There are two main strategies for producing AAV particles from AAV vectors and systems thereof, such as those described herein, which depend on how the adenovirus helper factors are provided (helper v. helper free). In an embodiment, a method of producing AAV particles from AAV vectors and systems thereof can include adenovirus infection into cell lines that stably harbor AAV replication and capsid encoding polynucleotides along with AAV vector containing the polynucleotide to be packaged and delivered by the resulting AAV particle (e.g.,one or more viral polynucleotide(s) of the present invention). In an embodiment, a method of producing AAV particles from AAV vectors and systems thereof can be a “helper free” method, which includes co-transfection of an appropriate producing cell line with three vectors (e.g., plasmid vectors): (1) an AAV vector that contains a polynucleotide of interest (e.g., one or more viral polynucleotide(s) of the present invention) between 2 ITRs; (2) a vector that carries the AAV Rep-Cap encoding polynucleotides; and (helper polynucleotides. One of skill in the art will appreciate various methods and variations thereof that are both helper and -helper free and as well as the different advantages of each system.Non-Viral Vectors

[0306] In an embodiment, the vector is a non-viral vector or vector system. The term of art “non-viral vector” and as used herein in this context refers to molecules and / or compositions that are vectors but that are not based on one or more component of a virus or virus genome (excluding any nucleotide to be delivered and / or expressed by the non-viral vector) that can be capable of incorporating polynucleotide(s) of the present invention and delivering said polynucleotide(s) to a cell and / or expressing the polynucleotide in the cell. It will be appreciated that this does not exclude vectors containing a polynucleotide designed to target a virus-based polynucleotide that is to be delivered. For example, if a gRNA to be delivered is directed against a virus component and it is inserted or otherwise coupled to an otherwise non-viral vector or carrier, this would not make said vector a “viral vector”. Non-viral vectors can include, without limitation, naked polynucleotides and polynucleotide (non-viral) based vector and vector systems.Naked Polynucleotides

[0307] In an embodiment one or more CAA polynucleotides of the present invention, e.g., one or more viral polynucleotides, described elsewhere herein can be included in a naked polynucleotide. The term “naked polynucleotide,” as used herein, refers to polynucleotides that are not associated with another molecule (e.g., proteins, lipids, and / or other molecules) that can often help protect it from environmental factors and / or degradation. As used herein, associated with includes, but is not limited to, linked to, adhered to, adsorbed to, enclosed in, enclosed in or within, mixed with, and the like. Naked polynucleotides that include one or more of the polynucleotides of the present invention described herein can be delivered directly to a host cell and optionally expressed therein. The naked polynucleotides can have any suitable two- and three- dimensional configurations. By way of non-limiting examples, naked polynucleotides can besingle-stranded molecules, double stranded molecules, circular molecules (e.g., plasmids and artificial chromosomes), molecules that contain portions that are single stranded and portions that are double stranded (e.g., ribozymes), and the like. In an embodiment, the naked polynucleotide contains only the polynucleotide(s) of the present invention. In an embodiment, the naked polynucleotide can contain other nucleic acids and / or polynucleotides in addition to the polynucleotide(s) of the present invention. The naked polynucleotides can include one or more elements of a transposon system. Transposons and system thereof are described in greater detail elsewhere herein.Non-Viral Polynucleotide Vectors

[0308] In an embodiment, one or more of the CAA polynucleotides of the present invention, such as a viral polynucleotide of the present invention described elsewhere herein, can be included in a non-viral polynucleotide vector. Suitable non-viral polynucleotide vectors include, but are not limited to, transposon vectors and vector systems, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, AR (antibiotic resistance)-free plasmids and miniplasmids, circular covalently closed vectors (e.g., minicircles, minivectors, miniknots), linear covalently closed vectors (“dumbbell shaped”), MIDGE (minimalistic immunologically defined gene expression) vectors, MiLV (micro-linear vector) vectors, Ministrings, mini-intronic plasmids, PSK systems (post-segregationally killing systems), ORT (operator repressor titration) plasmids, and the like. See, e.g., Hardee et al. 2017. Genes. 8(2):65.

