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WO2025097055A2 - Compositions et méthodes d'utilisation de lymphocytes t en immunothérapie - Google Patents

Compositions et méthodes d'utilisation de lymphocytes t en immunothérapie Download PDF

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WO2025097055A2
WO2025097055A2 PCT/US2024/054265 US2024054265W WO2025097055A2 WO 2025097055 A2 WO2025097055 A2 WO 2025097055A2 US 2024054265 W US2024054265 W US 2024054265W WO 2025097055 A2 WO2025097055 A2 WO 2025097055A2
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cell
cells
tcr
antigen
seq
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Steven Carr
Mirco Julian FRIEDRICH
Gabrielle HERNANDEZ
Jennifer ABELIN
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Massachusetts Institute of Technology
Broad Institute Inc
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Massachusetts Institute of Technology
Broad Institute Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4271—Melanoma antigens
    • A61K40/4273—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/30—Mixture of cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex

Definitions

  • TCRs T cell receptors
  • neoplasia 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.
  • MM multiple myeloma
  • plasma cell myeloma also known as plasma cell myeloma, myelomatosis, Kahler’s
  • plasma cell myeloma a type of white blood cell normally responsible for producing antibodies in which collections of the neoplastic plasma cells accumulate in the bone marrow.
  • 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, proteasome inhibitors, and immunomodulatory drugs have resulted in significant survival benefits, however relapse is inevitable and disease remains incurable with a median survival of 5 years.
  • Acute myeloid leukemia is a heterogeneous hematologic disorder characterized by clonal expansion of myeloid blasts in bone marrow, peripheral blood, and other tissues.
  • AML Acute myeloid leukemia
  • 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.
  • the techniques described herein relate to an isolated engineered immune cell including a T cell receptor (TCR) capable of recognizing a disease-associated antigen.
  • TCR T cell receptor
  • the techniques described herein relate to a cell, wherein the disease- associated antigen is a virus-associated antigen.
  • the techniques described herein relate to a cell, wherein the disease-associated antigen is a cancer-associated antigen.
  • the techniques described herein relate to a cell, wherein the cancer- associated antigens are associated with one or more hematological malignancies.
  • the techniques described herein relate to a cell, wherein the hematological malignancy is multiple myeloma (MM).
  • the techniques described herein relate to a cell, wherein the hematological malignancy is acute myeloid leukemia (AML).
  • the techniques described herein relate to a cell, wherein the hematological malignancy is chronic lymphocytic leukemia (CLL).
  • 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.
  • 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.
  • 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.
  • 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.
  • a method of treating cancer comprises administering the engineered immune cell to a subject in need thereof.
  • the subject suffers from a cancer that is a hematological malignancy.
  • the hematological malignancy is MM, AML, or CLL.
  • the techniques described herein relate to a vaccine including a cancer-associated antigen.
  • 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
  • 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.
  • the vaccine includes a polynucleotide encoding the conserved cancer antigen.
  • the polynucleotide is mRNA.
  • a method of treating cancer comprises administering the vaccine to a subject in need thereof.
  • the subject suffers from a hematological malignancy.
  • the hematological malignancy is multiple myeloma, acute myeloid leukemia, or chronic lymphocytic leukemia.
  • 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.
  • TCRs tumor-reactive T-cell receptors
  • 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.
  • 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).
  • IL-2 interleukin-2
  • TNF tumor necrosis factor
  • step (b) further includes assaying for expression of surface 4-IBB as an indicator of an antigen-activated T cell.
  • 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.
  • the techniques described herein relate to a method further including defining an antigen-reactive TCR signature based on the identified baseline transcriptional state.
  • 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 the detection of surface proteins using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq).
  • scRNA-seq high-throughput single-cell RNA sequencing
  • scTCR-seq single-cell TCR sequencing
  • CITE-seq cellular indexing of transcriptomes and epitopes by sequencing
  • 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.
  • scRNA single-cell RNA sequencing
  • 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).
  • step (b) further includes optical screening to quantify T cell activation and cytokine production.
  • 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.
  • 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.
  • BMR-T bone marrow resident T cells
  • scRNA-seq high- throughput single-cell RNA sequencing
  • scTCR- seq single-cell TCR sequencing
  • CITE-seq cellular indexing of transcriptomes and epitopes by sequencing
  • TCR-alpha and beta-chain sequencing TCRA/B-seq
  • 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 reactive T cells by matching each TCR to its baseline transcriptional state using the CDR3 nucleotide sequence as unique barcode of a given clone
  • 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. 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.
  • 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. 1A 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 cyto
  • 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).
  • FIG. 2G T cell subtype composition of BMR-T reactive to the outlined antigens. Total number of cells per antigen indicated.
  • FIG. 21 Scaled average expression heatmap of selected marker genes per recognized antigen.
  • 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.
  • 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. 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. 3F shows 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.
  • FIG. 3G shows flow cytometry analysis of CD29 surface protein expression on homed TCR-transgenic T cells in TDLN from k) (Left).
  • FIG. 3G 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.
  • FIGS. 4A-4J show the clinical relevance of tumor reactive T cells in multiple myeloma.
  • FIG. 4C shows prospective area under the curve (AUC) of receiver operator characteristic (ROC) shown (Methods).
  • 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).
  • TMB tumor mutational burden
  • Methods total count
  • 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.
  • 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.
  • 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.
  • BM bone marrow
  • PB peripheral blood
  • 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.
  • CAAs cancer-associated antigens
  • nuORFs novel or unannotated open reading frames.
  • FIG. 51 shows mRNA transfection and functional testing 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).
  • MHC major histocompatibility complex
  • FIGS. 6A-6I show MM-TCR signature identifies TCRs responsive to bispecific antibodies.
  • Statistical significance between antigen specificity groups was determined by one-way ANOVA with Tukey post-hoc test for multiple hypothesis testing correction.
  • FIGs. 6G-6H show bone marrow counts of T cells with TCRs detected among tumor reactive CD8+ BMR-T classified as effector-memory (EM; FIG. 6G) or progenitor- exhausted (PEX; FIG.
  • FIGS. 7A-7L show the expansion of tumor reactive BMR-T underlies response to immune checkpoint inhibition in AML.
  • R/R AML relapsed/refractory acute myeloid leukemia
  • 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 is indicated 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.
  • FIG. 8A-8F show identification of antigen-specific bone-marrow associated T cells using MHC immunopeptidomes.
  • 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. 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.
  • FIG. 9A-9I show myeloma reactive T cells target public or immunoglobulin-derived antigens.
  • 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.
  • FIG. 9C shows clonality (1/Shannon diversity) of bone marrow TCRs split by reactivity.
  • FIG. 9E shows a violin plot depicting cell-wise expression of cytotoxicity score split by antigen reactivity.
  • FIG. 9F shows a violin plot depicting cell-wise expression of dysfunction score split by antigen reactivity.
  • 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).
  • FIGS. 10A-10G show conserved transcriptional signatures of tumor-reactive BMTCs.
  • FIG. 10B shows a ridge plot of selected 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.
  • FIG. 10A shows conserved transcriptional signatures of tumor-reactive BMTCs.
  • FIG. 10B shows a ridge plot of selected marker genes per reactivity group (Myeloma
  • 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).
  • 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.
  • FIGS. 11A-11I show MM-TCR signature identifies TCRs responsive to bispecific antibodies.
  • FIG. 11D shows TCR clonal dynamics over time for representative RRMM 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. HI is shows clinical response status.
  • FIGS. 12A-12L show transfer of tumor-reactive TCRs by autologous stem cell transplantation.
  • 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 each clone occupied at that timepoint.
  • FIG. 12D shows representative gating strategy of PBSC samples subjected to multiparametric flow cytometry.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • UMAP Uniform Manifold Approximation and Projection
  • 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.
  • 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. 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
  • FIGS. 18A-18H show functional expansion of tumor reactive T cells on BMR-T in establishment cohort.
  • FIG. 18A show absolute T cell count in assay at baseline (dO) and post expansion (d28) per patient.
  • FIG. 18A shows 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).
  • 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.
  • FIGS. 19A-19B shows retrospective and prospective TCR signature benchmarking of MM-TCR signature versus published signatures of tumor-infdtrating lymphocytes.
  • FIGS. 20A-20F shows trajectory and fate mapping of tumor reactive and bystander BMR-T using RNA velocities and CellRank.
  • FIG. 20A-20B shows 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. 20D shows 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.
  • FIG. 20F shows 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.
  • FIGS. 21A-21E shows profiling and tumor reactivity classification of BMR-T in validation NDMM cohort.
  • FIG. 21C shows relative abundance of expansion-categories within cells of each patient.
  • FIG. 21E shows average clonotype proportion in sample as dot size by T cell subtype within each patient.
  • 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.
  • 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.
  • FIGS. 23A-23M shows tumor reactive T cells expand upon autologous stem cell transplantation.
  • 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.
  • 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).
  • 23G 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.
  • 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. 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
  • 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.
  • 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.
  • FIG. 25 shows TCR sequence sharing in tumor and virus reactive BMR-T.
  • 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).
  • 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 scaled BLOSUM45-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
  • FIG. 26B 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 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.
  • FIGS. 27A-27C show tumor reactive T cells expand upon autologous stem cell transplantation.
  • N 18 patients treated with bispecific BCMAxCD3 antibodies.
  • FIG. 28 shows fluorescence imaging of BMTCs targeting multiple myeloma by antigen-agnostic microfluidics screening.
  • FIGS. 29 and 30 show amplified TCRs from tumor-reactive BMTCs retrieved from antigen-agnostic microfluidics screening.
  • FIGS. 31A-31G show phenotype composition and cloning of TCRs targeting multiple myeloma retrieved from antigen-agnostic microfluidics screening.
  • FIGS 32A-32D, 33A-33D, and 34 show tumor specificity validation of TCRs targeting multiple myeloma retrieved from antigen-agnostic microfluidics screening.
  • FIG. 35 shows MHC class I blocking experiments of TCRs targeting multiple myeloma retrieved from antigen-agnostic microfluidics screening.
  • FIGS. 36A-36C shows fluorescence imaging of peripheral blood T cells targeting acute myeloid leukemia by antigen-agnostic microfluidics screening.
  • FIGS. 37A-37C show fluorescence imaging of peripheral blood T cells targeting chronic lymphocytic leukemia by antigen-agnostic microfluidics screening.
  • FIGS. 38A-38F show compartment tracing of antigen-specific TCRs in multiple myeloma patients.
  • FIGS. 39A and 39B show TCR sequence similarities in tumor-reactive BMTCs in multiple myeloma patients.
  • FIGS. 40A and 40B show TCR sequence similarities in virus-specific and random BMTCs in multiple myeloma patients.
  • 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).
  • 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.
  • FIGS. 44A and 44B show retrospective and prospective TCR signature benchmarking of MM-TCR signature versus published signatures of tumor-infiltrating lymphocytes.
  • FIGS. 45A-45F show CD29 (JTGB1 as marker gene of tumor specific T cells.
  • FIGS. 46A-46F show clinical trial cohort of TCRV0 multiple myeloma patients undergoing ASCT.
  • FIGS. 47A and 47B show tumor-reactive T cells expand upon ASCT.
  • FIG. 48 shows transfer of tumor-reactive T cells with ASCT.
  • FIG. 49 shows persistence of tumor-reactive T cells one year after ASCT.
  • 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,
  • subject refers to a vertebrate, preferably a mammal, more preferably a human.
  • 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.
  • the subject is a human.
  • TIL tumor infiltrating lymphocyte
  • TME tumor microenvironment
  • 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.
  • the gene expressions profiles can predict a consistent response to cell based therapies, antibody based therapeutics, including bi-specific antibodies, and disease-specific vaccines.
  • 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.
  • inventions 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 antigen receptor (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.
  • TIL tumorinfiltrating lymphocyte
  • 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).
  • hTERT human telomerase reverse transcriptase
  • MDM2B mouse double minute 2 homolog
  • CYP1B cytochrome P450 IB 1
  • HER2/neu HER2/neu
  • Wilms’ tumor gene 1 (WT1) livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), M
  • 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.
  • BCMA B cell maturation antigen
  • TACI transmembrane activator and CAML Interactor
  • BAFF-R B-cell activating factor receptor
  • the engineered immune cell comprises a TCR capable of recognizing a cancer-associated antigen.
  • 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.
  • the hematological malignancy is multiple myeloma.
  • the hematological malignancy is a leukemia.
  • the leukemia is acute myeloid leukemia.
  • 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 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 T lymphocyte 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.
  • 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.
  • the TCR is encoded by a nucleotide sequence that has been codon- optimized.
  • the alpha and/or beta chain further comprise a signal peptide.
  • the TCR is isolated or purified or is recombinant.
  • the TCR is human.
  • the TCR is monoclonal.
  • the TCR is singlechain.
  • the TCR comprises two chains.
  • nucleic acid molecules encoding any of the provided TCRs, or an alpha or beta chain thereof.
  • nucleotide sequence is codon-optimized.
  • a vector comprising a nucleic acid of any provided herein.
  • the vector is an expression vector.
  • the vector is a viral vector.
  • an engineered cell comprising the nucleic acid molecule of any provided herein or vector of any provided herein.
  • an engineered cell including the TCR of any provided herein.
  • the TCR is heterologous to the cell.
  • the engineered cell is a cell line.
  • the engineered cell is a primary cell obtained from a subject.
  • the subject is a mammalian subject.
  • the subject is human.
  • the engineered cell is a T cell.
  • the T cell is CD8+.
  • the T cell is CD4+.
  • TCRs are identified that recognize a tumor antigen.
  • tumor antigen 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.
  • 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.
  • TSA tumor-specific antigens
  • TAA tumor-specific membrane antigens
  • TAA tumor-associated antigens
  • embryonic antigens on tumors growth factor receptors, growth factor ligands, etc.
  • 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.
  • 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 chain CDR3 sequence selected from SEQ ID NO: 63-121 or 41903-41948.
  • the TCR comprise recognizes CTAG2 or IGKV.
  • TCR comprises an alpha or beta chain CDR3 sequences of TCR No. 11729 or 15343 from Table 8.
  • 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).
  • 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.
  • 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).
  • an antibody or an antigen-binding fragment e.g., scFv
  • an antigen such as an intact antigen, expressed on the surface of a cell.
  • 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.
  • 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.
  • the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs).
  • CARs chimeric antigen receptors
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and/or magnesium and/or many or all divalent cations.
  • 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.
  • a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer’s instructions.
  • the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS.
  • components of a blood cell sample are removed, and the cells directly resuspended in culture media.
  • 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.
  • 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.
  • the immune cells may include engineered receptors, such as chimeric antigen receptors (CARs) or specific T cell receptors (TCRs), to target cancer cells.
  • CARs chimeric antigen receptors
  • TCRs specific T cell receptors
  • TCR identified antigen-activated T cell receptor
  • TCRs that are clonal or specific to an antigen are identified.
  • the TCR CDR3 is used to generate a chimeric antigen receptor.
  • adoptive cell therapy 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.
  • 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 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.
  • TIL tumor infiltrating lymphocytes
  • allogenic cells immune cells are transferred (see, e.g., Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266).
  • allogenic cells can be edited to reduce alloreactivity and prevent graft-versus-host disease.
  • 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.
  • 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).
  • TSA tumor-specific antigen
  • 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.
  • 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).
  • TAA tumor-associated antigen
  • CAA cancer-associated antigen
  • 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.
  • 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.
  • hTERT human telomerase reverse transcriptase
  • MDM2 mouse double minute 2 homolog
  • CYP1B cytochrome P450 IB 1
  • HER2/neu HER2/neu
  • Wilms’ tumor gene 1 WT1
  • 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.
  • the antigen may be CD19.
  • 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.
  • 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
  • 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).
  • CLL1 may be targeted in acute myeloid leukemia.
  • MAGE A3, MAGE A6, SSX2, and/or KRAS may be targeted in solid tumors.
  • HPV E6 and/or HPV E7 may be targeted in cervical cancer or head and neck cancer.
  • 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.
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndromes
  • CML chronic myeloid leukemia
  • non-small cell lung cancer breast, pancreatic, ovarian or colorectal cancers
  • mesothelioma may be targeted in B cell malignancies, including non-Hodgkin lymphoma, diffuse large B-cell lymphoma, or acute lymphoblastic leukemia.
  • CD171 may be targeted in neuroblastoma, glioblastoma, or lung, pancreatic, or ovarian cancers.
  • 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.
  • MUC 16 may be targeted in MUC16ecto+ epithelial ovarian, fallopian tube or primary peritoneal cancer.
  • 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).
  • RRCC renal cell carcinoma
  • GBM gliomas
  • HNSCC head and neck cancers
  • 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).
  • TCR T cell receptor
  • 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).
  • TCR T cell receptor
  • CARs chimeric antigen receptors
  • TCRs T cells or natural killer cells
  • NK natural killer cells
  • 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).
  • 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.
  • 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.
  • the antigen-binding domain may comprise a receptor, such that the CAR is capable of binding to the ligand of the receptor.
  • the antigen-binding domain may comprise a ligand, such that the CAR is capable of binding the endogenous receptor of that ligand.
  • 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.
  • 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.
  • the hinge region may be modified to prevent off-target binding by FcRs or other potential interfering objects.
  • 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.
  • 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.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • 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.
  • First-generation CARs typically consist of a single-chain variable fragment of an antibody 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.
  • 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.
  • the primary signaling domain comprises a functional signaling domain of CD3( ⁇ or FcRy.
  • 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, I
  • the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the group consisting of: 4-1BB, CD27, and CD28.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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
  • 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.
  • scFv single chain variable region moiety
  • 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 molecule included 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.
  • 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.
  • GM-CSF human granulocyte-macrophage colony-stimulating factor
  • a plasmid encoding this sequence was digested with Xhol and Notl.
  • 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).
  • cells intended for adoptive cell therapies 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.
  • 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 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).
  • Example 1 and Table 1 of WO2015187528 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).
  • CD28-CD3 ⁇ 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.
  • a signal sequence human CD8-alpha or GM-CSF receptor
  • extracellular and transmembrane regions human CD8- alpha
  • 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
  • cells intended for adoptive cell therapies 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.
  • the antigen is CD19
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a 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.
  • 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.
  • the second target antigen is an MHC-class I molecule.
  • the intracellular signaling domain comprises a functional signaling portion of an immune checkpoint molecule, such as for example PD-1 or CTLA4.
  • the inclusion of such inhibitory CAR reduces the chance of the engineered immune cells attacking non-target (e.g., non-cancer) tissues.
