Methods for CAR-T delivery of synthetic peptide therapeutics

Abstract

The present disclosure provides engineered cells (e.g., T cells) comprising a chimeric antigen receptor (CAR) and a therapeutic peptide, and methods of use thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 166,073, filed March 25, 2021, which is incorporated by reference in its entirety herein.

[0002] Federally Sponsored Research and Development Statement This invention was made with Government support under CA228455 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention Chimeric antigen receptor T cells (CAR-T) have shown great potential in liquid malignancies, but responses in solid tumors have been limited so far. Similarly, bioactive peptide therapeutics have demonstrated promising preclinical activity in vitro and in vivo, but have yet to demonstrate significant clinical benefit. Both therapies face distinct obstacles to activity in larger patient populations. In the case of adoptive cell therapies (ACTs), such as CAR-T, diverse local tumor-intrinsic immunosuppressive mechanisms limit antitumor efficacy. Similarly, efficient delivery and localization of peptide therapeutics have proven to be significant obstacles.

[0004] There is a need in the art for improved CAR-T therapies that provide enhanced anti-tumor activity. The present invention addresses this need. Summary of the Invention

[0005] In one aspect, the present disclosure provides an engineered cell comprising a chimeric antigen receptor (CAR) and a therapeutic peptide. The CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain. The therapeutic peptide is a non-natural therapeutic peptide, and the CAR molecule and the therapeutic peptide are expressed from the same expression construct.

[0006] In another aspect, the present disclosure provides an engineered cell comprising a chimeric antigen receptor (CAR) and a therapeutic peptide. The CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, and the therapeutic peptide is a non-natural therapeutic peptide. The therapeutic peptide has one or more of the following properties: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway, (ii) the therapeutic peptide is a mimic of a cyclic dinucleotide, (iii) the therapeutic peptide is a mimic of a short chain fatty acid (SCFA), (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR), (v) the therapeutic peptide is a mimic of a steroid / hormone-like molecule, (vi) the therapeutic peptide promotes apoptosis of a target cell, (vii) the therapeutic peptide is an immunogenic epitope, and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in a target cell.

[0007] In certain embodiments, the therapeutic peptide is a mimic of cGAMP, cAMP, or cGMP. In certain embodiments, the therapeutic peptide is a mimic of TLR agonist. In certain embodiments, the therapeutic peptide is a mimic of flagellin, CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL). In certain embodiments, the therapeutic peptide is a second mitochondria derived activator of caspase (SMAC) mimic. In certain embodiments, the therapeutic peptide is an inhibitor of poly ADP-ribose polymerase (PARP).

[0008] In certain embodiments, the non-naturally occurring peptide is a peptide having 90% or less sequence identity to a naturally occurring peptide. In certain embodiments, the non-naturally occurring peptide is a peptide having 80% or less sequence identity to a naturally occurring peptide. In certain embodiments, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-16.

[0009] In certain embodiments, the therapeutic peptide is excreted from the engineered cells into extracellular vesicles. In certain embodiments, the therapeutic peptide is a mimic of a SCFA that binds to a G protein-coupled receptor (GPCR).

[0010] In certain aspects, the target cell is a tumor cell.

[0011] In certain embodiments, the engineered cells are T cells or NK cells.

[0012] In certain embodiments, the antigen-binding domain is selected from the group consisting of an antibody, an antigen-binding fragment (Fab), and a single chain variable fragment (scFv).

[0013] In certain embodiments, the binding domain is a T cell receptor (TCR).

[0014] In certain embodiments, the target antigen is selected from the group consisting of CD19, CD20, CD22, BCMA, CD123, CD133, EGFR, EGFRvIII, mesothelin, Her2, PSMA, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA 125, CTAG1B, mucin 1, and folate receptor-alpha. In certain embodiments, the target antigen is expressed on enterocytes.

[0015] In certain embodiments, the transmembrane domain is a transmembrane domain from a protein selected from the group consisting of CD8 alpha, CD3 zeta, CD3 epsilon, CD28, and ICOS.

[0016] In certain embodiments, the intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, TCR zeta, CD3 epsilon, CD3 gamma, CD3 delta, or ICOS. In certain embodiments, the intracellular signaling domain comprises a functional signaling domain and further comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain from 4-1BB or CD28.

[0017] In certain embodiments, the CAR comprises an anti-CD19 scFv, a CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

[0018] In certain embodiments, the therapeutic peptide is a SCFA mimetic or a mimetic of a steroid and / or hormone-like molecule, where the engineered cells are further modified to reduce activity of one or more effector functions.

[0019] In certain embodiments, the engineered cells are modified to reduce or prevent expression of one or more inflammatory cytokines, expression of granzyme B, or expression of perforin.

[0020] In certain embodiments, the CAR molecule and the therapeutic peptide are expressed from the same expression construct, wherein the expression construct further comprises an RNA molecule that activates a PRR. In certain embodiments, the RNA molecule is 7SL.

[0021] In another aspect, the disclosure provides a composition comprising any of the engineered cells contemplated herein.

[0022] In another aspect, the present disclosure provides a nucleic acid molecule encoding (i) a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, and (ii) a therapeutic peptide, wherein the therapeutic peptide is a non-naturally occurring peptide.

[0023] In certain embodiments, a stop codon separates the nucleic acid segment encoding the CAR and the nucleic acid segment encoding the therapeutic peptide.

[0024] In certain aspects, the therapeutic peptide encoded by the nucleic acid molecule has one or more of the following properties: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the therapeutic peptide is a mimic of a cyclic dinucleotide; (iii) the therapeutic peptide is a mimic of a short chain fatty acid (SCFA); (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) the therapeutic peptide is a mimic of a steroid / hormone-like molecule; (vi) the therapeutic peptide promotes apoptosis of a target cell; (vii) the therapeutic peptide is an immunogenic epitope; and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in a target cell.

[0025] In certain embodiments, the therapeutic peptide is a mimic of cGAMP, cAMP, or cGMP. In certain embodiments, the therapeutic peptide is a mimic of TLR agonist. In certain embodiments, the therapeutic peptide is a mimic of flagellin, CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL). In certain embodiments, the therapeutic peptide is a second mitochondria-derived caspase activator (SMAC) mimic. In certain embodiments, the therapeutic peptide is an inhibitor of poly ADP-ribose polymerase (PARP).

[0026] In certain embodiments, a nucleic acid encoding a non-naturally occurring peptide has 80% or less sequence identity to a nucleic acid encoding a naturally occurring peptide.

[0027] In certain aspects, the target cell is a tumor cell.

[0028] In certain embodiments, the antigen binding domain encoded by the nucleic acid molecule is selected from the group consisting of an antibody, a Fab, and an scFv.

[0029] In certain embodiments, the binding domain encoded by the nucleic acid molecule is a TCR.

[0030] In certain embodiments, the binding domain encoded by the nucleic acid molecule binds to a target antigen selected from the group consisting of CD19, CD20, CD22, BCMA, CD123, CD133, EGFR, EGFRvIII, mesothelin, Her2, PSMA, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA 125, CTAG1B, mucin 1, and folate receptor-alpha.

[0031] In certain embodiments, the transmembrane domain encoded by the nucleic acid molecule is a transmembrane domain from a protein selected from the group consisting of CD8 alpha, CD3 zeta, CD3 epsilon, CD28, and ICOS.

[0032] In certain embodiments, the intracellular signaling domain encoded by the nucleic acid molecule comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, TCR zeta, CD3 epsilon, CD3 gamma, CD3 delta, or ICOS. In certain embodiments, the intracellular signaling domain encoded by the nucleic acid molecule comprises a functional signaling domain and further comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain from 4-1BB or CD28.

[0033] In certain embodiments, the nucleic acid molecule encodes a CAR molecule comprising an anti-CD19 scFv, a CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

[0034] In certain embodiments, the nucleic acid molecule further comprises an RNA molecule that activates a PRR, hi certain embodiments, the RNA molecule is 7SL.

[0035] In another aspect, the disclosure provides an expression vector comprising any of the nucleic acid molecules contemplated herein.

[0036] In another aspect, the present disclosure provides a method for co-expressing a CAR and a therapeutic peptide in a cell, comprising delivering any of the expression vectors contemplated herein to a cell under conditions such that the CAR and the therapeutic peptide are expressed.

[0037] In another aspect, the disclosure provides a cell comprising any of the nucleic acid molecules or expression vectors contemplated herein.

[0038] In another aspect, the present disclosure provides a method for treating a disease or disorder in a subject.The method comprises administering to a subject an effective amount of T cells genetically modified to express a chimeric antigen receptor (CAR).The CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.The method further comprises stimulating the endogenous immune response against cancer via a non-natural therapeutic peptide.The non-natural therapeutic peptide is expressed in the modified T cells and / or administered in combination with the modified T cells. The non-naturally occurring therapeutic peptide has one or more of the following properties: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the therapeutic peptide is a mimic of a cyclic dinucleotide; (iii) the therapeutic peptide is a mimic of a short chain fatty acid (SCFA); (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) the therapeutic peptide is a mimic of a steroid / hormone-like molecule; (vi) the therapeutic peptide promotes apoptosis of a target cell; (vii) the therapeutic peptide is an immunogenic epitope; and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in a target cell.

[0039] In certain embodiments, the therapeutic peptide is a mimic of cGAMP, cAMP, or cGMP. In certain embodiments, the therapeutic peptide is a mimic of TLR agonist. In certain embodiments, the therapeutic peptide is a mimic of flagellin, CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL). In certain embodiments, the therapeutic peptide is a second mitochondria-derived caspase activator (SMAC) mimic. In certain embodiments, the therapeutic peptide is an inhibitor of poly ADP-ribose polymerase (PARP).

[0040] In certain embodiments, a non-naturally occurring peptide is a peptide having 80% or less sequence identity to any naturally occurring peptide.

[0041] In certain embodiments, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1-16.

[0042] In certain embodiments, the therapeutic peptide is an immunogenic epitope, and the immunogenic epitope is expressed on the surface of the cancer cells of the subject after administration to the subject.In certain embodiments, the therapeutic peptide is expressed in modified T cells, and the therapeutic peptide is discharged from modified T cells into one or more extracellular vesicles following administration of modified T cells to the subject.In certain embodiments, the therapeutic peptide is delivered to one or more antigen-presenting cells of the subject via one or more extracellular vesicles.

[0043] In another aspect, the present disclosure provides a method for enhancing the anti-cancer activity of T cells genetically modified to express chimeric antigen receptor (CAR).CAR comprises an antigen-binding domain that specifically binds to the antigen expressed on tumor cells, a transmembrane domain, and a signal transduction domain.The method comprises co-expressing a non-natural therapeutic peptide in T cells. The non-naturally occurring therapeutic peptide has one or more of the following properties: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the therapeutic peptide is a mimic of a cyclic dinucleotide; (iii) the therapeutic peptide is a mimic of a short chain fatty acid (SCFA); (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) the therapeutic peptide is a mimic of a steroid / hormone-like molecule; (vi) the therapeutic peptide promotes apoptosis of a target cell; (vii) the therapeutic peptide is an immunogenic epitope; and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in a target cell.

[0044] In another aspect, the disclosure provides a method for treating an inflammatory disease, an autoimmune disease, or cancer in a subject, the method comprising administering to the subject an effective amount of any of the engineered cells or compositions contemplated herein.

[0045] In certain embodiments, the cancer is a solid tumor cancer. In certain embodiments, the cancer is selected from the group consisting of lung cancer, small cell lung cancer, non-small cell lung cancer, mesothelioma, pancreatic cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, prostate cancer, colon cancer, gastric cancer, bladder cancer, esophageal cancer, and melanoma. In certain embodiments, the cancer is a blood cancer. In certain embodiments, the blood cancer is leukemia or lymphoma. In certain embodiments, the hematological cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic myelogenous leukemia, The tumor is selected from the group consisting of myeloproliferative neoplasms, follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, lymphoplasmacytic lymphoma, and plasma cell myeloma.

[0046] In certain embodiments, the autoimmune disease is inflammatory bowel disease. [Brief description of the drawings]

[0047] The foregoing and other features and advantages of this invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0048] [Figure 1] 1 depicts a schematic diagram of an exemplary embodiment of the present disclosure. [Diagram 2]FIG. 1 is a diagram of an exemplary CAR construct comprising a CAR molecule (19BBz) and a non-natural peptide (SIINFEKL) (SEQ ID NO:7) separated by a stop codon. [Diagram 3] 1 depicts flow cytometry plots showing peptide / MHC expression on 19BBz CAR transduced (CAR+) and non-transduced (CAR-) cells; and Ova-19BBz CAR transduced (CAR+) and non-transduced (CAR-) cells. [Figure 4] 1 depicts a flow cytometry plot showing that the CAR molecule is expressed despite containing an internal stop codon. CAR expression was determined by staining with an anti-human Fab'2 antibody. A proposed peptide transfer event is shown. [Diagram 5] Illustrative experimental design of a study to evaluate encapsulation of immunogenic peptides from CAR-T transduced cells into extracellular vesicles and transfer of immunogenic peptide epitopes to tumor cells. [Figure 6A] Depicts flow cytometry plots showing peptide / MHC expression on tumor cells (top row) and Granzyme B expression in OT-I T cells (bottom row) incubated with no CAR-T EVs, 18 μg EVs, 37.5 μg EVs, or 75 μg EVs. [Figure 6B] Depicts flow cytometry plots showing Ki67 expression (top row) and IFNγ expression (bottom row) in OT-I T cells incubated with no CAR-T EVs, 18 μg EVs, 37.5 μg EVs, or 75 μg EVs. [Figure 6C] Quantification of Ova-19BBz EV data shown in the bottom row of Figure 6A and Figure 6B is depicted. Ki67 expression (Figure 6C, left panel), Granzyme B expression (Figure 6C, center panel), and IFNγ expression (Figure 6C, right panel) were significantly increased in the presence of EVs compared to the equivalent amount of EVs from 19BBz (i.e., no Ova peptide)-expressing CAR-T cells. [Figure 7]1 is a bar graph showing relative cell death of OT-I T cells and B16 cells incubated with 0 μg EV, 18 μg EV, 37.5 μg EV, or 75 μg EV. [Figure 8] FIG. 1 is a diagram of the study design for in vivo studies of immunogenic peptide epitope expression and transfer in vivo using B16-hCD19 tumor cells. [Figure 9A] Shown are peptide / MHC expression on tumor cells (top two panels) or dendritic cells (bottom two panels) following administration of 19BBz or Ova-19BBz to tumor cell-implanted mice. [Figure 9B] Peptide tetramer staining of CD8+ T cells collected from tumors following administration of 19BBz (left panel) or Ova-19BBz (middle panel) is shown. Tetramer+ cells isolated from the Ova-19BBz group were also Ki67+ (right panel), indicating that SIINFEKL-specific cells were activated. [Figure 10A] The % of Ova+ tumor cells (left panel) and % of Ova+ endogenous immune cells (right panel) in 19BBz versus Ova-19BBz recipients are shown. [Figure 10B] The percentage of endogenous T cells staining positive with Ova tetramer (top left panel) and the percentage of CD8+ T cells expressing the proliferation marker Ki67 (top right panel) in 19BBz versus Ova-19BBz recipient mice are shown. Tumor weights in recipient mice at day 16 are shown in the bottom panel. [Figure 11] Figure 11A is a schematic of the study design for an in vivo study of immunogenic peptide epitope expression and transfer in which tumors were implanted using a 1:1 mixture of B16-CD19 and B16 WT cells. Figure 11B shows that at 21, 24, and 28 days after tumor implantation, tumor volumes (cm3) in animals treated with 19BBz were significantly higher than those in animals treated with Ova-19BBz; and tumor volumes grew more rapidly in 19BBz recipients compared to Ova-19BBz recipients. [Figure 12A] 1 is a diagram of an exemplary CAR construct comprising a CAR molecule (19BBz) and a non-natural peptide (SIINFEKL) separated by a stop codon; and an exemplary CAR construct comprising a SIINFEKL peptide, a stop codon, a 19BBz CAR molecule, and a 7SL RNA. The RNA promoter U6 separates the 19BBz CAR molecule from the 7SL. Also shown is a diagram of the study design for in vivo studies of immunogenic peptide epitope expression and transfer using Ova-19BBz, 19BBz-7SL, Ova-19BBz-7SL, or control (19BBz) CAR T cells and tumors implanted using a 1:1 mixture of KP-hCD19 cells and KP WT cells. [Figure 12B] Tumor volumes are shown in each group of untreated mice (Utxl), mice treated with 19BBz, Ova-l9BBz (Ova-19), 19BBz-7SL (19-7SL), or Ova-19BBz-7SL (Ova-19-7SL). [Figure 12C] Shown is survival over time for mice treated with 19BBz, Ova-19BBz (BBz-Ova), 19BBz-7SL (BBz-7SL), and Ova-19BBz-7SL (Ova-BBz-7SL); or untreated mice (utx). [Figure 13] Shown are Protein Data Bank crystal structures of a human STING molecule complexed with cGAMP (PDB structure 4EMT; left panel) or a STING structure with cGAMP removed to reveal a solo active STING structure with an empty cGAMP pocket. [Figure 14] STING with poly-Gly sampled to generate binding peptides (left panel) and STING with predicted binding peptides present at the cGAMP binding site (right panel) are shown. The predicted binding peptide shown is peptide ST2. [Figure 15]15 is a schematic depiction of the study design to evaluate the activity of identified STING agonist peptides as measured by CD86 as a readout for DC activation (left panel). The right panel of FIG. 15 shows the % of CD86+ cells as measured by flow cytometry after incubation with a negative control, a positive control (cGAMP) or the indicated STING peptide. [Figure 16] Schematic depiction of study design to evaluate the effect of STING agonist peptides when incubated with DCs and OT-1 T cells (left panel). Right panel shows fold change of granzyme B, IFNγ and Ki67 in WT cells incubated with liposome-encapsulated STING peptides. Liposomes alone were used as a negative control. In addition to WT, cells from STING knockout mice (KO) are used to demonstrate that T cell activation by ST2 is STING dependent. [Figure 17] A schematic depiction of the 19BBz CAR construct and the STING agonist peptide-19BBz CAR construct comprising the STING agonist peptides ST2 and 19BBz is provided (top). The bottom shows an exemplary experimental design for in vivo evaluation of cells expressing the 19BBz-ST2 construct. [Figure 18] We show that animals that received 19BBz-STING peptide-expressing CAR T cells exhibited significantly improved survival compared to animals that received conventional 19BBz CAR-T cells. [Figure 19] Relative cell death in B16 (left panel) or KP (right panel) tumor cells is shown upon incubation with liposomes alone (negative control) or the indicated concentrations of SMAC mimetic peptides Pepl, Pep3, Pep4, Pep5, or Pep6 in the presence of TNF. [Figure 20]Figure 1 shows the relative cell death of B16 cells incubated with liposomes alone (negative control) or increasing concentrations of the indicated SMAC mimetic peptide SMACm6 in the presence or absence of TNF. Concentrations are shown in μM. The efficacy of SMACm6 in inducing cell death is dependent on TNF signaling. [Figure 21] Schematic depiction of CAR-T constructs containing SMACm6 peptide and 19BBz (top) and experimental design to evaluate the effect of EVs released from expanded SMACm CAR-T cells on tumor cell lines (bottom). [Figure 22] Relative cell death of B16 (left panel) or KP (right panel) cells incubated with the indicated concentrations of EVs from 19BBz or 19BBz-SMACm6 cells is shown. [Diagram 23] Tumor volume (cm3; left two panels) and survival (right two panels) of animals treated with the indicated CAR-T cells (19BBz or l9-SMACm6) with or without anti-CTLA4 therapy are shown. [Figure 24] 1 is a diagram showing PARPi dsDNA induction of PDL1. [Diagram 25] Shown is PD-L1 expression in TSA breast cancer cells following incubation with liposome-encapsulated PARPi peptides (Pep1, Pep2, Pep1C, or Pep3), negative control (liposomes only) or positive control (PARP inhibitor olaparib). [Figure 26A] Shown are the designs of 19BBz CAR vectors expressing Ova (Ova-19BBz) or Ova+RN7SL1 (Ova-19-7SL) (top) and representative flow cytometry plots showing detection of SIINFEKL peptide on CAR+ and CAR- T cells (bottom). [Figure 26B]Detection of SIINFEKL peptide on B16 cells loaded with the indicated concentrations of EVs from Ova-19BBz CAR-T cells or 19BBz CAR-T cells (control) is shown (flow plot, top). OT-I T cells were then added and activation was measured (flow plot, bottom) and quantified using GZMB and Ki67 (dot plot, bottom). [Figure 26C] Figure 1 shows SIINFEKL peptide transfer to cancer and immune cells measured in mixed CD19+ / CD19- B16 tumors following in vivo treatment with 19BBz or Ova-19BBz (Ova) CAR-T cells. Representative flow cytometry plots of cancer cells are shown (left). [Figure 26D] 1 shows endogenous Ova-specific T cell expansion measured by tetramer and Ki67 expression. [Figure 26E] The frequencies of Ova-specific and Ki67+ CD8 T cells as measured by tetramer and Ki67 expression are shown. [Figure 26F] Shown is the growth of heterogeneous CD19+ / CD19- B16 tumors following treatment with the indicated CAR-T cells. [Figure 26G] Shows the growth of heterogeneous CD19+ / CD19- KP mixed tumors following anti-CTLA4+anti-PD1 CAR-T cell therapy. 5x105 OT-I T cells were transferred prior to tumor implantation. [Fig. 26H] Figure 1 shows the relative transfer of Ova peptide from CD45.1+ CAR- T cells to intratumoral CD45.2+ immune cells versus CD45.2- tumor cells in mixed CD19- and CD19+ B16 tumors as quantified by flow cytometry for SIINFEKL / MHC-I staining. Each bar represents an individual tumor. [Figure 27A] Depicts the experimental setup of a study to evaluate the requirement of MHC-I from CAR-T cells to activate endogenous T cells via delivery of antigenic peptides from CAR-T cell extracellular vesicles (EVs). [Figure 27B]Expression of the indicated T cell activation markers on OT-I T cells following addition of EVs from MHC-I-expressing (top) or MHC-I-deficient (bottom) CAR-T cells under the indicated culture conditions (x-axis) is shown. [Figure 27C] Representative flow cytometry plots of T cell activation markers on OT-I T cells are shown. [Figure 28A] 1 depicts the experimental setup of a study to evaluate the ability of extracellular vesicles from CAR-T cells engineered to deliver antigenic peptides to directly activate endogenous T cells. [Figure 28B] Representative flow cytometry plots of OT-I CD8 T cells from the top wells for the indicated T cell activation markers (top row) or OVA / MHC-I transferred from CAR-T cell EVs (bottom row) after addition of the indicated CAR-T cells to the bottom wells. Two independent replicates are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Detailed Description Chimeric antigen receptor (CAR) therapy, e.g., CAR-T cells, offers a new way to treat diseases such as cancer, but improvements to such therapy are needed. Desirable improvements to CAR therapy include enhancing the potency and / or persistence of immune responses against cancer, exploiting cell death-promoting pathways in tumor cells, identifying new and diverse neoantigens, counteracting or negating immune evasion and survival strategies of tumor cells or in the tumor environment, identifying and using novel tumor antigens to target immune responses against tumors, and / or otherwise inducing or enhancing anti-cancer effects.

