Chimeric antigen receptor constructs

Abstract

The present disclosure provides CAR (chimeric antigen receptor) constructs, T cell populations having the CAR(s), and methods of treating cancer with the T cell populations.

Description

Chimeric Antigen Receptor Constructs

[0001] This application claims priority to U.S. Provisional Application No.63 / 673,254, filed July 19, 2024, which is hereby incorporated by reference. Field of the Invention

[0002] The present disclosure provides CAR (chimeric antigen receptor) constructs, T cell populations having the CAR(s), and methods of treating cancer with the T cell populations. Background of the Invention

[0003] Chimeric antigen receptors (CARs) are synthetic receptors in which a targeting moiety is associated with one or more signaling domains in a single fusion molecule. T-cells having novel specificities have been generated through the genetic transfer of constructs encoding CARs that can specifically bind a target of interest. CARs have successfully been used to engineer T cells to be directed against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors.

[0004] In general, the extracellular targeting moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv) comprising the light and heavy variable fragments of a monoclonal antibody joined by a flexible linker. The intracellular signaling domain for first generation CARs is derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T cell cytotoxicity, however, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX-40 (CD 134), and4-1BB (CD137) have been added either singly (second generation CARs) or in combination (third generation CARs) to enhance survival and increase proliferation of CAR-modified T cells.

[0005] T cells have the power to dispose of normal or malignant cells as seen in viral and autoimmune diseases and as also seen in the rare spontaneous remissions of cancer. It is the goal of CAR-T studies to supply the specificities and affinities to a patient’s T cells, without regard for their "endogenous" T cell receptor (TCR) repertoire, by providing an antibody-defined, anti- malignant cell marker recognition to kill malignant cells based on their expression of antigens recognized by the CAR.

[0006] Adoptive immunotherapy by infusion of T cells engineered CARs for redirected tumoricidal activity has been explored for the treating of metastatic cancer. CARs are constructed by joining the antigen recognition domains of an antibody with the signaling domains of receptors from T cells. Modification of T cells with CAR genes equips T cells with retargeted antibody-type antitumor cytotoxicity. Because killing is Major Histocompatibility Complex (MHC)-unrestricted, the approach offers a general therapy for all patients bearing the same antigen. T cells engineered with antigen specific CARs are called “CAR-T cells” or “T- bodies” (Eshar, et al., 1993 Proc. Nat’l Acad. of Sci. USA 90(2):720-724). A first-generation CAR, immunoglobulin-T cell receptor (IgTCR), was engineered to contain a signaling domain (TCR-CD3ζ) that delivers an activation stimulus (signal 1) only (Gross, et al., 1989 Proc. Nat’l Acad. of Sci. USA 86(24): 10024-10028; Eshar, et al., 1993 Proc. Nat’l Acad. Of Sci. USA 90(2):720-724; Haynes, et al., 2001 J Immunol.166(1): 182-187). T cells engineered to express the first-generation CARs alone exhibit limited anti-tumor efficacy due to suboptimal activation. A second generation CAR, immunoglobulinCD28-CD3ζ-T cell receptor (IgCD28TCR), incorporated a costimulatory CD28 (signal 2) into the first-generation receptor that increased the anti-tumor capacity of the CAR-T cells (Finney, et al., 1998 J Immunol.161(6):2791-2797; Hornbach, et al., 2001 J Immunol.167(11):6123-6131; Maher, et al., 2002 Nat Biotechnol. 20(l):70-7; Emtage, et al., 2008 Clin Cancer Res.14(24):8112-812; Lo, et al., 2010 Clin Cancer Res.16(10):2769-2780).

[0007] The current protocol for treatment of patients using adoptive immunotherapy is based on autologous cell transfer. In this approach, T lymphocytes are recovered from patients, geneticallymodified or selected ex vivo, cultivated in vitro in order to amplify the number of cells if necessary and finally infused into the patient. In addition to lymphocyte infusion, the host (patient) may be manipulated in other ways that support the engraftment of the T cells or their participation in an immune response, for example pre-conditioning (with radiation or chemotherapy) and administration of lymphocyte growth factors (such as IL-2). Each patient receives an individually fabricated treatment, using the patient's own lymphocytes (i.e. an autologous therapy).

[0008] Alternatively, an allogeneic strategy can be used where T cells from a donor is infused into the patient. Allogeneic T cells from healthy donors can be transfected with a CAR construct in advance for treatment of cancer and avoid expensive and time-consuming preparation and testing of each individual patient’s T cells. To avoid incompatibility between donor cells and the patient, the donor may be HLA matched with the patient or the allogeneic T cells expressing the CAR construct may be further genetically altered to eliminate expression of the endogenous T cell receptor.