  • T cells expressing CARs may be further modified to reduce or eliminate expression of endogenous TCRs to reduce off-target effects.
  • 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.
  • TCR TCR upon engagement of its MHC-peptide ligand
  • MHC-peptide ligand MHC-peptide ligand
  • 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.
  • RNA interference e.g., shRNA, siRNA, miRNA, etc.
  • CRISPR CRISPR
  • TCR-a and TCR-P CD3 chains in primary T cells.
  • CAR also may comprise a switch mechanism for controlling expression and/or activation of the CAR.
  • 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.
  • 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.
  • 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
  • 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.
  • 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).
  • RNA molecules may be used to transform target immunoresponsive cells, such as protoplast fusion, lipofection, transfection or electroporation.
  • vectors 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, for example 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.
  • 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).
  • 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.
  • AaPC y-irradiated activating and propagating cells
  • 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).
  • 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.
  • 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).
  • 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).
  • TCR transgenic T cell receptor
  • CAR chimeric antigen receptor
  • Regulatory T cells 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.
  • Tregs are divided into “natural” Tregs that develop in the thymus or “induced” Tregs that are generated in the periphery.
  • Regulatory T cells suppress immune responses through multiple mechanisms including direct interaction with other immune cells or by producing immunosuppressive cytokines 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.
  • 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).
  • ACT adoptive T cell transfer
  • ACT adoptive T cell transfer
  • 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).
  • 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).
  • TCRs T cell receptors
  • 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).
  • 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).
  • 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).
  • a disease such as a neoplasia
  • a pathogen infection such as a neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant reaction.
  • the treatment is administered after lymphodepleting pretreatment in the form of chemotherapy (typically a combination of cyclophosphamide and fludarabine) or radiation therapy.
  • chemotherapy typically a combination of cyclophosphamide and fludarabine
  • Immune suppressor cells like Tregs and MDSCs may attenuate the activity of transferred cells by outcompeting them for the necessary cytokines.
  • lymphodepl eting pretreatment may eliminate the suppressor cells allowing the TILs to persist.
  • 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.
  • the immunosuppressive treatment provides for the selection and expansion of the immunoresponsive T cells within the patient.
  • the treatment is administered before primary treatment (e.g., surgery or radiation therapy) to shrink a tumor before the primary treatment.
  • primary treatment e.g., surgery or radiation therapy
  • the treatment is administered after primary treatment to remove any remaining cancer cells.
  • 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).
  • 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.
  • 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).
  • the cell compositions of the present invention are preferably administered by intravenous injection.
  • 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.
  • the effective amount of cells are administrated as a single dose.
  • 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.
  • 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
  • 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.
  • 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.
  • a transgenic safety switch in the form of a transgene that renders the cells vulnerable to exposure to a specific signal.
  • 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).
  • 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.
  • 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. Patent Publication No. 20130071414; PCT Patent Publication WO2011146862; PCT Patent Publication W02014011987; PCT Patent Publication W02013040371; Zhou et al.
  • 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.
  • 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 the differentiation and/or proliferation of functionally exhausted or
  • editing may result in inactivation of a gene.
  • inactivating a gene it is intended that the gene of interest is not expressed in a functional protein form.
  • 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).
  • 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.
  • HDR homology directed repair
  • editing of 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.
  • an exogenous gene such as an exogenous gene encoding a CAR or a TCR
  • 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.
  • 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).
  • transgenes in particular CAR or exogenous TCR transgenes
  • 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.
  • TRA T cell receptor alpha locus
  • TRB T cell receptor beta locus
  • TRBC1 locus T cell receptor beta constant 1 locus
  • TRBC1 locus T cell receptor beta constant 2 locus
  • T cell receptors 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.
  • 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.
  • 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.
  • 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).
  • GVHD graft versus host disease
  • 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.
  • TCR disruption generally results in the elimination of the CD3 signaling component and alters the means of further T cell expansion.
  • editing of cells is performed to knock-out or knock-down expression of an endogenous TCR in a cell.
  • NHEJ-based or HDR-based gene editing approaches are employed to disrupt the endogenous TCR alpha and/or beta chain genes.
  • 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.
  • 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.
  • 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.
  • 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.
  • editing of 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.
  • 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.
  • the immune checkpoint targeted is cytotoxic T lymphocyte-associated antigen (CTLA-4).
  • CTLA-4 cytotoxic T lymphocyte-associated antigen
  • the immune checkpoint targeted is another member of the CD28 and CTLA4 Ig superfamily such as BTLA, LAG3, ICOS, PDL1 or KIR.
  • the immune checkpoint targeted is a member of the TNFR superfamily such as CD40, 0X40, CD 137, GITR, CD27 or TIM-3.
  • SHP-1 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).
  • PTP inhibitory protein tyrosine phosphatase
  • 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.
  • CAR chimeric antigen receptor
  • 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).
  • 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).
  • metallothioneins are targeted by gene editing in adoptively transferred T cells.
  • targets of gene editing may be at least one targeted locus involved in the expression of an immune checkpoint protein.
  • 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
  • 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.
  • a genetically engineered antigen receptor that specifically binds to an antigen, which receptor may be a CAR
  • 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
  • the disruption of the gene may be mediated by a gene editing nuclease,
  • 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.
  • an agent such as CRISPR, TALEN or ZFN
  • 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.
  • 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).
  • a CAR methylcytosine dioxygenase genes
  • editing of 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.
  • 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).
  • MDM2 mouse double minute 2
  • editing of 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 be reduced or avoided.
  • one or more HLA class I proteins such as HLA- A, B and/or C, and/or B2M are knocked-out or knocked-down.
  • B2M is knocked-out or knocked-down.
  • 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.
  • 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.
  • 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).
  • an endogenous TCR for example, TRBC1, TRBC2 and/or TRAC
  • an immune checkpoint protein or receptor for example PD1, PD-L1 and/or CTLA4
  • MHC constituent proteins for example, HLA-A, B and/or C, and/or B2M, preferably B2M.
  • 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.
  • Immune cells may be obtained using any method known in the art.
  • allogenic T cells may be obtained from healthy subjects.
  • 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.
  • T cells are obtained by apheresis.
  • 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).
  • mechanically dissociating e.g., mincing
  • enzymatically dissociating e.g., digesting
  • aspiration e.g., as with a needle
  • the bulk population of T cells obtained from a tumor sample may comprise any suitable type of T cell.
  • the bulk population of T cells obtained from a tumor sample comprises tumor infiltrating lymphocytes (TLLs).
  • the tumor sample may be obtained from any mammal.
  • the tumor sample is obtained from a human.
  • the tumor sample is obtained from a subject to be treated.
  • 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.
  • PBMC peripheral blood mononuclear cells
  • 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.
  • 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.
  • 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.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • 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.
  • 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.
  • the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
  • biocompatible buffers such as, for example, Ca-free, Mg-free PBS.
  • undesirable components of the apheresis sample can be removed, and the cells can be directly resuspended in culture media.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM 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 selection techniques.
  • 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 DYNABEADSTM for an incubation time sufficient for positive selection of the desired T cells.
  • 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.
  • use of longer incubation times 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.
  • TIL tumor infiltrating lymphocytes
  • 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.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA- DR, and CD 8.
  • monocyte populations 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.
  • the invention uses paramagnetic particles of a size sufficient to be engulfed by phagocytotic monocytes.
  • the paramagnetic particles are commercially available beads, for example, those produced by Life Technologies under the trade name DynabeadsTM.
  • 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.
  • the irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin.
  • 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.
  • any magnetic separation methodology may be used including a variety of which are commercially available, (e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)).
  • DYNAL MPC® Magnetic Particle Concentrator
  • 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.
  • the concentration of cells and surface can be varied.
  • 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.
  • a concentration of 2 billion cells/ml is used.
  • a concentration of 1 billion cells/ml is used.
  • greater than 100 million cells/ml is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • 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.
  • 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.
  • 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.
  • T cells can also be frozen.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • 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.
  • T cells for use in the present invention also may be antigen-specific T cells.
  • tumor-specific T cells can be used.
  • antigen-specific T cells are isolated from a patient of interest, such as a patient afflicted with a cancer or an infectious disease.
  • 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).
  • 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).
  • 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 numerous assays 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).
  • P2m P2- microglobulin
  • cells are directly labeled with an epitope-specific reagent for isolation by flow cytometry followed by characterization of phenotype and TCRs.
  • 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.), FACSAriaTM, FACSArrayTM, FACSVantageTM, BDTM LSR II, and FACSCaliburTM (BD Biosciences, San Jose, Calif).
  • the method comprises selecting cells that also express CD3.
  • the method may comprise specifically selecting the cells in any suitable manner.
  • 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.
  • the antibody is chosen such that it specifically recognizes and binds to the particular biomarker being selected.
  • 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.
  • the flow cytometry is fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • TCRs expressed on T cells can be selected based on reactivity to autologous tumors.
  • 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.
  • activated T cells can be selected for based on surface expression of CD 107a.
  • the method further comprises expanding the numbers of T cells in the enriched cell population.
  • 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 about 100-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.
  • ex vivo T cell expansion can be performed by isolation of T cells and subsequent stimulation or activation followed by further expansion.
  • the T cells may be stimulated or activated by a single agent.
  • 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.
  • ESP Engineered Multivalent Signaling Platform
  • both primary and secondary agents are co-immobilized on a surface, for example a bead or a cell.
  • the molecule providing the primary activation signal may be a CD3 ligand
  • the co-stimulatory molecule may be a CD28 ligand or 4- IBB ligand.
  • 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.
  • 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 of activated 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.
  • 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.
  • an AKT inhibitor such as, e.g., one or a combination of two or more AKT inhibitors disclosed in claim 8 of W02017070395
  • IL-7 exogenous Interle
  • 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.
  • 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).
  • TLR agonists are delivered in a nanoparticle system (see, e.g., Buss and Bhatia, Proc Natl Acad Sci.
  • Autologous stem cell transplantation 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.
  • HSCs hematopoietic stem cells
  • the reinfused HSCs subsequently repopulate the bone marrow, facilitating the recovery of the patient's hematopoietic system.
  • 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 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.
  • the cancer associated antigen is a conserved cancer antigen.
  • 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.
  • 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.
  • the CAA (including but not limited to, a conserved cancer antigen) is a polynucleotide.
  • the CAA (including but not limited to, a conserved cancer antigen) is recognized by a TCR.
  • 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.
  • 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.
  • 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.
  • MHC Major histocompatibility complex
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are typically recognized by CD4+ T cells.
  • 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.
  • HLA human leukocyte antigen
  • typically human MHC can also be referred to as human leukocyte antigen (HLA).
  • MHC-peptide complex 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.
  • the MHC-peptide complex is present or displayed on the surface of cells.
  • the MHC-peptide complex can be specifically recognized by an antigen receptor, such as a TCR, TCR-like CAR or antigen-binding portions thereof.
  • the CAA(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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the one or more polynucleotides encoding the one or more CAAs is DNA.
  • the one or more polynucleotides encoding the one or more CAAs 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.
  • the immunogenic composition can stimulate an immune response in a subject to which it is administered.
  • the immune response is a cell-mediated immune response.
  • the immune response is a humoral immune response.
  • the immune response includes B-cell, plasma cell, and/or antibody production (collectively referred to as a B-cell response).
  • the immune response includes a T-cell production (also referred to as a T-cell response).
  • the T-cell response includes CD 4+ T-cell production, CD8+ T cell production, or both.
  • the immune response includes both a B-cell and T-cell response.
  • the immunogenic composition is formulated as a vaccine.
  • the immunogenic composition is formulated as a protein or peptide vaccine.
  • the immunogenic composition is formulated as a DNA vaccine.
  • the immunogenic composition is formulated as an RNA, such as an mRNA, vaccine.
  • the immunogenic composition or formulation thereof is a cancer vaccine.
  • the immunogenic composition can stimulate an immune response against a cancer.
  • the immune response stimulated by the cancer vaccine is effective to reduce or eliminate the cancer in subject.
  • the cancer is a blood cancer.
  • the cancer is a white blood cell cancer.
  • the cancer is multiple myeloma.
  • 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.
  • 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.
  • the CAA (including but not limited to, conserved cancer antigen) is a peptide or a polypeptide or a polynucleotide encoding said peptide or polypeptide.
  • the CAA (including but not limited to, conserved cancer antigen) is recognized by a TCR or component thereof.
  • 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.
  • 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.
  • the polynucleotides are codon optimized for expression in humans or non-human animals.
  • the one or more CAA polynucleotides or 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.
  • the one or more CAA polynucleotides or encoding polynucleotides has a sequence corresponding 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.
  • 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.
  • 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.
  • 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, GGTG
  • sequence identity generally refers 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.
  • 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,
  • the polypeptides may be any length that is reasonable for an epitope.
  • 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.
  • the polypeptides may have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.
  • 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.
  • polypeptides of the present invention herein may comprise one or more modifications (e.g., post-translational modifications).
  • the polypeptides may comprise cysteinylated Cysteine.
  • modifications include ubiquitination, phosphorylation, sulfonation, glycosylation, acetylation, methylation, ADP-ribosylation, methionine oxidation, cysteine oxidation, cysteine lipidation, farnesylation, geranylation, pyroglutamation, and deamidation.
  • 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.
  • amino acids 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.
  • the CAA polynucleotide of the present invention is mRNA, e.g., synthetic mRNA.
  • the synthetic mRNA may comprise coding sequence(s) for one or more CAA polypeptides herein.
  • the synthetic mRNA is or is encoded by a CAA encoding polynucleotide described elsewhere herein.
  • 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.
  • 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.
  • 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.
  • 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).
  • SLBP stem-loop binding protein
  • 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.
  • 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).
  • a greatest dimension or greatest average dimension e.g., diameter or greatest average diameter
  • 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.
  • the delivery vehicles may have a greatest dimension or greatest average dimension ranging between 25 nm and 200 nm.
  • the delivery vehicles may be or comprise particles.
  • 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).
  • 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.
  • 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).
  • TEM electron microscopy
  • AFM atomic force microscopy
  • DLS dynamic light scattering
  • XPS X-ray photoelectron spectroscopy
  • XRD powder X-ray diffraction
  • FTIR Fourier transform infrared spectroscopy
  • MALD1-TOF matrix-assisted laser desorption/i
  • Characterization 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.
  • 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.
  • vectors that can contain one or more of the CAA polynucleotides of the present invention described elsewhere herein.
  • 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.
  • 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.
  • a cell such as a producer cell
  • vectors and vector systems described herein are also within the scope of this disclosure.
  • the term “vector” refers to a tool that allows or facilitates the transfer of an entity from one environment to another.
  • 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.
  • a vector is capable of replication when associated with the proper control elements.
  • 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.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector is another type of vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g.
  • 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.
  • 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.
  • 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.
  • a nucleic acid e.g., a polynucleotide
  • 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.
  • 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 in a 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.
  • the vector can be a viral vector.
  • the viral vector is an is an adeno-associated virus (AAV), adenovirus vector, a retroviral vector, or lentiviral vector.
  • AAV adeno-associated virus
  • adenovirus vector adenovirus vector
  • retroviral vector a retroviral vector
  • lentiviral vector lentiviral vector
  • Vectors may be introduced and propagated in a prokaryote or prokaryotic cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • the suitable host cell is a eukaryotic cell.
  • 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.
  • 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.
  • the host cell is a suitable yeast cell.
  • the yeast cell can be from Saccharomyces cerevisiae.
  • the host cell is a suitable mammalian cell.
  • 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).
  • the vector can be a yeast expression vector.
  • yeast Saccharomyces cerevisiae examples 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).
  • 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.
  • 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).
  • CEN centromeric
  • ARS autonomous replication sequence
  • a promoter such as an RNA Polymerase III promoter
  • a terminator such as an RNA polymerase III terminator
  • an origin of replication e.g., auxotrophic, antibiotic, or other selectable markers
  • marker gene e.g., auxotrophic, antibiotic, or other selectable markers.
  • expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2p plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and
  • 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).
  • the vector is a mammalian expression vector.
  • 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.
  • 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.
  • suitable regulatory elements capable of controlling expression of the one or more polynucleotides and/or proteins in the mammalian cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • the vector can be a fusion vector or fusion expression vector.
  • 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.
  • 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.
  • 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.
  • a proteolytic cleavage site 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
  • GST glutathione S-transferase
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.).
  • RNA or DNA starting material 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.).
  • 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.
  • Mg2+, K+, etc. co-factors
  • 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 extract
  • 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 the like. 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.
  • the polynucleotides and/or vectors thereof described herein can include one or more regulatory elements that can be operatively linked to the polynucleotide.
  • 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).
  • 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).
  • 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).
  • 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.
  • pol III promoters include, but are not limited to, U6 and Hl promoters.
  • 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.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • 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).
  • 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.
  • the vector can contain a minimal promoter.
  • the minimal promoter is the Mecp2 promoter, tRNA promoter, or U6.
  • the minimal promoter is tissue specific.
  • the length of the vector polynucleotide the minimal promoters and polynucleotide sequences is less than 4.4Kb.
  • 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.
  • 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.
  • 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.
  • 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 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 layer cell specific promoters e.g. Oct4, NANOG, Synthetic Oct4, T brachyury, NES, SOX17, FOXA2, MIR122
  • 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.
  • 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.
  • 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.
  • 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.
  • a plant promoter i.e., a promoter operable in plant cells.
  • the use of different types of promoters is envisaged.
  • 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”).
  • ORF open reading frame
  • 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.
  • 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.
  • 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 et al., (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.
  • 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.
  • LITE Light Inducible Transcriptional Effector
  • 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.
  • 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.
  • 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.
  • 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.
  • organelles can include, but are not 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.
  • peroxisome e.g. (S/A/C)-(K/R/H)-(L/A), SLK, (R/K)-(L/V/I)-XXXXX-(H/Q)-(L/A/F).
  • Minimotif Miner http:minimotifminer.org, http://mitominer.mrc- mbu.cam.ac.uk/release-4.0/
  • 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.
  • 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 present invention.
  • the selectable marker or tag is a polynucleotide barcode or unique molecular identifier (UMI).
  • 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.
  • polypeptides of the present invention such as a viral polypeptide
  • 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.
  • 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,
  • GFP GFP, FLAG- and His-tags
  • UMI molecular barcode or unique molecular identifier
  • Other suitable markers will be appreciated by those of skill in the art.
  • 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).
  • suitable linkers are described elsewhere herein.
  • the vector or vector system can include one or more polynucleotides encoding one or more targeting moieties.
  • 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.