[0309] In an embodiment, the non-viral polynucleotide vector has a conditional origin of replication. In an embodiment, the non-viral polynucleotide vector is an ORT plasmid. In an embodiment, the non-viral polynucleotide vector has a minimalistic immunologically defined gene expression. In an embodiment, the non-viral polynucleotide vector has one or more post- segregationally killing system genes. In an embodiment, the non-viral polynucleotide vector is AR-free. In an embodiment, the non-viral polynucleotide vector is a minivector. In an embodiment, the non-viral polynucleotide vector includes a nuclear localization signal. In an embodiment, the non-viral polynucleotide vector includes one or more CpG motifs. In an embodiment, the non-viral polynucleotide vectors include one or more scaffold / matrix attachment regions (S / MARs). See e.g., Mirkovitch et al. 1984. Cell. 39:223-232, Wong et al. 2015. Adv. Genet. 89: 113-152, whose techniques and vectors can be adapted for use in the present invention. S / MARs are AT-rich sequences that play a role in the spatial organization of chromosomes throughDNA loop base attachment to the nuclear matrix. S / MARs are often found close to regulatory elements such as promoters, enhancers, and origins of DNA replication. Inclusion of one or S / MARs can facilitate a once-per-cell-cycle replication to maintain the non-viral polynucleotide vector as an episome in daughter cells. In an embodiment, the S / MAR sequence is located downstream of an actively transcribed polynucleotide (e.g., one or more polynucleotides of the present invention) included in the non-viral polynucleotide vector. In an embodiment, the S / MAR is a S / MAR from the beta-interferon gene cluster. See e.g., Verghese et al. 2014. Nucleic Acid Res. 42:e53; Xu et al. 2016. Sci. China Life Sci. 59: 1024-1033; Jin et al. 2016. 8:702-711; Koirala et al. 2014. Adv. Exp. Med. Biol. 801 :703-709; and Nehlsen et al. 2006. Gene Ther. Mol. Biol. 10:233-244, whose techniques and vectors can be adapted for use in the present invention.

[0310] In an embodiment, the non-viral vector is a transposon vector or system thereof. As used herein, “transposon” (also referred to as transposable element) refers to a polynucleotide sequence that is capable of moving form location in a genome to another. There are several classes of transposons. Transposons include retrotransposons and DNA transposons. Retrotransposons require the transcription of the polynucleotide that is moved (or transposed) to transpose the polynucleotide to a new genome or polynucleotide. DNA transposons are those that do not require reverse transcription of the polynucleotide that is moved (or transposed) to transpose the polynucleotide to a new genome or polynucleotide. In an embodiment, the non-viral polynucleotide vector is a retrotransposon vector. In an embodiment, the retrotransposon vector includes long terminal repeats. In an embodiment, the retrotransposon vector does not include long terminal repeats. In an embodiment, the non-viral polynucleotide vector is a DNA transposon vector. DNA transposon vectors include a polynucleotide sequence encoding a transposase. In an embodiment, the transposon vector is configured as a non-autonomous transposon vector, meaning that the transposition does not occur spontaneously on its own. In some of these embodiments, the transposon vector lacks one or more polynucleotide sequences encoding proteins required for transposition. In an embodiment, the non-autonomous transposon vectors lack one or more Ac elements.

[0311] In an embodiment a non-viral polynucleotide transposon vector system includes a first polynucleotide vector that contains the polynucleotide(s) of the present invention flanked on the 5’ and 3’ ends by transposon terminal inverted repeats (TIRs) and a second polynucleotide vector that includes a polynucleotide capable of encoding a transposase coupled to a promoter to driveexpression of the transposase. When both are expressed in the same cell the transposase is expressed from the second vector; transpose the material between the TIRs on the first vector (e.g., the polynucleotide(s) of the present invention); and integrate it into one or more positions in the host cell’s genome. In an embodiment, the transposon vector or system thereof is configured as a gene trap. In an embodiment, the TIRs are configured to flank a strong splice acceptor site followed by a reporter and / or other gene (e.g., one or more of the polynucleotide(s) of the present invention) and a strong poly A tail. When transposition occurs while using this vector or system thereof, the transposon inserts into an intron of a gene. This insertion of the reporter or other gene triggers a mis-splicing process, thereby activating the trapped gene.