[0009] CD19 is a 95 kD type II transmembrane glycoprotein of the immunoglobulin superfamily with a long C-terminal cytoplasmic domain that is expressed by cells of the B cell lineage. During ontogeny, CD19 appears on B-lineage committed stem cells and its expression increases with B cell maturation, with mature B cells expressing approximately three-fold the surface density of immature B cells. CD19 is also expressed on malignant B cells, and has been used as a target in anti-neoplastic therapies that originate from the B cell lineage, including for example Non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[0010] Indian Patent Application No.202021050877 discloses lentivirus particles packed with genes expressing chimeric antigen receptors against CD19.

[0011] International Publication Nos. WO 2015 / 185728, and WO 2022 / 081486 discloses CARs targeting CD-19.Summary of the Invention

[0012] The present invention is directed to improved chimeric antigen receptors (CARs), T cells having these CARs, and their use to treat cancer.

[0013] One embodiment is a CAR nucleic acid construct comprising a nucleic acid sequence encoding: (a) an antigen binding protein (e.g., a single-chain variable fragment (scFv)), (b) a hinge region comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the hinge region of hIgG4 (human IgG4) (such as SEQ ID NO: 212), (c) a transmembrane domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the transmembrane domain of hCD28 (human CD28) (such as SEQ ID NO: 213), (d) a first (co-stimulatory) intracellular domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the intracellular T-cell signaling domain of hCD28, 4-1BB, or CD4 (such as SEQ ID NO: 214, 215, or 216, respectively), and (e) a second (stimulatory) intracellular domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the intracellular T-cell signaling domain of hCd3-zeta (such as SEQ ID NO: 217), wherein optionally a double leucine within the CAR (such as within transmembrane domain) is replaced with any other amino acid(s) that are not leucine (such as double alanine) in order to maintain the scFv on the surface of the cell.

[0014] Yet another embodiment is a CAR nucleic acid construct comprising a nucleic acid sequence encoding: (a) an antigen binding protein (e.g., a single-chain variable fragment (scFv)), (b) a hinge region comprising the hinge region of hIgG4 (such as SEQ ID NO: 212), (c) a transmembrane domain comprising the transmembrane domain of hCD28 (such as SEQ ID NO: 213), (d) a first (co-stimulatory) intracellular domain comprising the intracellular T-cellsignaling domain of hCD28, 4-1BB, or CD4 (such as SEQ ID NO: 214, 215, or 216, respectively), and (e) a second (stimulatory) intracellular domain comprising the intracellular T-cell signaling domain of hCD3-zeta (such as SEQ ID NO: 217), wherein optionally a double leucine within the CAR (such as within transmembrane domain) is replaced with any other amino acid(s) that are not leucine (such as double alanine) in order to maintain the scFv on the surface of the cell.

[0015] Yet another embodiment is a chimeric antigen receptor (CAR) nucleic acid construct comprising a nucleic acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 71-134, 204, 207, and 210.

[0016] Yet another embodiment is a chimeric antigen receptor (CAR) nucleic acid construct comprising a nucleic acid sequence that is any one of SEQ ID NOs: 71-134, 204, 207, and 210.

[0017] In one embodiment, the CAR nucleic acid construct is an mRNA. In one embodiment, the mRNA includes a poly(A) tail.

[0018] In one embodiment of any of CARs or CAR nucleic acid constructs, the scFV is an scFv that binds CD19 (such as a human CD19 scFv). In another embodiment, the CAR comprises an anti-CD19 antibody capable of targeting tumor cells that express CD19.

[0019] In another embodiment, the scFv is bispecific. In yet another embodiment, the scFv is trispecific.

[0020] Another embodiment is a lipid nano-particle comprising a CAR nucleic acid construct (such as an mRNA) described herein. The lipid nano-particle may include a lipid (e.g., an ionizable cationic lipid) to enable intracellular delivery of a nucleic acid, e.g., mRNA, to the cytosolic compartment of a target cell type and optionally rapidly degrade into non-toxic components. The lipid may be an ionizable cationic lipid and / or a lipid-immune cell targeting group conjugate (e.g., a lipid-T-cell targeting group conjugate).