  • 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.
  • 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.
  • the carrier e g., polymer, lipid, inorganic molecule etc.
  • the targeting moiety can be attached to the carrier and any attached or associated polynucleotide(s) and/or polypeptides of the present invention to specific cells, tissues, organs, etc.
  • the polynucleotide encoding one or more polypeptides of the present invention described herein can be codon optimized.
  • 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.
  • 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.
  • codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • 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).
  • 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.
  • one or more codons e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • 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.
  • the vector polynucleotide can be codon optimized for expression in a specific celltype, tissue type, organ type, and/or subject type.
  • 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.
  • 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.
  • 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, tend
  • the polynucleotide is codon optimized for a specific tissue type.
  • tissue types can include, but are not limited to, muscle tissue, connective tissue, connective tissue, nervous tissue, and epithelial tissue.
  • codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein.
  • the polynucleotide is codon optimized for a specific organ.
  • 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.
  • codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein.
  • 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.
  • the vectors described herein can be constructed using any suitable process or technique.
  • 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.
  • a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”).
  • 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
  • a single expression construct may be used to target nucleic acid-targeting activity to multiple different, corresponding target sequences within a cell.
  • a single vector may comprise about 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.
  • 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.
  • 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.
  • the vector is a viral vector.
  • viral vector 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.
  • 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.
  • HdAd helper-dependent adenoviral
  • 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.
  • the viral vectors are configured to produce replication incompetent viral particles for improved safety of these systems.
  • 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.
  • the delivery system can provide one or more of the same protein or a mixture of such proteins.
  • 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.
  • 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.
  • 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
  • Siadenovirus
  • 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.
  • 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 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.
  • 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.
  • 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.
  • HlV human immunodeficiency virus
  • FlV feline immunodeficiency virus
  • SIV simian immunodeficiency virus
  • Mo-MLV Moloney Murine Leukaemia Virus
  • VMV Visna.maedi
  • 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).
  • 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.
  • 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 combinations thereof), 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.
  • 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.
  • the second-generation vector lacks one or more accessory virulence factors (e.g., vif, vprm, vpu, nef, and combinations thereof).
  • no single second-generation lentiviral vector includes all features necessary to express and package a polynucleotide into a virus particle.
  • 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.
  • 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.
  • SI self-inactivating
  • 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.
  • 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.
  • 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 can be used/and or adapted to the polypeptides and/or polynucleotides of the present invention described elsewhere herein.
  • 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.
  • an “envelope protein” or “outer protein” means a protein exposed at the surface of a viral particle that is not a capsid protein.
  • envelope or outer proteins typically comprise proteins embedded in the envelope of the virus.
  • 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.
  • LDLR 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.
  • RD114 feline endogenous virus envelope protein
  • modified Sindbis virus envelope proteins see, e.g., Morizono et al. 2010. J. Virol. 84(14) 6923-6934; Morizon
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • the TEFCA SEQ ID NO: 309
  • 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.
  • 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;
  • 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.
  • selectable marker genes e.g., antibiotic resistance genes
  • Psi (T) Psi
  • RRE rev response element
  • cPPT central polypurine tract
  • WPRE woodchuck hepatitis post-transcriptional regulatory element
  • SV40 polyadenylation signal pUC origin, SV40 origin, Fl origin, and combinations thereof.
  • 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.
  • 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.
  • the Gag, Pol, and accessory proteins are lentiviral and/or gammaretroviral.
  • 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
  • the vector can be an adenoviral vector.
  • 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.
  • the polynucleotide to be delivered via the adenoviral particle can be up to about 8 kb.
  • 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.
  • 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).
  • 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 systems have 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • a hybrid-adenoviral vector can include one or more features of a retrovirus and/or an adeno-associated virus.
  • 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.
  • 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 techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention.
  • AAV Adeno Associated Viral
  • the vector can be an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the AAV can integrate into a specific site on chromosome 19 of a human cell with no observable side effects.
  • 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.
  • the regulatory molecules can be promoters, enhancers, repressors and the like, which are described in greater detail elsewhere herein.
  • the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory proteins.
  • the one or more regulatory proteins can be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof.
  • 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.
  • the AAV capsid can contain 60 capsid proteins.
  • the ratio of VP1 :VP2:VP3 in a capsid can be about 1 : 1 : 10.
  • 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.
  • adenovirus helper factors can include, but are not limited, El A, E1B, E2A, E4ORF6, and VA RNAs.
  • a producing host cell line expresses one or more of the adenovirus helper factors.
  • the AAV vector or system thereof can be configured to produce AAV particles having a specific serotype.
  • the serotype can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9 or any combinations thereof.
  • the AAV can be AAV1, AAV-2, AAV-5 or any combination thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector.
  • 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)).
  • 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).
  • 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.
  • 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.
  • AAV Adeno Associated Virus
  • cap gene 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.
  • 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.
  • the C- terminal end of the polypeptide of the present invention is fused to the N- terminal end of the AAV capsid domain.
  • 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.
  • 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.
  • 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.
  • a linker such as a GlySer linker
  • the polypeptide of the present invention may be fused in frame within, i.e., internal to, the AAV capsid domain.
  • the AAV capsid domain again preferably retains its N- terminal and C- terminal ends.
  • a linker is preferred,
  • the polypeptide of the present invention is again part of (or fused to) the AAV capsid domain.
  • the positioning of the polypeptide of the present invention is such that the polypeptide of the present invention is at the external surface of the viral capsid once formed.
  • 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.
  • AAV Adeno- Associated Virus
  • the term “associated” means fused, bound to, or tethered to.
  • 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.
  • a biotinylation sequence (15 amino acids) therefore is fused to the polypeptide of the present invention.
  • 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.
  • 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.
  • a biotinylation sequence (15 amino acids) is fused to the AAV VP2 domain, in particular the N- terminus of the AAV VP2 domain.
  • 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.
  • a biotinylation sequence (15 amino acids) is fused to the polypeptide of the present invention, which is fused with the AAV VP2 domain fused with streptavidin, wherein, in preferred embodiments, the fusion is located at the N-terminus of the AVV capsid domain.
  • the polypeptide of the present invention and the AAV VP2 domain are fused.
  • the fusion is to the N- terminal end of the polypeptide of the present invention.
  • the AAV and polypeptide of the present invention are associated via fusion.
  • 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.
  • the polypeptide of the present invention comprises at least one Nuclear Localization Signal (NLS).
  • 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.
  • 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.
  • the adaptor comprises a binding protein which recognizes and binds (or is bound by) an RNA sequence specific for said binding protein.
  • 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).
  • 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.
  • 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.
  • the modified guide is an sgRNA.
  • the modified guide comprises a distinct RNA sequence; see, e.g., International Patent Application No. PCT7US14/70175, incorporated herein by reference.
  • distinct RNA sequence is an aptamer.
  • 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.
  • 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.
  • the polypeptide of 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.
  • 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.
  • DISC disabled infections single copy
  • 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.
  • the host cell can be a complementing cell.
  • HSV vector or system thereof can be capable of producing virus particles capable of delivering a polynucleotide cargo of up to 150 kb.
  • 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.
  • the vector is a poxvirus vector or system thereof.
  • the poxvirus vector results in cytoplasmic expression of one or more polynucleotides and/or polypeptides of the present invention.
  • the capacity of the poxvirus vector or system thereof is about 25 kb or more.
  • the poxvirus vector or system thereof includes one or more polynucleotides of the present invention described herein.
  • compositions and systems can be delivered to plant cells using viral vehicles.
  • 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).
  • 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).
  • 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
  • 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).
  • tobravirus e.g., tobacco rattle virus, tobacco mosaic virus
  • potexvirus e.g., potato virus X
  • hordeivirus e.g., barley stripe mosaic virus.
  • the replicating genomes of plant viruses can be non-integrative vectors.
  • 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.
  • suitable host cells include HEK 293 cells and its variants (HEK 293T and HEK 293TN cells).
  • 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.
  • the cells 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.
  • packaging of the polynucleotide to be delivered e.g., a viral polynucleotide of the present invention
  • virus particle assembly e.g., a viral polynucleotide of the present invention
  • Lentiviruses can be prepared from any lentiviral vector or vector system described herein.
  • 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.
  • 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.
  • PVDF 0.45um low protein binding
  • 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).
  • 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.
  • plasmid vectors e.g., plasmid vectors
  • the vector is a non-viral vector or vector system.
  • 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.
  • Non-viral vectors can include, without limitation, naked polynucleotides and polynucleotide (non-viral) based vector and vector systems.
  • 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.
  • naked polynucleotide 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.
  • 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.
  • naked polynucleotides can be single-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.
  • the naked polynucleotide contains only the polynucleotide(s) of the present invention.
  • 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.
  • one or more of the CAA polynucleotides of the present invention 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.
  • the non-viral polynucleotide vector has a conditional origin of replication.
  • the non-viral polynucleotide vector is an ORT plasmid.
  • the non-viral polynucleotide vector has a minimalistic immunologically defined gene expression.
  • the non-viral polynucleotide vector has one or more post- segregationally killing system genes.
  • the non-viral polynucleotide vector is AR-free.
  • the non-viral polynucleotide vector is a minivector.
  • the non-viral polynucleotide vector includes a nuclear localization signal.
  • the non-viral polynucleotide vector includes one or more CpG motifs.
  • 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 through DNA 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.
  • S/MARs can facilitate a once-per-cell-cycle replication to maintain the non-viral polynucleotide vector as an episome in daughter cells.
  • 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.
  • 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.
  • the non-viral vector is a transposon vector or system thereof.
  • transposon also referred to as transposable element
  • 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.
  • the non-viral polynucleotide vector is a retrotransposon vector.
  • the retrotransposon vector includes long terminal repeats.
  • the retrotransposon vector does not include long terminal repeats.
  • the non-viral polynucleotide vector is a DNA transposon vector.
  • DNA transposon vectors include a polynucleotide sequence encoding a transposase.
  • the transposon vector is configured as a non-autonomous transposon vector, meaning that the transposition does not occur spontaneously on its own.
  • the transposon vector lacks one or more polynucleotide sequences encoding proteins required for transposition.
  • the non-autonomous transposon vectors lack one or more Ac elements.
  • 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 drive expression of the transposase.
  • TIRs transposon terminal inverted repeats
  • 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.
  • the transposon vector or system thereof is configured as a gene trap.
  • 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.
  • 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.
  • transposon system 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.
  • Sleeping Beauty transposon system Tcl/mariner superfamily
  • piggyBac piggyBac superfamily
  • Tol2 superfamily hAT
  • Frog Prince Tcl/mariner superfamily
  • the delivery vehicles may comprise non-viral vehicles.
  • methods and vehicles capable of delivering nucleic acids and/or proteins may be used for delivering the systems compositions herein.
  • 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.
  • the delivery vehicles may comprise lipid particles, e.g., lipid nanoparticles (LNPs) and liposomes.
  • LNPs lipid nanoparticles
  • 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., TransfectamTM and LipofectinTM).
  • 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.
  • lipidmucleic acid complexes including targeted liposomes such as immunolipid complexes
  • crystal Science 270:404-410 (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 Lipid nanoparticles
  • LNPs may encapsulate nucleic acids within cationic lipid particles (e.g., liposomes), and may be delivered to cells with relative ease.
  • 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.
  • LNPs may be used for delivering DNA molecules (e.g., those comprising polynucleotides of the present invention and/or polypeptides they encode).
  • 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”-
  • DLinDAP 1,2- dilineoyl-3- dimethylammonium-propane
  • DLinDMA l,2-dilinoleyloxy-3-N,N- dimethylaminopropane
  • DLinK-DMA l,2-dilinoleyloxyketo-N,N-dimethyl-3-aminoprop
  • an LNP delivery vehicle can be used to deliver a virus particle containing a polynucleotides and/or polypeptides of the present invention.
  • 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.
  • 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.
  • the LNP also includes a shielding compound, which is removable from the lipid composition under in vivo conditions.
  • the shielding compound is a biologically inert compound.
  • the shielding compound does not carry any charge on its surface or on the molecule as such.
  • the shielding compounds are polyethylenglycoles (PEGs), hydroxyethylglucose (HEG) based polymers, polyhydroxyethyl starch (polyHES) and polypropylene.
  • PEGs polyethylenglycoles
  • HEG hydroxyethylglucose
  • polyHES polyhydroxyethyl starch
  • the PEG, HEG, polyHES, and a polypropylene weight between about 500 to 10,000 Da or between about 2000 to 5000 Da.
  • the shielding compound is PEG2000 or PEG5000.
  • the LNP can include one or more helper lipids.
  • the helper lipid can be a phosphor lipid or a steroid.
  • the helper lipid is between about 20 mol % to 80 mol % of the total lipid content of the composition.
  • the helper lipid component is between about 35 mol % to 65 mol % of the total lipid content of the LNP.
  • the LNP includes lipids at 50 mol% and the helper lipid at 50 mol% of the total lipid content of the LNP.
  • a lipid particle may be liposome.
  • Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer.
  • 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).
  • BBB blood brain barrier
  • 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.
  • DSPC l,2-distearoryl-sn-glycero-3 - phosphatidyl choline
  • sphingomyelin sphingomyelin
  • egg phosphatidylcholines monosialoganglioside, or any combination thereof.
  • 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.
  • DOPE 1,2- dioleoyl-sn-glycero-3- phosphoethanolamine
  • 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.
  • the virus particle(s) are adsorbed to the liposome, such as through electrostatic interactions, and/or is attached to the liposomes via a linker.
  • 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.
  • Trojan Horse liposome also known in the art as Molecular Trojan Horses
  • 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.
  • 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.
  • 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.
  • SNALPs may comprise synthetic cholesterol, l,2-distearoyl-sn-glycero-3-phosphocholine, PEG- eDMA, and 1,2- dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMAo).
  • 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.
  • 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.
  • the delivery vehicle comprises a lipidoid, such as any of those set forth in, for example, US 20110293703.
  • 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.
  • 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.
  • 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 lipids
  • ZALs zwitterionic amino lipids
  • Ca2Jr e.g., forming DNA/Ca2+microcomplexes
  • PEI polyethenimine
  • PLL poly(L-lysine)
  • the delivery vehicle is a sugar-based particle.
  • 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;
  • 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 fragments of DNA).
  • CPPs may be of different sizes, amino acid sequences, and charges.
  • CPPs translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or an organelle.
  • CPPs may be introduced into cells via different mechanisms, e.g., direct penetration in the membrane, endocytosis-mediated entry, and translocation through the formation of a transitory structure.
  • CPPs may have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively.
  • a third class of CPPs are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake.
  • Another type of CPPs is the trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1).
  • CPPs examples include to Penetratin, Tat (48-60), Transportan, and (R-AhX- R4) (Ahx refers to aminohexanoyl), Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin
  • Ahx refers to aminohexanoyl
  • FGF Kaposi fibroblast growth factor
  • polyarginine peptide Args sequence examples of CPPs and related applications also include those described in US Patent 8,372,951.
  • CPPs can be used for in vitro and ex vivo work quite readily, and extensive optimization for each cargo and cell type is usually required.
  • CPPs may be covalently attached to the Cas protein directly, which is then complexed with the gRNA and delivered to cells.
  • separate delivery of CPP-Cas and CPP-gRNA to multiple cells may be performed.
  • CPP may also be used to delivery RNPs.
  • CPPs may be used to deliver the compositions and systems to plants.
  • CPPs may be used to deliver the components to plant protoplasts, which are then regenerated to plant cells and further to plants.
  • the delivery vehicles comprise DNA nanoclews.
  • a DNA nanoclew refers to a sphere-like structure of DNA (e.g., with a shape of a ball of yarn).
  • the nanoclew may be synthesized by rolling circle amplification with palindromic sequences that aide in the selfassembly of the structure. The sphere may then be loaded with a payload.
  • An example of DNA nanoclew is described in Sun W et al, J Am Chem Soc. 2014 Oct 22; 136(42): 14722-5; and Sun W et al, Angew Chem Int Ed Engl. 2015 Oct 5;54(41): 12029-33.
  • DNA nanoclew may have palindromic sequences to be partially complementary to one or more of the polynucleotides of the present invention described elsewhere herein.
  • a DNA nanoclew may be coated, e.g., coated with PEI to induce endosomal escape.
  • the delivery vehicles comprise gold nanoparticles (also referred to AuNPs or colloidal gold).
  • Gold nanoparticles may form complex with cargos, e.g., polynucleotides and/or polypeptides of the present invention described elsewhere herein.
  • Gold nanoparticles may be coated, e.g., coated in a silicate and an endosomal disruptive polymer, PAsp(DET).
  • Exemplary gold nanoparticles include AuraSense Therapeutics’ Spherical Nucleic Acid (SNATM) constructs, and those described in Mout R, et al. (2017). ACS Nano 11 :2452-8; Lee K, et al. (2017). Nat Biomed Eng 1:889-901.
  • metal nanoparticles can also be complexed with cargo(s).
  • Such metal particles include, but are not limited to, tungsten, palladium, rhodium, platinum, and iridium particles.
  • Other non-limiting, exemplary metal nanoparticles are described in US 20100129793.
  • the delivery vehicles comprise iTOP.
  • iTOP refers to a combination of small molecules drives the highly efficient intracellular delivery of native proteins, independent of any transduction peptide.
  • iTOP may be used for induced transduction by osmocytosis and propanebetaine, using NaCl-mediated hyperosmolality together with a transduction compound (propanebetaine) to trigger macropinocytotic uptake into cells of extracellular macromolecules.
  • Examples of iTOP methods and reagents include those described in D’Astolfo DS, Pagliero RJ, Pras A, et al. (2015). Cell 161 :674-690.
  • the delivery vehicles comprise polymer-based particles (e.g., nanoparticles).
  • the polymer-based particles mimic a viral mechanism of membrane fusion.
  • the polymer-based particles may be a synthetic copy of Influenza virus machinery and form transfection complexes with various types of nucleic acids ((siRNA, miRNA, plasmid DNA or shRNA, mRNA) that cells take up via the endocytosis pathway, a process that involves the formation of an acidic compartment.
  • the low pH in late endosomes acts as a chemical switch that renders the particle surface hydrophobic and facilitates membrane crossing. Once in the cytosol, the particle releases its payload for cellular action.
  • the polymer-based particles comprise alkylated and carboxyalkylated branched polyethylenimine.
  • the polymer-based particles are VIROMER, e.g., VIROMER RNAi, VIROMER RED, VIROMER mRNA, VIROMER CRISPR.