[0312] Any suitable transposon system can be used. Suitable transposon and systems thereof include, but are not limited to: Sleeping Beauty transposon system (Tcl / mariner superfamily) (see e.g., Ivies et al. 1997. Cell. 91(4): 501-510), piggyBac (piggyBac superfamily) (see e.g., Li et al. 2013 110(25): E2279-E2287 and Yusa et al. 2011. PNAS. 108(4): 1531-1536), Tol2 (superfamily hAT), Frog Prince (Tcl / mariner superfamily) (see e.g., Miskey et al. 2003 Nucleic Acid Res. 31(23):6873-6881) and variants thereof.Non-Vector Delivery Vehicles

[0313] The delivery vehicles may comprise non-viral vehicles. In general, methods and vehicles capable of delivering nucleic acids and / or proteins may be used for delivering the systems compositions herein. Examples of non-viral vehicles include lipid nanoparticles, cell-penetrating peptides (CPPs), DNA nanoclews, metal nanoparticles, streptolysin O, multifunctional envelopetype nanodevices (MENDs), lipid-coated mesoporous silica particles, and other inorganic nanoparticles.Lipid Particles

[0314] The delivery vehicles may comprise lipid particles, e.g., lipid nanoparticles (LNPs) and liposomes. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, International Patent Publication Nos. WO 91 / 17424 and WO 91 / 16024. The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).Lipid nanoparticles (LNPs)

[0315] LNPs may encapsulate nucleic acids within cationic lipid particles (e.g., liposomes), and may be delivered to cells with relative ease. In some examples, lipid nanoparticles do not contain any viral components, which helps minimize safety and immunogenicity concerns. Lipid particles may be used for in vitro, ex vivo, and in vivo deliveries. Lipid particles may be used for various scales of cell populations.

[0316] In some examples. LNPs may be used for delivering DNA molecules (e.g., those comprising polynucleotides of the present invention and / or polypeptides they encode).

[0317] Components in LNPs may comprise cationic lipids 1,2- dilineoyl-3- dimethylammonium-propane (DLinDAP), l,2-dilinoleyloxy-3-N,N- dimethylaminopropane (DLinDMA), l,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLinKC2-DMA), (3- o-[2”-(methoxypolyethyleneglycol 2000) succinoyl]-l,2-dimyristoyl-sn-glycol (PEG-S-DMG), R-3- [(ro-methoxy-poly(ethylene glycol)2000) carbamoyl]-!, 2-dimyristyloxlpropyl-3-amine (PEG-C- DOMG, and any combination thereof. Preparation of LNPs and encapsulation may be adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011).

[0318] In an embodiment, an LNP delivery vehicle can be used to deliver a virus particle containing a polynucleotides and / or polypeptides of the present invention. In an embodiment, the virus particle(s) can be adsorbed to the lipid particle, such as through electrostatic interactions, and / or can be attached to the liposomes via a linker.

[0319] [In an embodiment, the LNP contains a nucleic acid, wherein the charge ratio of nucleic acid backbone phosphates to cationic lipid nitrogen atoms is about 1 : 1.5 - 7 or about 1 :4.

[0320] [In an embodiment, the LNP also includes a shielding compound, which is removable from the lipid composition under in vivo conditions. In an embodiment, the shielding compound is a biologically inert compound. In an embodiment, the shielding compound does not carry any charge on its surface or on the molecule as such. In an embodiment, the shielding compounds arepolyethylenglycoles (PEGs), hydroxyethylglucose (HEG) based polymers, polyhydroxyethyl starch (polyHES) and polypropylene. In an embodiment, the PEG, HEG, polyHES, and a polypropylene weight between about 500 to 10,000 Da or between about 2000 to 5000 Da. In an embodiment, the shielding compound is PEG2000 or PEG5000.