[0050] Enhanced CAR-T therapy, which includes expressing CAR molecules in cells together with exogenous RNA molecules, such as RNA molecules that stimulate the immune system, has been investigated, providing improved anti-cancer immune activity.However, such approaches cannot utilize many immune response pathways and features that cannot be coded on RNA molecules.This disclosure provides a compelling method to meet this need, and demonstrates that the approach provided herein shows surprisingly high efficacy.

[0051] Thus, in certain aspects, the present disclosure provides methods and compositions for delivering multiple anti-tumor immune components to tumors and tumor microenvironments that are tailored to disease-specific requirements.In certain embodiments, the present disclosure provides novel therapeutic molecules that are delivered by and enhance CAR therapy.In certain embodiments, the novel therapeutic molecules are synthetic, non-natural peptides.The present disclosure further provides methods for producing and using the novel compositions provided herein.The present disclosure further provides compositions and methods for making immunogenic epitope libraries and for transferring immunogenic peptides to tumor cells to enhance anti-cancer immunity.

[0052] It is to be understood that the methods described in this disclosure are not limited to the particular methods and / or experimental conditions disclosed herein, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0053] Moreover, the experiments described herein use conventional molecular cell biology and immunological techniques within the skill of those skilled in the art, unless otherwise indicated.Such techniques are well known to skilled workers and are fully described in the literature.See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook, and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0054] A. definition Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of any potential meaning uncertainty, the definitions provided herein take precedence over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Unless otherwise stated, the use of "or" means "and / or." The use of the term "including" and other forms such as "includes" and "included" are non-limiting.

[0055] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and widely used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art, as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as generally accomplished in the art or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein, as well as the test procedures and techniques thereof, are well known and widely used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0056] In order that this disclosure may be more readily understood, selected terms are defined below.

[0057] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0058] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are reasonable in practicing the disclosed methods.

[0059] As used herein, "activation" refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation.Activation can also be associated with induced cytokine production and detectable effector function.The term "activated T cells" refers specifically to T cells that undergo cell division.

[0060] As used herein, "alleviating" a disease means reducing the severity of one or more symptoms of the disease.

[0061] The term "antigen" as used herein is defined as a molecule that provokes an immune response. This immune response may include either or both antibody production or activation of specific immunologically competent cells. Those skilled in the art will appreciate that virtually any macromolecule can act as an antigen, including any protein or peptide.

[0062] Furthermore, the antigen can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0063] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is subsequently reintroduced into that individual.

[0064] "Costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0065] A "costimulatory signal," as used herein, refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0066] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause a further deterioration in the animal's health.

[0067] The term "downregulation," as used herein, refers to a decrease or elimination of gene expression of one or more genes.

[0068] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material or composition described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that, when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the present disclosure. The immune response can be readily assessed by a myriad of art-recognized methods. Those skilled in the art will understand that the amount of the composition administered herein will vary and can be readily determined based on a number of factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0069] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and which is usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0070] As used herein, "endogenous" refers to any material that originates or is produced within an organism, cell, tissue or system.

[0071] The term "epitope" as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response and induce a B-cell response and / or a T-cell response. An antigen can have one or more epitopes. Most antigens have many epitopes; that is, they are multivalent. In general, an epitope is approximately the size of about 10 amino acids and / or sugars. Preferably, an epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, even more preferably about 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. In general, one skilled in the art will understand that the overall three-dimensional structure of the molecule, rather than the specific linear sequence, is the primary criterion for the specificity of an antigen, and thus, this distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0072] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue or system.

[0073] The term "expanded" as used herein refers to an increase in number, such as an increase in the number of T cells. In one embodiment, ex vivo expanded T cells are increased in number relative to the number originally present in the culture. In another embodiment, ex vivo expanded T cells are increased in number relative to other cell types in the culture. As used herein, the term "ex vivo" refers to cells removed from an organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0074] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0075] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus and adeno-associated virus) that incorporate a recombinant polynucleotide.

[0076] "Identity" as used herein refers to the identity of subunit sequences between two amino acid molecules, such as between two polymer molecules, particularly between two polypeptide molecules. If two amino acid sequences have the same residue at the same position; for example, if a position in each of the two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that are matched or identical; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10 amino acid long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, the two amino acid sequences are 90% identical.

[0077] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0078] The term "immunosuppression" is used herein to refer to the overall reduction of the immune response.

[0079] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0080] "Lentivirus" as used herein refers to a genus of the Retroviridae family.Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors.HIV, SIV and FIV are all examples of lentiviruses.Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0081] The term "modified" as used herein refers to an altered state or structure of a molecule or cell of the present disclosure. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0082] The term "modulate" as used herein means to mediate a detectable increase or decrease in the level of response in a subject, compared to the level of response in the subject in the absence of treatment or compound, and / or compared to the level of response in an otherwise identical but untreated subject. This term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0083] In the context of this disclosure, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0084] The term "oligonucleotide" typically refers to a short polynucleotide. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), it is understood that this nucleotide sequence also includes an RNA sequence (i.e., A, U, C, G) (where "U" is replaced by "T").

[0085] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that the nucleotide sequence encoding the protein may, in some version, contain introns.

[0086] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0087] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide as used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and they can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., as well as synthetic means.

[0088] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, also commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, inter alia, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0089] The term "specifically binds" as used herein with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not, in and of itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not, in and of itself, change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" can be used in relation to the interaction of an antibody, protein or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a particular protein structure rather than the entire protein. If an antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0090] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structures.

[0091] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0092] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies, among others.

[0093] The term "subject" is intended to include organisms (e.g., mammals) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include farm animals and pets, such as, for example, ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is a human.

[0094] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to the presentation of antigen. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of alpha (a) and beta (β) chains, although in some cells, TCRs consist of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains, a variable domain and a constant domain. In some embodiments, TCRs can be engineered on any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0095] The term "therapeutic" as used herein means treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0096] "Graft" refers to a biocompatible lattice or donor tissue, organ or cells to be transplanted. Examples of grafts may include, but are not limited to, skin cells or tissue, bone marrow and solid organs such as the heart, pancreas, kidney, lung and liver. Grafts can also refer to any material to be administered to a host. For example, grafts can refer to nucleic acids or proteins.

[0097] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0098] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder from which a subject suffers.

[0099] A "vector" is a composition of material that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0100] Ranges: Throughout this disclosure, various aspects of the disclosure may be presented in the form of a range. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0101] B. Therapeutic Peptides As used herein, the term "therapeutic peptide" refers to a peptide delivered by or in combination with a CAR molecule and elicits and / or exerts and / or enhances a therapeutic response in a subject. In certain embodiments, the therapeutic response elicited or exerted or enhanced by a therapeutic peptide is an anti-cancer effect. For example, in certain embodiments, a therapeutic peptide binds to a target and triggers one or more pathways in a cell that increase or enhance an immune response and / or increase or enhance a death-promoting pathway in a tumor cell. In other embodiments, a therapeutic peptide is an immunogenic peptide, such as a neoantigen. In certain embodiments, delivery of a therapeutic peptide via the methods provided herein results in the transfer of the immunogenic peptide to tumor cells, thereby enhancing an anti-cancer immune response. For example, in certain embodiments, a therapeutic peptide provided herein is an immunogenic peptide that can be expressed from the same expression vector and / or can be expressed together with a CAR molecule. In a further embodiment, once the therapeutic peptide is expressed in a cell, the peptide is secreted into extracellular vesicles that are taken up by tumor cells or immune cells (e.g., antigen-presenting cells). In certain embodiments, the therapeutic peptide is secreted into extracellular vesicles so that it can be taken up, processed, and presented on tumor cells via MHC.

[0102] In certain aspects, the therapeutic peptides provided herein are synthetic and non-natural peptides. The non-natural therapeutic peptides provided herein can be novel peptides identified through the methods provided herein or other methods known in the art. In certain aspects, the therapeutic peptides provided herein are rationally or computationally designed peptides that can bind to a specific target. In certain aspects, the therapeutic peptides are highly specific to a specific target. In certain aspects, the therapeutic pathway is designed to act as a mimic, mimicking the binding of a ligand to a receptor. In certain aspects, the ligand that the therapeutic peptide mimics is not a peptide or protein. In certain aspects, the therapeutic peptide acts as an agonist or antagonist to trigger a pathway in a cell, e.g., an immune cell. Exemplary targets and pathways are detailed herein, but one skilled in the art will recognize that the present disclosure provides a method for enhancing anti-cancer effects through synthetic therapeutic peptides that target any desired pathway.

[0103] In certain embodiments, the therapeutic peptide is expressed in the same cell that expresses the CAR molecule. In certain embodiments, the therapeutic peptide and the CAR molecule are encoded on the same expression vector. In certain embodiments, the therapeutic peptide is expressed in the cell that expresses the CAR molecule, and then secreted from the cell into extracellular vesicles (EVs). In certain embodiments, the therapeutic peptide exerts its effect on the cell in which it is expressed (e.g., the cell that expresses the CAR molecule). In certain embodiments, the therapeutic peptide is discharged from the cell (e.g., in EVs) and exerts its effect in the extracellular space (e.g., tumor microenvironment), or exerts its effect directly or indirectly on adjacent cells, such as dendritic cells, T cells, or tumor cells.

[0104] In certain embodiments, the non-natural therapeutic peptides provided herein are rationally designed and tested for binding to target molecules using standard peptide binding assays. In other embodiments, the non-natural therapeutic peptides provided herein are generated using algorithmic and computational peptide binding predictions. In certain embodiments, the crystal structure of the target complexed with its ligand is compared to the crystal structure of the target in the absence of the ligand. A computational program then performs a process of iteratively modeling the binding predictions of the folded peptide. In this manner, synthetic candidate peptides are identified and generated. The synthetic candidate peptides can be tested for target binding. The synthetic candidate peptides can be further tested for functional effects such as immune stimulation, induction of tumor cell death, induction of surrogate markers of immune stimulation and / or cell death, or any other effect related to the target. In yet other embodiments, the therapeutic peptides provided herein are neoantigens identified through the generation of immunogenic epitope libraries from tumor samples. Candidate synthetic peptides can further be expressed from an expression vector expressing a CAR as provided herein and tested for in vivo efficacy, e.g., in a cancer model.

[0105] Table 1. Amino acid sequences of exemplary therapeutic peptides TIFF2024511109000001.tif107156

[0106] The amino acid sequences provided herein may be modified. Such modifications may be conservative sequence modifications. "Conservative sequence modifications" refer to amino acid modifications that do not significantly affect or alter the functional properties of a protein. Conservative substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain aspects, the amino acid sequences provided herein may be modified such that they have about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to a reference sequence.

[0107] Also included within the scope of the disclosure are sequences that are about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous (or have about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity) to the specific sequences provided herein.

[0108] In certain embodiments, the peptides provided herein are non-naturally occurring peptides.Non-naturally occurring peptides include, for example, peptides that have sequence identity of 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 89% or less, 88% or less, 87% or less, 86% or less, 85% or less, 84% or less, 83% or less, 82% or less, 81% or less, 80% or less, 79% or less, 78% or less, 77% or less, 76% or less, 75% or less, 74% or less, 73% or less, 72% or less, 71% or less, 70% or less, 69% or less, 68% or less, 67% or less, 66% or less, 65% or less, 64% or less, 63% or less, 62% or less, 61% or less, or 60% or less to naturally occurring peptides, depending on the length and context of the peptide. For non-natural proteins, directed evolution approaches or the generation of de novo peptides have been reported to provide novel and / or completely different functions compared to natural proteins. For example, it has been reported that mutating a small number of amino acids (e.g., 94% or higher homology to natural peptides) results in peptides with completely different functions compared to natural peptides (see, e.g., Adriano-Silva et al., Nature 565(186-191) (2019)).

[0109] In certain embodiments, the present disclosure provides therapeutic peptides contained in extracellular vesicles (EVs). Thus, in certain embodiments, the present disclosure provides EVs that contain any one or more of the therapeutic peptides provided herein. EVs are membrane-like micro- or nano-sized biological particles that are released by cells and can travel through intercellular space and contact adjacent cells. In certain embodiments, in the compositions and methods of the present disclosure, the therapeutic peptides provided herein are expressed in immune cells, then packaged into EVs and released from the cells. In certain embodiments, the EVs that contain one or more of the therapeutic peptides provided herein are taken up by adjacent cells, such as tumor cells or other immune cells. Once taken up by adjacent cells, the therapeutic peptides can exert their therapeutic effects, for example, as immune stimulatory molecules, modulators of death-promoting or apoptotic pathways, modulators of DNA repair mechanisms, antigens presented in the context of MHC, or other effects.

[0110] C. Exemplary Targets and Pathways The present disclosure advantageously provides compositions and methods for cell-based delivery of therapeutic agents to target cells, including persistent or long-term delivery in desired target tissues by cells expressing therapeutic peptides. Such methods and compositions significantly improve the therapy of diseases and disorders that can be or have been treated by the delivery of molecules that require repeated administration currently available. Thus, the present disclosure provides compositions and methods for effectively treating diseases and disorders while avoiding the long-term repeated administration of therapeutic agents that is often necessary for the treatment of certain diseases and disorders.

[0111] In certain embodiments, the disease or disorder is cancer or another cancer proliferation disorder. In such embodiments, the engineered cells provided herein advantageously enhance immune response and / or initiate proapoptotic programs and cell death in cancer cells or other cells associated with cell proliferation disorder. In other embodiments, the disease or disorder is an immune-mediated disease, such as an autoimmune disease (e.g., inflammatory bowel disease). In such embodiments, the engineered cells provided herein can be further modified to reduce effector cell function. For example, when an enhanced immune response is not desired, the engineered cells provided herein can be modified to reduce or eliminate the production of one or more inflammatory cytokines, granzyme B expression, perforin expression, antibody secretion and / or proliferation capacity.

[0112] One promising immunotherapy for cancer and other cell proliferation disorders involves the immune system's response to certain danger signals associated with pathogenic infection and / or cell or tissue damage. The innate immune system does not have antigen specificity but responds to diverse effector mechanisms such as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Receptors that recognize PAMPs are referred to as pattern recognition receptors (PRRs) and include Toll-like receptors (TLRs), nucleotide-binding domain leucine-rich repeat-containing proteins (NLRs; also referred to as NOD-like receptors), C-type lectin receptors (CLRs), and RNA helicases of retinoic acid-inducible gene-1 (RIG-1)-like receptors (RLRs). In certain aspects, the present disclosure provides enhanced targeted therapeutic strategies to treat diseases and disorders such as cancer by combining such immune stimulatory pathways with CAR-expressing cell therapy. In certain aspects, this is achieved via small molecules and therapeutic peptidomimetics that stimulate danger signal pathways.

[0113] Examples of PAMPs and DAMPs include free cytosolic DNA and RNA, e.g., double-stranded DNA (dsDNA). Cyclic dinucleotides are second messengers used in signal transduction in a wide variety of bacteria and agonists of innate immune responses in mammalian cells. Exemplary cyclic dinucleotides include cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), cyclic di-GMP, cyclic di-AMP, and cyclic AMP-GMP (cAMP-GMP, also known as cGAMP). The primary sensor of cytosolic DNA is cGAS (cyclic GMP AMP synthase). Upon recognition of cytosolic DNA, cGAS catalyzes the production of cGAMP, which tightly binds to the ER transmembrane adaptor protein Stimulator of Interferon Genes (STING). Upon binding of cGAMP to STING, STING undergoes a conformational change and translocates to the perinuclear compartment, inducing the activation of key transcription factors IRF-3 and NF-κB. This leads to strong induction of type I interferons and production of proinflammatory cytokines such as IL-6, TNF-α and IFN-γ. These molecules strongly increase T cell activation, for example, by enhancing the ability of dendritic cells and macrophages to take up, process, present and cross-present antigens to T cells, and by engaging their cognate receptors to induce the activation of interferon-responsive genes. Thus, stimulation of STING through cGAMP binding and other DAMP and PAMP-inducing pathways contributes greatly to adaptive immune cell activation.

[0114] TLRs are potent inducers of innate immunity and can detect PAMPs on the cell surface or in the lumen of intracellular vesicles such as endosomes or lysosomes. TLR activation occurs after ligand (agonist) binding, leading to recruitment of the adaptor molecules MyD88 and TRIF and subsequent activation of kinase-driven signaling pathways, resulting in IRF3 / 7 NF-κB activation and inflammatory responses as described above. TLR ligands include double-stranded RNA (dsRNA), which indicates the presence of viruses and activates TLR3; DNA containing unmethylated CpG motifs, found in viral and bacterial DNA, which activates TLR9; single-stranded RNA (ssRNA) and small interfering RNA (siRNA) molecules, also derived from viruses, which activate TLR7 and TLR8; flagellin, which is derived from mobile bacteria and activates TLR5; and lipopolysaccharide (LPS) and monophosphoryl lipid A (MPL) from gram-negative bacteria, which each activate TLR4.

[0115] NLRs and PRRs are found in the cytosol and are activated by bacterial products, such as peptides derived from peptidoglycan fragments and flagellin, type III secretion system rod components, toxins (e.g., Bacillus anthracis lethal toxin (LT); nigericin (Streptomyces hygroscopicus), erolysin (Aeromonas hydrophila), maitotoxin (Marina dinoflagellates), gramicidin (Brevibacillus subtilis) and α-toxin (Staphylococcus aureus)), and viral double-stranded DNA (dsDNA). NOD1 and NOD2 are two well-characterized NLRs that recognize structural motifs distinct from peptidoglycan. NALP3 and NALP1 recognize components derived from bacteria, such as toxins. IPAF and NAIP5 recognize flagellin. NLR activation leads to the formation of an "inflammasome" that engages caspase-1 and ultimately leads to the production of IL-1β.

[0116] RLRs (e.g., RIG-I) typically recognize viral dsRNA in the cytosol and then recruit the adaptor IFN-β promoter stimulator 1 (IPS-1; also called MAVS, VISA, and Cardif), leading to phosphorylation of the transcription factors IRF-3 and IRF7 and expression of type I IFN genes. Absent in melanoma 2 (AIM2) is an interferon-inducible protein that can bind to dsDNA in the cytosol and induce the autocleavage of caspase-1, thereby inflammasome activation. C-type lectin receptors (CLRs), such as Dectin-1, bind to fungal cell wall components (e.g., β-glucan), leading to activation of NF-κB.

[0117] In certain embodiments, the present disclosure provides small molecules and / or therapeutic peptides that can activate immune responses, such as innate immune response pathways, including but not limited to those described herein. Small molecules and / or therapeutic peptides can be mimics of molecules that stimulate innate immune response pathways, such as PRR mimics, cyclic dinucleotide mimics, and short chain fatty acid mimics. In certain embodiments, the present disclosure provides synthetic non-natural small molecules and synthetic non-natural therapeutic peptides that are mimics of any one or more of bacterial second messengers, TLR agonists, NLR agonists, RLR agonists, CLR agonists, or short chain fatty acids. For example, the provided small molecules and / or therapeutic peptides can be mimics of cGAMP, cAMP, cGMP, CpG, LPS, flagellin, bacterial toxins, peptidoglycan, or butyric acid.

[0118] Therefore, such compounds may be used in the treatment of human cancer. Given the efficacy of STING and other PRR-related pathways in stimulating immune response, these molecules are associated with a highly desirable strategy aimed at enhancing existing therapies, such as CAR-T cell therapy. However, STING binding partner cGAMP is a cyclic dinucleotide, and therefore cannot be encoded on RNA. Similarly, many other PRRs and related molecules cannot be encoded on RNA, and / or their binding partners can be more potently activated using synthetically designed peptide binders. The present disclosure provides novel methods, including peptidomimetics that can be expressed in and / or with CAR-expressing cells, and can be used to stimulate STING pathway or other immune activation pathways to enhance CAR therapy.

[0119] In certain embodiments, the therapeutic peptide is a PARP inhibitor.Poly(ADP-ribose) polymerase (PARP) is a nuclear and cytoplasmic enzyme that is involved in cellular processes including DNA repair, genome stability and programmed cell death.The key function of PARP is to detect DNA strand breaks and initiate repair reactions in cells.In response to DNA breaks, PARP-1 or PARP-2 binds to single and double DNA nicks.Since radiotherapy and many chemotherapy approaches for cancer treatment act by inducing DNA damage, PARP inhibitors are useful as chemotherapy and radiosensitizers for cancer treatment.In addition to being involved in DNA repair, PARP can also act as a mediator of cell death.Since PARP functions by degrading NAD+ into nicotinamide and ADP-ribose to form ADP-ribose polymers, PARP activation can lead to a large depletion of intracellular NAD+, which can lead to cell death.

[0120] Some cancers depend on PARP activity for DNA repair, and therefore PARP inhibitors have been developed and used as cancer therapeutics.Exemplary PARP inhibitors include olaparib, rucaparib, niraparib, talazoparib, veliparib, and pamiparib.In an embodiment, the present disclosure provides novel PARP inhibitors that are synthetically produced small molecules and / or therapeutic peptides.In certain embodiments, the PARP inhibitors provided herein can be used together with CAR-expressing cell therapy, for example, in cancer therapy.

[0121] In certain aspects, the therapeutic peptides provided herein are SMAC mimetics. Secondary mitochondrial-derived activator of caspases (SMAC) is a proapoptogenic mitochondrial protein that is released in the cytosol in response to certain apoptotic response stimuli. When present in the cytosol, SMAC interacts with and antagonizes inhibitors of apoptosis proteins (IAPs), leading to apoptosis through the release of caspases. Thus, SMAC sensitizes tumor cells to death by apoptosis. In some cancer cells with high IAP activity, SMAC activity can induce apoptosis on its own. In some cancer cells, SMAC increases the apoptotic effect of cell death factors such as TNF-related apoptosis-inducing ligand (TRAIL). Without wishing to be bound by theory, the SMAC pathway is particularly suitable for combination with CAR-T cell therapy, since activated CAR-T cells induce apoptosis of tumor cells, in part, through cell death inducers such as TRAIL. In certain aspects, the present disclosure provides novel mimetics of SMAC. In certain embodiments, SMAC mimetics can be used in conjunction with CAR-expressing cell therapy, e.g., in cancer therapy, to induce cancer cell death.