  • Example methods of delivering the polynucleotides and/or polypeptides of the present invention described elsewhere herein herein include those described in Bawage SS et al., bioRxiv 370460, Lagauzere, Sandra. (2017). Viromer® RED, a powerful tool for transfection of keratinocytes.
  • the delivery vehicles may be streptolysin O (SLO).
  • SLO is a toxin produced by Group A streptococci that works by creating pores in mammalian cell membranes. SLO may act in a reversible manner, which allows for the delivery of proteins (e.g., up to 100 kDa) to the cytosol of cells without compromising overall viability. Examples of SLO are provided in Sierig G, et al. (2003). Infect Immun 71 :446-55; Walev I, et al. (2001). Proc Natl Acad Sci U S A 98:3185-90; Teng KW, et al. (2017). Elife 6:e25460. Multifunctional Envelope-Type Nanodevice (MEND)
  • MEND Multifunctional Envelope-Type Nanodevice
  • the delivery vehicles may comprise multifunctional envelope-type nanodevice (MENDs).
  • MENDs may comprise condensed plasmid DNA, a PLL core, and a lipid film shell.
  • a MEND may further comprise cell -penetrating peptide (e.g., stearyl octaarginine).
  • the cell penetrating peptide may be in the lipid shell.
  • the delivery vehicles may comprise lipid-coated mesoporous silica particles.
  • Lipid- coated mesoporous silica particles may comprise a mesoporous silica nanoparticle core and a lipid membrane shell.
  • the silica core may have a large internal surface area, leading to high cargo loading capacities.
  • pore sizes, pore chemistry, and overall particle sizes are modified for loading different types of cargos.
  • the lipid coating of the particle also may be modified to maximize cargo loading, increase circulation times, and provide precise targeting and cargo release.
  • Exemplary lipid-coated mesoporous silica particles include those described in Du X, et al. (2014). Biomaterials 35:5580-90; Durfee PN, et al. (2016). ACS Nano 10:8325-45.
  • the delivery vehicles may comprise inorganic nanoparticles.
  • Exemplary inorganic nanoparticles include carbon nanotubes (CNTs) (e.g., as described in Bates K and Kostarelos K. (2013). Adv Drug Deliv Rev 65:2023-33.), bare mesoporous silica nanoparticles (MSNPs) (e.g., as described in Luo GF, et al. (2014). Sci Rep 4:6064), and dense silica nanoparticles (SiNPs) (as described in Luo D and Saltzman WM. (2000). Nat Biotechnol 18:893-5).
  • CNTs carbon nanotubes
  • MSNPs bare mesoporous silica nanoparticles
  • SiNPs dense silica nanoparticles
  • the delivery vehicles may comprise exosomes.
  • Exosomes include membrane bound extracellular vesicles, which can be used to contain and delivery various types of biomolecules, such as proteins, carbohydrates, lipids, and nucleic acids, and complexes thereof (e.g., RNPs).
  • examples of exosomes include those described in Schroeder A, et al., J Intern Med. 2010 Jan;267(l):9-21; El-Andaloussi S, et al., Nat Protoc. 2012 Dec;7(12):2112-26; Uno Y, et al., Hum Gene Ther. 2011 Jun;22(6):711-9; Zou W, et al., Hum Gene Ther. 2011 Apr;22(4):465-75.
  • the exosome forms a complex (e.g., by binding directly or indirectly) to one or more components of the cargo.
  • a molecule of an exosome is fused with first adapter protein and a component of the cargo is fused with a second adapter protein.
  • the first and the second adapter protein may specifically bind each other, thus associating the cargo with the exosome.
  • Exemplary exosomes include those described in Ye Y, et al., Biomater Sci. 2020 Apr 28.
  • exosomes include any of those set forth in Alvarez- Erviti et al. 2011, Nat Biotechnol 29: 341; El-Andaloussi et al. (Nature Protocols 7:2112- 2126(2012); and Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 el30).
  • SNAs Spherical Nucleic Acids
  • the delivery vehicle can be a SNA.
  • SNAs are three dimensional nanostructures that comprise densely functionalized and highly oriented nucleic acids that are covalently attached to the surface of spherical nanoparticle cores.
  • the core of the spherical nucleic acid imparts the conjugate with specific chemical and physical properties and acts as a scaffold for assembling and orienting the oligonucleotides into a dense spherical arrangement that gives rise to many of their functional properties, distinguishing them from other forms of matter.
  • the core is a crosslinked polymer.
  • Non-limiting, exemplary SNAs include any of those set forth in Cutler et al., J. Am. Chem. Soc.
  • the delivery vehicle is a self-assembling nanoparticle.
  • the self-assembling nanoparticles contain one or more polymers.
  • the self-assembling nanoparticles are PEGylated.
  • Self-assembling nanoparticles are known in the art. Non-limiting, exemplary self-assembling nanoparticles include any of those set forth in Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19, Bartlett et al. (PNAS, September 25, 2007, vol. 104, no. 39; Davis et al., Nature, Vol 464, 15 April 2010.
  • the delivery vehicle is a supercharged protein.
  • supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge.
  • Non-limiting, exemplary supercharged proteins include any of those set forth in Lawrence et al., 2007, Journal of the American Chemical Society 129, 10110-10112.
  • the delivery vehicle allows for targeted delivery to a specific cell, tissue, organ, or system.
  • the delivery vehicle includes one or more targeting moieties that directs targeted delivery of the cargo(s).
  • the delivery vehicle comprises a targeting moiety, such as active targeting of a lipid entity of the invention, e.g., lipid particle or nanoparticle or liposome or lipid bilayer of the invention comprising a targeting moiety for active targeting.
  • An actively targeting lipid particle or nanoparticle or liposome or lipid bilayer delivery system (generally as to embodiments of the invention, “lipid entity of the invention” delivery systems) are prepared by conjugating targeting moieties, including small molecule ligands, peptides and monoclonal antibodies, on the lipid or liposomal surface; for example, certain receptors, such as folate and transferrin (Tf) receptors (TfR), are overexpressed on many cancer cells and have been used to make liposomes tumor cell specific. Liposomes that accumulate in the tumor microenvironment can be subsequently endocytosed into the cells by interacting with specific cell surface receptors.
  • the targeting moiety have an affinity for a cell surface receptor and to link the targeting moiety in sufficient quantities to have optimum affinity for the cell surface receptors; and determining these embodiments are within the ambit of the skilled artisan.
  • active targeting there are a number of cell-, e.g., tumor-, specific targeting ligands.
  • targeting ligands on liposomes can provide attachment of liposomes to cells, e.g., vascular cells, via a noninternalizing epitope; and this can increase the extracellular concentration of that which is being delivered, thereby increasing the amount delivered to the target cells.
  • a strategy to target cell surface receptors, such as cell surface receptors on cancer cells, such as overexpressed cell surface receptors on cancer cells is to use receptor-specific ligands or antibodies.
  • Many cancer cell types display upregulation of tumor-specific receptors. For example, TfRs and folate receptors (FRs) are greatly overexpressed by many tumor cell types in response to their increased metabolic demand.
  • lipid entity of the invention Folate-linked lipid particles or nanoparticles or liposomes or lipid by layers of the invention (“lipid entity of the invention”) deliver their cargo intracellularly through receptor-mediated endocytosis. Intracellular trafficking can be directed to acidic compartments that facilitate cargo release, and, most importantly, release of the cargo can be altered or delayed until it reaches the cytoplasm or vicinity of target organelles. Delivery of cargo using a lipid entity of the invention having a targeting moiety, such as a folate-linked lipid entity of the invention, can be superior to nontargeted lipid entity of the invention.
  • a lipid entity of the invention coupled to folate can be used for the delivery of complexes of lipid, e.g., liposome, e.g., anionic liposome and virus or capsid or envelope or virus outer protein, such as those herein discussed such as adenovirous or AAV.
  • Tf is a monomeric serum glycoprotein of approximately 80 KDa involved in the transport of iron throughout the body.
  • Tf binds to the TfR and translocates into cells via receptor-mediated endocytosis.
  • the expression of TfR can be higher in certain cells, such as tumor cells (as compared with normal cells and is associated with the increased iron demand in rapidly proliferating cancer cells.
  • the invention comprehends a TfR-targeted lipid entity of the invention, e.g., as to liver cells, liver cancer, breast cells such as breast cancer cells, colon such as colon cancer cells, ovarian cells such as ovarian cancer cells, head, neck, and lung cells, such as head, neck and non-small-cell lung cancer cells, cells of the mouth such as oral tumor cells.
  • a lipid entity of the invention can be multifunctional, i.e., employ more than one targeting moiety such as CPP, along with Tf; a bifunctional system, e.g., a combination of Tf and poly-L-arginine which can provide transport across the endothelium of the blood-brain barrier.
  • EGFR is a tyrosine kinase receptor belonging to the ErbB family of receptors that mediates cell growth, differentiation and repair in cells, especially non-cancerous cells, but EGF is overexpressed in certain cells such as many solid tumors, including colorectal, non-smallcell lung cancer, squamous cell carcinoma of the ovary, kidney, head, pancreas, neck and prostate, and especially breast cancer.
  • the invention comprehends EGFR-targeted monoclonal antibody(ies) linked to a lipid entity of the invention.
  • HER-2 is often overexpressed in patients with breast cancer, and is also associated with lung, bladder, prostate, brain and stomach cancers.
  • HER-2 encoded by the ERBB2 gene.
  • the invention comprehends a HER-2-targeting lipid entity of the invention, e.g., an anti-HER-2-antibody(or binding fragment thereof)-lipid entity of the invention, a HER-2-targeting-PEGylated lipid entity of the invention (e.g., having an anti-HER-2- antibody or binding fragment thereof), a HER-2-targeting-maleimide-PEG polymer- lipid entity of the invention (e.g., having an anti-HER-2-antibody or binding fragment thereof).
  • the receptor-antibody complex can be internalized by formation of an endosome for delivery to the cytoplasm.
  • ligand/target affinity and the quantity of receptors on the cell surface are advantageous.
  • PEGylation can act as a barrier against interaction with receptors.
  • the use of antibody-lipid entity of the invention targeting can be advantageous. Multivalent presentation of targeting moieties can also increase the uptake and signaling properties of antibody fragments.
  • ligand density e.g., high ligand densities on a lipid entity of the invention may be advantageous for increased binding to target cells).
  • lipid entity of the invention Preventing early by macrophages can be addressed with a sterically stabilized lipid entity of the invention and linking ligands to the terminus of molecules such as PEG, which is anchored in the lipid entity of the invention (e.g., lipid particle or nanoparticle or liposome or lipid bilayer).
  • the microenvironment of a cell mass such as a tumor microenvironment can be targeted; for instance, it may be advantageous to target cell mass vasculature, such as the tumor vasculature microenvironment.
  • the invention comprehends targeting VEGF.
  • VEGF and its receptors are well-known proangiogenic molecules and are well-characterized targets for anti angiogenic therapy.
  • VEGFRs or basic FGFRs have been developed as anticancer agents and the invention comprehends coupling any one or more of these peptides to a lipid entity of the invention, e.g., phage IVO peptide(s) (e.g., via or with a PEG terminus), tumor-homing peptide APRPG (SEQ ID NO: 310) such as APRPG- PEG-modified (SEQ ID NO: 310).
  • a lipid entity of the invention e.g., phage IVO peptide(s) (e.g., via or with a PEG terminus), tumor-homing peptide APRPG (SEQ ID NO: 310) such as APRPG- PEG-modified (SEQ ID NO: 310).
  • APRPG tumor-homing peptide APRPG
  • VCAM the vascular endothelium plays a key role in the pathogenesis of inflammation, thrombosis and atherosclerosis.
  • CAMs are involved in inflammatory disorders, including cancer, and are a logical target, E- and P-selectins, VCAM-1 and ICAMs. Can be used to target a lipid entity of the invention., e.g., with PEGylation.
  • Matrix metalloproteases belong to the family of zinc-dependent endopeptidases. They are involved in tissue remodeling, tumor invasiveness, resistance to apoptosis and metastasis. There are four MMP inhibitors called TIMP1-4, which determine the balance between tumor growth inhibition and metastasis; a protein involved in the angiogenesis of tumor vessels is MT 1 -MMP, expressed on newly formed vessels and tumor tissues.
  • TIMP1-4 MMP inhibitors
  • the proteolytic activity of MT 1 -MMP cleaves proteins, such as fibronectin, elastin, collagen, and laminin, at the plasma membrane and activates soluble MMPs, such as MMP -2, which degrades the matrix.
  • an antibody or fragment thereof such as a Fab' fragment can be used in the practice of the invention such as for an antihuman MT1-MMP monoclonal antibody linked to a lipid entity of the invention, e.g., via a spacer such as a PEG spacer.
  • aP-integrins or integrins are a group of transmembrane glycoprotein receptors that mediate attachment between a cell and its surrounding tissues or extracellular matrix.
  • Integrins contain two distinct chains (heterodimers) called a- and P-subunits.
  • the tumor tissue-specific expression of integrin receptors can be utilized for targeted delivery in the invention, e.g., whereby the targeting moiety can be an RGD peptide such as a cyclic RGD.
  • Aptamers are ssDNA or RNA oligonucleotides that impart high affinity and specific recognition of the target molecules by electrostatic interactions, hydrogen bonding and hydrophobic interactions as opposed to the Watson-Crick base pairing, which is typical for the bonding interactions of oligonucleotides.
  • Aptamers as a targeting moiety can have advantages over antibodies: aptamers can demonstrate higher target antigen recognition as compared with antibodies; aptamers can be more stable and smaller in size as compared with antibodies; aptamers can be easily synthesized and chemically modified for molecular conjugation; and aptamers can be changed in sequence for improved selectivity and can be developed to recognize poorly immunogenic targets.
  • Such moieties as a sgc8 aptamer can be used as a targeting moiety (e.g., via covalent linking to the lipid entity of the invention, e.g., via a spacer, such as a PEG spacer).
  • the invention also comprehends intracellular delivery. Since liposomes follow the endocytic pathway, they are entrapped in the endosomes (pH 6.5-6) and subsequently fuse with lysosomes (pH ⁇ 5), where they undergo degradation that results in a lower therapeutic potential. The low endosomal pH can be taken advantage of to escape degradation. Fusogenic lipids or peptides, which destabilize the endosomal membrane after the conformational transition/activation at a lowered pH.
  • Unsaturated dioleoylphosphatidylethanolamine readily adopts an inverted hexagonal shape at a low pH, which causes fusion of liposomes to the endosomal membrane.
  • This process destabilizes a lipid entity containing DOPE and releases the cargo into the cytoplasm; fusogenic lipid GALA (SEQ ID NO: 311), cholesteryl-GALA (SEQ ID NO: 311) and PEG-GALA (SEQ ID NO: 311) may show a highly efficient endosomal release; a pore-forming protein listeriolysin O may provide an endosomal escape mechanism; and histidine-rich peptides have the ability to fuse with the endosomal membrane, resulting in pore formation, and can buffer the proton pump causing membrane lysis.
  • the invention comprehends a lipid entity of the invention modified with CPP(s), for intracellular delivery that may proceed via energy dependent macropinocytosis followed by endosomal escape.
  • the invention further comprehends organelle-specific targeting.
  • a lipid entity of the invention surface-functionalized with the triphenylphosphonium (TPP) moiety or a lipid entity of the invention with a lipophilic cation, rhodamine 123 can be effective in delivery of cargo to mitochondria.
  • DOPE/sphingomyelin/stearyl-octa-arginine can delivers cargos to the mitochondrial interior via membrane fusion.
  • a lipid entity of the invention surface modified with a lysosomotropic ligand, octadecyl rhodamine B can deliver cargo to lysosomes.
  • Ceramides are useful in inducing lysosomal membrane permeabilization; the invention comprehends intracellular delivery of a lipid entity of the invention having a ceramide.
  • the invention further comprehends a lipid entity of the invention targeting the nucleus, e.g., via a DNA-intercalating moiety.
  • the invention also comprehends multifunctional liposomes for targeting, i.e., attaching more than one functional group to the surface of the lipid entity of the invention, for instance to enhances accumulation in a desired site and/or promotes organelle-specific delivery and/or target a particular type of cell and/or respond to the local stimuli such as temperature (e.g., elevated), pH (e.g., decreased), respond to externally applied stimuli such as a magnetic field, light, energy, heat or ultrasound and/or promote intracellular delivery of the cargo. All of these are considered actively targeting moieties.
  • the local stimuli such as temperature (e.g., elevated), pH (e.g., decreased)
  • respond to externally applied stimuli such as a magnetic field, light, energy, heat or ultrasound and/or promote intracellular delivery of the cargo. All of these are considered actively targeting moieties.
  • each possible targeting or active targeting moiety herein-discussed there is an embodiment of the invention wherein the delivery system comprises such a targeting or active targeting moiety.
  • Table A provides exemplary targeting moieties that can be used in the practice of the invention an as to each an embodiment of the invention provides a delivery system that comprises such a targeting moiety.
  • embodiments disclosed herein are directed to the immunogenic compositions disclosed herein formulated as vaccines.
  • a vaccine is a biological preparation that provides active acquired immunity to a particular infectious or malignant disease. Tumor specific antigens may be produced in vitro as peptides or polypeptides, which may then be formulated into a vaccine or immunogenic composition and administered to a subject.
  • Such in vitro production may occur by a variety of methods known to one of ordinary skill in the art such as, for example, peptide synthesis or expression of a peptide/polypeptide from a DNA or RNA molecule in any of a variety of bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptide/polypeptide.
  • the present invention also contemplates the use of nucleic acid molecules as vehicles for delivering antigenic peptides/polypeptides to the subject in need thereof, in vivo, in the form of, e.g., DNA/RNA vaccines (see, e.g., WO2012/159643, and WO2012/159754, hereby incorporated by reference in their entirety).
  • antigenic peptides may be administered to a patient in need thereof by use of an mRNA vaccine (see, e.g., Sahin, U, Kariko, K and Tureci, O (2014). mRNA-based therapeutics - developing a new class of drugs. Nat Rev Drug Discov 13: 759-780; Weissman D, Kariko K. mRNA: Fulfilling the Promise of Gene Therapy. Mol Ther. 2015;23(9): 1416-1417. doi: 10.1038/mt.2015.138; Kowalski PS, Rudra A, Miao L, Anderson DG. Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery. Mol Ther. 2019;27(4):710-728.