[0021] Yet another embodiment is a CAR comprising: (a) an antigen binding protein (e.g., a single-chain variable fragment (scFv)), (b) a hinge region comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the hinge region of hIgG4 (such as SEQ ID NO: 212), (c) a transmembrane domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the transmembrane domain of hCD28 (such as SEQ ID NO: 213), 4-1BB, or CD4, (d) a first (co-stimulatory) intracellular domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the intracellular T-cell signaling domain of hCD28 (such as SEQ ID NO: 214), and (e) a second (stimulatory) intracellular domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the intracellular T-cell signaling domain of hCD3-zeta (such as SEQ ID NO: 217), wherein optionally a double leucine within the CAR (such as within transmembrane domain) is replaced with any other amino acid(s) that are not leucine (such as double alanine) in order to maintain the scFv on the surface of the cell.

[0022] Yet another embodiment is a CAR comprising: (a) an antigen binding protein (e.g., a single-chain variable fragment (scFv)), (b) a hinge region comprising the hinge region of hIgG4 (such as SEQ ID NO: 212), (c) a transmembrane domain comprising the transmembrane domain of hCD28 (such as SEQ ID NO: 213), 4-1BB, or CD4, (d) a first (co-stimulatory) intracellular domain comprising the intracellular T-cell signaling domain of hCD28 (such as SEQ ID NO: 214), and (e) a second (stimulatory) intracellular domain comprising the intracellular T-cell signaling domain of hCD3-zeta (such as SEQ ID NO: 217), wherein optionally a double leucine within the CAR (such as within transmembrane domain) is replaced with any other amino acid(s) that are not leucine (such as double alanine) in order to maintain the scFv on the surface of the cell.

[0023] The CAR includes (i) the hinge region between the scFv (antigen binding protein) and the transmembrane domain, and (ii) the first intracellular domain between the transmembrane domain and the second intracellular domain.

[0024] Yet another embodiment is a CAR comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 138-201, 205, 208 and 211.

[0025] Yet another embodiment is a CAR comprising an amino acid sequence that is any one of SEQ ID NOs: 138-201, 205, 208 and 211.

[0026] Yet another embodiment is a CAR comprising (a) an antigen binding protein (e.g., a single-chain variable fragment (scFv)), (b) a hinge region, (c) a transmembrane domain, (d) optionally a first (co-stimulatory) intracellular domain, and (e) a second (stimulatory) intracellular domain, where a double leucine within the CAR (such as within transmembrane domain) is replaced with any other amino acid(s) that are not leucine (such as double alanine) in order to maintain the scFv on the surface of the cell. In one embodiment, the double leucine, which is replaced, is located in the transmembrane domain. In another embodiment, the double leucine is replaced with double alanine. Yet another embodiment is a nucleic acid construct encoding the aforementioned CAR.

[0027] Yet another embodiment is a method of decreasing the internalization of a receptor protein into the cell. The method comprises replacing any double leucine (LL) within the receptor protein with any other amino acid(s) that is not leucine, thereby maintaining the receptor protein on the surface of the cell. In one embodiment, the receptor protein is a CAR protein. In another embodiment, the double leucine (LL) is replaced with a double alanine (AA). In yet another embodiment, the double leucine (LL) is located within the transmembrane domain of the receptor protein. In yet another embodiment, the receptor protein includes a cytosolic domain. The cytosolic domain may be selected from a co-stimulatory domain and a signaling domain. In one embodiment, the co-stimulatory domain is 4-1BB and / or CD4. In another embodiment, the signaling domain is CD3 zeta. In yet another embodiment, the receptor protein has the amino acid sequence of any one of SEQ ID NOs: 157-159, 163, 166, and 169 (2-CAR-10-hCD28-LL>AA, 3-CAR-10-hCD28-LL>AA-LH, 4-CAR-10-hCD28-LL>AA-YMNM, 8-CAR-10-CD4- LL>AA, 11-CAR-10-LL>AA-CD4LL>AA, and 14-CAR-10-4-1BB-LL>AA).

[0028] Yet another embodiment is a host cell, or a population of host cells, which express any CAR described herein.

[0029] Yet another embodiment is a host cell, or a population of host cells, comprising a CAR nucleic acid construct described herein.

[0030] Yet another embodiment is a host cell, or a population of host cells, transfected with a lipid nano-particle described herein.

[0031] In one embodiment, the host cell or population of host cells are selected from a PBMC- derived T cell (or population thereof), a placental derived T cell (or a population thereof), and a cord blood derived T cell (or population thereof). In another embodiment, the host cell or cells are pan T cells or gd T cells.

[0032] Yet another embodiment is a method of treating a cancer in a patient, comprising administering to the patient a population of host cells as described herein.