[0122] Short-chain fatty acids (SCFAs) are another category of immune modulators. SCFAs are free fatty acids containing less than six carbons. They are produced by the gut microbiota as bacterial fermentation products and include formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and 2-methylbutanoic acid. SCFAs can regulate immune system differentiation and function by binding to metabolite-sensing G protein-coupled receptors (e.g., GPR41, GPR43, and GPR109A) expressed on intestinal epithelial cells, adipocytes, myeloid lineage cells, and / or other cells located in the gut. SCFAs also function through HDAC inhibitor pathways. It has been reported that SCFAs have anti-inflammatory activity in regulating intestinal inflammation and thus help suppress chronic intestinal inflammatory responses, for example, by affecting T cell differentiation and cytokine production indirectly via macrophages, DCs, and other cells. SCFAs have been shown to promote the production of cytokines such as IL-10 and / or IL-1β, and therefore there is interest in utilizing the effects of SCFAs in inflammatory bowel disease (IBD).However, there are challenges with the delivery of SCFAs and the repeated administration that may be required to initiate and / or maintain their effects.In certain embodiments, the present disclosure provides a method for the delivery of SCFA mimics via CAR-expressing cell therapy.An important advantage of the delivery of SCFA mimics via CAR-expressing cell therapy provided herein is the persistent expression of SCFA mimics in the intestinal microenvironment (e.g., by CAR-expressing cells).

[0123] In certain embodiments, the present disclosure provides engineered cells comprising a CAR construct comprising a CAR and a therapeutic peptide that is a SCFA mimetic. In certain embodiments, the present disclosure provides a method for treating a disorder of the gastrointestinal system, comprising administering to a subject in need thereof an engineered cell provided herein comprising a CAR and a therapeutic peptide that is a SCFA mimetic. In certain embodiments, the present disclosure provides a method for treating an inflammatory disorder, comprising administering to a subject in need thereof an engineered cell provided herein comprising a CAR and a therapeutic peptide that is a SCFA mimetic. For example, in certain embodiments, the inflammatory disorder is an inflammatory bowel disease (IBD), such as ulcerative colitis or Crohn's disease. In such situations, it may be desirable to reduce inflammation that may be induced by other engineered cells. Thus, in certain embodiments, the engineered cells are further modified to knock out and / or reduce the expression or activity of one or more effector functions. For example, in certain embodiments, the engineered cells have been modified to knock out or reduce expression of one or more genes required for inflammatory cytokine expression, granzyme B expression, perforin expression, and / or other effector functions associated with inflammation.

[0124] In certain embodiments, the therapeutic peptides of the present disclosure are mimics of steroids, hormones, and / or hormone-like molecules. For example, in certain embodiments, the therapeutic peptides are mimics of steroids or hormones used in the therapy of diseases or disorders. Exemplary diseases and disorders include growth deficiency disorders, thyroid disorders, infertility, and cancers such as breast cancer and prostate cancer. Exemplary hormones or hormone-like molecules of the therapeutic peptides provided herein can be human growth hormone, luteinizing hormone releasing hormone (LHRH) antagonist, estrogen, estradiol, and progesterone. Exemplary steroids include betamethasone, hydrocortisone, methylprednisolone, prednisolone, and triamcinolone. In certain embodiments, it is desired to reduce inflammation or avoid excessive inflammation in diseases and disorders that are amenable to treatment with steroids, hormones, and / or hormone-like molecules. In such embodiments, the engineered cells may be further modified to knock out and / or reduce the expression or activity of one or more effector functions (e.g., to knock out or reduce expression of one or more genes required for inflammatory cytokine expression, granzyme B expression, perforin expression, and / or other effector functions associated with inflammation).

[0125] D. Chimeric Antigen Receptor The present disclosure provides compositions and methods for engineered cells (e.g., immune cells or their precursors, e.g., T cells) that contain chimeric antigen receptors (CARs) and therapeutic peptides. Thus, in some embodiments, immune cells are genetically modified to express CARs. The CARs of the present disclosure include an antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0126] The antigen binding domain can be operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, both of which are described elsewhere herein, for expression in a cell. In one embodiment, a first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding the transmembrane domain, which is further operably linked to a third nucleic acid sequence encoding the intracellular domain.

[0127] The antigen-binding domain described herein can be combined with any of the transmembrane domains described herein, any of the intracellular or cytoplasmic domains described herein, or any of the other domains described herein that can be included in the CAR of the present disclosure.The subject CAR of the present disclosure can also include a hinge domain as described herein.The subject CAR of the present disclosure can also include a spacer domain as described herein.In some embodiments, each of the antigen-binding domain, the transmembrane domain, and the intracellular domain is separated by a linker.

[0128] The present invention should be constructed to include any CAR known in the art and / or disclosed herein.Exemplary CARs include, but are not limited to, those disclosed herein, US8916381B1, US9394368B2, US20140050708A1, US9598489B2, US9365641B2, US20210079059A1, US9783591B2, WO2016028896A1, US9446105B2, WO2016014576A1, US20210284752A1, WO2016014565A2, WO2016014535A1 and US9272002B2, and any other CARs generally disclosed in the art.

[0129] Antigen-binding domain The antigen-binding domain of CAR is the extracellular region of CAR for binding to specific target antigens, including proteins, carbohydrates and glycolipids.In some embodiments, CAR comprises affinity for target antigens on target cells.Target antigens can comprise any kind of protein or epitope thereof associated with target cells.For example, CAR can comprise affinity for target antigens on target cells that indicate a certain disease state of target cells.

[0130] In certain embodiments, the target cell antigen is a tumor-associated antigen (TAA).Examples of tumor-associated antigens (TAA) include, but are not limited to, differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS. In preferred embodiments, the antigen binding domain of the CAR targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, PSCA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.

[0131] In certain embodiments, the target antigen is selected from the group consisting of CD19, CD20, CD22, BCMA, CD123, CD133, EGFR, EGFRvIII, mesothelin, Her2, PSMA, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA 125, CTAG1B, mucin 1, and folate receptor-alpha.

[0132] In certain aspects, the target antigen is expressed on intestinal cells.

[0133] Depending on the desired antigen to be targeted, the CAR can be engineered to contain an appropriate antigen-binding domain specific to the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as the antigen-binding moiety for incorporation into the CAR.

[0134] In one embodiment, the target cell antigen is CD19. Thus, in one embodiment, the CAR of the present disclosure has affinity for CD19 on target cells. This should not be interpreted as limiting in any way, as long as CARs with affinity for any target antigen are suitable for use in the composition or method of the present disclosure.

[0135] As described herein, the CAR of the present disclosure having affinity for a specific target antigen on a target cell can include a target-specific binding domain. In some embodiments, the target-specific binding domain is a murine target-specific binding domain, e.g., the target-specific binding domain is of murine origin. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin. In one embodiment, the CAR of the present disclosure having affinity for CD19 on a target cell can include a CD19 binding domain.

[0136] In some embodiments, the CAR of the present disclosure may have affinity for one or more target antigens on one or more target cells. In some embodiments, the CAR may have affinity for one or more target antigens on a target cell. In such embodiments, the CAR is a bispecific or multispecific CAR. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR that comprises one or more target-specific binding domains that have affinity for the same target antigen can also bind to completely different epitopes of the target antigen. When there are multiple target-specific binding domains in a CAR, the binding domains may be arranged tandemly or separated by a linker peptide. For example, in a CAR that comprises two target-specific binding domains, the binding domains are covalently connected to each other on a single polypeptide chain through an oligo- or polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0137] In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), and a single chain variable fragment (scFv). In some embodiments, the CD19 binding domain of the present disclosure is selected from the group consisting of a CD19-specific antibody, a CD19-specific Fab, and a CD19-specific scFv. In one embodiment, the CD19 binding domain is a CD19-specific antibody. In one embodiment, the CD19 binding domain is a CD19-specific Fab. In one embodiment, the CD19 binding domain is a CD19-specific scFv.

[0138] The antigen-binding domain can include any domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. In some embodiments, the antigen-binding domain portion comprises a mammalian antibody or a fragment thereof. The choice of antigen-binding domain can depend on the type and number of antigens present on the surface of the target cell.

[0139] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein in which the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin (e.g., mouse or human) are covalently linked to form a VH::VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked by a peptide-encoded linker connecting the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., CD19 binding domain) comprises an scFv having, from the N-terminus to the C-terminus, the VH-linker-VL configuration. In some embodiments, the antigen-binding domain comprises an scFv having, from the N-terminus to the C-terminus, the VL-linker-VH configuration. Those skilled in the art will be able to select a suitable configuration for use in the present disclosure.

[0140] The linker is usually glycine-rich for flexibility, as well as serine- or threonine-rich for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. (GS) n , (GSGGS) n (SEQ ID NO:17), (GGGS) n (SEQ ID NO:18), and (GGGGS) nA variety of linker sequences are known in the art, including, but not limited to, glycine serine (GS) linkers such as (SEQ ID NO:19), where n represents an integer of at least 1. Exemplary linker sequences include: The amino acid sequence of the linker may include, but is not limited to, TIFF2024511109000002.tif19153. One of skill in the art will be able to select a linker sequence suitable for use in the present invention. In one embodiment, the antigen binding domain of the present invention (e.g., PSMA binding domain) comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are linked to the nucleic acid sequence Amino acid sequence that can be encoded by TIFF2024511109000003.tif4162 It is separated by a linker sequence having TIFF2024511109000004.tif4128.

[0141] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from nucleic acids containing VH and VL coding sequences as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 136(8):2252-61). 2 (10:31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0142] As used herein, "Fab" refers to the fragment of an antibody structure that binds an antigen but is monovalent and does not have the Fc portion; for example, digestion of an antibody with the enzyme papain results in two Fab fragments and one Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind antigen).

[0143] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, where the fragment has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains linked by S-S bonds, a portion of a heavy chain for binding to antigen and a light (L) chain, with the remaining heavy chain portions linked together. The "F(ab')2" fragment can be split into two separate Fab' fragments.

[0144] In some embodiments, the antigen binding domain may be derived from the same species as the species that the CAR will ultimately be used in. For example, for use in humans, the antigen binding domain of the CAR may comprise a human antibody or a fragment thereof. In some embodiments, the antigen binding domain may be derived from a different species that the CAR will ultimately be used in. For example, for use in humans, the antigen binding domain of the CAR may comprise a mouse antibody or a fragment thereof.

[0145] Exemplary antigen binding domains include, but are not limited to, those found in US8916381B1, US9394368B2, US20140050708A1, US9598489B2, US9365641B2, US20210079059A1, US9783591B2, WO2016028896A1, US9446105B2, WO2016014576A1, US20210284752A1, WO2016014565A2, WO2016014535A1, US9272002B2, and any antigen binding domain from any CAR generally disclosed in the art. Further exemplary antigen binding domains are those having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:41, 42 or 43.

[0146] TIFF2024511109000005.tif129165

[0147] Transmembrane domain The CAR of the present disclosure may comprise a transmembrane domain that connects the antigen-binding domain of the CAR with the intracellular domain of the CAR. The transmembrane domain of the subject CAR is a region that can penetrate the plasma membrane of a cell (e.g., an immune cell or its precursor). In some embodiments, the transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is sandwiched between the antigen-binding domain and the intracellular domain of the CAR.

[0148] In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains with the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

[0149] The transmembrane domain may be derived from either natural or synthetic sources.When the source is natural, the domain can be derived from any membrane-binding or transmembrane protein, for example, type I transmembrane protein.When the source is synthetic, the transmembrane domain can be any artificial sequence, for example, artificial hydrophobic sequence, that facilitates the insertion of CAR into cell membrane. Examples of transmembrane domains of particular use in the present disclosure include, but are not limited to, transmembrane domains derived from (i.e., comprising at least the transmembrane region of) the alpha, beta or zeta chains of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9. In some embodiments, the transmembrane domain may be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine. Preferably, a triad of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is selected from the group consisting of CD8 alpha, CD3 zeta, CD3 epsilon, CD28, and ICOS.

[0150] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that can be included in the subject CAR.

[0151] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the subject of the present disclosure may also comprise a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component of the CAR. The hinge region may comprise a domain selected from an antibody Fc fragment, an antibody hinge region, an antibody CH2 region, an antibody CH3 region, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, the CD8a hinge, an artificial hinge composed of a polypeptide that may be as small as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0152] In some embodiments, the CAR of the present disclosure includes a hinge region that connects the antigen binding domain to the transmembrane domain, which then connects it to the intracellular domain. The hinge region is preferably capable of assisting the antigen binding domain in recognizing and binding to the target antigen on the target cell (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, which allows the antigen binding domain to have a structure that optimally recognizes the specific structure and density of the target antigen on a cell, such as a tumor cell (Hudecek et al., supra). The flexibility of the hinge region allows it to adopt many different conformations.

[0153] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region).

[0154] The hinge region can have a length of about 4 amino acids to about 50 amino acids, for example, about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of more than 5 aa, more than 10 aa, more than 15 aa, more than 20 aa, more than 25 aa, more than 30 aa, more than 35 aa, more than 40 aa, more than 45 aa, more than 50 aa, more than 55 aa, or more.

[0155] A suitable hinge region can be readily selected and can be any of a number of suitable lengths, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of more than 20 amino acids (e.g., 30, 40, 50, 60, or more).

[0156] For example, the hinge region may be a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 17) and (GGGS) n(SEQ ID NO:18), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and therefore can serve as neutral tethers between components. Glycine polymers can be used; glycine has significantly more access to the φ-ψ space than alanine and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO:20), GGSGG (SEQ ID NO:21), GSGSG (SEQ ID NO:22), GSGGG (SEQ ID NO:23), GGGSG (SEQ ID NO:24), GSSSG (SEQ ID NO:25), and the like.

[0157] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. The amino acid sequences of immunoglobulin hinge regions are known in the art; see, for example, Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As a non-limiting example, an immunoglobulin hinge region may have the following amino acid sequence: TIFF2024511109000006.tif12128 (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); TIFF2024511109000007.tif34165, etc.

[0158] The hinge region can comprise the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 hinge region. In one embodiment, the hinge region can comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge can be replaced by Tyr, thereby causing the hinge region to have the sequence see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In certain embodiments, the hinge region can comprise an amino acid sequence derived from human CD8 or a variant thereof.

[0159] Intracellular signaling domains The subject CAR of the present disclosure also comprises an intracellular signaling domain. The terms "intracellular signaling domain" and "intracellular domain" are used interchangeably herein. The intracellular signaling domain of a CAR is responsible for activating at least one effector function of the cell (e.g., immune cell) in which the CAR is expressed. The intracellular signaling domain transmits an effector function signal and instructs the cell (e.g., immune cell) to carry out its specialized function, such as damaging and / or destroying a target cell.

[0160] Examples of intracellular domains for use in the present disclosure include, but are not limited to, the cytoplasmic portion of a surface receptor, a costimulatory molecule, and any molecule that acts in concert to initiate signaling in a T cell, as well as any derivatives or variants of these elements, and any synthetic sequences having the same functional capabilities.

[0161] Examples of intracellular signaling domains include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologs, such as the η chain, FcsRI γ and β chains, MB 1 (Iga) chain, B29 (Ig) chain, human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell signaling, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain can be human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.

[0162] In certain embodiments, the intracellular signaling domain of the CAR comprises at least one signaling domain derived from any portion of one or more costimulatory molecules, e.g., CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0163] Other examples of intracellular domains include TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma Rila, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT These include fragments or domains derived from one or more molecules or receptors, including, but not limited to, AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0164] Additional examples of intracellular domains include the intracellular signaling domains of several different other immune signaling receptors, including but not limited to, first, second and third generation T cell signaling proteins, including, but not limited to, CD3, B7 family costimulatory receptors and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, the intracellular signaling domain can include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), such as the signaling domains of NKp30(B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0165] In certain embodiments, the intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, TCR zeta, CD3 epsilon, CD3 gamma, CD3 delta, or ICOS. In certain embodiments, the intracellular signaling domain comprises a functional signaling domain and further comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain from 4-1BB or CD28.

[0166] Intracellular signaling domains suitable for use in the subject CARs of the present disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; altered transcription of a target gene; altered activity of a protein; altered cellular behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of a cell signaling response, etc.) in response to activation of the CAR (i.e., activated by an antigen and a dimerization agent). In some embodiments, the intracellular signaling domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane-bound CAR, but is instead diffused in the cytoplasm.

[0167] The intracellular signaling domain suitable for use in the subject CAR of the present disclosure comprises an immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptide. In some embodiments, the ITAM motif is repeated twice within the intracellular signaling domain, where the first and second instances of the ITAM motif are separated from each other by 6-8 amino acids. In one embodiment, the intracellular signaling domain of the subject CAR comprises three ITAM motifs.

[0168] In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0169] Suitable intracellular signaling domains can be ITAM motif-containing moieties derived from ITAM motif-containing polypeptides. For example, suitable intracellular signaling domains can be ITAM motif-containing domains derived from any ITAM motif-containing protein. Therefore, suitable intracellular signaling domains do not need to contain the entire sequence of the entire protein from which they are derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0170] In one aspect, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX activating protein 12; KAR associated protein; TYRO protein tyrosine kinase binding protein; killer activating receptor associated protein; killer activating receptor associated protein, etc.). In one aspect, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity gamma chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one aspect, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.).In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR comprises the cytoplasmic signaling domain of human CD3 zeta.

[0171] Typically, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain so long as it transmits an effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0172] The intracellular signaling domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that can be included in a CAR.

[0173] E. Engineered cells The present disclosure provides engineered cells (e.g., modified immune cells or their precursors; e.g., T cells) that comprise chimeric antigen receptors (CARs) and therapeutic peptides. In certain embodiments, the therapeutic peptides are non-natural therapeutic peptides. In certain embodiments, the CAR molecule and the therapeutic peptides are expressed from the same expression construct. Also provided are cells that comprise any of the nucleic acid molecules envisaged herein or any of the expression vectors envisaged herein.

[0174] In certain aspects, the cells comprise a therapeutic peptide having one or more of the following properties: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway, (ii) the therapeutic peptide is a mimic of a cyclic dinucleotide, (iii) the therapeutic peptide is a mimic of a short chain fatty acid (SCFA), (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR), (v) the therapeutic peptide is a mimic of a steroid / hormone-like molecule, (vi) the therapeutic peptide promotes apoptosis of the target cell, (vii) the therapeutic peptide is an immunogenic epitope, and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in the target cell.

[0175] In certain embodiments, the therapeutic peptide is a mimic of cGAMP, cAMP, or cGMP. In certain embodiments, the therapeutic peptide is a mimic of a TLR agonist. In certain embodiments, the therapeutic peptide is a mimic of flagellin, CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL). In certain embodiments, the therapeutic peptide is a second mitochondria-derived caspase activator (SMAC) mimic. In certain embodiments, the therapeutic peptide is an inhibitor of poly ADP-ribose polymerase (PARP). In certain embodiments, the therapeutic peptide is a mimic of a SCFA that binds to a G protein-coupled receptor (GPCR). In certain embodiments, the therapeutic peptide is a SCFA mimic or a mimic of a steroid and / or hormone-like molecule, where the engineered cells are further modified to reduce the activity of one or more effector functions.

[0176] In certain embodiments, the non-naturally occurring peptide is a peptide having 90% or less sequence identity to a naturally occurring peptide. In certain embodiments, the non-naturally occurring peptide is a peptide having 80% or less sequence identity to a naturally occurring peptide. In certain embodiments, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-16.

[0177] In certain embodiments, the therapeutic peptide is excreted from the engineered cell into extracellular vesicles. In certain embodiments, the target cell is a tumor cell. In certain embodiments, the engineered cell is a T cell or a NK cell.

[0178] In certain embodiments, the engineered cells are modified to reduce or prevent expression of one or more inflammatory cytokines, expression of granzyme B, or expression of perforin.

[0179] In certain embodiments, the CAR molecule and the therapeutic peptide are expressed from the same expression construct, wherein the expression construct further comprises an RNA molecule that activates a PRR. In certain embodiments, the RNA molecule is 7SL.

[0180] Thus, provided are cells, compositions and methods that enhance immune cell (e.g., T cell) function in adoptive cell therapy, such as by enhancing the activity and efficacy of administered engineered cells, while maintaining the persistence or exposure of transferred cells over time, e.g., providing improved efficacy. In some embodiments, the engineered cells exhibit enhanced expansion and / or persistence when administered in vivo to a subject, compared to certain available methods. In some embodiments, the provided cells exhibit enhanced persistence when administered in vivo to a subject. In some embodiments, the persistence of the engineered cells in a subject when administered is greater than the persistence that would be achieved by alternative methods, e.g., methods in which T cells are administered engineered cells without CAR and therapeutic peptide. In some embodiments, persistence is increased by at least or about at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 60, 70, 80, 90, 100 or more times.

[0181] In some embodiments, the degree or extent of persistence of the administered cells can be detected or quantified after administration to a subject. For example, in some aspects, quantitative PCR (qPCR) is used to assess the amount of cells expressing the CAR and therapeutic peptide in the blood or serum or organ or tissue (e.g., disease site) of a subject. In some aspects, persistence is quantified as copies of DNA or plasmid encoding the exogenous receptor per microgram of DNA, or as the number of receptor-expressing cells per microliter of sample, e.g., blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of sample. In some embodiments, flow cytometry assays can also be performed to detect cells expressing the receptor, typically using antibodies specific for the receptor. Cell-based assays can also be used to detect the number or percentage of functional cells, e.g., cells capable of binding to and / or neutralizing disease or pathological cells or cells expressing an antigen recognized by the receptor, and / or inducing a response, e.g., a cytotoxic response, against the cells. In any such embodiment, the range or level of expression of another marker associated with the CAR can be used to distinguish between the administered cells and endogenous cells in the subject.

[0182] F. Cell Source In some embodiments, the source of cells (e.g. immune cells; e.g. T cells) for ex vivo manipulation is obtained from a subject. The source of target cells for ex vivo manipulation can also include, for example, autologous or heterologous donor blood, umbilical cord blood, or bone marrow. For example, the source of immune cells can be derived from the subject to be treated with the modified immune cells of the present disclosure, and can be, for example, the subject's blood, the subject's umbilical cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the subject is a human.

[0183] Immune cells can be obtained from several sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of innate or adaptive immunity, such as lymphocytes, typically myeloid or lymphoid cells, including T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and multipotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. The cells are typically primary cells, such as primary cells that are directly isolated from the subject and / or isolated and frozen from the subject.

[0184] In certain embodiments, the cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), or a dendritic cell. In some embodiments, the cell is a monocyte or a granulocyte, e.g., a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil. In one embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell that has been generated from a subject and engineered to alter (e.g., induce mutations in) or manipulate expression of one or more target genes, e.g., differentiated into a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0185] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturation, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, 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, mucosal-associated invariant T (MAIT) cells, innate 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. In certain embodiments, any number of T cell lines available in the art may be used.

[0186] In some embodiments, the method includes steps of isolating immune cells from a subject, preparing, treating, culturing, and / or manipulating them. In some embodiments, the preparation of the engineered cells includes one or more culturing and / or preparation steps. The cells for manipulation as described may be isolated from a sample, such as a biological sample, for example a sample obtained or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject with a disease or condition, or a subject in need of or to whom cell therapy will be administered. The subject is, in some embodiments, a human in need of a particular therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, treated, and / or manipulated. Thus, the cells are, in some embodiments, primary cells, for example, primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissues, and organ samples, including processed samples derived therefrom.