  • an mRNA vaccine see, e.g., Sahin, U, Kariko, K and Tureci, O (2014). mRNA-based therapeutics - developing a new class of drugs. Nat Rev Drug Discov 13: 759-780; Weissman D, Kariko K. mRNA: Fulfilling
  • mRNA encoding for an antigenic peptide is delivered using lipid nanoparticles (see, e.g., Reichmuth, et al., 2016) and administered directly to tumor tissue.
  • mRNA encoding for an antigenic peptide is delivered using biomaterial-mediated sequestration (see, e.g., Khalil, et al., 2020) and administered directly to tumor tissue.
  • antigens are administered to a patient in need thereof by use of a plasmid.
  • plasmids that usually consist of a strong viral promoter to drive the in vivo transcription and translation of the gene (or complementary DNA) of interest (Mor, et al ., (1995), The Journal of Immunology 155 (4): 2039-2046).
  • Intron A may sometimes be included to improve mRNA stability and hence increase protein expression (Leitner et al. (1997), The Journal of Immunology 159 (12): 6112-6119).
  • Plasmids also include a strong polyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences (Alarcon et al., (1999), Adv. Parasitol. Advances in Parasitology 42: 343-410; Robinson et al., (2000). Adv. Virus Res. Advances in Virus Research 55: 1-74; Bohmet al., (1996). Journal of Immunological Methods 193 (1): 29-40.). Multi cistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).
  • a strong polyadenylation/transcriptional termination signal such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences (Alarcon et al., (1999), Adv. Parasitol. Advances
  • the plasmid is the “vehicle” from which the immunogen is expressed, optimizing vector design for maximal protein expression is essential (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).
  • One way of enhancing protein expression is by optimizing the codon usage of pathogenic mRNAs for eukaryotic cells.
  • Another consideration is the choice of promoter.
  • promoters may be the SV40 promoter or Rous Sarcoma Virus (RSV).
  • Plasmids may be introduced into animal tissues by a number of different methods. The two most popular approaches are injection of DNA in saline, using a standard hypodermic needle, and gene gun delivery.
  • Immune responses to this method of delivery can be affected by many factors, including needle type, needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the animal being injected (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343- 410).
  • Gene gun delivery the other commonly used method of delivery, ballistically accelerates plasmid DNA (pDNA) that has been adsorbed onto gold or tungsten microparticles into the target cells, using compressed helium as an accelerant (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410; Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).
  • pDNA plasmid DNA
  • Alternative delivery methods may include aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa, (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88) and topical administration of pDNA to the eye and vaginal mucosa (Lewis et al., (1999) Advances in Virus Research (Academic Press) 54: 129-88).
  • Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors.
  • DNA or RNA may also be delivered to cells following mild mechanical disruption of the cell membrane, temporarily permeabilizing the cells. Such a mild mechanical disruption of the membrane can be accomplished by gently forcing cells through a small aperture (Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells, Sharei et al, PLOS ONE
  • the method of delivery determines the dose of DNA required to raise an effective immune response.
  • Saline injections require variable amounts of DNA, from 10 pg-1 mg, whereas gene gun deliveries require 100 to 1000 times less DNA than intramuscular saline injection to raise an effective immune response.
  • 0.2 pg - 20 pg are required, although quantities as low as 16 ng have been reported. These quantities vary from species to species, with mice, for example, requiring approximately 10 times less DNA than primates.
  • Saline injections require more DNA because the DNA is delivered to the extracellular spaces of the target tissue (normally muscle), where it has to overcome physical barriers (such as the basal lamina and large amounts of connective tissue, to mention a few) before it is taken up by the cells, while gene gun deliveries bombard DNA directly into the cells, resulting in less “wastage” (See e g., Sedegah et al., (1994). Proceedings of the National Academy of Sciences of the United States of America 91 (21): 9866- 9870; Daheshiaet al., (1997). The Journal of Immunology 159 (4): 1945-1952; Chen et al., (1998).
  • a neoplasia vaccine or immunogenic composition may include separate DNA plasmids encoding, for example, one or more antigenic peptides/polypeptides as identified in according to the invention.
  • the exact choice of expression vectors can depend upon the peptide/polypeptides to be expressed, and is well within the skill of the ordinary artisan.
  • the expected persistence of the DNA constructs is expected to provide an increased duration of protection.
  • One or more antigenic peptides of the invention may be encoded and expressed in vivo using a viral based system (e.g., an adenovirus system, an adeno associated virus (AAV) vector, a poxvirus, or a lentivirus).
  • a viral based system e.g., an adenovirus system, an adeno associated virus (AAV) vector, a poxvirus, or a lentivirus.
  • the neoplasia vaccine or immunogenic composition includes a viral based vector for use in a human patient in need thereof, such as, for example, an adenovirus (see, e.g., Baden et al. First-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001). J Infect Dis.
  • Plasmids that can be used for adeno associated virus, adenovirus, and lentivirus delivery have been described previously (see e g., U.S. Patent Nos. 6,955,808 and 6,943,019, and U.S. Patent application No. 20080254008, hereby incorporated by reference).
  • the peptides and polypeptides of the invention can also be expressed by a vector, e.g., a nucleic acid molecule as herein-discussed, e.g., RNA or a DNA plasmid, a viral vector such as a poxvirus, e.g., orthopox virus, avipox virus, or adenovirus, AAV or lentivirus.
  • a vector e.g., a nucleic acid molecule as herein-discussed, e.g., RNA or a DNA plasmid, a viral vector such as a poxvirus, e.g., orthopox virus, avipox virus, or adenovirus, AAV or lentivirus.
  • a vector e.g., a nucleic acid molecule as herein-discussed, e.g., RNA or a DNA plasmid, a viral vector such as a poxvirus,
  • retrovirus is a lentivirus.
  • high transduction efficiencies have been observed in many different cell types and target tissues.
  • the tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells.
  • 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.
  • Cell type specific promoters can be used to target expression in specific cell types.
  • Lentiviral vectors are retroviral vectors (and hence both lentiviral and retroviral vectors may be used in the practice of the invention). Moreover, lentiviral vectors are preferred as they are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system may therefore depend on the target tissue. Retroviral vectors are comprised 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 desired nucleic acid into the target cell to provide permanent expression.
  • Widely used retroviral vectors that may be used in the practice of the invention include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., (1992) J. Virol. 66:2731-2739; Johann et al., (1992) J. Virol. 66: 1635-1640; Sommnerfelt et al., (1990) Virol. 176:58-59; Wilson et al., (1998) J. Virol.
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immuno deficiency virus
  • HAV human immuno deficiency virus
  • lentiviral vectors are based on the equine infectious anemia virus (EIAV) (see, e g., Balagaan, (2006) J Gene Med; 8: 275 - 285, Published online 21 November 2005 in Wiley InterScience (www.interscience.wiley.com).).
  • EIAV equine infectious anemia virus
  • the vectors may have cytomegalovirus (CMV) promoter driving expression of the target gene.
  • CMV cytomegalovirus
  • the invention contemplates amongst vector(s) useful in the practice of the invention: viral vectors, including retroviral vectors and lentiviral vectors.
  • 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 delivery to the Brain, see, e.g., US Patent Publication Nos. US20110293571; US20040013648, US20070025970, US20090111106 and US Patent No. US7259015. In another embodiment lentiviral vectors are used to deliver vectors to the brain of those being treated for a disease.
  • the delivery is via an lentivirus.
  • Zou et al. administered about 10 pl of a recombinant lentivirus having a titer of 1 x 109 transducing units (TU)/ml by an intrathecal catheter.
  • These sort of dosages can be adapted or extrapolated to use of a retroviral or lentiviral vector in the present invention.
  • the viral preparation is concentrated by ultracentrifugation.
  • the resulting preparation should have at least 108 TU/ml, preferably from 108 to 109TU/ml, more preferably at least 109 TU/ml.
  • Other methods of concentration such as ultrafiltration or binding to and elution from a matrix may be used.
  • the amount of lentivirus administered may be 1.x.105 or about 1.x.105 plaque forming units (PFU), 5.x.105 or about 5.x.105 PFU, 1.x.106 or about l ,xlO6 PFU, 5.x.106 or about 5.x.106 PFU, 1.x.107 or about 1.X.107PFU, 5.x.107 or about 5.X.107 PFU, 1.x.108 or about 1 .X.108 PFU, 5.x.108 or about 5.X.108 PFU, 1 .x.109 or about 1.X.109 PFU, 5.x.109 or about 5.x.109 PFU, 1 .x.1010 or about 1 .x.1010 PFU or 5.x.1010 or about 5.x.1010 PFU as total single dosage for an average human of 75 kg or adjusted for the weight and size and species of the subject.
  • PFU plaque forming units
  • Suitable dosages for a virus can be determined empirically.
  • an adenovirus vector is also useful in the practice of the invention.
  • One advantage is the ability of recombinant adenoviruses to efficiently transfer and express recombinant genes in a variety of mammalian cells and tissues in vitro and in vivo, resulting in the high expression of the transferred nucleic acids. Further, the ability to productively infect quiescent cells, expands the utility of recombinant adenoviral vectors. In addition, high expression levels ensure that the products of the nucleic acids will be expressed to sufficient levels to generate an immune response (see e.g., U.S. Patent No. 7,029,848, hereby incorporated by reference).
  • adenovirus vectors useful in the practice of the invention mention is made of US Patent No. 6,955,808.
  • the adenovirus vector used can be selected from the group consisting of the Ad5, Ad35, Adi 1, C6, and C7 vectors.
  • Ad5 The sequence of the Adenovirus 5 (“Ad5”) genome has been published. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285; the contents if which is hereby incorporated by reference).
  • Ad35 vectors are described in U.S. Pat. Nos.
  • Adi 1 vectors are described in U.S. Pat. No. 6,913,922.
  • C6 adenovirus vectors are described in U.S. Pat. Nos. 6,780,407; 6,537,594; 6,309,647; 6,265, 189; 6,156,567; 6,090,393; 5,942,235 and 5,833,975.
  • C7 vectors are described in U.S. Pat. No. 6,277,558.
  • Adenovirus vectors that are El-defective or deleted, E3- defective or deleted, and/or E4-defective or deleted may also be used.
  • adenoviruses having mutations in the El region have improved safety margin because El -defective adenovirus mutants are replication-defective in non-permissive cells, or, at the very least, are highly attenuated.
  • Adenoviruses having mutations in the E3 region may have enhanced the immunogenicity by disrupting the mechanism whereby adenovirus down-regulates MHC class I molecules.
  • Adenoviruses having E4 mutations may have reduced immunogenicity of the adenovirus vector because of suppression of late gene expression. Such vectors may be particularly useful when repeated re-vaccination utilizing the same vector is desired.
  • Adenovirus vectors that are deleted or mutated in El, E3, E4, El and E3, and El and E4 can be used in accordance with the present invention.
  • “gutless” adenovirus vectors, in which all viral genes are deleted can also be used in accordance with the present invention.
  • Such vectors require a helper virus for their replication and require a special human 293 cell line expressing both El a and Cre, a condition that does not exist in natural environment.
  • Such “gutless” vectors are non-immunogenic and thus the vectors may be inoculated multiple times for revaccination.
  • the “gutless” adenovirus vectors can be used for insertion of heterologous inserts/genes such as the transgenes of the present invention, and can even be used for co-delivery of a large number of heterologous inserts/genes.
  • the delivery is via an adenovirus, which may be at a single booster dose containing at least 1 x 105 particles (also referred to as particle units, pu) of adenoviral vector.
  • the dose preferably is at least about 1 x 106 particles (for example, about 1 x 106-1 x 1012particles), more preferably at least about 1 x 107 particles, more preferably at least about 1 x 108 particles (e.g., about 1 x 108-1 x 1011 particles or about 1 x 108- 1 x 1012 particles), and most preferably at least about 1 x 109 particles (e.g., about 1 x 109-1 x
  • the dose comprises no more than about 1 x 1014 particles, preferably no more than about 1 x 1013 particles, even more preferably no more than about 1 x 1012 particles, even more preferably no more than about 1 x
  • the dose may contain a single dose of adenoviral vector with, for example, about 1 x 106 particle units (pu), about 2 x 106pu, about 4 x 106 pu, about 1 x 107 pu, about 2 x 10 pu, about 4 x 10 pu, about 1 x 10 pu, about 2 x 10 pu, about 4 x 10 pu, about 1 x 109 pu, about
  • the adenovirus is delivered via multiple doses.
  • AAV In terms of in vivo delivery, AAV is advantageous over other viral vectors due to low toxicity and low probability of causing insertional mutagenesis because it doesn’t integrate into the host genome.
  • AAV has a packaging limit of 4.5 or 4.75 Kb. Constructs larger than 4.5 or 4.75 Kb result in significantly reduced virus production.
  • promoters that can be used to drive nucleic acid molecule expression.
  • AAV ITR can serve as a promoter and is advantageous for eliminating the need for an additional promoter element.
  • the following promoters can be used: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc.
  • promoters For brain expression, the following promoters can be used: Synapsinl for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Promoters used to drive RNA synthesis can include: Pol III promoters such as U6 or HI . The use of a Pol II promoter and intronic cassettes can be used to express guide RNA (gRNA).
  • gRNA guide RNA
  • the AAV can be AAV1, AAV2, AAV5 or any combination thereof.
  • AAV8 is useful for delivery to the liver. The above promoters and vectors are preferred individually.
  • the delivery is via an AAV.
  • a therapeutically effective dosage for in vivo delivery of the AAV to a human is believed to be in the range of from about 20 to about 50 ml of saline solution containing from about 1 x 1010 to about 1 x 1050 functional AAV/ml solution. The dosage may be adjusted to balance the therapeutic benefit against any side effects.
  • the AAV dose is generally in the range of concentrations from about 1 x 10 to 1 x 10 genomes AAV, from about 1 x 10 to 1 x 10 genomes AAV, from about 1 x 1010 to about 1 x 1016 genomes, or about 1 x 1011 to about 1 x 1016 genomes AAV.
  • a human dosage may be about 1 x 1013 genomes AAV. Such concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution.
  • AAV is used with a titer of about 2 x 1013 viral genomes/milliliter, and each of the striatal hemispheres of a mouse receives one 500 nanoliter injection.
  • Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. See, for example, U.S. Patent No. 8,404,658 B2 to Hajjar, et al., granted on March 26, 2013, at col. 27, lines 45-60.
  • effectively activating a cellular immune response for a neoplasia vaccine or immunogenic composition can be achieved by expressing the relevant antigens in a vaccine or immunogenic composition in a non-pathogenic microorganism.
  • a non-pathogenic microorganism are Mycobacterium bovis BCG, Salmonella and Pseudomona (See, U.S. Patent No. 6,991,797, hereby incorporated by reference in its entirety).
  • a Poxvirus is used in the neoplasia vaccine or immunogenic composition.
  • Effective vaccine or immunogenic compositions advantageously include a strong adjuvant to initiate an immune response.
  • poly-ICLC an agonist of TLR3 and the RNA helicase -domains of MDA5 and RIG3, has shown several desirable properties for a vaccine or immunogenic composition adjuvant. These properties include the induction of local and systemic activation of immune cells in vivo, production of stimulatory chemokines and cytokines, and stimulation of antigen-presentation by DCs.
  • poly-ICLC can induce durable CD4+ and CD8+ responses in humans.
  • the antigen peptides may be combined with an adjuvant (e.g., poly- ICLC) or another anti - neoplastic agent.
  • an adjuvant e.g., poly- ICLC
  • another anti - neoplastic agent e.g., poly- ICLC
  • these antigens are expected to bypass central thymic tolerance (thus allowing stronger anti -tumor T cell response), while reducing the potential for autoimmunity (e.g., by avoiding targeting of normal self- antigens).
  • An effective immune response advantageously includes a strong adjuvant to activate the immune system (Speiser and Romero, Seminars in Immunol 22: 144 (2010)).
  • TLRs Toll-like receptors
  • poly-ICLC a synthetic doublestranded RNA mimic
  • poly-ICLC has been shown to be safe and to induce a gene expression profile in peripheral blood cells comparable to that induced by one of the most potent live attenuated viral vaccines, the yellow fever vaccine YF-17D (Caskey et al, J Exp Med 208:2357 (2011)).
  • Hiltonol® a GMP preparation of poly-ICLC prepared by Oncovir, Inc, is utilized as the adjuvant. In other embodiments, other adjuvants described herein are envisioned.
  • Targeting Moiety Target Molecule Target Cell or Tissue folate folate receptor cancer cells transferrin transferrin receptor cancer cells
  • ASSHN SEQ ID NO: endothelial progenitor cells; anti ⁇
  • the delivery vehicle can allow for responsive delivery of the cargo(s), e.g., one or more polynucleotides and/or polypeptides of the present invention described elsewhere herein.
  • Responsive delivery refers to delivery of cargo(s) by the delivery vehicle in response to an external stimuli.
  • suitable stimuli include, without limitation, an energy (light, heat, cold, and the like), a chemical stimuli (e.g. chemical composition, etc.), and a biologic or physiologic stimuli (e.g. environmental pH, osmolarity, salinity, biologic molecule, etc.).
  • the targeting moiety can be responsive to an external stimuli and facilitate responsive delivery. In other embodiments, responsiveness is determined by a non-targeting moiety component of the delivery vehicle.
  • the delivery vehicle can be stimuli-sensitive, e.g., sensitive to an externally applied stimuli, such as magnetic fields, ultrasound or light; and pH-triggering can also be used, e.g., a labile linkage can be used between a hydrophilic moiety such as PEG and a hydrophobic moiety such as a lipid entity of the invention, which is cleaved only upon exposure to the relatively acidic conditions characteristic of the a particular environment or microenvironment such as an endocytic vacuole or the acidotic tumor mass.
  • an externally applied stimuli such as magnetic fields, ultrasound or light
  • pH-triggering can also be used, e.g., a labile linkage can be used between a hydrophilic moiety such as PEG and a hydrophobic moiety such as a lipid entity of the invention, which is cleaved only upon exposure to the relatively acidic conditions characteristic of the a particular environment or microenvironment such as an endocytic vacuole or the acidotic tumor mass
  • pH-sensitive copolymers can also be incorporated in embodiments of the invention can provide shielding; diortho esters, vinyl esters, cysteine- cleavable lipopolymers, double esters and hydrazones are a few examples of pH-sensitive bonds that are quite stable at pH 7.5, but are hydrolyzed relatively rapidly at pH 6 and below, e.g., a terminally alkylated copolymer of N-isopropyl acrylamide and methacrylic acid that copolymer facilitates destabilization of a lipid entity of the invention and release in compartments with decreased pH value; or, the invention comprehends ionic polymers for generation of a pH- responsive lipid entity of the invention (e.g., poly(methacrylic acid), poly(diethylaminoethyl methacrylate), poly(acrylamide) and poly(acrylic acid)).