[0321] In an embodiment, the LNP can include one or more helper lipids. In an embodiment, the helper lipid can be a phosphor lipid or a steroid. In an embodiment, the helper lipid is between about 20 mol % to 80 mol % of the total lipid content of the composition. In an embodiment, the helper lipid component is between about 35 mol % to 65 mol % of the total lipid content of the LNP. In an embodiment, the LNP includes lipids at 50 mol% and the helper lipid at 50 mol% of the total lipid content of the LNP.

[0322] Other non-limiting, exemplary LNP delivery vehicles are described in U.S. Patent Publication Nos. US 20160174546, US 20140301951, US 20150105538, US 20150250725, Wang et al., J. Control Release, 2017 Jan 31. pii: S0168-3659(17)30038-X. doi: 10.1016 / j.jconrel.2017.01.037. [Epub ahead of print]; Altinoglu et al., Biomater Sci., 4(12): 1773- 80, Nov. 15, 2016; Wang et al., PNAS, 113(11):2868-73 March 15, 2016; Wang et al., PloS One, 10(11): e0141860. doi: 10.1371 / journal. pone.0141860. eCollection 2015, Nov. 3, 2015; Takeda et al., Neural Regen Res. 10(5):689-90, May 2015; Wang et al., Adv. Heal the Mater., 3(9): 1398-403, Sep. 2014; and Wang et al., Agnew Chem Int Ed Engl., 53(11):2893-8, Mar. 10, 2014; James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) published online 11 May 2014, doi: 10.1038 / nnano.2014.84; Coelho et al., N Engl J Med 2013; 369:819-29;Aleku et al., Cancer Res., 68(23): 9788-98 (Dec. 1, 2008), Strumberg et al., Int. J. Clin. Pharmacol. Then, 50(1): 76-8 (Jan. 2012), Schultheis et al., J. Clin. Oncol., 32(36): 4141-48 (Dec. 20, 2014), and Fehring et al., Mol. Ther., 22(4): 811-20 (Apr. 22, 2014); Novobrantseva, Molecular Therapy-Nucleic Acids (2012) l, e4; doi:10.1038 / mtna.2011.3;W02012135025; US 20140348900; US 20140328759; US 20140308304; WO 2005 / 105152; WO 2006 / 069782; WO 2007 / 121947; US 2015 / 082080; US 20120251618; 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035; 1519714; 1781593 and 1664316;Liposomes

[0323] In an embodiment, a lipid particle may be liposome. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipid bilayer. In an embodiment, liposomes are biocompatible, nontoxic, deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB).

[0324] Liposomes can be made from several different types of lipids, e.g., phospholipids. A liposome may comprise natural phospholipids and lipids such as l,2-distearoryl-sn-glycero-3 - phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines, monosialoganglioside, or any combination thereof.

[0325] Several other additives may be added to liposomes to modify their structure and properties. For instance, liposomes may further comprise cholesterol, sphingomyelin, and / or 1,2- dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), e.g., to increase stability and / or to prevent the leakage of the liposomal inner cargo.

[0326] In an embodiment, a liposome delivery vehicle can be used to deliver a virus particle containing a polynucleotides and / or polypeptides of the present invention described elsewhere herein. In an embodiment, the virus particle(s) are adsorbed to the liposome, such as through electrostatic interactions, and / or is attached to the liposomes via a linker.

[0327] In an embodiment, the liposome is a Trojan Horse liposome (also known in the art as Molecular Trojan Horses), see e.g. http: / / cshprotocols.cshlp.Org / content / 2010 / 4 / pdb.prot5407.long, the teachings of which can be applied and / or adapted to generated and / or deliver the polynucleotides and / or polypeptides of the present invention described elsewhere herein.