[0187] In some aspects, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product.Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom.Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0188] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from a heterologous source, e.g., mouse, rat, non-human primate, and pig. In some embodiments, the isolation of cells includes one or more preparation steps and / or cell separation steps that are not based on affinity. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove undesired components, enrich for desired components, and lyse or remove cells that are sensitive to a particular reagent. In some examples, the cells are separated based on one or more properties, such as density, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0189] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects cells other than red blood cells and platelets. In some embodiments, blood cells collected from a subject are washed, for example to remove the plasma fraction and place the cells in a buffer or medium suitable for the subsequent processing step. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in the medium. In some embodiments, the method includes a density-based cell separation method, such as preparation of white blood cells from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

[0190] In one embodiment, the immune cells are cells obtained from the circulating blood of an individual obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and place the cells in a suitable buffer or medium, such as phosphate-buffered saline (PBS), or the washing solution may be calcium-free and magnesium-free, or may be free of many, if not all, divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, etc. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0191] In some embodiments, the isolation method comprises the separation of different cell types based on the expression or presence in cells of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids.In some embodiments, any known method may be used for such marker-based separation.In some embodiments, the separation is affinity or immunoaffinity-based separation.For example, in some aspects, the separation comprises the separation of cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such marker, generally followed by a washing step, and separation of the cells that bind to the antibody or binding partner from the cells that do not bind to the antibody or binding partner.

[0192] Such separation steps can be based on positive selection, where cells bound to the reagent are retained for further use, and / or negative selection, where cells that are not bound to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, such that separation is best performed based on markers expressed by cells other than the desired population. Separation does not need to result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to increasing the number or percentage of such cells, but does not need to result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, refers to decreasing the number or percentage of such cells, but does not need to result in the complete removal of all such cells.

[0193] In some examples, multiple rounds of separation steps are performed, with the positively or negatively selected fraction from one step being subjected to another separation step, such as a subsequent positive or negative selection. In some examples, cells expressing multiple markers simultaneously can be depleted in a single separation step, such as by incubating cells with multiple antibodies or binding partners, each specific to the marker targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on various cell types.

[0194] In some embodiments, one or more of the T cell populations are positive for (marker+) or express high levels of one or more particular markers, e.g., surface markers. high ) cells, or cells that are negative for one or more markers ( -) or expresses it at a relatively low level (marker low ) cells are enriched or depleted. For example, in some aspects, a particular subpopulation of T cells, e.g., cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are markers that are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels on certain other populations of T cells (e.g., memory cells). In one embodiment, the cells (CD8+ cells, or T cells, such as CD3+ cells) are enriched for cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L (i.e., positively selected for), and / or are depleted for cells that are positive for or express high surface levels of CD45RA (e.g., negatively selected for). In some embodiments, the cells are enriched for or depleted for cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In some examples, the CD8+ T cells are enriched for cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L. For example, CD3+,CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0195] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on non-T cells such as B cells, monocytes, or other white blood cells, e.g., CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively high degree on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is performed to increase efficacy, e.g., to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such subpopulations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0196] In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for CD62L-CD8+ and / or CD62L+CD8+ fractions using anti-CD8 and anti-CD62L antibodies, etc. In some embodiments, CD4+ T cell population and CD8+ T cell subpopulation, such as a subpopulation enriched for central memory (TCM) cells. In some embodiments, enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, the isolation of a CD8+ population enriched for TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, the enrichment of central memory T (TCM) cells is performed starting from a negative fraction of cells selected based on CD4 expression, which is subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are performed simultaneously in some aspects, and sequentially in either order in other aspects. In some aspects, the same selection step based on CD4 expression used to prepare a CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, whereby both the positive and negative fractions from CD4-based separation are retained and used in subsequent steps of the method, optionally following one or more additional positive or negative selection steps.

[0197] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for the separation of cells for positive and / or negative selection.

[0198] In some embodiments, the cells are incubated and / or cultured prior to or in association with the genetic manipulation. The incubation step can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the composition or cells are incubated in the presence of stimulatory conditions or stimuli. Such conditions include conditions designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic manipulation, such as the introduction of a recombinant antigen receptor. The conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents, such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulatory conditions or stimuli include one or more agents, such as ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent initiates or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such an agent can include, for example, an antibody bound to a solid support such as a bead, for example, an antibody specific for a TCR component and / or a costimulatory receptor, for example, anti-CD3, anti-CD28, and / or one or more cytokines. Optionally, the expansion method can further include adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulator includes IL-2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0199] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cord. In any event, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0200] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including but not limited to CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using isolated antibodies, biological samples containing antibodies, such as ascites fluid, antibodies bound to a physical support, or antibodies bound to cells.

[0201] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies against surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can be used to enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0202] To isolate the desired population of cells by positive or negative selection, cell concentration and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between cells and beads. For example, in one embodiment, a cell concentration of 2 billion cells / ml is used. In one embodiment, a cell concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, cell concentrations of 125 or 150 million / ml can be used. Using higher concentrations can result in increased cell yield, cell activation, and cell expansion.

[0203] T cells can also be frozen after a washing step that does not require a monocyte removal step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. Although numerous freezing solutions and parameters are known in the art and would be useful in this context, in a non-limiting example, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing medium. The cells are then frozen to -80°C at a rate of 1°C / min and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing and immediate uncontrolled freezing at -20°C or in liquid nitrogen may also be used.

[0204] In one embodiment, the population of T cells is comprised in cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified populations of T cells, and T cell lines. In another embodiment, peripheral blood mononuclear cells comprise the T cell population. In yet another embodiment, purified T cells comprise the T cell population.

[0205] In certain embodiments, regulatory T cells (Tregs) can be isolated from a sample. The sample can include, but is not limited to, umbilical cord blood or peripheral blood. In certain embodiments, Tregs are isolated by flow cytometry sorting. The sample can be enriched for Tregs before isolation by any means known in the art. The isolated Tregs can be cryopreserved and / or expanded before use. Methods for isolating Tregs are described in U.S. Patent Nos. 7,754,482, 8,722,400, and 9,555,105, and U.S. Patent Application No. 13 / 639,927, the contents of which are incorporated herein in their entirety.

[0206] G. Nucleic acids and expression vectors The present disclosure provides a nucleic acid encoding a chimeric antigen receptor (CAR) and a therapeutic peptide.Any CAR that is specifically disclosed elsewhere herein is contemplated.In certain embodiments, the therapeutic peptide is a non-natural peptide.Any therapeutic peptide that is specifically disclosed elsewhere herein is contemplated.

[0207] In certain embodiments, a stop codon separates the nucleic acid segment encoding the CAR and the nucleic acid segment encoding the therapeutic peptide.

[0208] In certain embodiments, linkers may be used to allow multiple proteins to be encoded by the same nucleic acid sequence (e.g., polycistronic or bicistronic sequences), which are translated as a polyprotein that dissociates into separate protein components. In some embodiments, the linker comprises a nucleic acid sequence that encodes an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry to a start codon, such as ATG, of a protein coding region, leading to cap-independent translation of the gene. A variety of internal ribosome entry sites are known to those skilled in the art, including but not limited to IRES available from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES available from, for example, cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select the appropriate IRES for use in the present disclosure.

[0209] In some embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide. As used herein, "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows multiple proteins to be encoded as a polyprotein, which dissociates into its constituent proteins after translation. The use of the term "self-cleavage" is not intended to imply a proteolytic cleavage reaction. A variety of self-cleaving peptides or 2A peptides are known to those skilled in the art, including, but not limited to, those found in members of the Picornaviridae virus family, such as foot and mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), Thosea asigna virus (TaV), and porcine teschovirus-1 (PTV-1); and cardioviruses such as tylovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A", "E2A", "P2A" and "T2A", respectively. One of skill in the art would be able to select an appropriate self-cleaving peptide for use in the present disclosure.

[0210] In some embodiments, the linker further comprises a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-Golgi and processes protein precursors prior to secretion. Furin cleaves at the COOH-terminus of its consensus recognition sequence. One skilled in the art will be able to select an appropriate furin cleavage site for use in the present disclosure.

[0211] In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, a linker comprising a nucleic acid sequence encoding furin and F2A, a linker comprising a nucleic acid sequence encoding furin and E2A, a linker comprising a nucleic acid sequence encoding furin and P2A, and a linker comprising a nucleic acid sequence encoding furin and T2A. Those skilled in the art will be able to select the appropriate combination for use in the present disclosure. In such embodiments, the linker may further comprise a spacer sequence between furin and the 2A peptide. A variety of spacer sequences are known in the art, including, but not limited to, glycine serine (GS) spacers, such as (GS)n, (GSGGS)n (SEQ ID NO:17) and (GGGS)n (SEQ ID NO:18), where n represents an integer of at least 1. Exemplary spacer sequences can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO:20), GGSGG (SEQ ID NO:21), GSGSG (SEQ ID NO:22), GSGGG (SEQ ID NO:23), GGGSG (SEQ ID NO:24), GSSSG (SEQ ID NO:25), etc. One of skill in the art would be able to select appropriate spacer sequences for use in the present disclosure.

[0212] In some aspects, the nucleic acids of the disclosure may be operably linked to transcriptional regulatory elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those of skill in the art.

[0213] In certain embodiments, the nucleic acid encoding the CAR is operably linked to a promoter. In certain embodiments, the promoter is a phosphoglycerate kinase-1 (PGK) promoter.

[0214] Suitable promoters for expression in bacterial cells include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. Suitable promoters for expression in eukaryotic cells include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoter; promoters present in long terminal repeats from retroviruses; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters from a first organism for use in a second organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) is well known in the art.Such reversible promoters, and systems based on such reversible promoters but also including additional regulatory proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, alcohol transactivator protein (A1cR) responsive promoter, etc.), tetracycline-regulated promoters, (e.g., promoter systems including TetActivator, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-associated regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0215] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK cell-specific promoter.For example, the CD4 gene promoter can be used; see, for example, Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416.As another example, the CD8 gene promoter can be used.NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al. Blood (2011) 117:1565.

[0216] Suitable promoters for expression in yeast cells are constitutive promoters such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, etc.; or regulatable promoters such as the GAL1 promoter, the GAL10 promoter, the ADH2 promoter, the PHOS promoter, the CUP1 promoter, the GALT promoter, the MET25 promoter, the MET3 promoter, the CYC1 promoter, the HIS3 promoter, the ADH1 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 (e.g., for use in Pichia). Selection of the appropriate vector and promoter is well within the level of one of ordinary skill in the art. Promoters suitable for use in prokaryotic host cells include, but are not limited to, the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, e.g., the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, and the like; the araBAD promoter; in vivo regulated promoters such as the ssaG promoter or related promoters (see, e.g., U.S. Patent Application Publication No. 20040131637), the pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), the nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), and the like (see, for example, Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol.(1992) 10:888-892); sigma70 promoters, e.g., the consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); stationary phase promoters, e.g., the dps promoter, the spv promoter, and the like; promoters from pathogenicity island SPI-2 (see, e.g., WO96 / 17951); actA promoters (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); rpsM promoters (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); tet promoters (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res.(1984) 12:7035). Strong promoters suitable for use in prokaryotes, such as Escherichia coli, include, but are not limited to, Trc, Tac, T5, T7, and Plambda. Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25).

[0217] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, EF-1 alpha promoter, and human gene promoters such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0218] In some embodiments, the locus or construct or transgene containing a suitable promoter is irreversibly switched via induction of an inducible system. Suitable systems for induction of irreversible switches are well known in the art, for example, induction of irreversible switches may utilize Cre-lox mediated recombination (see, for example, Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc., known in the art may be used to generate irreversibly switchable promoters. Methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein are used to generate irreversibly switched promoters and are well known in the art. See, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0219] In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR-inducing expression cassette. In one embodiment, the CAR-inducing expression cassette is for the production of a transgenic polypeptide product that is released upon CAR signaling. See, for example, Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine that is operably linked to a T cell activation response promoter. In some embodiments, the cytokine that is operably linked to a T cell activation response promoter is present on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12.

[0220] The nucleic acids of the present disclosure may be present in an expression vector and / or a cloning vector. Expression vectors may include a selection marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. Numerous suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating recombinant constructs of interest. The following vectors are provided by way of example, but should not be construed as limiting in any way: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Prokaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0221] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655); adeno-associated viruses (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci.USA (1993) 90: 10613-10617); SV40; herpes simplex virus; viral vectors based on human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0222] Additional expression vectors suitable for use include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid virus vectors, transposon-mediated vectors, etc. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0223] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0224] In some embodiments, an expression vector (e.g., lentiviral vector) may be used to introduce a CAR into immune cells or their precursor cells (e.g., T cells). Thus, an expression vector (e.g., lentiviral vector) of the present disclosure may comprise a nucleic acid encoding a CAR and a therapeutic peptide. In some embodiments, an expression vector (e.g., lentiviral vector) will comprise additional elements that aid in the functional expression of the CAR encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding a CAR further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation factor-1-alpha promoter (EF-1α promoter). Use of the EF-1α promoter may increase the efficiency in the expression of downstream transgenes (e.g., nucleic acid sequences encoding a CAR and a therapeutic peptide). A physiological promoter (e.g., EF-1α promoter) may be less likely to induce integration-mediated genotoxicity and may negate the ability of a retroviral vector to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those of skill in the art and may be incorporated into the vectors of the present disclosure. In some embodiments, the vector (e.g., lentiviral vector) further comprises non-essential cis-acting sequences that may improve titer and gene expression. A non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear transport. Other non-essential cis-acting sequences are known to those skilled in the art and may be incorporated into the vector (e.g., lentiviral vector) of the present disclosure. In some embodiments, the vector further comprises a post-transcriptional regulatory element. The post-transcriptional regulatory element may improve the translation of RNA, improve the expression of the transgene, and stabilize the RNA transcript. One example of a post-transcriptional regulatory element is the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the vector for the present disclosure further comprises a WPRE sequence.Various post-transcriptional regulator elements are known to those skilled in the art and may be incorporated into the vectors (e.g., lentiviral vectors) of the present disclosure. The vectors of the present disclosure may further comprise additional elements such as a rev response element (RRE) for transport of RNA, a packaging sequence, and 5' and 3' long terminal repeats (LTRs). The term "long terminal repeats" or "LTRs" refers to a domain of base pairs located at the end of retroviral DNA, including the U3, R and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promotion, initiation and polyadenylation of gene transcripts) and viral replication. In one embodiment, the vectors (e.g., lentiviral vectors) of the present disclosure comprise a 3' U3 deleted LTR. Thus, the vectors (e.g., lentiviral vectors) of the present disclosure may comprise any combination of elements described herein to increase the efficiency of functional expression of transgenes. For example, a vector of the disclosure (e.g., a lentiviral vector) can include a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, a 3' U3 deleted LTR' in addition to nucleic acids encoding a CAR and a therapeutic peptide.

[0225] The vector of the present disclosure can be a self-inactivating vector. The term "self-inactivating vector" as used herein refers to a vector in which the 3' LTR enhancer promoter region (U3 region) is modified (e.g., by deletion or substitution). A self-inactivating vector can prevent viral transcription beyond the first round of viral replication. As a result, a self-inactivating vector can infect and then be integrated into the host genome (e.g., mammalian genome) only once, but cannot be further passaged. Therefore, a self-inactivating vector can greatly reduce the risk of generating replication-competent virus.

[0226] In some embodiments, the nucleic acid of the present disclosure can be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled in the art; any known method can be used to synthesize RNA comprising sequences encoding the CAR and therapeutic peptide of the present disclosure. Methods for introducing RNA into host cells are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising nucleotide sequences encoding the CAR and therapeutic peptide of the present disclosure into host cells can be performed in vitro or ex vivo or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated with RNA comprising nucleotide sequences encoding the CAR and therapeutic peptide of the present disclosure in vitro or ex vivo.

[0227] To evaluate the expression of a polypeptide or a portion thereof, the expression vector to be introduced into cells may also contain one or both of a selection marker gene or a reporter gene to facilitate the identification and selection of expressing cells from the cell population to be transfected or infected by the viral vector.In some embodiments, the selection marker may be carried on a separate piece of DNA and used in a co-transfection procedure.Both the selection marker and the reporter gene may be adjacent to the appropriate regulatory sequence to allow expression in the host cell.Useful selection markers include, but are not limited to, antibiotic resistance genes.

[0228] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by recipient organisms or tissues, and code for polypeptides whose expression is manifested by some easily detectable properties, such as enzymatic activity. The expression of reporter genes is evaluated at a suitable time after DNA is introduced into recipient cells. Suitable reporter genes may include, but are not limited to, luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes that code for secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0229] H. Treatment Method The engineered cells described herein (e.g., T cells comprising CAR and therapeutic peptide) can be included in a composition for immunotherapy. The composition can include a pharmaceutical composition and can further include a pharma- ceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising engineered T cells can be administered.

[0230] In one aspect, the present disclosure provides a method for adoptive cell transfer therapy comprising administering to a subject in need thereof an engineered T cell of the present disclosure. In another aspect, the present disclosure provides a method for treating a disease or condition in a subject comprising administering to a subject in need thereof a population of engineered T cells.

[0231] Methods for administration of immune cells for adoptive cell therapy are known in the art and may be used with the provided methods and compositions.For example, adoptive T cell therapy is described in, for example, Gruenberg et al., US Patent Application Publication No. 2003 / 0170238; Rosenberg, US Patent No. 4,690,915; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85).See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments, cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, where cells are isolated and / or otherwise prepared from the subject who will receive cell therapy or a sample derived from such a subject.Thus, in some aspects, cells are derived from a subject, e.g., a patient, in need of treatment, and the cells are administered to the same subject after isolation and processing.

[0232] In some embodiments, cell therapy, such as adoptive T cell therapy, is carried out by allogeneic transfer, in which cells are isolated and / or otherwise prepared from a subject other than the subject that will or will eventually receive cell therapy, such as the first subject.In such embodiments, the cells are then administered to a different subject of the same species, such as the second subject.In some embodiments, the first and second subjects are genetically identical.In some embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0233] In some embodiments, the subject has been treated with a disease or condition, e.g., a tumor-targeting therapeutic agent, prior to administration of the cell or cell-containing composition. In some aspects, the subject is refractory or non-responsive to other therapeutic agents. In some embodiments, the subject has persistent or recurrent disease after treatment with another therapeutic intervention, e.g., chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, administration effectively treats the subject even though the subject has become resistant to another therapy.

[0234] In some embodiments, the subject is responsive to another therapeutic agent, and treatment with the therapeutic agent reduces the disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits recurrence of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk of relapse, e.g., at high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of relapse or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0235] In some embodiments, the subject has persistent or recurrent disease after treatment with another therapeutic intervention, including, for example, chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, administration effectively treats the subject despite the subject having become refractory to another therapy.

[0236] The engineered cells of the present disclosure can be administered to animals, preferably mammals, and even more preferably humans, to treat cancer. In addition, the cells of the present disclosure can be used for the treatment of any condition related to cancer, particularly cell-mediated immune response against tumor cells, when it is desirable to treat or alleviate the disease. The types of cancer to be treated by the modified cells or pharmaceutical compositions of the present disclosure include carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid tumors, benign and malignant tumors, and malignant diseases, such as sarcomas, carcinomas, and melanomas. Other exemplary cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancer can be a non-solid tumor (such as a blood tumor) or a solid tumor. Adult tumors / cancers and pediatric tumors / cancers are also included. In one embodiment, the cancer is a solid tumor or a hematological tumor. In one embodiment, the cancer is a type of carcinoma. In one aspect, the cancer is a sarcoma. In one aspect, the cancer is a leukemia. In one aspect, the cancer is a solid tumor.

[0237] A solid tumor is an abnormal mass of tissue that does not usually contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcoma and carcinoma, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovium, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphatic tumors, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, and sebaceous gland carcinoma. Cancers include, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors, such as gliomas (such as brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases.

[0238] Carcinomas amenable to treatment by the methods disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma including transitional cell carcinoma (malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma including small cell lung carcinoma and non-small cell lung carcinoma, adrenal cortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0239] Sarcomas that may be treated by the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0240] In certain exemplary embodiments, the modified immune cells of the present disclosure are used to treat myeloma or myeloma-related conditions. Examples of myeloma or myeloma-related conditions include, but are not limited to, light chain myeloma, non-secretory myeloma, monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma (e.g., solitary plasmacytoma, multiple solitary plasmacytoma, extramedullary plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, the method of the present disclosure is used to treat multiple myeloma. In one embodiment, the method of the present disclosure is used to treat refractory myeloma. In one embodiment, the method of the present disclosure is used to treat relapsed myeloma.

[0241] In certain exemplary aspects, the modified immune cells of the present disclosure are used to treat melanoma or melanoma-related conditions. Examples of melanoma or melanoma-related conditions include, but are not limited to, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentigo melanoma, amelanotic melanoma, or cutaneous melanoma (e.g., cutaneous melanoma, ocular melanoma, vulvar melanoma, vaginal melanoma, rectal melanoma). In one aspect, the method of the present disclosure is used to treat cutaneous melanoma. In one aspect, the method of the present disclosure is used to treat refractory melanoma. In one aspect, the method of the present disclosure is used to treat recurrent melanoma.

[0242] In yet another exemplary embodiment, the modified immune cells of the present disclosure are used to treat sarcomas or sarcoma-related conditions. Examples of sarcomas or sarcoma-related conditions include, but are not limited to, angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma. In one embodiment, the method of the present disclosure is used to treat synovial sarcoma. In one embodiment, the method of the present disclosure is used to treat liposarcoma, such as myxoid / round cell liposarcoma, differentiated / dedifferentiated liposarcoma, and pleomorphic liposarcoma. In one embodiment, the method of the present disclosure is used to treat myxoid / round cell liposarcoma. In one embodiment, the method of the present disclosure is used to treat refractory sarcomas. In one embodiment, the method of the present disclosure is used to treat recurrent sarcomas.

[0243] The cells of the present disclosure to be administered can be autologous to the subject undergoing treatment.

[0244] Administration of the cells of the present disclosure may be performed by any convenient method known to those skilled in the art. The cells of the present disclosure may be administered to a subject by aerosol inhalation, injection, oral ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In other examples, the cells of the present disclosure are directly injected into a site of inflammation in a subject, a site of local disease in a subject, a lymph node, an organ, a tumor, etc.

[0245] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types. Thus, the dosage of cells is based in some embodiments on the total number of cells (or number per kg body weight) and the desired ratio of individual populations or subtypes, such as the ratio of CD4+ to CD8+. In some embodiments, the dosage of cells is based on the desired total number of cells (or number per kg body weight) in an individual population or of an individual cell type. In some embodiments, the dosage is based on a combination of such features, such as the desired total number of cells, the desired ratio, and the desired total number of cells in an individual population.