  • ionic polymers for generation of a pH- responsive lipid entity of the invention e.g., poly(me
  • Temperature-triggered delivery is also within the ambit of the invention. Many pathological areas, such as inflamed tissues and tumors, show a distinctive hyperthermia compared with normal tissues. Utilizing this hyperthermia is an attractive strategy in cancer therapy since hyperthermia is associated with increased tumor permeability and enhanced uptake. This technique involves local heating of the site to increase microvascular pore size and blood flow, which, in turn, can result in an increased extravasation of embodiments of the invention.
  • Temperaturesensitive lipid entity of the invention can be prepared from thermosensitive lipids or polymers with a low critical solution temperature. Above the low critical solution temperature (e.g., at site such as tumor site or inflamed tissue site), the polymer precipitates, disrupting the liposomes to release.
  • Lipids with a specific gel-to-liquid phase transition temperature are used to prepare these lipid entities of the invention; and a lipid for a thermosensitive embodiment can be dipalmitoylphosphatidylcholine.
  • Thermosensitive polymers can also facilitate destabilization followed by release, and a useful thermosensitive polymer is poly (N-isopropylacrylamide).
  • Another temperature triggered system can employ lysolipid temperature-sensitive liposomes.
  • the invention also comprehends redox-triggered delivery.
  • GSH is a reducing agent abundant in cells, especially in the cytosol, mitochondria, and nucleus.
  • the GSH concentrations in blood and extracellular matrix are just one out of 100 to one out of 1000 of the intracellular concentration, respectively.
  • This high redox potential difference caused by GSH, cysteine and other reducing agents can break the reducible bonds, destabilize a lipid entity of the invention and result in release of payload.
  • the disulfide bond can be used as the cleavable/reversible linker in a lipid entity of the invention, because it causes sensitivity to redox owing to the disulfideto-thiol reduction reaction; a lipid entity of the invention can be made reduction sensitive by using two (e.g., two forms of a disulfide-conjugated multifunctional lipid as cleavage of the disulfide bond (e.g., via tris(2- carboxyethyl)phosphine, dithiothreitol, L-cysteine or GSH), can cause removal of the hydrophilic head group of the conjugate and alter the membrane organization leading to release of payload.
  • two e.g., two forms of a disulfide-conjugated multifunctional lipid as cleavage of the disulfide bond (e.g., via tris(2- carboxyethyl)phosphine, dithiothreitol, L-cysteine
  • Calcein release from reduction-sensitive lipid entity of the invention containing a disulfide conjugate can be more useful than a reduction-insensitive embodiment.
  • Enzymes also can be used as a trigger to release payload. Enzymes, including MMPs (e.g., MMP2), phospholipase A2, alkaline phosphatase, transglutaminase, or phosphatidylinositolspecific phospholipase C, have been found to be overexpressed in certain tissues, e.g., tumor tissues.
  • an MMP2-cleavable octapeptide (Gly-Pro- Leu-Gly-Ile-Ala-Gly-Gln (SEQ ID NO: 316)) can be incorporated into a linker, and can have antibody targeting, e.g., antibody 2C5.
  • the invention also comprehends light-or energy-triggered delivery, e.g., the lipid entity of the invention can be light-sensitive, such that light or energy can facilitate structural and conformational changes, which lead to direct interaction of the lipid entity of the invention with the target cells via membrane fusion, photo-isomerism, photofragmentation or photopolymerization; such a moiety therefor can be benzoporphyrin photosensitizer.
  • Ultrasound can be a form of energy to trigger delivery; a lipid entity of the invention with a small quantity of particular gas, including air or perfluorated hydrocarbon can be triggered to release with ultrasound, e.g., low-frequency ultrasound (LFUS).
  • LFUS low-frequency ultrasound
  • a lipid entity of the invention can be magnetized by incorporation of magnetites, such as Fe3O4 or y-Fe2O3, e.g., those that are less than 10 nm in size. Targeted delivery can be then by exposure to a magnetic field.
  • magnetites such as Fe3O4 or y-Fe2O3, e.g., those that are less than 10 nm in size.
  • Targeted delivery can be then by exposure to a magnetic field.
  • the present disclosure provides cells and organisms comprising the compositions, such as the CAA polynucleotides, polypeptides, vectors, delivery vehicles, etc. described herein.
  • the cells are producer cells and are capable of generating virus particles or other delivery vehicles (e.g., exosomes) containing the one or more polynucleotides and/or polypeptides of the present invention.
  • the cells may be in tissue, organ, or isolated cells. Such cells may be of a unique type of cells or a group of different types of cells such as cultured cell lines, primary cells and proliferative cells.
  • the cells may be prokaryotic cells, lower eukaryotic cells such as yeast, and other eukaryotic cells such as insect cells, plant, and mammalian (e.g., human or non-human) cells as well as cells capable of producing the vector of the invention (e.g., 293, HER96, PERC.6 cells, Vero, HeLa, CEF, duck cell lines, etc.).
  • the cells may include cells which can be or has been the recipient of the vector described herein as well as progeny of such cells.
  • Host cells can be cultured in conventional fermentation bioreactors, flasks, and petri plates. Culturing can be carried out at a temperature, pH, and oxygen content appropriate for a given cell.
  • the cells e.g., engineered cells
  • the cells are eukaryotic cells, such as mammalian cells, e.g., human cells.
  • the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells.
  • exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).
  • the cells are human cells.
  • the cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigenspecificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • the cells may be allogeneic and/or autologous.
  • T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
  • TN naive T
  • TSCM stem cell memory T
  • TCM central memory T
  • TEM effector memory T
  • TIL tumor-infiltrating lymphocyte
  • embodiments disclosed herein are directed to methods of treating cancer by administering to a subject in need thereof the immunogenic compositions and vaccine compositions disclosed herein.
  • the immunogenic compositions and vaccine may comprise the cancer-associated antigens described herein.
  • the method comprises administering a vaccine comprising a disease-associated antigen selected from SEQ ID NO: 325-41854, and/or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.
  • the subject is suffering from a hematological malignancy.
  • the hematological malignancy is selected from multiple myeloma, acute myeloid leukemia, or chronic lymphocytic leukemia.
  • the vaccine is polypeptide-based and comprises the polypeptide selected from SEQ ID NO: 325-41854, and/or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.
  • the vaccine is DNA-based and comprises a DNA polynucleotide sequence encoding a polypeptide from SEQ ID NO: 325-41854, and/or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.
  • the vaccine is RNA-based and comprises an RNA polynucleotide sequence encoding a polypeptide from SEQ ID NO: 325-41854, and/or TATGATAGC, CAGGCGTCT, TTGGCTTCT, GGTGCATCC, AGTGCATCC, AAAGACAGT, GCTGCATCT, TGGGCATCA, AGTACTTAT, GCTGCGTCC, GAGGTCACC.
  • the methods may comprise administering a pharmaceutically effective (e.g., therapeutically effective amount or prophylactically effective amount)) amount of an immunogenic composition or pharmaceutical formulation thereof (including but not limited to a peptide, DNA, or mRNA vaccine) herein to a subject, e.g., a subject in need thereof.
  • a pharmaceutically effective e.g., therapeutically effective amount or prophylactically effective amount
  • an immunogenic composition or pharmaceutical formulation thereof including but not limited to a peptide, DNA, or mRNA vaccine
  • the method comprises administering the composition(s), the polynucleotide(s), and/or the vector(s) herein to a subject.
  • a pharmaceutically effective amount refers to an amount that can elicit a biological, medicinal, or immunological response in a tissue, system, or subject (e.g., animal or human) that can prevent or alleviate one or more of the local or systemic symptoms or features of a disease or condition being treated.
  • Described in certain example embodiments herein are methods of inducing a B-cell response and/or T-cell response to a virus in a subject in need thereof, comprising administering, to the subject, the immunogenic composition or the therapeutic composition, or a pharmaceutical formulation thereof of the present invention described elsewhere herein.
  • the B cell response comprises antibody production.
  • Described in certain example embodiments herein are methods of treating a viral infection in a subject in need thereof comprising administering, to the subject in need thereof, the immunogenic composition or the therapeutic composition, or a pharmaceutical formulation thereof of the present invention as described elsewhere herein in combination with an antiviral therapeutic.
  • Described in certain example embodiments herein are methods an infection status of a subject comprising contacting immune cells derived from a subject with the immunogenic composition or a pharmaceutical formulation thereof of the present invention as described elsewhere herein; and detecting crossreactivity of the immune cells to the immunogenic composition.
  • a “signature” may encompass any gene or genes, protein or proteins, or epigenetic element(s) whose expression profile or whose occurrence is characteristic of multiple myeloma reactive T cells.
  • the terms “signature”, “expression profile”, or “expression program” may be used interchangeably.
  • multiple myeloma reactive T cells may be identified and isolated based on the detection of a multiple myeloma-reactive T cell molecular signature as disclosed in Table 6.
  • the signature profile may be used in microfluidics- based forward screening of single T cells against autologous tumor cells to identify and facilitate the isolation and expansion of MM reactive T cells in bone marrow or peripheral blood samples from therapy -naive multiple myeloma patients.
  • a gene expression signature of multiple myeloma reactive T cells comprises the genes GNLY, ZNF683, GZMH, FGFBP2, GZMB, NKG7, CCL5, HOPX, KLRD1, EFHD2, CD8A, CTSW, CST7, ITGB1, and BHLHE40 (sigMM).
  • a gene expression signature of multiple myeloma reactive T cells comprises the genes GNLY, ZNF683, GZMH, FGFBP2, and GZMB (sigMM_2).
  • a gene expression signature of multiple myeloma reactive T cells comprises the genes GNLY, ZNF683, GZMH, FGFBP2, GZMB, NKG7, CCL5, HOPX, KLRD1, and EFHD2 (sigMM_3).
  • a gene expression signature of multiple myeloma reactive T cells comprises the genes GNLY, ZNF683, GZMH, FGFBP2, GZMB, NKG7, CCL5, HOPX, KLRD1, EFHD2, CD8A, CTSW, CST7, ITGB1, BHLHE40, LYAR, S100A4, GZMA, MXRA7, and KLRK1 (sigMM 4).
  • a gene expression signature of multiple myeloma reactive T cells comprises the genes 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, and LGALS1 (sigMM_5).
  • a gene expression signature of multiple myeloma reactive T cells comprises the genes 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, and MATK, (sigMM 6).
  • a gene expression signature of multiple myeloma reactive T cells comprises the genes 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, (sigMM_7).
  • a gene expression signature of multiple myeloma reactive T cells comprises the genes 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 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 (sigMM_8).
  • a gene expression signature of multiple myeloma reactive T cells 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, CLICl, KLF6, Clorf21, SYNE2, HLA-DPB1, HLA-DPA1, DSTN, and CD99, (sigMM_7).
  • a gene expression signature of multiple myeloma reactive T cells comprises two 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, 1TGA4, FCRL6, TGFB1, CCL4, ZEB2, AO AH, AHNAK, S100A10, LGALS1, PRF1, ITGB2, CD52, TPST2, PRSS23, ANXA1, CYBA, C12orf75, LAIR2, MATK, S100A6, TNFAIP3, CLICl, KLF6, Clorf21, SYNE2, HLA-DPB1, HLA-DPA1, DSTN, and CD99, (sigMM_7).
  • a gene expression signature of multiple myeloma reactive T cells comprises three 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, CLICl, KLF6, Clorf21, SYNE2, HLA-DPB1, HLA-DPA1, DSTN, and CD99, (sigMM_7).
  • a gene expression signature of multiple myeloma reactive T cells comprises 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, CLICl, 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,
  • a gene expression signature of multiple myeloma reactive T cells comprises two 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, CLICl, 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,
  • a gene expression signature of multiple myeloma reactive T cells comprises three 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, CL1C1, HLA-DPA1, HLA-DPB1, TNFA1P3, 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
  • the detection of the disclosed gene signatures characteristic of multiple myeloma reactive T cells in a patient’s bone marrow or peripheral blood is predictive of a better treatment outcome, for example, with adjuvant chemotherapy, bi-specific antibodies, checkpoint inhibitors.
  • compositions that can contain an amount, effective amount, and/or least effective amount, and/or therapeutically effective amount of one or more compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof (which are also referred to as the primary active agent or ingredient elsewhere herein) described in greater detail elsewhere herein and a pharmaceutically acceptable carrier or excipient.
  • pharmaceutical formulation refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.
  • pharmaceutically acceptable carrier or excipient refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non-toxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • a “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient.
  • the compound can optionally be present in the pharmaceutical formulation as a pharmaceutically acceptable salt.
  • the pharmaceutical formulation can include, such as an active ingredient, a polynucleotide, polypeptide, vector, delivery vehicle, and/or cell of the present invention described in greater detail elsewhere herein.
  • the active ingredient is present as a pharmaceutically acceptable salt of the active ingredient.
  • pharmaceutically acceptable salt refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts.
  • Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2- hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-(CH2)n-COOH where n is 0-4, and the like.
  • acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, s
  • pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium, and ammonium.
  • a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent.
  • compositions described herein can be administered to a subject in need thereof via any suitable method or route, which typically depends on the disease to be treated and/or the active ingredient(s).
  • compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof described in greater detail elsewhere herein can be provided to a subject in need thereof as an ingredient, such as an active ingredient or agent, in a pharmaceutical formulation.
  • an ingredient such as an active ingredient or agent
  • pharmaceutical formulations containing one or more of the compounds and salts thereof, or pharmaceutically acceptable salts thereof described herein.
  • Suitable salts include, hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate.
  • the subject in need thereof has or is suspected of having a viral infection or a symptom thereof.
  • agent refers to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a biological and/or physiological effect on a subject to which it is administered to.
  • active agent or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to.
  • active agent or active ingredient refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed.
  • An agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed.
  • An agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed.
  • the pharmaceutical formulation can include a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include, but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.
  • the pharmaceutical formulations can be sterilized, and if desired, mixed with agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active compound.
  • agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active compound.
  • the pharmaceutical formulation can also include an effective amount of secondary active agents, including but not limited to, biologic agents or molecules including, but not limited to, e.g., polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, chemotherapeutics, and combinations thereof.
  • the pharmaceutical formulation comprises an effective amount of one or more chemotherapeutics, immunomodulators, or both.
  • Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g., IL-2, IL-7, and IL-12) , cytokines (e.g. interferons (e.g. IFN-a, IFN-P, IFN-s, IFN-K, IFN-co, and IFN-y), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g.
  • the immunomodulator is a checkpoint blockade modulator. In an embodiment, the immunomodulator is a checkpoint blockade inhibitor.
  • chemotherapeutics include, but are not limited to, paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, Cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, de
  • the amount of the primary active agent and/or optional secondary agent is an effective amount, least effective amount, and/or therapeutically effective amount.
  • effective amount refers to the amount, concentration, etc. of the primary and/or optional secondary agent included in the pharmaceutical formulation that achieve one or more therapeutic effects or desired effect.
  • “least effective,” “least effective concentration,” and/or the like amount refers to the lowest amount, concentration, etc. of the primary and/or optional secondary agent that achieves the one or more therapeutic or other desired effects.
  • therapeutically effective amount refers to the amount, concentration, etc.
  • the one or more therapeutic effects are inducing an immune response in a subject to which they are delivered, inducing a B- and/or T- cell response in a subject to which it is delivered, treating or preventing a viral infection in a subject to which it is delivered.
  • the effective amount, least effective amount, and/or therapeutically effective amount of the primary and optional secondary active agent described elsewhere herein contained in the pharmaceutical formulation can be any non-zero amount ranging from about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,
  • the effective amount, least effective amount, and/or therapeutically effective amount can be an effective concentration, least effective concentration, and/or therapeutically effective concentration, which can each be any non-zero amount ranging from about O to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
  • the effective amount, least effective amount, and/or therapeutically effective amount of the primary and optional secondary active agent be any nonzero amount ranging from about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,
  • the primary and/or the optional secondary active agent present in the pharmaceutical formulation can be any non-zero amount ranging from about 0 to 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27,
  • the amount or effective amount, particularly where an infective particle is being delivered e g., a virus particle having the primary or secondary agent as a cargo
  • the effective amount of virus particles can be expressed as a titer (plaque forming units per unit of volume) or as a MOI (multiplicity of infection).
  • the effective amount can be about 1X10 1 particles per pL, nL, pL, mL, or L to 1X1O 20 / particles per pL, nL, pL, mL, or L or more, such as about 1x10 1 , IxlO 2 , IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 , IxlO 7 , IxlO 8 , IxlO 9 , IxlO 10 , IxlO 11 , IxlO 12 , IxlO 13 , IxlO 14 , IxlO 13 , IxlO 16 , IxlO 17 , IxlO 18 , IxlO 19 , to/or about IxlO 20 particles per pL, nL, pL, mL, or L.
  • the effective titer can be about 1X10 1 transforming units per pL, nL, pL, mL, or L to 1X1O 20 / transforming units per pL, nL, pL, mL, or L or more, such as about IxlO 1 , IxlO 2 , IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 , IxlO 7 , IxlO 8 , IxlO 9 , IxlO 10 , IxlO 11 , IxlO 12 , IxO 13 , IxlO 14 , IxlO 15 , IxlO 16 , IxlO 17 , IxlO 18 , IxlO 19 , to/or about IxlO 20 transforming units per pL, nL, pL, mL, or L or any numerical value or subrange within these ranges.
  • the MOI of the pharmaceutical formulation can range from about 0.1 to 10 or more, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4,
  • the amount or effective amount of the one or more of the active agent(s) described herein contained in the pharmaceutical formulation can range from about 1 pg/kg to about 10 mg/kg based upon the body weight of the subject in need thereof or average bodyweight of the specific patient population to which the pharmaceutical formulation can be administered.
  • the effective amount of the secondary active agent will vary depending on the secondary agent, the primary agent, the administration route, subject age, disease, stage of disease, among other things, which will be one of ordinary skill in the art.
  • the secondary active agent can be included in the pharmaceutical formulation or can exist as a stand-alone compound or pharmaceutical formulation that can be administered contemporaneously or sequentially with the compound, derivative thereof, or pharmaceutical formulation thereof.
  • the effective amount of the secondary active agent when optionally present, is any non-zero amount ranging from about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
  • the effective amount of the secondary active agent is any non-zero amount ranging from about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • the pharmaceutical formulations described herein can be provided in a dosage form.