[0328] Other non-limiting, exemplary liposomes include those as set forth in Wang et al., ACS Synthetic Biology, 1, 403-07 (2012); Wang et al., PNAS, 113(11) 2868-2873 (2016); Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679; WO 2008 / 042973; US Pat. No. 8,071,082; WO 2014 / 186366; 20160257951; US20160129120; US 20160244761; 20120251618; WO2013 / 093648; Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE.RTM. (e.g., LIPOFECTAMINE.RTM. 2000, LIPOFECTAMINE RTM. 3000, LIPOFECTAMINE RTM. RNAiMAX, LIPOFECTAMINE.RTM. LTX), SAINT-RED (Synvolux Therapeutics, Groningen Netherlands), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif), and Eufectins (JBL, San Luis Obispo, Calif).Stable nuclei c-acid-lipid particles (SNALPs)

[0329] In an embodiment, the lipid particles are stable nucleic acid lipid particles (SNALPs). SNALPs may comprise an ionizable lipid (DLinDMA) (e.g., cationic at low pH), a neutral helper lipid, cholesterol, a diffusible polyethylene glycol (PEG)-lipid, or any combination thereof. In some examples, SNALPs may comprise synthetic cholesterol, dipalmitoylphosphatidylcholine, 3- N-[(w-methoxy polyethylene glycol)2000)carbamoyl]-l,2- dimyrestyloxypropylamine, and cationic l,2-dilinoleyloxy-3-N,Ndimethylaminopropane. In some examples, SNALPs may comprise synthetic cholesterol, l,2-distearoyl-sn-glycero-3-phosphocholine, PEG- eDMA, and 1,2- dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMAo).

[0330] Other non-limiting, exemplary SNALPs that can be used to deliver the polynucleotides and / or polypeptides of the present invention described elsewhere herein can be any such SNALPs as described in Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005, Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006; Geisbert et al., Lancet 2010; 375: 1896-905; Judge, J. Clin. Invest. 119:661-673 (2009); and Semple et al., Nature Niotechnology, Volume 28 Number 2 February 2010, pp. 172-177.Other Lipids

[0331] The lipid particles may also comprise one or more other types of lipids, e.g., cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2- DMA), DLin-KC2-DMA4, C12- 200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG.

[0332] In an embodiment, the delivery vehicle comprises a lipidoid, such as any of those set forth in, for example, US 20110293703.

[0333] In an embodiment, the delivery vehicle can be or include an amino lipid, such as any of those set forth in, for example, Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529 -8533.

[0334] In an embodiment, the delivery vehicle comprises a lipid envelope, such as any of those set forth in, for example, Korman et al., 2011. Nat. Biotech. 29: 154-157.Lipoplexes / pol plexes

[0335] In an embodiment, the delivery vehicles comprise lipoplexes and / or polyplexes. Lipoplexes may bind to negatively charged cell membrane and induce endocytosis into the cells. Lipoplexes may be complexes comprising lipid(s) and non-lipid components. Exemplary lipoplexes and polyplexes include FuGENE-6 reagent, a non-liposomal solution containing lipidsI l land other components, zwitterionic amino lipids (ZALs), Ca2Jr (e.g., forming DNA / Ca2+microcomplexes), polyethenimine (PEI) (e.g., branched PEI), and poly(L-lysine) (PLL).Sugar-Based Particles

[0336] In an embodiment, the delivery vehicle is a sugar-based particle. In an embodiment, the sugar-based particles comprise GalNAc, such as any of those described in WO2014118272; US 20020150626; Nair, IK et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961; Ostergaard et al., Bioconjugate Chem., 2015, 26 (8), pp 1451-1455;Cell Penetrating Peptides

[0337] In an embodiment, the delivery vehicles comprise cell penetrating peptides (CPPs). CPPs are short peptides that facilitate cellular uptake of various molecular cargo (e.g., from nanosized particles to small chemical molecules and large...

Claims

CLAIMSWhat is claimed is:

1. An isolated engineered immune cell comprising a T cell receptor (TCR) capable of recognizing a disease-associated antigen.

2. The cell of claim 1, wherein the disease-associated antigen is a virus-associated antigen.

3. The cell of claim 2, wherein the disease-associated antigen is a cancer-associated antigen.

4. The cell of claim 3, wherein the cancer-associated antigens are associated with one or more hematological malignancies.