[0246] In some embodiments, CD8 + T cells and CD4 + A population or subtype of cells, such as T cells, is administered at or within an acceptable variance of a desired dose of total cells, e.g., a desired dose of T cells. In some aspects, the desired dose is a desired number of cells, or a desired number of cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or exceeds a minimum number of cells or a minimum number of cells per unit body weight. In some aspects, among the total cells administered at a desired dose, individual populations or subtypes are administered at or above a desired output ratio (CD4 + vs. CD8 + (e.g., a ratio of 0.1 to 0.5), e.g., within a certain allowed variation or error of such ratio.

[0247] In some embodiments, the cells are administered at or within a tolerable difference of the desired dose of one or more of the individual populations or subtypes of cells, e.g., the desired dose of CD4+ cells and / or the desired dose of CD8+ cells. In some aspects, the desired dose is the desired number of cells of a subtype or population, or the desired number of such cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is or exceeds the minimum number of cells of a population or subtype, or the minimum number of cells of a population or subtype per unit body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose and a desired ratio of total cells, and / or based on one or more, e.g., each, of the individual subtypes or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed dose or minimum dose of T cells and CD4 + Cells vs. CD8 + Based on the desired ratio of cells and / or CD4 + Cells and / or CD8 + Based on a desired fixed or minimum dose of cells.

[0248] In certain embodiments, a distinct population of cells, or subtypes of cells, is in the range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 ... 0 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and optionally about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges.

[0249] In some embodiments, the dose of total cells and / or the dose of individual subpopulations of cells is greater than or equal to 1×10 cells. 5 pcs or approx. 1×10 5 pieces / kg ~ approx. 1×10 11 cells / kg, 10 cells 4 ~10 pieces 11 Pieces or about 10 11 pieces / kilogram (kg) body weight, e.g., 10 cells 5 ~10 6 In the range of 1 x 10 cells / kg body weight, for example, 5 pcs or approx. 1×10 5 cells / kg, cells 1.5×10 5 cells / kg, cells 2×10 5 cells / kg, or 1 x 10 cells 6 For example, in some embodiments, the cells are 10 T cells / kg body weight. 4 Pieces or about 10 4 ~10 pieces 9 Pieces or about 109 cells / kilogram (kg) body weight, e.g., 10 T cells 5 ~10 pieces 6 / kg body weight or within a certain margin of error, e.g., 1 x 10 T cells 5 pcs or approx. 1×10 5 cells / kg, T cells 1.5×10 5 cells / kg, T cells 2×10 5 cells / kg, or 1 x 10 T cells 6 In other exemplary embodiments, the dosage range of modified cells suitable for use in the methods of the present disclosure is about 1×10 cells / kg body weight. 5 pcs / kg~cells approx. 1×10 6 cells / kg, approximately 1×10 cells 6 pcs / kg~cells approx. 1×10 7 cells / kg, approximately 1×10 cells 7 cells / kg~cells approx. 1×10 8 cells / kg, approximately 1×10 cells 8 pcs / kg~cells approx. 1×10 9 cells / kg, approximately 1×10 cells 9 cells / kg~cells approx. 1×10 10 cells / kg, approximately 1×10 cells 10 cells / kg~cells approx. 1×10 11 In an exemplary embodiment, a dosage suitable for use in the methods of the present disclosure is about 1×10 cells / kg. 8 In an exemplary embodiment, a dosage suitable for use in the methods of the present disclosure is about 1×10 cells / kg. 7 In other embodiments, a suitable dosage is about 1×10 total cells / kg. 7 ~ Total cells: approx. 5 x 10 7 In some embodiments, a suitable dosage is about 1×10 total cells. 8 ~ Total cells: approx. 5 x 10 8 In some embodiments, a suitable dosage is about 1.4 x 10 total cells. 7 Total cells: approx. 1.1 x 10 9 In an exemplary embodiment, a suitable dosage for use in the methods of the present disclosure is about 7×10 total cells. 9 There are 10.

[0250] In some embodiments, the cells are 4 or about 10 4 ~10 9 or about 10 9 CD4 count / kilogram (kg) of body weight + and / or CD8 + Cells, e.g., 10 5 ~10 6 CD4 count / kg body weight + and / or CD8 + in cells, or within a certain margin of error, e.g., 1 x 10 5 Or about 1 x 10 5 CD4 cells / kg + and / or CD8 + cells, 1.5 x 10 5 CD4 cells / kg + and / or CD8 + cells, 2 x 10 5 CD4 cells / kg + and / or CD8 + cells, or 1 x 10 6 CD4 count / kg body weight + and / or CD8 + In some embodiments, the cells are administered at a concentration of about 1×10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 More than 1×10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 CD4 + cells, and / or at least about 1 x 10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 CD8 + cells, and / or at least about 1 x 10 6 pieces, approximately 2.5×106 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 In some embodiments, the cells are administered at about 10 T cells or within a certain margin of error. 8 ~10 pieces 12 Pieces or about 10 10 ~10 pieces 11 T cells, approximately 10 8 ~10 pieces 12 Pieces or about 10 10 ~10 pieces 11 CD4 + cells, and / or about 10 8 ~10 pieces 12 Pieces or about 10 10 ~10 pieces 11 CD8 + The dose is administered at or within a certain range of error in cells.

[0251] In some embodiments, the cells are administered at a desired output ratio or within a tolerated range of multiple cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, e.g., in some embodiments, the desired ratio (e.g., CD4 + Cells vs. CD8 +The ratio of cells is 5:1 or about 5:1 to 5:1 or about 5:1 (or greater than about 1:5 and less than about 5:1), or 1:3 or about 1:3 to 3:1 or about 3:1 (or greater than about 1:3 and less than about 3:1), for example, 2:1 or about 2:1 to 1:5 or about 1:5 (or greater than about 1:5 and less than about 2:1, for example, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 , or 1:5, or about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2.5:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1:1, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, or about 1:5. In some aspects, tolerated differences are within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value between these ranges.

[0252] In some embodiments, the dose of modified cells is administered to the subject in need thereof in a single dose or multiple doses.In some embodiments, the dose of modified cells is administered in multiple doses, for example, once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days.In exemplary embodiments, a single dose of modified cells is administered to the subject in need thereof.In exemplary embodiments, a single dose of modified cells is administered to the subject in need thereof by rapid intravenous infusion.

[0253] The dosage appropriate for preventing or treating a disease may depend on the type of disease being treated, the type of cells or recombinant receptor, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, prior therapy, the subject's clinical history and response to the cells, and the judgment of the attending physician. The compositions and cells are, in some embodiments, administered to a subject at one time or over a series of treatments, as appropriate.

[0254] In some embodiments, the cells are administered as part of a combination treatment, e.g., simultaneously with another therapeutic intervention, e.g., an antibody or engineered cell or receptor, or an agent, e.g., a cytotoxic or therapeutic agent, or sequentially in any order. The cells are co-administered in some embodiments simultaneously or sequentially in any order with one or more additional therapeutic agents or in association with another therapeutic intervention. In some situations, the cells are co-administered with another therapy close enough in time that the cell population enhances the effect of the one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered before the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the method includes administration of a chemotherapeutic agent.

[0255] In certain embodiments, the engineered cells of the present disclosure (e.g., modified cells comprising a CAR and a therapeutic peptide) may be administered to a subject in combination with an immune checkpoint antibody. (e.g., an anti-PD1 antibody, an anti-CTLA-4 antibody, or an anti-PDL1 antibody). For example, the modified cells may be administered in combination with, for example, an antibody or antibody fragment that targets PD-1 (programmed death 1 protein). Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly known as lambrolizumab, also known as MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®), or an antigen-binding fragment thereof. In certain embodiments, the modified cells may be administered in combination with an anti-PD-L1 antibody, or an antigen-binding fragment thereof. Examples of anti-PD-L1 antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, atezolizumab), and MEDI4736 (durvalumab, Imfinzi). In certain embodiments, the modified cells may be administered in combination with an anti-CTLA-4 antibody or an antigen-binding fragment thereof. An example of an anti-CTLA-4 antibody includes, but is not limited to, ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may be used, including but not limited to small molecules, siRNA, miRNA, and CRISPR systems. The immune checkpoint modulator may be administered before, after, or simultaneously with the modified cells comprising the CAR and therapeutic peptide. In certain embodiments, combination treatments including immune checkpoint modulators may enhance the therapeutic efficacy of therapies including the modified cells of the present disclosure.

[0256] Following administration of the cells, in some embodiments, the biological activity of the engineered cell population is measured, for example, by any of a number of known methods. Parameters for evaluation include specific binding of engineered or natural T cells or other immune cells to antigens, for example, in vivo by imaging, or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as, for example, cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying the expression and / or secretion of one or more cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects, the biological activity is measured by evaluating clinical outcomes, such as reduction in tumor burden or tumor burden.

[0257] In certain embodiments, the subject is provided with a secondary treatment, including, but not limited to, chemotherapy, radiation, surgery, and drug therapy.

[0258] In some embodiments, conditioning therapy can be administered to the subject prior to CAR T cell therapy. In some embodiments, conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, conditioning therapy comprises administering an effective amount of fludarabine to the subject. In a preferred embodiment, conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administering conditioning therapy prior to CAR T cell therapy can enhance the efficacy of CAR T cell therapy. Methods for conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

[0259] In some embodiments, certain dosing regimens of the present disclosure include a lymphodepletion step prior to administration of the modified T cells. In exemplary embodiments, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0260] In some embodiments, the lymphodepletion step comprises administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day or 500 mg / m 2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m 2 In some embodiments, the lymphodepletion step is at about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m 2 / day.

[0261] In some embodiments, the lymphodepletion step comprises administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day). In an exemplary embodiment, the lymphodepletion step comprises administration of fludarabine at a dose of about 300 mg / m 2 / day dose of cyclophosphamide and approximately 30 mg / m 2 The treatment involves administration of fludarabine at a dose of 100 mg / day.

[0262] In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg / m for 3 days. 2 / day and fludarabine dosing was 30 mg / m for 3 days. 2 / day.

[0263] Dosing of lymphodepleting chemotherapy may be scheduled for days -6 to -4 (with a -1 day window, i.e., dosing on days -7 to -5) relative to T cell (e.g., CAR-T, TCR-T, modified T cells, etc.) infusion on day 0.

[0264] In an exemplary embodiment, for subjects with cancer, the subject is administered 300 mg / m 2 of erythropoietin by intravenous infusion 3 days prior to administration of the engineered T cells. 2 In an exemplary embodiment, for subjects with cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at 300 mg / m by intravenous infusion for 3 days prior to administration of the modified T cells. 2 will undergo lymphodepleting chemotherapy, including cyclophosphamide.

[0265] In an exemplary embodiment, for a subject with cancer, the subject receives about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day). In an exemplary embodiment, for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m 2 Patients will receive lymphodepleting chemotherapy containing a dose of fludarabine for three days.

[0266] In an exemplary embodiment, for a subject with cancer, the subject receives about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day). In an exemplary embodiment, for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of about 300 mg / m 2 / day dose of cyclophosphamide and 30 mg / m 2 Patients will receive lymphodepleting chemotherapy containing a dose of fludarabine for three days.

[0267] The cells of the present disclosure can be administered at dosages and routes and times to be determined in appropriate preclinical and clinical experiments and tests. The cell composition can be administered multiple times at dosages within these ranges. The administration of the cells of the present disclosure can be combined with other methods useful for treating the desired disease or condition as determined by those skilled in the art.

[0268] It is known in the art that one of the adverse effects following infusion of CAR T cells is the onset of immune activation known as cytokine release syndrome (CRS). CRS is immune activation leading to elevated inflammatory cytokines. CRS is a known on-target toxicity and its occurrence is likely to correlate with efficacy. Clinical and laboratory measures range from mild CRS (systemic symptoms and / or grade 2 organ toxicity) to severe CRS (sCRS; grade ≧3 organ toxicity, aggressive clinical intervention and / or potentially life-threatening). Clinical features include high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal dysfunction, liver failure, and disseminated intravascular coagulation. Dramatic elevation of cytokines including interferon-gamma, granulocyte-macrophage colony-stimulating factor, IL-10, and IL-6 has been shown following CAR T cell infusion. One sign of CRS is elevated cytokines including IL-6 (severely elevated), IFN-gamma, TNF-alpha (moderately elevated), and IL-2 (mildly elevated). Elevations of clinically available markers of inflammation including ferritin and C-reactive protein (CRP) have also been observed to correlate with CRS syndrome. The presence of CRS generally correlates with expansion of adoptively transferred cells and progressive immune activation. It has been demonstrated that the severity of CRS is determined by the disease burden at the time of infusion, as patients with high tumor burden experience greater sCRS.

[0269] Therefore, the present disclosure provides a CRS management strategy suitable for alleviating the physiological symptoms of uncontrolled inflammation after the diagnosis of CRS without compromising the anti-tumor efficacy of engineered cells (e.g., CAR T cells).CRS management strategies are known in the art.For example, systemic corticosteroids may be administered to rapidly reverse the symptoms of sCRS (e.g., grade 3 CRS) without compromising the initial anti-tumor response.

[0270] In some embodiments, anti-IL-6R antibody may be administered.An example of anti-IL-6R antibody is the monoclonal antibody tocilizumab, also known as atlizumab, approved by the US Food and Drug Administration (commercially available as Actemra or RoActemra).Tocilizumab is a humanized monoclonal antibody against interleukin-6 receptor (IL-6R).Administration of tocilizumab has demonstrated almost immediate regression of CRS.

[0271] CRS is generally managed based on the severity of the syndrome observed, and interventions are individualized accordingly. Decisions for management of CRS can be based on clinical signs and symptoms, not just laboratory values, and response to interventions.

[0272] Mild to moderate cases are generally treated with symptom management using fluid therapy, nonsteroidal anti-inflammatory drugs (NSAIDs) and antihistamines as needed for adequate relief of symptoms. More severe cases include patients with any degree of hemodynamic instability; in any hemodynamic instability, administration of tocilizumab is recommended. First-line management of CRS may be tocilizumab IV over 60 minutes at a labelled dose of 8 mg / kg (not to exceed 800 mg / dose) in some embodiments; tocilizumab may be repeated Q8 hours. If the response to the first dose of tocilizumab is suboptimal, additional doses of tocilizumab may be considered. Tocilizumab may be administered alone or in combination with corticosteroid therapy. Patients with persistent or progressive CRS symptoms with inadequate clinical improvement or poor response to tocilizumab within 12-18 hours may be treated with high-dose corticosteroid therapy, typically hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. In patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be administered high-dose corticosteroid therapy early in the course of CRS. Guidance for CRS management may be based on published criteria (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).

[0273] Concurrent with the clinical signs of CRS, features consistent with macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007). MAS appears to be a response to immune activation resulting from CRS and should therefore be considered a manifestation of CRS. MAS is similar to HLH, also a response to immune stimulation. The clinical syndrome of MAS is characterized by high-grade non-releasing fever, cytopenias affecting at least two of the three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, as well as reduced circulating natural killer (NK) activity.

[0274] Thus, the engineered cells comprising the CAR and therapeutic peptide of the present disclosure when used in the methods of treatment as described herein enhance the ability of the engineered cells to perform their functions. Thus, the present disclosure provides methods for enhancing the function of engineered cells (e.g., immune cells) for use in the methods of treatment as described herein.

[0275] In one aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject any one of the engineered cells disclosed herein. Yet another aspect of the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an engineered cell produced by any one of the methods disclosed herein.

[0276] Another aspect includes a method for treating a disease or disorder in a subject, comprising administering to the subject an effective amount of T cells genetically modified to express chimeric antigen receptor (CAR). CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The method further comprises stimulating the endogenous immune response against cancer via a non-natural therapeutic peptide. The non-natural therapeutic peptide is expressed in the modified T cells and / or administered in combination with the modified T cells. The non-naturally occurring therapeutic peptide has one or more of the following properties: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the therapeutic peptide is a mimic of a cyclic dinucleotide; (iii) the therapeutic peptide is a mimic of a short chain fatty acid (SCFA); (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) the therapeutic peptide is a mimic of a steroid / hormone-like molecule; (vi) the therapeutic peptide promotes apoptosis of a target cell; (vii) the therapeutic peptide is an immunogenic epitope; and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in a target cell.

[0277] In certain embodiments, the therapeutic peptide is a mimic of cGAMP, cAMP, or cGMP. In certain embodiments, the therapeutic peptide is a mimic of a TLR agonist. In certain embodiments, the therapeutic peptide is a mimic of flagellin, CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL). In certain embodiments, the therapeutic peptide is a second mitochondria-derived activator of caspases (SMAC) mimic. In certain embodiments, the therapeutic peptide is an inhibitor of poly ADP-ribose polymerase (PARP). In certain embodiments, the non-natural peptide is a peptide having 80% or less sequence identity to any naturally occurring peptide. In certain embodiments, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-16.

[0278] In certain embodiments, the therapeutic peptide is an immunogenic epitope, and after administration to a subject, the immunogenic epitope is expressed on the surface of the cancer cells of the subject.In certain embodiments, the therapeutic peptide is expressed in modified T cells, and after administration of modified T cells to a subject, the therapeutic peptide is discharged from modified T cells into one or more extracellular vesicles.In certain embodiments, the therapeutic peptide is delivered to one or more antigen-presenting cells of the subject via one or more extracellular vesicles.

[0279] Also provided herein is a method for enhancing the anti-cancer activity of T cells genetically modified to express chimeric antigen receptor (CAR), wherein CAR comprises an antigen-binding domain that specifically binds to the antigen expressed on tumor cells, a transmembrane domain, and an intracellular signaling domain.The method comprises co-expressing non-natural therapeutic peptide in T cells. The non-naturally occurring therapeutic peptide has one or more of the following properties: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the therapeutic peptide is a mimic of a cyclic dinucleotide; (iii) the therapeutic peptide is a mimic of a short chain fatty acid (SCFA); (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) the therapeutic peptide is a mimic of a steroid / hormone-like molecule; (vi) the therapeutic peptide promotes apoptosis of a target cell; (vii) the therapeutic peptide is an immunogenic epitope; and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in a target cell.

[0280] Also provided herein are methods for treating an inflammatory disease, an autoimmune disease, or cancer in a subject, comprising administering to the subject an effective amount of any of the engineered cells or compositions contemplated herein.

[0281] I. Methods for Producing Engineered Cells Provided herein is a method for producing or generating the engineered cells (e.g., immune cells or their precursors; e.g., T cells) of the present disclosure for tumor immunotherapy, e.g., adoptive immunotherapy. The cells are generally engineered by introducing one or more nucleic acids encoding CAR and therapeutic peptides into the cells.

[0282] Also provided are methods for co-expressing a CAR and a therapeutic peptide in a cell, comprising delivering any of the expression vectors contemplated herein to a cell under conditions such that the CAR and the therapeutic peptide are expressed.

[0283] In some embodiments, CAR and therapeutic peptide are introduced into cells by expression vector. Expression vectors comprising nucleic acid sequences encoding CAR and therapeutic peptide of the present disclosure are provided herein. Suitable expression vectors include lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA including but not limited to transposon-mediated vectors, such as integrases such as Sleeping Beauty, Piggybak and Phi31. Some other suitable expression vectors include herpes simplex virus (HSV) and retrovirus expression vectors.

[0284] In certain embodiments, the nucleic acid encoding the CAR is introduced into the cell via viral transduction. In certain embodiments, the viral transduction comprises contacting the immune cell or progenitor cell with a viral vector comprising the nucleic acid encoding the CAR. In certain embodiments, the viral vector is an adeno-associated virus (AAV) vector. In certain embodiments, the AAV vector comprises a 5'ITR and a 3'ITR. In certain embodiments, the AAV vector comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In certain embodiments, the AAV vector comprises a polyadenylation (polyA) sequence. In certain embodiments, the polyA sequence is a bovine growth hormone (BGH) polyA sequence.

[0285] Adenoviral expression vectors are based on adenovirus and have a low capacity for integration into genomic DNA, but high efficiency for transfection into host cells. Adenoviral expression vectors contain sufficient adenoviral sequences to (a) assist in packaging of the expression vector, and (b) ultimately express CAR in host cells. In some embodiments, the adenoviral genome is a 36 kb linear double-stranded DNA into which a foreign DNA sequence (e.g., a nucleic acid encoding a CAR) may be inserted to replace a large piece of adenoviral DNA to generate an expression vector of the present disclosure (see, e.g., Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).

[0286] Another expression vector is based on adeno-associated virus (AAV) and utilizes the adenovirus conjugation system. This AAV expression vector has a high integration frequency into the host genome. This vector can infect non-dividing cells, and is therefore useful for delivering genes into mammalian cells, for example, in tissue culture or in vivo. AAV vector has a wide host range for infection. Details regarding the generation and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0287] Retroviral expression vectors can integrate into host genomes, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and package into special cell lines.Retroviral vectors are constructed by inserting nucleic acid (e.g., nucleic acid encoding CAR and therapeutic peptide) into a specific location in the viral genome to produce a virus that is replication-defective.Retroviral vectors can infect a wide variety of cell types, but the integration and stable expression of CAR and therapeutic peptide require the division of host cells.

[0288] Lentiviral vectors are composite retroviruses derived from lentiviruses and contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol and env (see, for example, U.S. Patent Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors have been generated by multiple attenuation of HIV pathogenicity genes, for example, genes env, vif, vpr, vpu and nef are deleted, making the vector biologically safe. Lentiviral vectors can infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, for example, of nucleic acids encoding CAR and therapeutic peptides (see, for example, U.S. Patent No. 5,994,136).

[0289] The expression vector containing the nucleic acid of the present disclosure can be introduced into the host cell by any means known to those skilled in the art. The expression vector may contain a viral sequence for transfection if desired. Alternatively, the expression vector may be introduced by fusion, electroporation, biolistics, transfection, lipofection, etc. The host cell may be grown and expanded in culture before the introduction of the expression vector, followed by treatment suitable for the introduction and integration of the vector. The host cell may then be expanded and screened by a marker present in the vector. Various markers that may be used are known in the art and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms "cell", "cell line" and "cell culture" may be used interchangeably. In some embodiments, the host cell is an immune cell or a precursor thereof, such as a T cell, a NK cell, or a NKT cell.

[0290] The present disclosure also provides engineered cells that contain and stably express the CAR of the present disclosure. In some embodiments, the engineered cells are engineered T lymphocytes (T cells), naive T cells (TN), memory T cells (e.g., central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), and macrophages that can generate therapeutically relevant progeny. In certain embodiments, the engineered cells are autologous cells. In certain embodiments, the engineered cells are resistant to T cell exhaustion. In certain embodiments, the engineered cells are resistant to T cell dysfunction.

[0291] Modified cells (e.g., containing CAR) can be produced by stably transfecting an expression vector containing the nucleic acid of the present disclosure into a host cell. Additional methods for generating modified cells of the present disclosure include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, phototransformation, gene electrotransfer and / or hydrodynamic delivery) and / or particle-based methods (e.g., impalefection and / or magnetofection using gene guns). Transfected cells expressing the CAR and therapeutic peptide of the present disclosure can be expanded ex vivo.