[0033] Yet another embodiment is a method of inhibiting growth of a tumor expressing CD19 in a patient (e.g., having cancer), comprising administering to the patient a population of T cells comprising a CAR as described herein (such as one where the scFv binds CD19).

[0034] Yet another embodiment is a method for treating a mammal (e.g., a human) having an autoimmune disease, wherein the method comprises administering to the mammal identified as having an autoimmune disease an effective amount of a population of T cells comprising a CAR as described herein. In one embodiment, the T cells express a first chimeric antigen receptor polypeptide having a first antigen binding domain that binds a first antigen on a CD11c+Tbet+B cell with low affinity, where the binding activates the T cell, and where the T cell expresses a second chimeric antigen receptor polypeptide having a second antigen binding domain that binds a second antigen on a CD11c+Tbet+B cell and stimulates the T cell. In one embodiment, the method reduces the number of age-associated B-cells.

[0035] In one embodiment, the autoimmune disease results from production of autoantibodies by age-associated B cells. The autoimmune disease may be lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, Crohn's disease, Celiac disease, or polyarteritis nodosa.

[0036] In one embodiment, the population of T cells are isolated from peripheral blood mononuclear cells (PBMCs).

[0037] In another embodiment, the population of T cells are isolated from placental tissue or cord blood.

[0038] In yet another embodiment, the population of T cells are allogeneic with respect to the patient.

[0039] In one embodiment, the cancer is a hematological cancer.

[0040] In another embodiment, the cancer is non-Hodgkin's lymphoma (NHL), B chronic lymphocytic leukemia (B-CLL), or B acute lymphocytic leukemia (ALL).

[0041] The CAR(s) of the present invention may be incorporated into T-cells by transfecting T- cells with lipid nanoparticles containing mRNA encoding the CAR(s), such as those lipid nanoparticles described herein.

[0042] In one embodiment, the nucleic acid construct further includes a nucleic acid sequence encoding a signal peptide at the N-terminus of the CAR that may be removed when the synthesized CAR is localized to the cell membrane.

[0043] In some embodiments, the CAR nucleic acid construct can optionally include a sequence encoding a peptide tag for detection of the synthesized CAR, for example, a myc tag (or another peptide tag.

[0044] A nucleic acid molecule comprising a sequence encoding a CAR as disclosed herein can be, as nonlimiting examples, a DNA molecule or RNA molecule (e.g., an mRNA), and can optionally include one or more modified nucleotides and / or one or more backbone modifications. The nucleic acid molecule can be a linear nucleic acid molecule or can be a circular nucleic acid molecule, and can be double-stranded or single-stranded. A nucleic acid molecule as provided herein can be a vector that includes the CAR-encoding sequence. The nucleic acid molecule can be a viral vector or a viral genome (in circular or linear form). In some embodiments the vector may be a lentiviral or retroviral transfer vector, for example, a replication-defective lentiviral or retroviral (e.g., gammaretroviral) transfer vector, that includes a lentiviral or retroviral packaging sequence. Further included herein is a lentivirus or retrovirus that includes a CAR encoding nucleic acid sequence. In some embodiments the retrovirus is a gammaretrovirus. The CAR- encoding sequence of a nucleic acid molecule as provided herein can be operably linked to expression control sequences, such as but not limited to a promoter. A promoter operably linked to a CAR-encoding sequence is preferably a promoter active in a human cell and in some embodiments can be a retroviral promoter, e.g., an LTR promoter of a retrovirus.

[0045] The CAR can be a polypeptide inserted into the membrane of a transgenic cell, such as an engineered host cell as disclosed herein.

[0046] The present disclosure further provides a host cell, or a population of host cells, that includes a CAR construct as described herein.

[0047] The CAR construct may further include a signal peptide-encoding sequence at the N- terminus of the CAR-encoding sequence. The CAR construct can optionally also include a sequence encoding a peptide tag for detection of the synthesized CAR, such as, for example, a myc tag or another peptide tag.

[0048] A cell engineered to express a nucleic acid molecule that includes a sequence encoding a CAR with a scFv targeting CD19 as provided herein can specifically bind CD19. Exemplary nucleic acid sequences encoding CARs include SEQ ID NOs:4-67, 70-134, 203, 204, 206, 207, 209, and 210.