5. The cell of claim 3, wherein the hematological malignancy is multiple myeloma (MM).

6. The cell of claim 3, wherein the hematological malignancy is acute myeloid leukemia (AML).

7. The cell of claim 3, wherein the hematological malignancy is chronic lymphocytic leukemia (CLL).

8. The cell of claim 1, wherein the disease-associated antigen is selected from SEQ ID NO: 325-41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.

9. The cell of claim 1, wherein the TCR comprises SEQ ID NOs: 1-121, and / or a TCR alpha chain CDR3 sequence selected from SEQ ID NO: 1-62, 41855-41902 or TCR beta chain CDR3 sequence selected from SEQ ID NO: 63-121 or 41903-41948.

10. The cell of claim 1, wherein the cell is a CD8 T cell.

11. The cell of claim 10, wherein the CD8 T cell is isolated from a subject to be treated.

12. The cell of claim 10, wherein the cell comprises one or more modifications to one or more genes that modify an immune reactivity of the cell.

13. The cell of claim 1, wherein the cell is a CAR T cell.

14. A method of treating cancer comprising delivering the cell of any one of claims 1 to 13.

15. The method of claim 14, wherein the cancer is a hematological malignancy.

16. The method of claim 15, wherein the hematological malignancy is MM, AML, or CLL.

17. A method of bone marrow transplant for use in treating hematological malignancies comprising transfusing a composition comprising the cells of any one of claims 1 to 13 into a subject suffering from a hematological malignancy.

18. The method of claim 17, wherein the hematological malignancy is MM, AML, or CLL.

19. A vaccine comprising a cancer-associated antigen.

20. The vaccine of claim 19, wherein the antigen is recognized by a TCR selected from SEQ ID NOs: 1-121 and / or a TCR alpha chain CDR3 sequence selected from SEQ ID NO: 1-62, or 41855-41902 or TCR beta chain CDR3 sequence selected from SEQ ID NO: 63-121 or 41903- 41948.

21. The vaccine of claim 19, wherein the antigen is selected from SEQ ID NO: 325- 41854, and / or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.

22. The vaccine of claim 19, wherein the vaccine comprises a polynucleotide encoding the conserved cancer antigen.

23. The vaccine of claim 22, wherein the polynucleotide is mRNA.

24. The vaccine of claim 19, wherein the vaccine comprises the antigen and optionally a carrier or adjuvant.

25. A method of treating cancer in a subject comprising administering the cancer vaccine of any one of claims 19 to 24.

26. A method for detecting tumor-reactive T-cell receptors (TCRs):(a) characterizing the phenotype and clonality of a population of isolated T cells to define a baseline transcriptional state;(b) segregating single isolated T cells from the population of isolated T cells into individual discrete volumes and exposing the single isolated T cells to a tumor cell;(c) identifying and retrieving single isolated T cells from the individual discrete volumes and conducting TCR alpha and beta chain sequencing; and(d) identifying antigen-reactive T cells by matching each TCR to its baseline transcriptional state using the CDR3 amino acid sequence as an endogenous barcode of each TCR.

27. The method of claim 26, wherein step (b) further comprises capture beads to detect T cell-derived cytokines and wherein single isolated T cells are retrieved for step (c) if T cell cytokines are detected.

28. The method of claim 27, wherein the T cell-derived cytokines comprise interleukin-2 (IL-2), interferon-gamma, and tumor necrosis factor (TNF).

29. The method of claim 26, wherein step (b) further comprises assaying for expression of surface 4-IBB as an indicator of an antigen-activated T cell.

30. The method of claim 26, further comprises exposing a subset of the population of isolated T cells to stimulation with tumor or viral antigens and obtaining TCR sequencing TCRs using TCRV(Beta)-seq, and integrating the TCRV(beta)-seq with the baseline transcriptional state using the CDR3 amino acid sequence.

31. The method of claim 26 further comprising defining an antigen-reactive TCR signature based on the identified baseline transcriptional state.

32. The method of claim 26, wherein characterizing the phenotype and clonality of the cells comprises using high-throughput single-cell RNA sequencing (scRNA-seq), single-cell TCR sequencing (scTCR-seq) coupled with the detection of surface proteins using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq).