[0292] Physical methods for introducing expression vectors into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Chemical methods for introducing expression vectors into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0293] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform can be used as the sole solvent since it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of single and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. Before the formation of closed structures, the lipid components undergo self-rearrangement, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). Also included are compositions that have structures in solution that differ from the normal vesicular structure. For example, lipids may adopt micellar structures or simply exist as non-uniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also envisioned.

[0294] Regardless of the method used to introduce exogenous nucleic acid into host cell or the method used to expose cell to the inhibitor of the present disclosure, various assays can be carried out to confirm the presence of nucleic acid in host cell.Such assays include, for example, molecular biological assays well known to those skilled in the art, such as Southern blotting, Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot), or by the assays described herein to identify agents that fall within the scope of the present disclosure.

[0295] In one embodiment, the nucleic acid introduced into the host cell is RNA. In another embodiment, the RNA is mRNA, including in vitro transcribed RNA or synthetic RNA. RNA can be produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). Interesting DNA from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using suitable primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other suitable DNA source.

[0296] PCR may be used to generate templates for in vitro transcription of mRNA that is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have a region that is substantially complementary to the DNA region to be used as a template for PCR. "Substantially complementary" as used herein refers to a nucleotide sequence that is complementary to most or all of the bases in the primer sequence. A substantially complementary sequence is capable of annealing or hybridizing with the DNA target of interest under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify gene portions that are normally transcribed in cells (open reading frames), including 5' UTRs and 3' UTRs. Primers can also be designed to amplify portions of genes that code for a particular domain of interest. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or part of 5' UTRs and 3' UTRs. Primers useful for PCR are generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a nucleotide region that is substantially complementary to nucleotides on a DNA template that is upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to the 5th position of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a nucleotide region that is substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to the 3' position of the DNA sequence to be amplified relative to the coding strand.

[0297] Chemical structures capable of promoting RNA stability and / or translation efficiency may also be used. The RNA preferably has a 5' UTR and a 3' UTR. In one embodiment, the 5' UTR is zero to 3000 nucleotides long. The length of the 5' UTR and 3' UTR sequences to be added to the coding region can be altered by different methods, including but not limited to designing primers for PCR that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the length of the 5' UTR and 3' UTR required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0298] 5'UTR and 3'UTR can be the naturally occurring endogenous 5'UTR and 3'UTR for the gene of interest. Alternatively, the UTR sequence that is not endogenous to the gene of interest can be added by incorporating the UTR sequence in the forward primer and reverse primer, or by any other modification of the template. The use of the UTR sequence that is not endogenous to the gene of interest can be useful for modifying the stability and / or translation efficiency of RNA. For example, it is known that AU-rich elements in 3'UTR sequence can reduce the stability of mRNA. Therefore, 3'UTR can be selected or designed to increase the stability of transcribed RNA based on the properties of UTR that are well known in the art.

[0299] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, if a 5' UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5' UTR sequence. Although the Kozak sequence can increase the translation efficiency of some RNA transcripts, it does not appear to be necessary for all RNAs to allow efficient translation. The necessity of the Kozak sequence for many mRNAs is known in the art. In another embodiment, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In another embodiment, various nucleotide analogs can be used in the 3' UTR or 5' UTR to prevent exonuclease degradation of mRNA.

[0300] To allow RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter should be attached to the DNA template upstream of the sequence to be transcribed.When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter will be incorporated upstream of the open reading frame to be transcribed in the PCR product.In one embodiment, the promoter is a T7 polymerase promoter as described elsewhere herein.Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters.The consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0301] In one embodiment, mRNA has both a cap on the 5' end and a 3' poly(A) tail, which determine ribosome binding, translation initiation and the stability of mRNA in cells.On circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatemeric products that are not suitable for expression in eukaryotic cells.Transcription of plasmid DNA linearized at the end of 3'UTR produces mRNA of normal size, which is not effective for eukaryotic transfection even when it is polyadenylated after transcription.

[0302] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of a transcript beyond the final base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0303] The polyA / T segment of the transcribed DNA template can be generated during PCR by using a reverse primer containing a polyT tail (size can be 50-5000T), such as a 100T tail, or after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The poly(A) tail also provides stability to RNAs and reduces their degradation. In general, the length of the poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100-5000 adenosines.

[0304] The poly(A) tail of the RNA can be further extended by the use of a poly(A) polymerase, such as Escherichia coli (E. coli) polyA polymerase (E-PAP), after in vitro transcription. In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides results in an approximately two-fold increase in the translation efficiency of the RNA. In addition, attachment of different chemical groups to the 3' end can increase the stability of the mRNA. Such attachments can contain modified / artificial nucleotides, aptamers and other compounds. For example, poly(A) polymerase can be used to incorporate ATP analogs into the poly(A) tail. The ATP analogs can further increase the stability of the RNA.

[0305] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein comprises a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0306] In some embodiments, the RNA is electroporated into cells as in vitro transcribed RNA. Any solute suitable for cell electroporation can be included, which can contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and detergents.

[0307] In some embodiments, the nucleic acid encoding the CAR of the present disclosure will be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known in the art; any known method can be used to synthesize RNA comprising sequences encoding CAR and therapeutic peptide. Methods for introducing RNA into host cells are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising nucleotide sequences encoding CAR and therapeutic peptide into host cells can be performed in vitro, ex vivo, or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated with RNA comprising nucleotide sequences encoding CAR and therapeutic peptide in vitro or ex vivo.

[0308] The disclosed methods can be applied to modulating T cell activity in basic research and therapy in the areas of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including assessing the ability of genetically modified T cells to kill targeted cancer cells.

[0309] The method also provides the ability to control the expression level over a wide range, for example by varying the amount of promoter or input RNA, allowing expression levels to be individually adjusted.Furthermore, the PCR-based technique of mRNA production makes it extremely easy to design mRNAs with different structures and combinations of their domains.

[0310] One advantage of the RNA transfection method of the present disclosure is that RNA transfection is essentially transient and vector-free.RNA transgenes can be delivered to lymphocytes and expressed therein after a short period of in vitro cell activation as a minimal expression cassette without the need for any additional viral sequence.Under these conditions, the integration of transgenes into host cell genome is unlikely to occur.Due to the transfection efficiency of RNA and the ability of RNA to uniformly modify the entire lymphocyte population, cloning of cells is unnecessary.

[0311] Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, both of which have been successfully tested in various animal models. In vitro transcribed RNA is transfected into cells by lipofection or electroporation. To achieve long-term expression of the transferred IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications.

[0312] Several IVT vectors are known from the literature that are utilized in standard procedures as templates for in vitro transcription and that have been genetically modified to produce stabilized RNA transcripts. Currently, the protocols used in the art are based on plasmid vectors with the following structure: a 5' RNA polymerase promoter that allows RNA transcription, followed by the gene of interest flanked by untranslated regions (UTRs) either at the 3' and / or 5', as well as a 3' polyadenylation cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). The polyadenylation cassette thus corresponds to the subsequent poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced in cells from such a construct.

[0313] In another aspect, RNA construct is delivered into cell by electroporation.See, for example, the formulation and methodology of electroporation of nucleic acid construct into mammalian cell as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, US 2004 / 0092907A1.Various parameters, including the electric field strength required for electroporation of any known cell type, are generally known from related research literature and numerous patents and applications in this field.See, for example, US Patent No. 6,678,556, US Patent No. 7,171,264, and US Patent No. 7,173,116. Devices for therapeutic applications of electroporation are commercially available, for example, MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S. Pat. No. 6,233,482; electroporation can also be used for in vitro transfection of cells, for example, as described in US20070128708A1. Electroporation can also be utilized to deliver nucleic acid into cells in vitro. Thus, electroporation-mediated administration of nucleic acid, including expression constructs, into cells using any of the many available devices and electroporation systems known to those skilled in the art represents an exciting new means to deliver RNA of interest to target cells.

[0314] In some embodiments, the cells (e.g., T cells) can be incubated or cultivated before, during, and / or after introducing the nucleic acid molecule encoding the CAR and the therapeutic peptide. In some embodiments, the cells (e.g., T cells) can be incubated or cultivated before, during, and / or after introducing the nucleic acid molecule encoding the CAR and the therapeutic peptide, for example, before, during, or after transducing the cell with a viral vector (e.g., lentiviral vector) encoding the CAR and the therapeutic peptide. In some embodiments, the method includes activating or stimulating the cell with a stimulatory or activating agent (e.g., anti-CD3 / anti-CD28 antibody) before introducing the nucleic acid molecule encoding the CAR and the therapeutic peptide. In some embodiments, the stimulatory or activating agent and / or cytokine is not removed before introducing the agent. One skilled in the art can determine the order in which each of one or more nucleic acid sequences is introduced into the host cell.

[0315] J. Increase in immune cells The cells, whether before or after engineering (e.g., expressing a CAR and a therapeutic peptide), can be engineered using methods such as those described in, e.g., U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067 The T cells can be activated and expanded in number using methods such as those described in US Patent Publication Nos. 318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and US Patent Publication No. 20060121005. For example, the T cells of the present disclosure can be expanded by contact with a surface to which an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface are attached. In particular, the T cell population can be stimulated by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. For costimulation of accessory molecules on the T cell surface, a ligand that binds to the accessory molecule is used. For example, T cells can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which can be used in the present disclosure, although other methods and reagents known in the art can be used as well (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

[0316] The expansion of T cells by the methods disclosed herein can be about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold or more, and any and all whole integers and partial integers therebetween. In one embodiment, T cells are expanded in the range of about 20-fold to about 50-fold.

[0317] Following culture, the T cells can be incubated in the cell medium in the culture device for a period of time or until the cells reach confluence or a high cell density for optimal passaging before passaging the cells to another culture device. The culture device can be any culture device commonly used for in vitro culture of cells. Preferably, the level of confluence is 70% or more before passaging the cells to another culture device. More preferably, the level of confluence is 90% or more. The period of time can be any time suitable for in vitro cell culture. The T cell medium can be replaced at any time during the culture of the T cells. Preferably, the T cell medium is replaced about every 2-3 days. The T cells are then harvested from the culture device, after which the T cells can be used immediately or cryopreserved and stored for later use. In one aspect, the present disclosure includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed before introducing the nucleic acid into the T cells.

[0318] In another embodiment, the method includes isolating T cells and expanding the T cells. In another embodiment, the disclosure further includes cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed to electroporate RNA encoding the chimeric membrane protein.

[0319] Another procedure for ex vivo expansion of cells is described in U.S. Patent No. 5,199,942 (incorporated herein by reference). Expansion as described in U.S. Patent No. 5,199,942 can be an alternative or an addition to other expansion methods described herein. Briefly, ex vivo culture and expansion of T cells includes the addition of cell growth factors or other factors such as those described in U.S. Patent No. 5,199,942, such as flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding T cells includes culturing T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.

[0320] The culturing step as described herein (following contact with an agent as described herein or electroporation) can be very short, e.g., less than 24 hours, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as further described herein (contact with an agent as described herein) can be longer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0321] Various terms are used to describe cultured cells. Cell culture generally refers to cells taken from a living organism and grown under controlled conditions. Primary cell culture is a culture of cells, tissues or organs taken directly from an organism before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger cell population. When cells are expanded in culture, the proliferation rate of the cells is typically measured by the time required for the number of cells to double, otherwise known as the doubling time.

[0322] Each round of subculture is called a passage. When cells are subcultured, they are said to be passaged. A particular cell population or cell line is sometimes referred to or characterized by the number of times they have been subcultured. For example, a cultured cell population that has been subcultured 10 times may be referred to as a P10 culture. A primary culture, i.e., the first culture after isolating cells from tissue, is called P0. After the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those skilled in the art that there may be many population doublings during the subculture period; therefore, the population doubling number of a culture is greater than the number of passages. The expansion of cells between subcultures (i.e., the number of population doublings) depends on many factors, including but not limited to the seeding density, substrate, medium, and subculture interval.

[0323] In one embodiment, cells may be cultured for a few hours (about 3 hours) to about 14 days or any integer value of time therebetween. Conditions suitable for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Media 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta and TNF-α or any other additive for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts for the growth and expansion of T cells, with the addition of amino acids, sodium pyruvate and vitamins. Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures, not in the cultures of cells to be injected into the subject. The target cells are maintained under the conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2).

[0324] The medium used to culture T cells can include an agent that can costimulate T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. The cells isolated by the method disclosed herein can be expanded approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more. In one embodiment, T cells are expanded in the range of about 20-fold to about 50-fold, or more. In one embodiment, human regulatory T cells are expanded via KT64.86 artificial antigen presenting cells (aAPCs) coated with anti-CD3 antibodies. Methods for expanding and activating T cells can be found in U.S. Patent Nos. 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein in their entireties.

[0325] In one embodiment, the method of expanding T cells can further include isolating the expanded T cells for further application.In another embodiment, the method of expanding can further include subsequent electroporation of the expanded T cells before culturing.Subsequent electroporation can include introducing a nucleic acid encoding an agent into the expanded T cell population, such as transducing the expanded T cells with a nucleic acid, transfecting the expanded T cells, or electroporating the expanded T cells, and the agent further stimulates the T cells.The agent can stimulate the T cells by further expanding, stimulating effector function or another T cell function, etc.

[0326] K. T cell receptor The present disclosure provides compositions and methods for engineered cells (e.g., immune cells or precursors thereof, e.g., T cells) that contain an exogenous T cell receptor (TCR) and a therapeutic peptide. Thus, in some embodiments, the cells are modified to contain a specific T cell receptor (TCR) gene (e.g., a nucleic acid encoding an alpha / beta TCR). TCRs or antigen-binding portions thereof include those that recognize peptide epitopes or T cell epitopes of target polypeptides, such as antigens of tumors, viruses, or autoimmune proteins. In certain embodiments, the TCR has binding specificity for a tumor-associated antigen, e.g., human NY-ESO-1.

[0327] TCR is a disulfide-linked heterodimeric protein composed of six different membrane-bound chains that are involved in the activation of T cells in response to antigen. There are alpha / beta TCRs and gamma / delta TCRs. Alpha / beta TCRs include TCR alpha chains and TCR beta chains. T cells expressing TCRs that include TCR alpha chains and TCR beta chains are commonly referred to as alpha / beta T cells. Gamma / delta TCRs include TCR gamma chains and TCR delta chains. T cells expressing TCRs that include TCR gamma chains and TCR delta chains are commonly referred to as gamma / delta T cells. The TCRs of the present disclosure are TCRs that include TCR alpha chains and TCR beta chains.

[0328] The TCR alpha and beta chains each consist of two extracellular domains, a variable region and a constant region. The TCR alpha and beta chain variable regions are required for the affinity of the TCR to the target antigen. Each variable region contains three hypervariable or complementarity determining regions (CDRs) that provide binding to the target antigen. The constant regions of the TCR alpha and beta chains are proximal to the cell membrane. The TCR further comprises a transmembrane region and a short cytoplasmic tail. The CD3 molecule assembles together with the TCR heterodimer. The CD3 molecule contains a characteristic sequence motif for tyrosine phosphorylation known as the immunoreceptor tyrosine-based activation motif (ITAM). Proximal signaling events are mediated through the CD3 molecule, and thus the TCR-CD3 complex interaction plays a key role in mediating cell recognition events.

[0329] Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHC) on antigen-presenting cells, which present antigenic peptides to T cells and interact with the TCR to induce a series of intracellular signaling cascades. TCR engagement initiates both positive and negative signaling cascades that result in cell proliferation, cytokine production and / or activation-induced cell death.

[0330] The TCR of the present disclosure can be a wild-type TCR, a high affinity TCR, and / or a chimeric TCR. A high affinity TCR can be the result of modification to a wild-type TCR that confers a higher affinity to target antigen compared to the wild-type TCR. A high affinity TCR can be an affinity-matured TCR. Methods for modifying TCR and / or affinity maturing TCR are known to those skilled in the art. Techniques for engineering and expressing TCRs include, but are not limited to, the production of TCR heterodimers containing native disulfide bridges linking each subunit (Garboczi, et al., (1996), Nature 384(6605): 134-41; Garboczi, et al., (1996), J Immunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91: 11408-11412; Davodeau et al., (1993), J. Biol. Chem. 268(21): 15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; U.S. Patent No. 6,080,840).

[0331] In some embodiments, the exogenous TCR is a complete TCR or an antigen-binding portion or fragment thereof. In some embodiments, the TCR is an intact or full-length TCR, including αβ or γδ forms of the TCR. In some embodiments, the TCR is an antigen-binding portion that is shorter than the full-length TCR but binds to a specific peptide bound in an MHC molecule, e.g., binds to an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of the TCR can contain only a portion of the structural domain of the full-length or intact TCR, but is still capable of binding to a peptide epitope that the complete TCR binds, e.g., an MHC-peptide complex. In some cases, the antigen-binding portion contains sufficient variable domains of the TCR, e.g., the variable α and β chains of the TCR, to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain the complementarity determining regions (CDRs) involved in the recognition of peptides, MHC and / or MHC-peptide complexes.

[0332] In some embodiments, the variable domain of TCR contains hypervariable loops or CDRs, which are generally the primary contributors to antigen recognition and binding capacity and specificity. In some embodiments, the CDRs of TCR or a combination thereof form all or substantially all of the antigen binding site of a given TCR molecule. The various CDRs in the variable region of TCR chains are generally separated by framework regions (FRs), which generally exhibit less variability between TCR molecules compared to CDRs (see, for example, Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; also see Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity, or is the most important of the three CDRs on a given TCR variable region for antigen recognition and / or interaction with the processed peptide portion of a peptide-MHC complex. In some circumstances, CDR1 of the alpha chain can interact with the N-terminal portion of a particular antigen peptide. In some circumstances, CDR1 of the beta chain can interact with the C-terminal portion of the peptide. In some circumstances, CDR2 is the primary CDR that contributes most strongly to or is responsible for interaction with or recognition of the MHC portion of an MHC-peptide complex. In some embodiments, the variable region of the beta chain can contain an additional hypervariable region (CDR4 or HVR4) that is generally involved in superantigen binding but not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0333] In some embodiments, the TCR comprises a variable alpha domain (V α ) and / or the variable beta domain (V β) or an antigen-binding fragment thereof. In some embodiments, the α-chain and / or β-chain of the TCR can also contain a constant domain, a transmembrane domain and / or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3 Ed., Current Biology Publications, p. 4:33, 1997). In some embodiments, the α-chain constant domain is encoded by the TRAC gene (IMGT nomenclature) or a variant thereof. In some embodiments, the β-chain constant region is encoded by the TRBC1 or TRBC2 gene (IMGT nomenclature) or a variant thereof. In some embodiments, the constant domain is adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains, each of which contains a CDR.

[0334] It is within the skill of one of ordinary skill in the art to determine or identify the various domains or regions of a TCR. In some aspects, the residues of a TCR are known or can be identified according to the International Immunogenetics Information System (IMGT) numbering system (see, e.g., www.imgt.org; see also Lefranc et al. (2003) Developmental and Comparative Immunology, 2&;55-77; and The T Cell Factsbook 2nd Edition, Lefranc and LeFranc Academic Press 2001). Using this system, the CDR1 sequence in the TCR Va chain and / or Vβ chain corresponds to amino acids present between residue numbers 27-38 (inclusive), the CDR2 sequence in the TCR Va chain and / or Vβ chain corresponds to amino acids present between residue numbers 56-65 (inclusive), and the CDR3 sequence in the TCR Va chain and / or Vβ chain corresponds to amino acids present between residue numbers 105-117 (inclusive). The IMGT numbering system should not be construed as limiting in any way, as there are other numbering systems known to those of skill in the art, and as it is within the skill of one of skill in the art to use any of the available numbering systems to identify various domains or regions of a TCR.

[0335] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), linked such as by a disulfide bond(s). In some embodiments, the constant domain of the TCR may contain a short connecting sequence in which cysteine ​​residues form a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, the TCR may have an additional cysteine ​​residue in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domain. In some embodiments, each of the constant and variable domains contains a disulfide bond formed by a cysteine ​​residue.

[0336] In some embodiments, the TCR for engineering cells as described is generated from known TCR sequence(s), such as sequences of Vα,β chains, for which substantially full-length coding sequences are readily available. Methods for obtaining full-length TCR sequences, including V chain sequences, from cellular sources are well known. In some embodiments, the nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of the nucleic acid encoding the TCR in or isolated from a given cell(s), or by synthesis of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, such as a cell, such as a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, the T cell can be obtained from an in vivo isolated cell. In some embodiments, the T cell can be a cultured T cell hybridoma or clone. In some embodiments, the TCR or an antigen-binding portion thereof can be generated synthetically from knowledge of the sequence of the TCR. In some embodiments, high affinity T cell clones against target antigens (e.g., cancer antigens) are identified, isolated from patients, and introduced into cells. In some embodiments, TCR clones against target antigens are generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). See, for example, tumor antigens (see, for example, Parkhurst et al. (2009) Clin Cancer Res. 15: 169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808). In some embodiments, phage display is used to isolate TCRs against target antigens (see, for example, Varela-Rohena et al. (2008) Nat Med. 14: 1390-1395 and Li (2005) Nat Biotechnol. 23:349-354).

[0337] In some embodiments, the TCR or antigen-binding portion thereof is modified or engineered. In some embodiments, directed evolution methods are used to generate TCRs with altered properties, such as higher affinity for a particular MHC-peptide complex. In some embodiments, directed evolution is achieved by display methods, including but not limited to yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, the display approach involves engineering or modifying a known, parent or reference TCR. For example, in some cases, a wild-type TCR can be used as a template to produce mutagenized TCRs in which one or more residues in the CDRs are mutated, and mutants with desired altered properties, such as higher affinity for a desired target antigen, are selected.

[0338] In some embodiments as described, the TCR can contain an introduced disulfide bond(s). In some embodiments, there are no native disulfide bonds. In some embodiments, one or more of the native cysteines (e.g., in the constant domains of the α and β chains) that form the native interchain disulfide bonds are replaced with another residue, such as serine or alanine. In some embodiments, the introduced disulfide bond can be formed by mutating non-cysteine ​​residues on the alpha and beta chains to cysteines, for example, in the constant domains of the α and β chains. Exemplary non-native disulfide bonds of TCRs are described in International Publication Nos. WO2006 / 000830 and WO2006 / 037960. In some embodiments, cysteines can be introduced at residues Thr48 of the α chain and Ser57 of the β chain, Thr45 of the α chain and Ser77 of the β chain, Tyr10 of the α chain and Serl7 of the β chain, Thr45 of the α chain and Asp59 of the β chain, and / or Serl5 of the α chain and Glul5 of the β chain. In some embodiments, the presence of a non-native cysteine ​​residue in a recombinant TCR (e.g., resulting in one or more non-native disulfide bonds) can favor production of the desired recombinant TCR in a cell into which it is introduced over expression of a mismatched TCR pair containing a native TCR chain.