  • the dosage form can be administered to a subject in need thereof.
  • the dosage form can be effective generate specific concentration, such as an effective concentration, at a given site in the subject in need thereof.
  • dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the primary active agent, and optionally present secondary active ingredient, and/or a pharmaceutical formulation thereof calculated to produce the desired response or responses in association with its administration.
  • the given site is proximal to the administration site.
  • the given site is distal to the administration site.
  • the dosage form contains a greater amount of one or more of the active ingredients present in the pharmaceutical formulation than the final intended amount needed to reach a specific region or location within the subject to account for loss of the active components such as via first and second pass metabolism.
  • the dosage forms can be adapted for administration by any appropriate route.
  • Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, parenteral, subcutaneous, intramuscular, intravenous, internasal, and intradermal.
  • Such dosage forms can be prepared by any method known in the art.
  • Dosage forms adapted for oral administration can discrete dosage units such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or non- aqueous liquids; edible foams or whips, or in oil-in-water liquid emulsions or water-in-oil liquid emulsions.
  • the pharmaceutical formulations adapted for oral administration also include one or more agents which flavor, preserve, color, or help disperse the pharmaceutical formulation.
  • Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as a foam, spray, or liquid solution.
  • the oral dosage form can be administered to a subject in need thereof. Where appropriate, the dosage forms described herein can be microencapsulated.
  • the dosage form can also be prepared to prolong or sustain the release of any ingredient.
  • compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof described herein can be the ingredient whose release is delayed.
  • the primary active agent is the ingredient whose release is delayed.
  • an optional secondary agent can be the ingredient whose release is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et. al.
  • suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.
  • cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate
  • polyvinyl acetate phthalate acrylic acid polymers and copolymers
  • methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany),
  • Coatings may be formed with a different ratio of water-soluble polymer, water insoluble polymers, and/or pH dependent polymers, with or without water insoluble/water soluble non-polymeric excipient, to produce the desired release profile.
  • the coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, “ingredient as is” formulated as, but not limited to, suspension form or as a sprinkle dosage form.
  • the dosage forms described herein can be a liposome.
  • primary active ingredient(s), and/or optional secondary active ingredient(s), and/or pharmaceutically acceptable salt thereof where appropriate are incorporated into a liposome.
  • the pharmaceutical formulation is thus a liposomal formulation.
  • the liposomal formulation can be administered to a subject in need thereof.
  • Dosage forms adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
  • the pharmaceutical formulations are applied as a topical ointment or cream.
  • a primary active ingredient, optional secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate can be formulated with a paraffinic or water-miscible ointment base.
  • the primary and/or secondary active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • Dosage forms adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.
  • Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders.
  • a primary active ingredient, optional secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate can be in a dosage form adapted for inhalation is in a particle-size-reduced form that is obtained or obtainable by micronization.
  • the particle size of the size reduced (e.g., micronized) compound or salt or solvate thereof is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art.
  • Dosage forms adapted for administration by inhalation also include particle dusts or mists.
  • Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions/suspensions of an active (primary and/or secondary) ingredient, which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators.
  • the nasal/inhalation formulations can be administered to a subject in need thereof.
  • the dosage forms are aerosol formulations suitable for administration by inhalation.
  • the aerosol formulation contains a solution or fine suspension of a primary active ingredient, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate and a pharmaceutically acceptable aqueous or non-aqueous solvent.
  • Aerosol formulations can be presented in single or multi-dose quantities in sterile form in a sealed container.
  • the sealed container is a single dose, multi-dose nasal, or an aerosol dispenser fitted with a metering valve (e.g., metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted.
  • a metering valve e.g., metered dose inhaler
  • the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon.
  • a suitable propellant under pressure such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon.
  • the aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer.
  • the pressurized aerosol formulation can also contain a solution or a suspension of a primary active ingredient, optional secondary active ingredient, and/or pharmaceutically acceptable salt thereof.
  • the aerosol formulation also contains co-solvents and/or modifiers incorporated to improve, for example, the stability and/or taste and/or fine particle mass characteristics (amount and/or profile) of the formulation.
  • Administration of the aerosol formulation can be once daily or several times daily, for example 2, 3, 4, or 8 times daily, in which 1, 2, 3 or more doses are delivered each time.
  • the aerosol formulations can be administered to a subject in need thereof.
  • the pharmaceutical formulation is a dry powder inhalable-formulations.
  • a dosage form can contain a powder base such as lactose, glucose, trehalose, mannitol, and/or starch.
  • a primary active agent, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate is in a particle-size reduced form.
  • a performance modifier such as L-leucine or another amino acid, cellobiose octaacetate, and/or metals salts of stearic acid, such as magnesium or calcium stearate.
  • the aerosol formulations are arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the compositions, compounds, vector(s), molecules, cells, and combinations thereof described herein.
  • Dosage forms adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Dosage forms adapted for rectal administration include suppositories or enemas. The vaginal formulations can be administered to a subject in need thereof.
  • Dosage forms adapted for parenteral administration and/or adapted for injection can include aqueous and/or non-aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.
  • the dosage forms adapted for parenteral administration can be presented in a single-unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials.
  • the doses can be lyophilized and re-suspended in a sterile carrier to reconstitute the dose prior to administration.
  • Extemporaneous injection solutions and suspensions can be prepared In an embodiment, from sterile powders, granules, and tablets.
  • the parenteral formulations can be administered to a subject in need thereof.
  • the dosage form contains a predetermined amount of a primary active agent, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate per unit dose.
  • the predetermined amount of primary active agent, secondary active ingredient, and/or pharmaceutically acceptable salt thereof where appropriate can be an effective amount, a least effect amount, and/or a therapeutically effective amount.
  • the predetermined amount of a primary active agent, secondary active agent, and/or pharmaceutically acceptable salt thereof where appropriate can be an appropriate fraction of the effective amount of the active ingredient.
  • the pharmaceutical formulation(s) described herein are part of a combination treatment or combination therapy.
  • the combination treatment can include the pharmaceutical formulation described herein and an additional treatment modality.
  • the additional treatment modality can be a chemotherapeutic, a biological therapeutic, surgery, radiation, diet modulation, environmental modulation, a physical activity modulation, and combinations thereof.
  • the co-therapy or combination therapy can additionally include but not limited to, polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, chemotherapeutics, and combinations thereof.
  • the co-therapy and/or combination therapy comprises an effective amount of one or more chemotherapeutics, immunomodulators, or both. Exemplary chemotherapeutics and immunomodulators for Co- and Combination therapies are previously discussed in connection with additional active agents.
  • the pharmaceutical formulations or dosage forms thereof described herein can be administered one or more times hourly, daily, monthly, or yearly (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times hourly, daily, monthly, or yearly).
  • the pharmaceutical formulations or dosage forms thereof described herein can be administered continuously over a period of time ranging from minutes to hours to days.
  • Devices and dosages forms are known in the art and described herein that are effective to provide continuous administration of the pharmaceutical formulations described herein.
  • the first one or a few initial amount(s) administered can be a higher dose than subsequent doses. This is typically referred to in the art as a loading dose or doses and a maintenance dose, respectively.
  • the pharmaceutical formulations can be administered such that the doses over time are tapered (increased or decreased) overtime so as to wean a subject gradually off of a pharmaceutical formulation or gradually introduce a subject to the pharmaceutical formulation.
  • the pharmaceutical formulation can contain a predetermined amount of a primary active agent, secondary active agent, and/or pharmaceutically acceptable salt thereof where appropriate.
  • the predetermined amount can be an appropriate fraction of the effective amount of the active ingredient.
  • Such unit doses may therefore be administered once or more than once a day, month, or year (e.g., 1, 2, 3, 4, 5, 6, or more times per day, month, or year).
  • Such pharmaceutical formulations may be prepared by any of the methods well known in the art.
  • Sequential administration is administration where an appreciable amount of time occurs between administrations, such as more than about 15, 20, 30, 45, 60 minutes or more.
  • the time between administrations in sequential administration can be on the order of hours, days, months, or even years, depending on the active agent present in each administration.
  • Simultaneous administration refers to administration of two or more formulations at the same time or substantially at the same time (e.g., within seconds or just a few minutes apart), where the intent is that the formulations be administered together at the same time.
  • the pharmaceutical formulations and/or immunogenic composition described herein are mRNA vaccines.
  • one or more CAA (including but not limited to conserved cancer antigen) polynucleotides or polynucleotides encoding the one or more CAA (including but not limited to conserved cancer antigen) polypeptides of the present invention described herein are included in an mRNA vaccine composition.
  • the polypeptides are immunogenic polypeptides.
  • the mRNA vaccine composition can be administered to a subject in need thereof.
  • the vaccine is administered to a subject in an effective amount to induce an immune response in the subject.
  • compositions that include one or more isolated messenger ribonucleic (mRNA) polynucleotides encoding at least one CAA polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to the antigenic polypeptide), such as any of those polynucleotides described in greater detail elsewhere herein, where the isolated mRNA is formulated in a lipid nanoparticle.
  • immunogenic polypeptide encompasses immunogenic fragments of the antigenic polypeptide (an immunogenic fragment that is induces (or is capable of inducing) an immune response to a cancer.
  • the cancer is a blood cancer.
  • the cancer is a white blood cell cancer. In an embodiment, the cancer is multiple myeloma.
  • the mRNA encoding at least one CAA polypeptide or immunogenic fragment thereof can include an open reading frame that encodes the at least one CAA antigenic polypeptide or immunogenic fragment thereof. In an embodiment, the mRNA encoding at least one CAA antigenic polypeptide or immunogenic fragment thereof can include a non-canconical open reading frame that encodes the at least one CAA polypeptide or immunogenic fragment thereof. In an embodiment, the open reading frame encodes at least two, at least five, or at least ten CAA polypeptides and/or immunogenic fragments thereof. In an embodiment, the open reading frame encodes at least 100 antigenic polypeptides. In an embodiment, the open reading frame encodes 2-100 CAA polypeptides and/or immunogenic fragments thereof.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, wherein the plurality of lipid nanoparticles optionally has a mean particle size of between 80 nm and 160 nm; and wherein the lipid nanoparticles comprise one or more polynucleotides encoding at least one viral antigenic polypeptide or an immunogenic fragment thereof.
  • the mRNA vaccine is multivalent.
  • the mRNA of the mRNA vaccine is codon-optimized.
  • an RNA (e.g., mRNA) vaccine further includes an adjuvant.
  • the isolated mRNA is not self-replicating.
  • the isolated mRNA comprises and/or encodes one or more 5 ’terminal cap (or cap structure), 3 ’terminal cap, 5 ’untranslated region, 3 ’untranslated region, a tailing region, or any combination thereof.
  • the capping region of the isolated mRNA region may be from 1 to 10, e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length.
  • the cap is absent.
  • a 5'-cap structure is capO, capl, ARCA, inosine, Nl-methyl- guanosine, 2 '-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, or 2-azido-guanosine.
  • the 5 ’terminal cap is 7mG(5')ppp(5')NlmpNp, m7GpppG cap, N 7 - methylguanine.
  • the 3 ’terminal cap is a 3'-O-methyl-m7GpppG.
  • the 3'-UTR is an alpha-globin 3'-UTR.
  • the 5'- UTR comprises a Kozak sequence.
  • the tailing sequence may range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides).
  • the tailing region is or includes a polyA tail. Where the tailing region is a polyA tail, the length may be determined in units of or as a function of polyA Binding Protein binding. In this embodiment, the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein. PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides.
  • polyA tails of about 80 nucleotides and 160 nucleotides are functional.
  • the poly-A tail is at least 160 nucleotides in length.
  • the at least one viral antigenic polypeptide linked to or fused to a signal peptide in an embodiment, the isolated mRNA encoding a viral antigenic polypeptide or immunogenic fragment thereof further includes a polynucleotide sequence encoding a signal peptide.
  • the signal peptide is selected from: a HuIgGk signal peptide (METPAQLLFLLLLWLPDTTG (SEQ ID NO: 317)); IgE heavy chain epsilon- 1 signal peptide (MDWTWILFLVAAATRVHS (SEQ ID NO: 318)); Japanese encephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS (SEQ ID NO: 319)), VSVg protein signal sequence (MKCLLYLAFLFIGVNCA (SEQ ID NO: 320)) and Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA (SEQ ID NO: 321)).
  • the signal peptide is fused to the N-terminus of at least one viral antigenic polypeptide.
  • a signal peptide is fused to the C-terminus of at least one viral antigenic polypeptide.
  • the polynucleotides of the mRNA vaccine composition are structurally modified and/or chemically modified.
  • a “structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to affect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”.
  • the same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”.
  • the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.
  • the polynucleotide, e.g., an mRNA of an mRNA vaccine composition described herein comprises at least one chemical modification.
  • the polynucleotide, e.g., an mRNA of an mRNA vaccine composition does not comprise a chemical or structural modification.
  • the at least one chemical modification is selected from pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5- methylcytosine, 5-methyluridine, 2-thio-l -methyl- 1-deaza-pseudouri dine, 2-thio-l -methylpseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l -methyl - pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyl uridine.
  • the chemical modification is in the 5-position of the uracil. In an embodiment, the chemical modification is a N1 -methylpseudouridine. In an embodiment, the chemical modification is a N1 -ethylpseudouridine.
  • the mRNA polynucleotide includes a stabilization element.
  • the stabilization element is a histone stem-loop.
  • the stabilization element is a nucleic acid sequence having increased GC content relative to wild type sequence.
  • the mRNA polynucleotide may include a sequence encoding a self-cleaving peptide.
  • the self-cleaving peptide may be, but is not limited to, a 2A peptide.
  • the 2A peptide has the protein sequence: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 322), fragments or variants thereof.
  • the 2A peptide cleaves between the last glycine and last proline.
  • the polynucleotides of the present invention includes a polynucleotide sequence encoding the 2A peptide having the protein sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 322) fragments or variants thereof.
  • polynucleotide sequence encoding the 2A peptide is GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCCTGGACCT (SEQ ID NO: 323).
  • the polynucleotide sequence of the 2A peptide may be modified or codon optimized by the methods described herein and/or are known in the art.
  • this sequence is used to separate the coding region of two or more polypeptides of interest.
  • the sequence encoding the 2A peptide is between a first coding region A and a second coding region B (A-2Apep-B).
  • the presence of the 2 A peptide results in the cleavage of one long protein into protein A, protein B and the 2A peptide.
  • Protein A and protein B may be the same or different peptides or polypeptides of interest.
  • the 2A peptide are used in the polynucleotides of the present invention to produce two, three, four, five, six, seven, eight, nine, ten, or more proteins.
  • the length of an mRNA included in the mRNA vaccine is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, about 1,900, about 2,000, about 2,500, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 20,000, about 30,000, about 40,000, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000 or up to and including about 100,000 nucleotides).
  • the length of an mRNA included in the mRNA vaccine includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500
  • the polynucleotides are linear.
  • the polynucleotides of the present invention that are circular are known as “circular polynucleotides” or “circP.”
  • “circular polynucleotides” or “circP” means a single stranded circular polynucleotide which acts substantially like, and has the properties of, an R A.
  • the term “circular” is also meant to encompass any secondary or tertiary configuration of the circP.
  • RNA modifications for mRNA vaccines and production of mRNA can be as described e.g., U.S. Pat. 8,278,036, 8,691,966, 8,748,089, 9,750,824, 10,232,055, 10,703,789, 10,702,600, 10,577,403, 10,442,756, 10,266,485, 10,064,959, 9,868,692, 10,064,959, 10,272,150 ;U.S. Publications, US20130197068, US20170043037, US20130261172, US20200030460, US20150038558, US20190274968, US20180303925, US20200276300; International Patent Application Publication Nos. WO/2018/081638A1, WO/2017/176330A1, which are incorporated herein by reference.
  • the mRNA vaccine includes one or more additional mRNAs that encode a polypeptide adjuvant. In an embodiment, the mRNA vaccine includes one or more additional mRNAs that encode a non-viral antigen, such as an antigen to another disease causing agent.
  • the one or more additional mRNAs that encode a polypeptide adjuvant encode a flagellin polypeptide.
  • at least one flagellin polypeptide e.g., encoded flagellin polypeptide
  • at least one flagellin polypeptide has at least 80%, at least 85%, at least 90%, or at least 95% identity to a flagellin polypeptide having a sequence identified by any one of SEQ ID NO: 54-56 of U.S. Pat.
  • At least one flagellin polypeptide and at least one viral and/or additional antigenic polypeptide are encoded by a single RNA (e.g., mRNA) polynucleotide. In other embodiments, at least one flagellin polypeptide and at least one viral and/or additional antigenic polypeptide are each encoded by a different RNA polynucleotide.
  • the isolated mRNAs and other polynucleotides of the mRNa vaccine can be formulated in a lipid nanoparticle.
  • the lipid nanoparticle is a cationic lipid nanoparticle.
  • the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.
  • the cationic lipid is a biodegradable cationic lipid.
  • the biodegradable cationic lipid comprises an ester linkage.
  • the biodegradable cationic lipid comprises DLin-DMA with an internal ester, DLin-DMA with a terminal ester, DLin-MC3-DMA with an internal ester, or DLin-MC3-DMA with a terminal ester.
  • a lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
  • a cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.
  • a cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z,15Z)- N,N-dimethyl-2-nonylhenicosa-12,15-dien-l-amine (L608), and N,N-dimethyl-l-[(lS,2R)-2- o
  • the neutral lipid is 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), the sterol is cholesterol, and the PEG-modified lipid is l,2-dimyristoyl-racalycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) or PEG- cDMA.
  • DSPC 1,2- distearoyl-sn-glycero-3-phosphocholine
  • the sterol is cholesterol
  • the PEG-modified lipid is l,2-dimyristoyl-racalycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) or PEG- cDMA.
  • the lipid nanoparticle is any nanoparticle described in U.S. Pat. No. 10,442,756, and/or comprises any compound described in U.S. Pat. No. 10,442,756, including but not limited to a nanoparticle according to any one of Formulas (IA) or (II) described therein.
  • the lipid nanoparticle is any nanoparticle described in e.g., U.S. Pat. No. 10,266,485, and/or comprises any compound described in U.S. Pat. No. 10,266,485, including but not limited to a nanoparticle according to Formula (II) described therein.
  • the lipid nanoparticle is a nanoparticle described in U.S. Pat. No. 9,868,692, and/ or comprises a compound described in e.g., U.S. Pat. No.
  • a lipid nanoparticle comprises compounds of Formula (I) and/or Formula (II) as described in U.S. Pat. No. 10272150.