33. The method of claim 26, wherein determining one or more epitopes on the cells to define the clonotype comprises using high-throughput single-cell RNA sequencing (scRNA) and single-cell TCR sequencing.

34. The method of claim 26, wherein determining one or more epitopes on the cells comprises using cellular indexing of the transcriptomes and epitopes by sequencing (CITE-seq).

35. The method of claim 26, wherein step (b) further comprises optical screening to quantify T cell activation and cytokine production.

36. The method of claim 26, further comprising expanding the identified antigenspecific T cells in a cell population and delivering the cell population to a subject in need thereof.

37. The cell of claim 1, wherein the TCR comprises a sequence corresponding to TCR11729 or TCR15434 as shown in Table 8, with TCR11729 recognizing the CTAG2 and TCR15434 recognizing IGKV.

38. The vaccine of claim 20, wherein the TCR comprises a sequence corresponding to TCR11729 or TCR15434 as shown in Table 8, with TCR11729 recognizing CTAG2 and TCR15434 recognizing IGKV.

39. The method of claim 26, wherein the TCR detected comprises a sequence corresponding to TCR11729 or TCR15434 as shown in Table 8 with TCR11729 recognizing the CTAG2 antigen and TCR15434 recognizing a patient-specific mutation in the IGKV gene.

40. A method for treating multiple myeloma in a subject in need thereof comprising administering to the subject a cancer therapy in an amount effective to prevent or reduce the progression of the multiple myeloma only if expression of a gene signature is detected in a biological sample comprising the subject’s T cells, wherein said gene signature comprises one or more genes chosen from GNLY, ZNF683, GZMH, FGFBP2, GZMB, NKG7, CCL5, HOPX, KLRD1, EFHD2, CD8A, CTSW, CST7, ITGB1, BHLHE40, LYAR, S100A4, GZMA, MXRA7, KLRK1, SH3BGRL3, ITGA4, FCRL6, TGFB1, CCL4, ZEB2, AOAH, AHNAK, S100A10, LGALS1, PRF1, ITGB2, CD52, TPST2, PRSS23, ANXA1, CYBA, C12orf75, LAIR2, MATK, S100A6, TNFAIP3, CLIC1, KLF6, Clorf21, SYNE2, HLA-DPB1, HLA-DPA1, DSTN, and CD99, or one or more genes chosen from EFHD2, SH3BGRL3, CD52, ZNF683, S100A10, S100A6, S100A4, FCRL6, TAGLN2, Clorf21, PLEK, GNLY, CD8A, ZEB2, ITGA4, BHLHE40, LYAR, FGFBP2, HOPX, GZMA, CLIC1, HLA-DPA1, HLA-DPB1, TNFAIP3, AOAH, ANXA1, KLF6, ITGB1, PRF1, AHNAK, CTSW, PRSS23, KLRD1, KLRK1, LINC02446, RPS26, C12orf75, RGCC, GZMH, GZMB, NFKBIA, SYNE2, FOS, PPP2R5C, CRIP1, AKAP13, CYBA, CCL5, CCL4, MXRA7, GADD45B, MATK, ZFP36, TGFB1, NKG7, LAIR2, DSTN, CST7, ITGB2, TPST2, LGALS1, CD99, and FLNA.

41. The method of claim 40, wherein the cancer therapy comprises adjuvant chemotherapy, antibody drug conjugates, bispecific antibodies, a checkpoint inhibitor and / or a proteosome inhibitor.

42. The method of claim 41, wherein the bispecific antibody comprises a BCMA / CD3 T-cell engaging bispecific antibody, a GPRC5DxCD3 bispecific antibody, an anti-CD3 / CD38 bispecific antibodies, or an Fc receptor-like 5xCD3 bispecific antibody.

43. The method of claim 41, wherein the checkpoint inhibitor comprises a PD-1 inhibitor comprising nivolumab and pembrolizumab, or a PD-L1 inhibitor comprising durvalumab and atezolizumab.

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