[0339] In some embodiments, the TCR chain contains a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some aspects, each chain of the TCR (e.g., alpha or beta) can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR associates, for example, via the cytoplasmic tail, with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. In some cases, this structure allows the TCR to associate with other molecules, such as CD3 and its subunits. For example, a TCR containing a constant domain with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signal transduction apparatus or complex. The intracellular tail of the CD3 signal transduction subunit (e.g., CD3y, CD35, CD3s, and CD3ζ chain) contains one or more immunoreceptor activation tyrosine motifs or ITAMs that are involved in the signal transduction capacity of the TCR complex.

[0340] In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). The TCR can be cell-bound or solution-bound. In some embodiments, for purposes of the provided methods, the TCR is cell-bound expressed on the surface of a cell. In some embodiments, the dTCR contains a first polypeptide in which a sequence corresponding to a TCR alpha chain variable region sequence is fused to the N-terminus of a sequence corresponding to a TCR alpha chain constant region extracellular sequence, and a second polypeptide in which a sequence corresponding to a TCR beta chain variable region sequence is fused to the N-terminus of a sequence corresponding to a TCR beta chain constant region extracellular sequence, the first and second polypeptides being linked by a disulfide bond. In some embodiments, the bond can correspond to a natural interchain disulfide bond present in a natural dimeric alpha beta TCR. In some embodiments, interchain disulfide bonds do not exist in native TCRs. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequence of the dTCR polypeptide pair. In some cases, both native and non-native disulfide bonds may be desirable. In some embodiments, the TCR contains a transmembrane sequence for membrane anchoring. In some embodiments, the dTCR contains a TCR α chain containing a variable α domain, a constant α domain, and a first dimerization motif attached to the C-terminus of the constant α domain, and a TCR β chain containing a variable β domain, a constant β domain, and a first dimerization motif attached to the C-terminus of the constant β domain, where the first and second dimerization motifs readily interact to form a covalent bond between the amino acids in the first dimerization motif and the amino acids in the second dimerization motif, linking the TCR α chain and the TCR β chain together.

[0341] In some embodiments, the TCR is a scTCR, which is a single amino acid chain containing an α chain and a β chain that can bind to an MHC-peptide complex.Typically, scTCRs can be generated using methods known to those skilled in the art, see, for example, International Publication Nos. WO 96 / 13593, WO 96 / 18105, W099 / 18129, WO04 / 033685, WO2006 / 037960, WO2011 / 044186; U.S. Patent No. 7,569,664; and Schlueter, CJ et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, the scTCR contains a first segment comprised of an amino acid sequence corresponding to a TCR alpha chain variable region, a second segment comprised of an amino acid sequence corresponding to a TCR beta chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR beta chain constant domain extracellular sequence, and a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment. In some embodiments, the scTCR contains a first segment comprised of an amino acid sequence corresponding to a TCR beta chain variable region, a second segment comprised of an amino acid sequence corresponding to a TCR alpha chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR alpha chain constant domain extracellular sequence, and a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment. In some embodiments, the scTCR contains a first segment comprised of an alpha chain variable region sequence fused to the N-terminus of an alpha chain extracellular constant domain sequence, a second segment comprised of a beta chain variable region sequence fused to the N-terminus of a sequence of a beta chain extracellular constant and transmembrane sequence, and optionally a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment. In some embodiments, the scTCR contains a first segment comprised of a TCR beta chain variable region sequence fused to the N-terminus of a beta chain extracellular constant domain sequence, a second segment comprised of an alpha chain variable region sequence fused to the N-terminus of a sequence comprising an alpha chain extracellular constant domain sequence and a transmembrane sequence, and optionally a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.In some embodiments, for a scTCR to bind to an MHC-peptide complex, the a-chain and the β-chain must be paired such that their variable region sequences are oriented for such binding. Various methods to promote pairing of α and β in a scTCR are well known in the art. In some embodiments, a linker sequence is included that links the a-chain and the β-chain to form a single polypeptide chain. In some embodiments, the linker should have a length sufficient to span the distance between the C-terminus of the α-chain and the N-terminus of the β-chain (or vice versa), while also ensuring that the linker length is not so long as to block or reduce binding of the scTCR to the target peptide-MHC complex. In some embodiments, the linker of the scTCR that links the first and second TCR segments can be any linker capable of forming a single polypeptide chain while retaining TCR binding specificity. In some embodiments, the linker sequence can have, for example, the formula -P-AA-P-, where P is proline and AA represents an amino acid sequence, where the amino acids are glycine and serine. In some embodiments, the first and second segments are paired such that their variable region sequences are oriented for such binding. Thus, in some cases, the linker has sufficient length to span the distance between the C-terminus of the first segment and the N-terminus of the second segment (or vice versa), but is not so long as to block or reduce binding of the scTCR to the target ligand. In some embodiments, the linker can contain 10-45 amino acids or about 10-45 amino acids, e.g., 10-30 amino acids or 26-41 amino acid residues, e.g., 29, 30, 31 or 32 amino acids. In some embodiments, the scTCR contains disulfide bonds between residues of a single amino acid chain, which in some cases can promote stability of the pairing between the α and β regions of the single chain molecule (see, e.g., U.S. Pat. No. 7,569,664). In some embodiments, the scTCR contains a covalent disulfide bond linking residues from the immunoglobulin region of the constant domain of the α chain and residues from the immunoglobulin region of the constant domain of the β chain of the single chain molecule.In some embodiments, the disulfide bond corresponds to a natural disulfide bond present in a natural dTCR. In some embodiments, there is no disulfide bond in a natural TCR. In some embodiments, the disulfide bond is a non-natural disulfide bond introduced, for example, by incorporating one or more cysteines into the constant region extracellular sequence of the first and second chain regions of the scTCR polypeptide. Exemplary cysteine ​​mutations include any as described above. In some cases, both natural and non-natural disulfide bonds may be present.

[0342] In some embodiments, any TCR, including dTCR or scTCR, can be linked to a signaling domain that produces an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of a cell. In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the transmembrane domain can be a Ca or CP transmembrane domain. In some embodiments, the transmembrane domain can be of non-TCR origin, such as a transmembrane region from CD3z, CD28, or B7.1. In some embodiments, the TCR contains a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR contains a CD3z signaling domain. In some embodiments, the TCR can form a TCR complex with CD3. In some embodiments, the TCR or its antigen-binding portion can be a recombinantly produced native protein or a variant thereof that has one or more properties, such as binding properties, altered. In some embodiments, the TCR can be derived from one of a variety of animal species, such as human, mouse, rat, or other mammals.

[0343] In some embodiments, TCR comprises affinity for target antigen on target cell. Target antigen can comprise any kind of protein or epitope thereof associated with target cell. For example, TCR can comprise affinity for target antigen on target cell that shows a certain disease state of target cell. In some embodiments, target antigen is processed and presented by MHC.

[0344] L. Pharmaceutical Compositions and Formulations Also provided are populations of cells (e.g., immune cells; e.g., T cells) of the present disclosure, compositions containing such cells and / or enriched with such cells, for example, compositions in which the cells expressing CAR and therapeutic peptides account for at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells or a certain type of cells, such as T cells or CD8+ cells or CD4+ cells, in the composition.The compositions are, among others, pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy.Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

[0345] Also provided are compositions that contain cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions that contain the number of cells for administration at a given dose or fraction thereof.Pharmaceutical compositions and formulations generally contain one or more optional pharmaceutically acceptable carriers or excipients.In some embodiments, the composition contains at least one additional therapeutic agent.

[0346] The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation will be administered. "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some aspects, the choice of carrier is determined, in part, by the particular cell and / or by the method of administration. Thus, there is a wide variety of suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0347] In some aspects, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0348] The formulation may comprise an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition treated by the cells, preferably with complementary activities to the cells, each of which does not adversely affect the other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharma- ceutical active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition, in some embodiments, contains the cells in an amount effective to treat or prevent the disease or condition, such as a therapeutically or prophylactically effective amount. The therapeutic or prophylactic effectiveness is, in some embodiments, monitored by periodically evaluating the subject being treated. The desired dosage can be delivered by administration of a single bolus of cells, by administration of multiple boluses of cells, or by administration of continuous infusions of cells.

[0349] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. The composition is provided in some embodiments as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially for administration by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact period with a particular tissue. The liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0350] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, for example, in a solvent mixed with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, glucose, dextrose, etc. The composition can contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents and / or coloring agents, depending on the desired route of administration and preparation. In some aspects, standard textbooks may be consulted for preparing appropriate preparations.

[0351] Various additives can be added to enhance the stability and sterility of the composition, including antibacterial preservatives, antioxidants, chelating agents, and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of injectable pharmaceutical dosage forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0352] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0353] M. List of aspects The following list of aspects is provided, the numbering of which should not be construed as indicating any level of importance. Aspect 1 provides: An engineered cell comprising a chimeric antigen receptor (CAR) and a therapeutic peptide, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and the therapeutic peptide is a non-naturally occurring therapeutic peptide, and wherein the CAR molecule and the therapeutic peptide are expressed from the same expression construct. Aspect 2 provides: An engineered cell comprising a chimeric antigen receptor (CAR) and a therapeutic peptide, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and the therapeutic peptide is a non-naturally occurring therapeutic peptide, and the therapeutic peptide has the following properties: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the therapeutic peptide is a mimetic of a cyclic dinucleotide; (iii) the therapeutic peptide is a mimetic of a short chain fatty acid (SCFA); (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) the therapeutic peptide is a mimetic of a steroid / hormone-like molecule; (vi) the therapeutic peptide promotes apoptosis of a target cell; (vii) the therapeutic peptide is an immunogenic epitope, and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in the target cell An engineered cell having one or more of the following: Aspect 3 provides: The engineered cell of any of the above embodiments, wherein the therapeutic peptide is a mimic of cGAMP, cAMP, or cGMP. Aspect 4 provides: 3. The engineered cell of embodiment 1 or 2, wherein the therapeutic peptide is a mimic of a TLR agonist. Aspect 5 provides the following: The engineered cell of embodiment 4, wherein the therapeutic peptide is a mimic of flagellin, a CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL). Aspect 6 provides the following: 3. The engineered cell of embodiment 1 or 2, wherein the therapeutic peptide is a second mitochondria derived activator of caspase (SMAC) mimetic. Aspect 7 provides the following: 3. The engineered cell of embodiment 1 or 2, wherein the therapeutic peptide is an inhibitor of poly ADP ribose polymerase (PARP). Aspect 8 provides the following: 8. The engineered cell according to any one of aspects 1 to 7, wherein the non-naturally occurring peptide is a peptide having 90% or less sequence identity to a naturally occurring peptide. Aspect 9 provides the following: 8. The engineered cell according to any one of aspects 1 to 7, wherein the non-naturally occurring peptide is a peptide having 80% or less sequence identity to a naturally occurring peptide. Aspect 10 provides the following: 10. The engineered cell of any one of embodiments 1 to 9, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 16. Aspect 11 provides the following: 11. The engineered cell according to any one of embodiments 1 to 10, wherein the therapeutic peptide is excreted from the engineered cell in extracellular vesicles. Aspect 12 provides the following: 12. The engineered cell of any one of embodiments 1-11, wherein the therapeutic peptide is a mimetic of a SCFA that binds to a G protein-coupled receptor (GPCR). Aspect 13 provides the following: 13. The engineered cell according to any one of embodiments 1 to 12, wherein the target cell is a tumor cell. Aspect 14 provides the following: 14. The engineered cell according to any one of embodiments 1 to 13, which is a T cell or a NK cell. Aspect 15 provides the following: 15. The engineered cell of any one of embodiments 1 to 14, wherein the antigen-binding domain is selected from the group consisting of an antibody, an antigen-binding fragment (Fab), and a single chain variable fragment (scFv). Aspect 16 provides the following: 15. The engineered cell of any one of embodiments 1 to 14, wherein the binding domain is a T cell receptor (TCR). Aspect 17 provides the following: 17. The engineered cell of any one of embodiments 1-16, wherein the target antigen is selected from the group consisting of CD19, CD20, CD22, BCMA, CD123, CD133, EGFR, EGFRvIII, mesothelin, Her2, PSMA, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA 125, CTAG1B, mucin 1, and folate receptor-alpha. Aspect 18 provides the following: 20. The engineered cell of embodiment 17, wherein the target antigen is expressed on an intestinal cell. Aspect 19 provides the following: 19. The engineered cell of any one of embodiments 1-18, wherein the transmembrane domain is a transmembrane domain from a protein selected from the group consisting of CD8 alpha, CD3 zeta, CD3 epsilon, CD28, and ICOS. Aspect 20 provides the following: 20. The engineered cell of any one of embodiments 1-19, wherein the intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, TCR zeta, CD3 epsilon, CD3 gamma, CD3 delta, or ICOS. Aspect 21 provides the following: 20. The engineered cell of any one of embodiments 1-19, wherein the intracellular signaling domain comprises a functional signaling domain and further comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain from 4-1BB or CD28. Aspect 22 provides the following: 22. The engineered cell of any one of embodiments 1-21, wherein the CAR comprises an anti-CD19 scFv, a CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. Aspect 23 provides the following: The engineered cell of any one of embodiments 1-13, wherein the therapeutic peptide is a SCFA mimetic or a mimetic of a steroid and / or hormone-like molecule, and the engineered cell is further modified to reduce activity of one or more effector functions. Aspect 24 provides the following: 24. The engineered cell of embodiment 23, wherein the engineered cell has been modified to reduce or prevent expression of one or more inflammatory cytokines, expression of granzyme B, or expression of perforin. Aspect 25 provides the following: 25. The engineered cell of any one of embodiments 1-24, wherein the CAR molecule and the therapeutic peptide are expressed from the same expression construct, wherein the expression construct further comprises an RNA molecule that activates a PRR. Aspect 26 provides the following: 26. The engineered cell of embodiment 25, wherein the RNA molecule is 7SL. Aspect 27 provides the following: A composition comprising an engineered cell according to any one of embodiments 1 to 26. Aspect 28 provides the following: (i) a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain; (ii) a therapeutic peptide; A nucleic acid molecule encoding a therapeutic peptide, wherein the therapeutic peptide is a non-naturally occurring peptide. Aspect 29 provides the following: 30. The nucleic acid molecule of embodiment 28, wherein a stop codon separates the nucleic acid segment encoding the CAR and the nucleic acid segment encoding the therapeutic peptide. Aspect 30 provides the following: The therapeutic peptide encoded by the nucleic acid molecule has the following characteristics: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the therapeutic peptide is a mimetic of a cyclic dinucleotide; (iii) the therapeutic peptide is a mimetic of a short chain fatty acid (SCFA); (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) the therapeutic peptide is a mimetic of a steroid / hormone-like molecule; (vi) the therapeutic peptide promotes apoptosis of a target cell; (vii) the therapeutic peptide is an immunogenic epitope, and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in the target cell 30. The nucleic acid molecule of embodiment 28 or embodiment 29, having one or more of: Aspect 31 provides the following: The nucleic acid molecule of embodiment 30, wherein the therapeutic peptide is a mimetic of cGAMP, cAMP, or cGMP. Aspect 32 provides the following: The nucleic acid molecule of embodiment 30, wherein the therapeutic peptide is a mimetic of a TLR agonist. Aspect 33 provides the following: 33. The nucleic acid molecule of embodiment 32, wherein the therapeutic peptide is a mimic of flagellin, a CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL). Aspect 34 provides the following: 31. The nucleic acid molecule of embodiment 30, wherein the therapeutic peptide is a second mitochondrial-derived activator of caspase (SMAC) mimetic. Embodiment 35 provides the following: 31. The nucleic acid molecule of embodiment 30, wherein the therapeutic peptide is an inhibitor of poly ADP ribose polymerase (PARP). Aspect 36 provides the following: 36. The nucleic acid molecule according to any one of embodiments 28 to 35, wherein the nucleic acid encoding the non-natural peptide has a sequence identity of 80% or less to a nucleic acid encoding a naturally occurring peptide. Aspect 37 provides the following: 37. The nucleic acid molecule according to any one of embodiments 28 to 36, wherein the target cell is a tumor cell. Embodiment 38 provides the following: 38. The nucleic acid molecule according to any one of aspects 28 to 37, wherein the antigen-binding domain encoded by the nucleic acid molecule is selected from the group consisting of an antibody, a Fab, and an scFv. Aspect 39 provides the following: 38. The nucleic acid molecule according to any one of aspects 28 to 37, wherein the binding domain encoded by the nucleic acid molecule is TCR. Aspect 40 provides the following: 40. The nucleic acid molecule of any one of aspects 28 to 39, wherein the binding domain encoded by the nucleic acid molecule binds to a target antigen selected from the group consisting of CD19, CD20, CD22, BCMA, CD123, CD133, EGFR, EGFRvIII, mesothelin, Her2, PSMA, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA 125, CTAG1B, mucin 1, and folate receptor-alpha. Aspect 41 provides the following: 41. The nucleic acid molecule according to any one of aspects 28 to 40, wherein the transmembrane domain encoded by the nucleic acid molecule is a transmembrane domain from a protein selected from the group consisting of CD8 alpha, CD3 zeta, CD3 epsilon, CD28, and ICOS. Aspect 42 provides the following: 42. The nucleic acid molecule of any one of embodiments 28 to 41, wherein the intracellular signaling domain encoded by the nucleic acid molecule comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, TCR zeta, CD3 epsilon, CD3 gamma, CD3 delta, or ICOS. Aspect 43 provides the following: 43. The nucleic acid molecule of any one of aspects 28 to 42, wherein the intracellular signaling domain encoded by the nucleic acid molecule comprises a functional signaling domain and further comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain from 4-1BB or CD28. Aspect 44 provides the following: 44. The nucleic acid molecule of any one of embodiments 28 to 43, encoding a CAR molecule comprising an anti-CD19 scFv, a CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. Aspect 45 provides the following: 45. The nucleic acid molecule of any one of embodiments 28 to 44, further comprising an RNA molecule that activates a PRR. Aspect 46 provides the following: The engineered cell of embodiment 45, wherein the RNA molecule is 7SL. Aspect 47 provides the following: An expression vector comprising the nucleic acid molecule according to any one of embodiments 28 to 46. Aspect 48 provides the following: 48. A method for co-expressing a CAR and a therapeutic peptide in a cell, comprising delivering to the cell an expression vector of embodiment 47 under conditions such that the CAR and the therapeutic peptide are expressed. Aspect 49 provides the following: A cell comprising a nucleic acid molecule according to any one of aspects 28 to 45 or an expression vector according to aspect 47. Embodiment 50 provides the following: 1. A method for treating a disease or disorder in a subject comprising administering to the subject an effective amount of T cells genetically modified to express a chimeric antigen receptor (CAR), the CAR comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, the method further comprising stimulating an endogenous immune response against cancer via a non-naturally occurring therapeutic peptide, wherein the non-naturally occurring therapeutic peptide is expressed in and / or administered in combination with the modified T cells, and wherein the non-naturally occurring therapeutic peptide has the following characteristics: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the therapeutic peptide is a mimetic of a cyclic dinucleotide; (iii) the therapeutic peptide is a mimetic of a short chain fatty acid (SCFA); (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) the therapeutic peptide is a mimetic of a steroid / hormone-like molecule; (vi) the therapeutic peptide promotes apoptosis of a target cell; (vii) the therapeutic peptide is an immunogenic epitope, and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in the target cell The method of claim 1, further comprising one or more of the steps of: Aspect 51 provides the following: The method of embodiment 50, wherein the therapeutic peptide is a mimic of cGAMP, cAMP, or cGMP. Embodiment 52 provides the following: The method of embodiment 50, wherein the therapeutic peptide is a mimic of a TLR agonist. Embodiment 53 provides the following: The method of embodiment 50, wherein the therapeutic peptide is a mimic of flagellin, a CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL). Embodiment 54 provides the following: The method of embodiment 50, wherein the therapeutic peptide is a second mitochondrial-derived activator of caspase (SMAC) mimetic. Embodiment 55 provides the following: The method of embodiment 50, wherein the therapeutic peptide is an inhibitor of poly ADP ribose polymerase (PARP). Embodiment 56 provides the following: 51. The method of embodiment 50, wherein the non-naturally occurring peptide is a peptide having 80% or less sequence identity to any naturally occurring peptide. Embodiment 57 provides the following: The method of embodiment 50, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-16. Embodiment 58 provides the following: The method of embodiment 50, wherein the therapeutic peptide is an immunogenic epitope, wherein after administration to a subject, the immunogenic epitope is expressed on the surface of cancer cells of the subject. Embodiment 59 provides the following: The method of any one of embodiments 51-58, wherein a therapeutic peptide is expressed in the modified T cell, and wherein following administration of the modified T cell to a subject, the therapeutic peptide is excreted from the modified T cell in one or more extracellular vesicles. Embodiment 60 provides the following: The method of embodiment 59, wherein the therapeutic peptide is delivered to one or more antigen-presenting cells of the subject via one or more extracellular vesicles. Aspect 61 provides the following: 1. A method for enhancing anti-cancer activity of T cells genetically engineered to express a chimeric antigen receptor (CAR), the CAR comprising an antigen binding domain that specifically binds to an antigen expressed on a tumor cell, a transmembrane domain, and a signaling domain, the method comprising co-expressing a non-naturally occurring therapeutic peptide in the T cells, the non-naturally occurring therapeutic peptide having the following properties: (i) the therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the therapeutic peptide is a mimetic of a cyclic dinucleotide; (iii) the therapeutic peptide is a mimetic of a short chain fatty acid (SCFA); (iv) the therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) the therapeutic peptide is a mimetic of a steroid / hormone-like molecule; (vi) the therapeutic peptide promotes apoptosis of a target cell; (vii) the therapeutic peptide is an immunogenic epitope, and / or (viii) the therapeutic peptide blocks one or more DNA repair mechanisms in the target cell The method of claim 1, further comprising one or more of the steps of: Embodiment 62 provides the following: A method for treating an inflammatory disease, an autoimmune disease, or cancer in a subject comprising administering to the subject an effective amount of an engineered cell according to any one of aspects 1 to 26, or a composition according to aspect 27, or a cell according to aspect 49. Embodiment 63 provides the following: The method of any one of aspects 50 to 61, wherein the cancer is a solid tumor cancer. Embodiment 64 provides the following: 64. The method of embodiment 63, wherein the cancer is selected from the group consisting of lung cancer, small cell lung cancer, non-small cell lung cancer, mesothelioma, pancreatic cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, prostate cancer, colon cancer, gastric cancer, bladder cancer, esophageal cancer, and melanoma. Embodiment 65 provides the following: The method of any one of aspects 50 to 61, wherein the cancer is a blood cancer. Embodiment 66 provides the following: The method of embodiment 65, wherein the hematological cancer is leukemia or lymphoma. Aspect 67 provides the following: Blood cancers include chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia, myeloproliferative neoplasms, and follicular 66. The method of embodiment 65, wherein the tumor is selected from the group consisting of myeloma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, lymphoplasmacytic lymphoma, and plasma cell myeloma. Embodiment 68 provides the following: The method of embodiment 62, wherein the autoimmune disease is inflammatory bowel disease.