  • the mRNA vaccine is formulated in a lipid nanoparticle that comprises a compound selected from Compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112 and 122 of U.S. Pat. No. 10,272,150.
  • lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
  • the lipid nanoparticle has a mean diameter of 50-200 nm.
  • a lipid nanoparticle comprises compounds of Formula (I) and/or Formula (II), as discussed below.
  • a lipid nanoparticle comprises Compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112, or 122 as set forth in U.S. Pat. No. 10272150.
  • the lipid nanoparticle has a poly dispersity value of less than 0.4 (e.g., less than 0.3, 0.2 or 0.1).
  • a plurality of lipid nanoparticles such as when contained in a formulation, has a mean PDI of between 0.02 and 0.2.
  • a plurality of lipid nanoparticles such as when contained in a formulation comprising one or more polynucleotide(s), has a mean lipid to polynucleotide ratio (wt/wt) of between 10 and 20.
  • the lipid nanoparticle has a net neutral charge at a neutral pH value.
  • compositions described herein can be used to induce an antigen specific immune response to a virus or a viral variant. Exemplary viruses are described elsewhere herein.
  • the methods of inducing an antigen specific immune response in a subject include administering to the subject any of the RNA (e.g., mRNA) vaccine as provided herein in an amount effective to produce an antigen-specific immune response.
  • an antigen-specific immune response comprises a T cell response and/or a B cell response.
  • a method of producing an antigen-specific immune response comprises administering to a subject a single dose (no booster dose) of a RNA (e.g., mRNA) vaccine of the present disclosure.
  • a RNA e.g., mRNA
  • the RNA (e.g., mRNA) vaccine is a combination vaccine comprising a combination of an mRNA vaccine described herein and at least one other mRNA vaccine.
  • the at least one other mRNA vaccine can be against the same or a different virus or disease-causing agent.
  • a method further comprises administering to the subject a second (booster) dose of an RNA (e.g., mRNA) vaccine. Additional doses of an RNA (e.g., mRNA) vaccine may be administered.
  • RNA e.g., mRNA
  • the subject exhibits a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) following the first dose or the second (booster) dose of the vaccine.
  • Seroconversion is the period during which a specific antibody develops and becomes detectable in the blood. After seroconversion has occurred, a virus can be detected in blood tests for the antibody. During an infection or immunization, antigens enter the blood, and the immune system begins to produce antibodies in response. Before seroconversion, the antigen itself may or may not be detectable, but antibodies are considered absent. During seroconversion, antibodies are present but not yet detectable. Any time after seroconversion, the antibodies can be detected in the blood, indicating a prior or current infection.
  • an RNA (e.g., mRNA) vaccine described herein is administered to a subject by intradermal, subcutaneous, or intramuscular injection.
  • the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition.
  • the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition in combination with electroporation.
  • the anti-antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In an embodiment, the anti-antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control. [0521] In an embodiment, the anti -antigenic polypeptide antibody titer produced in a subject is increased at least 2 times relative to a control. In an embodiment, the anti-antigenic polypeptide antibody titer produced in the subject is increased at least 5 times relative to a control. In an embodiment, the anti-antigenic polypeptide antibody titer produced in the subject is increased at least 10 times relative to a control. In an embodiment, the anti-antigenic polypeptide antibody titer produced in the subject is increased 2-10 times relative to a control.
  • control is an anti-antigenic polypeptide antibody titer produced in a subject who has not been administered an RNA (e.g., mRNA) vaccine of the present disclosure.
  • control is an anti -antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated or inactivated vaccine against a virus or wherein the control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered a recombinant or purified viral protein vaccine.
  • control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered a virus-like particle (VLP) vaccine comprising structural proteins of the virus.
  • VLP virus-like particle
  • RNA (e.g., mRNA) vaccine of the present disclosure can be administered to a subject in an effective amount (e.g., an amount effective to induce an immune response in the subject).
  • the RNA (e.g., mRNA) vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject.
  • the effective amount is a total dose of 25 pg to 1000 pg, or 50 pg to 1000 pg. In an embodiment, the effective amount is a total dose of 100 pg. In an embodiment, the effective amount is a dose of 25 pg administered to the subject a total of two times. In an embodiment, the effective amount is a dose of 100 pg administered to the subject a total of two times. In an embodiment, the effective amount is a dose of 400 pg administered to the subject a total of two times. In an embodiment, the effective amount is a dose of 500 pg administered to the subject a total of two times.
  • the efficacy (or effectiveness) of an RNA (e.g., mRNA) vaccine is greater than 60%.
  • AR disease attack rate
  • RR relative risk
  • vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. lQ Q Jun. 1; 201 (11): 1607-10).
  • Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial.
  • Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real -world’ outcomes of hospitalizations, ambulatory visits, or costs.
  • a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared.
  • the efficacy (or effectiveness) of an RNA (e.g., mRNA) vaccine is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.
  • the vaccine immunizes the subject against one or more cancers.
  • the cancer is a blood cancer.
  • the cancer is a white blood cell cancer.
  • the cancer is multiple myeloma.
  • the cancer is acute myeloid leukemia (AML).
  • the cancer is chronic lymphocytic leukemia (CLL). Exemplary viruses and variants are described elsewhere herein.
  • the subject to which the mRNA vaccine of the present disclosure is administered is about 5 years old or younger.
  • the subject may be between the ages of about 1 year and about 5 years (e.g., about 1, 2, 3, 5 or 5 years), or between the ages of about 6 months and about 1 year (e.g., about 6, 7, 8, 9, 10, 11 or 12 months).
  • the subject is about 12 months or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months or 1 month).
  • the subject is about 6 months or younger.
  • the subject to which the mRNA vaccine of the present disclosure is administered was bom full term (e.g., about 37-42 weeks).
  • the subject was born prematurely, for example, at about 36 weeks of gestation or earlier (e.g., about 36, about 35, about 34, about 33, about 32, about 31, about 30, about 29, about 28, about 27, about 26 or about 25 weeks).
  • the subject may have been born at about 32 weeks of gestation or earlier.
  • the subject was born prematurely from about 32 weeks to about 36 weeks of gestation.
  • an RNA (e.g., mRNA) vaccine may be administered later in life, for example, at the age of about 6 months to about 5 years, or older.
  • the subject to which the mRNA vaccine of the present disclosure is administered is pregnant (e.g., in the first, second or third trimester) when administered an RNA (e.g., mRNA) vaccine.
  • RNA e.g., mRNA
  • the subject to which the mRNA vaccine of the present disclosure is administered is a young adult between the ages of about 20 years and about 50 years (e.g., about 20, about 25, about 30, about 35, about 40, about 45 or about 50 years old).
  • the subject to which the mRNA vaccine of the present disclosure is administered is an elderly subject about 60 years old, about 70 years old, or older (e.g., about 60, about 65, about 70, about 75, about 80, about 85, about 90, or about 100 or more years old).
  • the subject to which the mRNA vaccine of the present disclosure is administered has cancer.
  • the subject to which the mRNA vaccine of the present disclosure is administered has a blood cancer.
  • the subject to which the mRNA vaccine of the present disclosure is administered has a white blood cell cancer.
  • the subject to which the mRNA vaccine of the present disclosure is administered has a multiple myeloma.
  • the subject to which the mRNA vaccine of the present disclosure is administered has acute myeloid leukemia (AML).
  • the subject to which the mRNA vaccine of the present disclosure is administered has chronic lymphocytic leukemia (CLL).
  • the subject to which the mRNA vaccine of the present disclosure is administered is immunocompromised (has an impaired immune system, e g., has an immune disorder or autoimmune disorder).
  • the mRNA vaccine of the present disclosure is delivered to a subj ect at a dosage of between 10 pg/kg and 400 pg/kg of the nucleic acid vaccine is administered to the subject.
  • the dosage of the RNA polynucleotide is 1-5 pg, 5-10 pg, 10-15 pg, 15-20 pg, 10-25 pg, 20-25 pg, 20-50 pg, 30-50 pg, 40-50 pg, 40-60 pg, 60-80 pg, 60-100 pg, 50- 100 pg, 80-120 pg, 40-120 pg, 40-150 pg, 50-150 pg, 50-200 pg, 80-200 pg, 100-200 pg, 120-250 pg, 150-250 pg, 180-280 pg, 200-300 pg, 50-300 pg, 80-300 pg, 100-300 pg, 40-300 pg, 100-
  • the subject can receive 1, 2, 3, 4, 5, 6, 7, or more doses.
  • the subject can receive one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional doses, referred to in the art as “booster” doses.
  • the booster doses can follow the initial dose at any suitable time interval such as within days, weeks, months, or even years.
  • multiple booster doses are needed close in time after the initial dose (such as within 1, 2, 3, or 4 weeks after the initial dose) followed by a larger gap in time (e.g., months or years before subsequent booster doses are needed).
  • a first dose of the mRNA vaccine is administered to the subject on day zero.
  • a second dose of the mRNA vaccine a is administered to the subject on day 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 or more days after the first dose.
  • a third dose of the mRNA vaccine is administered to the subject on day 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 or more days after the first and/or second dose.
  • the mRNA vaccine confers an antibody titer superior to the criterion for seroprotection for a cancer for an acceptable percentage of human subjects.
  • the cancer is a blood cancer.
  • the cancer is a white blood cell cancer.
  • the cancer is multiple myeloma.
  • the antibody titer produced by the mRNA vaccines of the invention is a neutralizing antibody titer.
  • the neutralizing antibody titer is greater than a protein vaccine.
  • the neutralizing antibody titer produced by the mRNA vaccines of the invention is greater than an adjuvanted protein vaccine.
  • the neutralizing antibody titer produced by the mRNA vaccines of the invention is 1,000-10,000, 1,200-10,000, 1,400-10,000, 1,500-10,000, 1,000- 5,000, 1,000-4,000, 1,800-10,000, 2000-10,000, 2,000-5,000, 2,000-3,000, 2,000-4,000, 3,000- 5,000, 3,000-4,000, or 2,000-2,500.
  • a neutralization titer is typically expressed as the highest serum dilution required to achieve a 50% reduction in the number of plaques.
  • a unit of use vaccine comprises between 10 ug and 400 ug of one or more RNA polynucleotides encoding the CAA polypeptide(s) and/or immunogenic fragment(s) thereof and a pharmaceutically acceptable carrier or excipient, formulated for delivery to a human subject.
  • the vaccine further comprises a cationic lipid nanoparticle.
  • aspects of the invention provide methods of creating, maintaining, or restoring antigenic memory to a cancer in an individual or population of individuals comprising administering to said individual or population an mRNA vaccine described herein.
  • aspects of the invention provide methods of creating, maintaining, or restoring antigenic memory to a blood cancer in an individual or population of individuals comprising administering to said individual or population an mRNA vaccine described herein.
  • aspects of the invention provide methods of creating, maintaining, or restoring antigenic memory to a white blood cell cancer in an individual or population of individuals comprising administering to said individual or population an mRNA vaccine described herein.
  • aspects of the invention provide methods of creating, maintaining, or restoring antigenic memory to multiple myeloma, acute myeloid leukemia (AML), and/or chronic lymphocytic leukemia (CLL) in an individual or population of individuals comprising administering to said individual or population an mRNA vaccine described herein.
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • the methods of vaccinating a subject comprising administering to the subject a single dosage of between 25 ug/kg and 400 ug/kg of an mRNA vaccine comprising one or more RNA polynucleotides encoding a CAA polypeptide and/or an immunogenic fragment thereof in an effective amount to vaccinate the subject.
  • the mRNA vaccines comprising one or more RNA polynucleotides encoding a CAA polypeptide and/or an immunogenic fragment thereof, wherein the RNA comprises at least one chemical modification, wherein the vaccine has at least 10-fold less RNA polynucleotide than is required for an unmodified mRNA vaccine to produce an equivalent antibody titer.
  • the RNA polynucleotide is present in a dosage of 25-100 micrograms.
  • the mRNA vaccine comprises an LNP formulated RNA polynucleotide having an open reading frame comprising no nucleotide modifications (unmodified), the open reading frame one or more RNA polynucleotides encoding a CAA polypeptide and/or an immunogenic fragment thereof, wherein the vaccine has at least 10-fold less RNA polynucleotide than is required for an unmodified mRNA vaccine not formulated in a LNP to produce an equivalent antibody titer.
  • the RNA polynucleotide is present in a dosage of 25-100 micrograms.
  • the mRNA vaccine comprises an LNP formulated RNA polynucleotide having an open reading frame comprising one or more modifications, the open reading frame one or more RNA polynucleotides encoding a CAA polypeptide and/or an immunogenic fragment thereof, wherein the vaccine has at least 10-fold less RNA polynucleotide than is required for an unmodified mRNA vaccine not formulated in a LNP to produce an equivalent antibody titer.
  • the RNA polynucleotide is present in a dosage of 25- 100 micrograms.
  • the method includes vaccinating a subject with a combination vaccine including at least two nucleic acid sequences encoding respiratory antigens, wherein at least one encodes a CAA or immunogenic fragment thereof wherein the dosage for the vaccine is a combined therapeutic dosage wherein the dosage of each individual nucleic acid encoding an antigen is a sub therapeutic dosage.
  • the combined dosage is 25 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject.
  • the combined dosage is 100 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject.
  • the combined dosage is 50 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In an embodiment, the combined dosage is 75 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In an embodiment, the combined dosage is 150 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In an embodiment, the combined dosage is 400 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In an embodiment, the sub therapeutic dosage of each individual nucleic acid encoding an antigen is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 micrograms.
  • vaccines of the invention produce prophylactically- and/or therapeutically-efficacious levels, concentrations and/or titers of antigen-specific antibodies in the blood or serum of a vaccinated subject.
  • antibody titer refers to the amount of antigen-specific antibody produces in the subject, e.g., a human subject.
  • antibody titer is expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result.
  • antibody titer is determined or measured by enzyme-linked immunosorbent assay (ELISA).
  • antibody titer is determined or measured by neutralization assay, e.g., by microneutralization assay. In certain aspects, antibody titer measurement is expressed as a ratio, such as 1 :40, 1: 100, etc.
  • an efficacious vaccine produces an antibody titer of greater than 1 :40, greater that 1 : 100, greater than 1 :400, greater than 1 : 1000, greater than 1 :2000, greater than 1 :3000, greater than 1:4000, greater than 1 :500, greater than 1:6000, greater than 1 :7500, greater than 1 : 10000.
  • the antibody titer is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination.
  • the titer is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the titer is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.)
  • antigen-specific antibodies are measured in units of pg/ml or are measured in units of IU/L (International Units per liter) or mIU/ml (milli International Units per ml).
  • an efficacious vaccine produces >0.5 pg/ml, >0.1 pg/ml, >0.2 pg/ml, >0.35 pg/ml, >0.5 pg/ml, >1 pg/ml, >2 pg/ml, >5 pg/ml or >10 pg/ml.
  • an efficacious vaccine produces >10 mIU/ml, >20 mIU/ml, >50 mIU/ml, >100 mIU/ml, >200 mIU/ml, >500 mIU/ml or >1000 mIU/ml.
  • the antibody level or concentration is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination.
  • the level or concentration is produced or reached following a single dose of vaccine administered to the subject.
  • the level or concentration is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.)
  • antibody level or concentration is determined or measured by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • antibody level or concentration is determined or measured by neutralization assay, e.g., by microneutralization assay
  • TCRs that target antigens derived from the corrupted immunoglobulin in multiple myeloma cells were also identified. Furthermore, the detection of tumor-reactive TCRs prior to treatment correlated with enhanced clinical responses to induction chemotherapy and bispecific antibody administration. Highlighting the therapeutic importance of autologous stem cell transplantation, it was shown an increase in tumor-reactive TCRs within stem cell grafts. These TCRs are selectively transplanted and exhibit long-term persistence upon re-infusion into patients.
  • NDMM was chosen as a proof-of-principle entity due to the presence of well- established and highly expressed surface protein markers to enable sorting of malignant cells.
  • NDMM furthermore less frequently disrupts the bone marrow microenvironment and suppresses lymphopoiesis compared to other malignant hematological diseases, which enabled testing of several thousand bone marrow-derived T cells per patient.
  • scRNA- seq high-throughput single-cell RNA sequencing
  • scVDJ-seq single-cell V(D)J sequencing
  • CITE-seq cellular indexing of transcriptomes and epitopes by sequencing
  • FIG. 13A & 13B Identification of tumor-reactive TCRs was performed in parallel using 1) a microfluidics- based forward screening approach of single BMTCs exposed to single autologous tumor cells (FIG. 1A, step 2) and 2) a functional expansion of tumor-reactive T cells assay 4 on BMTCs.
  • This antigen-dependent approach was informed by prior antigen discovery in patient tumor samples by prediction of whole genome sequencing (WG-seq) and RNA-seq-derived cancer associated antigens (CAAs) and neoepitopes as well as class I HLA immunoprecipitation (IP) followed by LC-MS/MS analysis. (FIG. 1A, step 2).
  • TCR alpha and beta chain sequencing TCRA/B-seq
  • functional expansion cultures were sequenced after 10 days of tumor- or virus epitope stimulation using ultradeep TCRVP-seq (FIG. 1A, step 3).
  • the data from both assays were then integrated and used to identify and phenotype antigen-reactive T cells by matching each TCR to its baseline transcriptional state using the CDR3 amino acid sequence as a unique endogenous barcode of a given clone (FIG. 1A, step 4).

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Abstract

La présente divulgation concerne, selon certains aspects, des méthodes, des cellules et des compositions pour préparer des cellules immunitaires modifiées isolées comprenant des récepteurs de lymphocytes T (TCR) pouvant reconnaître un antigène associé à une maladie. Selon certains aspects, les cellules immunitaires sont des lymphocytes T destinés à être utilisés en immunothérapie. Dans un mode de réalisation, la divulgation concerne des méthodes de préparation de lymphocytes T, comprenant l'isolement, le traitement, l'incubation et l'ingénierie génétique de cellules et de populations de cellules. L'invention concerne également les lymphocytes T modifiés isolés et les compositions produites par les procédés de la présente divulgation. Selon certains aspects, les méthodes préparent des lymphocytes T pour une thérapie adoptive. Dans un mode de réalisation, l'antigène associé à une maladie est un antigène associé au cancer.
PCT/US2024/054265 2023-11-02 2024-11-01 Compositions et méthodes d'utilisation de lymphocytes t en immunothérapie Pending WO2025097055A2 (fr)

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