[0354] The contents of the articles, patents and patent applications, and all other documents and electronically available information mentioned or cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0355] Although the present disclosure has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various modifications and equivalent substitutions may be made without departing from the true spirit and scope of the present disclosure. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the appended claims. Although specific embodiments have been described in detail herein, the same will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0356] Experimental Examples The invention will now be described with reference to the following examples, which are provided for purposes of illustration only, and the invention is not limited to these examples, but includes all variations that are evident as a result of the teachings provided herein.

[0357] Example 1: CAR-T cells engineered to express and deliver immunogenic peptide epitopes A study was performed to evaluate whether CAR-T cells could be engineered to express and transport immunogenic peptide epitopes. SIINFEKL peptide (SEQ ID NO:7) was utilized as the test peptide. Figure 2 provides a schematic diagram of the viral construct for SIINFEKL peptide delivery. The construct includes 19BBz CAR molecule (anti-CD19 scFv, and 4-1BB and CD3 zeta intracellular domains) and SIINFEKL peptide ("Ova-19BBz CAR"). The short peptide length tolerates internal stop codons, which facilitates efficient expression of both peptide and CAR molecule. The construct of SIINFEKL peptide as shown in Figure 2 was incorporated into CD3+ T cells isolated from a subject. The cells were then evaluated by flow cytometry to determine whether CAR and / or peptide were expressed in the engineered cells. Figure 3 shows that both peptide and CAR molecule were efficiently expressed. Peptide expression was detected by SIINFEKL peptide / MHC specific antibody. Furthermore, peptide / MHC complexes were detected not only on CAR+ cells but also on non-transduced (non-CAR-expressing) cells, indicating successful transfer of antigen peptides to other cells in the population (Figure 4).

[0358] Further studies were performed to evaluate the transfer of antigenic peptides from CAR-expressing cells to tumor and / or immune cells. As shown in the schematic depiction in Figure 5, Ova-19BBz CAR T cells were expanded in culture and cells and EVs were harvested after expansion. For expansion, T cells were isolated from mouse spleen cells using a negative selection T cell isolation kit. Following isolation, T cells were stimulated with CD3 / CD28 beads and 24 hours later, transduced with retrovirus (MSGV backbone) encoding the indicated CAR molecule. 48 hours after transduction, cells were de-beaded and examined for CAR transduction.

[0359] Extracellular vesicles (EVs) were collected from the medium via ultracentrifugation. EVs were then collected at 1 × 10 6OT-I T cells were incubated in vitro with various concentrations of B16 cells (melanoma cell line). After 72 hours, peptide / MHC loading and OT-I T cell activation were assessed by flow cytometry. T cell activation was determined by staining for Granzyme B. As shown in Figure 6A, a dose response of increased Ova / MHC expression on tumors as well as increased Granzyme B expression in OT-I T cells was observed with increasing concentrations of EVs from Ova-CAR-T expressing cells. Similarly, Ki67 (proliferation) and IFNγ expression on OT-I T cells were also increased in a dose-dependent manner in the presence of EVs released from Ova-CAR-T expressing cells as shown in Figure 6B. Figure 6C provides quantification of Ki67 expression (left panel), Granzyme B expression (middle panel) and IFNγ expression shown in Figures 6A and 6B. The data showed that CAR-T cells expressed and delivered the peptide to tumor cells, where it was presented in the context of MHC. Moreover, this peptide delivery enhanced antitumor T cell responses in a dose-dependent and statistically significant manner.

[0360] Utilizing the same experimental setup (Figure 5), tumor cell death was also assessed at various concentrations of Ova-CAR T EV loading by flow cytometry. Relative cell death is shown in Figure 7. At increasing EV concentrations (0, 18, 37.5, and 75 μg), incubation of B16 cells with EVs and OT-I T cells resulted in a statistically significant increase in tumor cell death levels. Thus, in vitro studies demonstrated that CAR-T cells engineered to express immunogenic peptides can transfer immunogenic peptides to tumor cells and elicit enhanced T cell responses and tumor death compared to CAR-T cells that do not express immunogenic peptides.

[0361] To examine whether CAR-T cells express and transfer immunogenic peptide epitopes in vivo, mice were implanted with a 1:1 mixture of B16 WT and B16-hCD19 tumor cells (50,000 cells) and then 4.5 × 10 6Ova-19BBz CAR-T cells. After 4 days, tumors were harvested and peptide loading of tumor cells and DCs was assessed by flow cytometry. Results are provided in Figure 9A. In animals receiving control CAR-T cells (19BBz), no peptide / MHC loading was detected in tumor or dendritic cells (gated on live cells / CD45.2+, F4 / 80-, CD11c+ / MHCII+). However, peptide / MHC positive cells were detected in both tumor and dendritic cells isolated from animals receiving Ova-19BBz CAR-T cells. Ova peptide loading also resulted in an expansion of Ova-specific CD8+ T cells as detected by positive tetramer staining in Ova-19BBz recipients (Figure 9B, center panel) compared to 19BBz control recipients (Figure 9B, left panel). Tetramer-positive Ova-specific T cells were also positive for Ki67, indicating activation (FIG. 9B, right panel). FIG. 10A shows the percentage of Ova+ tumor cells (left panel) and endogenous immune cells (right panel) in 19BBz and Ova-19BBz recipient mice. As above, no Ova+ tumor cells or endogenous immune cells were detected in animals receiving control 19BBz cells, whereas a large number of tumor cells and endogenous immune cells presented Ova in Ova-19BBz recipients. Quantification of the percentage of endogenous T cells stained positive for Ova tetramer and the percentage of CD8 T cells expressing Ki67 is provided in the top two panels of FIG. 10B. Tumor weights of the two different groups on day 16 are shown in the bottom panel of FIG. 10B. Tumor weights in Ova-19BBz recipients were significantly lower than those in 19BBz recipients (p=0.002).

[0362] Using a similar in vivo experimental setup, but using a 1:1 mixture of B16-hCD16 and B16 WT cells for tumor implantation, we further evaluated the ability of Ova-19BBz CAR-T cells to control tumor growth in vivo. Mice were implanted with a B16-hCD19 / B16 tumor cell mixture and then treated with Ova-19BBz CAR-T cells or control 19BBz CAR-T cells on day 12 post-implantation (Figure 11A). Tumor cell growth was monitored over time and tumor volume (cm 3 ) in the 19BBz group. Figure 11B shows that by day 21 after tumor implantation (8 days after CAR-T cell administration), tumor volumes were significantly higher in the 19BBz group compared to the Ova-19BBz group. At days 24 and 28 after tumor implantation, the difference in tumor volume between the two groups continued to increase over time (p<0.001).

[0363] In summary, this study demonstrated that incorporating immunogenic peptide epitopes into CAR constructs and expressing the CAR molecules and peptides in cells can enhance CAR-T therapy by transferring immunogenic peptides to tumor cells and dendritic cells and enhancing antitumor T cell responses. This immunogenic peptide transfer leads to enhanced antigen-specific T cell activation and antitumor activity, including significant inhibition of tumor size. Thus, this study demonstrated that the combined method of expressing immunogenic peptides and CAR-T cell therapy significantly improves the efficacy of CAR-T cell therapy.

[0364] To investigate the effect of Ova-19BBz CAR-T cells in the context of poorly immunogenic cancer, mice were implanted with mixed KP tumors and then treated with Ova-19BBz CAR-T cells on days 5 and 12 after implantation in combination with anti-CTLA4 and anti-PD1 antibodies on days 8, 11, and 14 (Figure 12A). Growth and survival were monitored. In addition, the study was designed to evaluate the effect of a CAR containing a combination of Ova peptide and RNA RN7SL1 (7SL) in poorly immunogenic cancer; see construct in Figure 12A. 7SL is a highly structured RNA that functions as an intratumoral PAMP and activates PRR signaling. 7SL CAR-T cells (e.g., BBz-7SL) are discussed, for example, in International Patent Application No. PCT / US2019 / 012675 and Johnson et al., J. Immunol vol. 202(1) 134-4 (2019). As shown in Figures 12B and 12C, in the context of very low immunogenic KP tumors, the combination of Ova peptide with 7SL PAMP resulted in significantly reduced tumor volume and significantly improved survival over time compared to 19BBz CAR-T cells, as well as compared to Ova-19BBz or BBz-7SL CAR-T cells. Therefore, the combination of immunogenic peptide and 7SL significantly improved the antitumor effect of CAR-T cells in this context.

[0365] Many solid human tumors lack sufficient neoantigens and / or an adequate antitumor T cell repertoire, either of which may limit the endogenous T cell response initiated by CAR-T cells expressing RN7SL1. To address this, CAR-T cells were engineered to co-deliver RN7SL1 with a peptide antigen of choice. As a proof of concept, 19BBz CAR-T cells were engineered to express the SIINFEKL peptide alone (Ova-19BBz) or together with RN7SL1 (Ova-19-7SL) (Figure 26A, top). Using an antibody that detects SIINFEKL complexed to MHC class I (Porgador et al., (1997) Immunity 6, 715-726), we demonstrated that the peptide was effectively presented on Ova-19BBz CAR-T cells and was also expressed by CAR-T cells. - SIINFEKL was also detected on T cells (Figure 26A, bottom), demonstrating successful expression and deployment of this peptide antigen. Indeed, when EVs from Ova-19BBz CAR-T ...

Claims

1. An engineered cell comprising a chimeric antigen receptor (CAR) and a non-naturally occurring therapeutic peptide, wherein the CAR comprises an antigen binding domain having affinity for a target antigen on a target cell, a transmembrane domain, and an intracellular signaling domain, and wherein the CAR and the non-naturally occurring therapeutic peptide are expressed from the same expression construct in the engineered cell.

2. An engineered cell comprising a chimeric antigen receptor (CAR) and a non-naturally occurring therapeutic peptide, wherein the CAR comprises an antigen binding domain having affinity for a target antigen on a target cell, a transmembrane domain, and an intracellular signaling domain, and the non-naturally occurring therapeutic peptide has the following properties: (i) the non-natural therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the non-natural therapeutic peptide is a mimetic of a cyclic dinucleotide; (iii) the non-natural therapeutic peptide is a mimetic of a short chain fatty acid (SCFA); (iv) the non-natural therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) The non-natural therapeutic peptide is a mimetic of a steroid / hormone-like molecule; (vi) the non-natural therapeutic peptide promotes apoptosis of a target cell; (vii) the non-natural therapeutic peptide is an immunogenic epitope, and / or (viii) The non-natural therapeutic peptide blocks one or more DNA repair mechanisms in the target cell. An engineered cell having one or more of the following:

3. A non-natural therapeutic peptide, (a) a mimic of cGAMP, cAMP, or cGMP; (b) a mimic of a TLR agonist; (c) a mimic of flagellin, a CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL); (d) a second mitochondria derived activator of caspase (SMAC) mimic; or (e) an inhibitor of poly ADP-ribose polymerase (PARP); 3. The engineered cell of claim 1 or claim 2.

4. 4. The engineered cell of any one of claims 1 to 3, wherein the non-natural therapeutic peptide has 90% or less sequence identity to the naturally occurring peptide.

5. 5. The engineered cell of any one of claims 1 to 4, wherein the non-natural therapeutic peptide has 80% or less sequence identity to the naturally occurring peptide.

6. The engineered cell described in any one of claims 1 to 5, wherein the non-natural therapeutic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 16.

7. The engineered cell described in any one of claims 1 to 6, wherein the non-natural therapeutic peptide is excreted from the engineered cell in extracellular vesicles.

8. The engineered cell of any one of claims 1 to 7, wherein the non-natural therapeutic peptide is a mimetic of a short chain fatty acid (SCFA) that binds to a G protein-coupled receptor (GPCR).

9. The engineered cell of any one of claims 1 to 8, wherein the target cell is a tumor cell.

10. 10. The engineered cell of any one of claims 1 to 9, which is a T cell or a NK cell.

11. 11. The engineered cell of any one of claims 1 to 10, wherein the antigen-binding domain is selected from the group consisting of an antibody, an antigen-binding fragment (Fab), and a single chain variable fragment (scFv).

12. The engineered cell of any one of claims 1 to 11, wherein the antigen-binding domain is a T cell receptor (TCR).

13. 13. The engineered cell of any one of claims 1-12, wherein the target antigen is selected from the group consisting of CD19, CD20, CD22, BCMA, CD123, CD133, EGFR, EGFRvIII, mesothelin, Her2, PSMA, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA 125, CTAG1B, mucin 1, and folate receptor-alpha.

14. 14. The engineered cell of claim 13, wherein the target antigen is expressed on an intestinal cell.

15. 15. The engineered cell of any one of claims 1 to 14, wherein the transmembrane domain is a transmembrane domain from a protein selected from the group consisting of CD8 alpha, CD3 zeta, CD3 epsilon, CD28, and ICOS.

16. 16. The engineered cell of any one of claims 1-15, wherein the intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, TCR zeta, CD3 epsilon, CD3 gamma, CD3 delta, and ICOS.

17. 17. The engineered cell of any one of claims 1-16, wherein the intracellular signaling domain comprises a functional signaling domain and further comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain from 4-1BB or CD28.

18. The engineered cell of any one of claims 1 to 17, wherein the CAR comprises an anti-CD19 scFv, a CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

19. The engineered cell of any one of claims 1 to 18, wherein the non-natural therapeutic peptide is a SCFA mimetic or a mimetic of a steroid and / or hormone-like molecule, and the engineered cell has been modified to reduce activity of one or more effector functions.

20. 20. The engineered cell of claim 19, wherein the engineered cell has been modified to reduce or prevent expression of one or more inflammatory cytokines, expression of granzyme B, or expression of perforin.

21. 21. The engineered cell of any one of claims 1-20, wherein the CAR and the non-naturally occurring therapeutic peptide are expressed from the same expression construct, wherein the expression construct further comprises an RNA molecule that activates a pattern recognition receptor (PRR).

22. 22. The engineered cell of claim 21 , wherein the RNA molecule is 7SL.

23. A composition comprising the engineered cell of any one of claims 1 to 22.

24. (i) a chimeric antigen receptor (CAR) comprising an antigen-binding domain having affinity for a target antigen on a target cell, a transmembrane domain, and an intracellular signaling domain; (ii) a non-natural therapeutic peptide; A nucleic acid molecule encoding

25. The nucleic acid molecule of claim 24, wherein a stop codon separates the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the non-naturally occurring therapeutic peptide.

26. A non-natural therapeutic peptide having the following properties: (i) the non-natural therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the non-natural therapeutic peptide is a mimetic of a cyclic dinucleotide; (iii) the non-natural therapeutic peptide is a mimetic of a short chain fatty acid (SCFA); (iv) the non-natural therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) The non-natural therapeutic peptide is a mimetic of a steroid / hormone-like molecule; (vi) the non-natural therapeutic peptide promotes apoptosis of a target cell; (vii) the non-natural therapeutic peptide is an immunogenic epitope, and / or (viii) The non-natural therapeutic peptide blocks one or more DNA repair mechanisms in the target cell.

26. The nucleic acid molecule of claim 24 or claim 25, comprising one or more of:

27. ​​A non-natural therapeutic peptide, (a) a mimic of cGAMP, cAMP, or cGMP; (b) a mimic of a TLR agonist; (c) a mimic of flagellin, a CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL); (d) a second mitochondrial-derived caspase activator (SMAC) mimetic; or (e) an inhibitor of poly ADP-ribose polymerase (PARP); 27. The nucleic acid molecule of claim 26.

28. 28. The nucleic acid molecule of any one of claims 24 to 27, wherein the nucleic acid sequence encoding the non-natural therapeutic peptide has 80% or less sequence identity to the nucleic acid sequence encoding the naturally occurring peptide.

29. The nucleic acid molecule of any one of claims 24 to 28, wherein the target cell is a tumor cell.

30. The nucleic acid molecule of any one of claims 24 to 29, wherein the antigen-binding domain is selected from the group consisting of an antibody, a Fab, and an scFv.

31. A nucleic acid molecule described in any one of claims 24 to 30, wherein the antigen binding domain is a T cell receptor (TCR).

32. A nucleic acid molecule described in any one of claims 24 to 31, wherein the antigen-binding domain binds to a target antigen selected from the group consisting of CD19, CD20, CD22, BCMA, CD123, CD133, EGFR, EGFRvIII, mesothelin, Her2, PSMA, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA 125, CTAG1B, mucin 1, and folate receptor-alpha.

33. A nucleic acid molecule described in any one of claims 24 to 32, wherein the transmembrane domain is a transmembrane domain from a protein selected from the group consisting of CD8 alpha, CD3 zeta, CD3 epsilon, CD28, and ICOS.

34. A nucleic acid molecule described in any one of claims 24 to 33, wherein the intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, TCR zeta, CD3 epsilon, CD3 gamma, CD3 delta, and ICOS.

35. A nucleic acid molecule described in any one of claims 24 to 34, wherein the intracellular signaling domain comprises a functional signaling domain and further comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain from 4-1BB or CD28.

36. A nucleic acid molecule described in any one of claims 24 to 35, wherein the CAR comprises an anti-CD19 scFv, a CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

37. A nucleic acid molecule described in any one of claims 24 to 36, further encoding an RNA molecule that activates a pattern recognition receptor (PRR).

38. 38. The nucleic acid molecule of claim 37, wherein the RNA molecule is 7SL.

39. An expression vector comprising the nucleic acid molecule of any one of claims 24 to 38.

40. 40. A method for co-expressing a CAR and a non-naturally occurring therapeutic peptide in a cell, comprising delivering to the cell the expression vector of claim 39 under conditions such that the CAR and the non-naturally occurring therapeutic peptide are expressed.

41. A cell comprising the nucleic acid molecule of any one of claims 24 to 38 or the expression vector of claim 39.

42. A pharmaceutical composition for treating cancer in a subject, comprising: (a) an effective amount of T cells genetically modified to express a chimeric antigen receptor (CAR) and, optionally, (b) a non-naturally occurring therapeutic peptide, the CAR comprises an antigen-binding domain having affinity for a target antigen on a target cell, a transmembrane domain, and an intracellular signaling domain; If the T cells have not been genetically modified to express a non-naturally occurring therapeutic peptide, the pharmaceutical composition is combined with a non-naturally occurring therapeutic peptide; The non-natural therapeutic peptides stimulate the endogenous immune response against cancer and have the following properties: (i) the non-natural therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the non-natural therapeutic peptide is a mimetic of a cyclic dinucleotide; (iii) the non-natural therapeutic peptide is a mimetic of a short chain fatty acid (SCFA); (iv) the non-natural therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) The non-natural therapeutic peptide is a mimetic of a steroid / hormone-like molecule; (vi) the non-natural therapeutic peptide promotes apoptosis of a target cell; (vii) the non-natural therapeutic peptide is an immunogenic epitope, and / or (viii) The non-natural therapeutic peptide blocks one or more DNA repair mechanisms in the target cell. A pharmaceutical composition comprising one or more of:

43. A non-natural therapeutic peptide, (a) a mimic of cGAMP, cAMP, or cGMP; (b) a mimic of a TLR agonist; (c) a mimic of flagellin, a CpG motif, peptidoglycan, lipopolysaccharide (LPS), or monophosphoryl lipid A (MPL); (d) a second mitochondrial-derived caspase activator (SMAC) mimetic; or (e) an inhibitor of poly ADP-ribose polymerase (PARP); 43. The pharmaceutical composition of claim 42.

44. 44. The pharmaceutical composition of claim 42 or 43, wherein the non-natural therapeutic peptide has 80% or less sequence identity to any naturally occurring peptide.

45. A pharmaceutical composition described in any one of claims 42 to 44, wherein the non-natural therapeutic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 to 16.

46. A pharmaceutical composition described in any one of claims 42 to 45, wherein the non-natural therapeutic peptide is an immunogenic epitope, and the immunogenic epitope is expressed on the surface of a cancer cell of the subject.

47. A pharmaceutical composition described in any one of claims 42 to 46, wherein the non-natural therapeutic peptide is expressed in the modified T cell and the non-natural therapeutic peptide is excreted from the modified T cell into one or more extracellular vesicles of the subject.

48. The pharmaceutical composition of claim 47, wherein the non-natural therapeutic peptide is delivered to one or more antigen-presenting cells of the subject via one or more extracellular vesicles.

49. A pharmaceutical composition for enhancing anti-cancer activity of T cells in a subject, the pharmaceutical composition comprising an effective amount of T cells genetically modified to express a chimeric antigen receptor (CAR), the CAR comprising an antigen binding domain that specifically binds to a target antigen expressed on a target tumor cell, a transmembrane domain, and an intracellular signaling domain, the T cells co-express a non-naturally occurring therapeutic peptide, the non-naturally occurring therapeutic peptide having the following properties: (i) the non-natural therapeutic peptide is an activator of the stimulator of interferon genes (STING) pathway; (ii) the non-natural therapeutic peptide is a mimetic of a cyclic dinucleotide; (iii) the non-natural therapeutic peptide is a mimetic of a short chain fatty acid (SCFA); (iv) the non-natural therapeutic peptide is an agonist of a pattern recognition receptor (PRR); (v) The non-natural therapeutic peptide is a mimetic of a steroid / hormone-like molecule; (vi) the non-natural therapeutic peptide promotes apoptosis of the target tumor cells; (vii) the non-natural therapeutic peptide is an immunogenic epitope, and / or (viii) The non-natural therapeutic peptide blocks one or more DNA repair mechanisms in the target tumor cells. A pharmaceutical composition comprising one or more of:

50. 42. A pharmaceutical composition for treating an inflammatory disease, an autoimmune disease, or cancer in a subject, comprising an effective amount of the engineered cell of any one of claims 1 to 22, or the composition of claim 23, or the cell of claim 41.

51. The pharmaceutical composition of any one of claims 42 to 49, wherein the cancer is a solid tumor cancer.

52. 52. The pharmaceutical composition of claim 51, wherein the cancer is selected from the group consisting of lung cancer, small cell lung cancer, non-small cell lung cancer, mesothelioma, pancreatic cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, prostate cancer, colon cancer, gastric cancer, bladder cancer, esophageal cancer, and melanoma.

53. The pharmaceutical composition of any one of claims 42 to 49, wherein the cancer is a hematological cancer.

54. 54. The pharmaceutical composition of claim 53, wherein the blood cancer is leukemia or lymphoma.

55. Blood cancers include chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia, myeloproliferative neoplasms, and follicular lymphoma.

54. The pharmaceutical composition of claim 53, wherein the tumor is selected from the group consisting of myeloma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, lymphoplasmacytic lymphoma, and plasma cell myeloma.

56. 51. The pharmaceutical composition of claim 50, wherein the autoimmune disease is inflammatory bowel disease.

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