[0049] The host cell or population of host cells can be, for example, T cells, and can be pan T cells which may be PBMC-derived T cells, placenta-derived T cells, or cord blood derived T cells, or the T cells may be a subpopulation of T cells isolated from PBMCs, placenta, or cord blood. In various embodiments, the host cells are cells transduced with a lentvirus or retrovirus (e.g., a gammaretrovirus) having a nucleic acid sequence that encodes a CAR as provided herein. The nucleic acid sequence encoding the CAR can be operably linked to a promoter which directs expression of the CAR construct in the transduced host cell or population of host cells. The promoter in some embodiments can be a retroviral promoter, for example, a promoter of the LTR of the retroviral vector.

[0050] The CAR-encoding nucleic acid sequence may be inserted into the genome of the cell. In some examples, the host cells, for example T cells, are transduced via a retrovirus engineered to include a gene encoding a CAR as disclosed herein, preferably operably linked to a promoter, which may be a retroviral promoter (e.g., a promoter of a retroviral LTR) and the transduced host cells include a retrovirally-mediated insertion of the nucleic acid sequence encoding the CAR. The transduced host cells may also include additional retroviral sequences associated with the CAR construct, such as for example, at least a portion of a retroviral (e.g., gammaretroviral) packaging signal.

[0051] The host cell or a population of host cells provided herein express the CAR construct, where the expressed CAR on the host cell or population of host cells specifically binds to one or more targets (e.g., CD19). In one embodiment, the T cell or a population of T cells that express the CAR construct, preferentially kill (are cytotoxic toward) cells e.g., target cells) that express the target (e.g., CD19).

[0052] A host cell or population of host cells that includes a CAR construct as provided herein can have one or more genes that encodes a T cell receptor (TCR) subunit that is disrupted. For example, one or more of a TCRa or TCRP gene may be mutated such that no functional subunit is produced. TCR subunit genes may be disrupted for example with CRISPR / Cas methods, Talens, or zinc finger nucleases.

[0053] In some embodiments, a host cell or population of host cells that express the CAR, can be a T cell (or a population thereof), which may be, for example, a peripheral blood derived T cell (or a population thereof), a placenta-derived T cell (or a population thereof) or a cord blood derived T cell (or a population thereof).

[0054] The present disclosure provides a method for treating a cancer or inhibiting tumor growth in a subject in need of a treatment comprising: administering to the subject a population of T cells expressing a CAR (e.g., a CD19 CAR) as provided herein. The T cells administered to the subject are host cells as described herein that can include a nucleic acid molecule that encodes a CAR as provided herein. The T cells can be, for example, peripheral blood-derived T cells, or may be T cells derived from placenta and / or cord blood. The CAR expressed by the T cells can optionally include a peptide tag for detection of the expressed CAR, such as, for example, a myc tag or another peptide tag.

[0055] The present disclosure provides methods for conducting adoptive cell therapy by administering to a subject genetically engineered cells expressing any of the CARs described herein. The methods provided herein include, for example, administering to a human subject a host cell which expresses a CAR described herein (or a host cell transduced to include a nucleic acid sequence encoding a CAR as described herein).

[0056] The methods described herein can be used cancer, including, but not limited to, hematologic breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, renal cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma. In various embodiments, the cancer is a hematologic cancer selected from the group consisting of acute lymphocytic leukemia (ALL), B chronic lymphocytic leukemia (B-CLL), non-Hodgkin's lymphoma (NHL), and Burkitt's lymphoma (BL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML). In some embodiments the hematologic cancer is selected from a group consisting of non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), and acute lymphocytic leukemia (ALL).Detailed Description of the Invention

[0057] The disclosed chimeric antigen receptor (CAR) constructs can preferentially bind to tumor cells such as those that express CD19. Transformed T cells are provided that include the CAR constructs that encode an scFv antibody (e.g., one targeted to CD19).

[0058] The sequences references by SEQ ID NO: can be found in Table I at the end of the specification. Definitions

[0059] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art.

[0060] The methods and techniques provided herein are generally performed according to conventional procedures well known in the art and as described in various general and more specific references that are cited and discussed herein unless otherwise indicated.

[0061] Throughout this application various publications, patents, and / or patent applications are referenced. The disclosures of the publications, patents and / or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0062] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[0063] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms “a”, “an” and “the”, and singular use ofany word, include plural referents unless expressly and unequivocally limited on one referent.

[0056] It is understood the use of the alternative (e.g., “or”) herein is taken to mean either one or both or any combination thereof of the alternatives.

[0064] The term “and / or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0065] As used herein, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0066] The term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends, in part, on how the value is measured or determined. For example, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

[0067] The term "isolated" refers to a protein (e.g., an antibody) or polynucleotide that is substantially free of other cellular material. A protein may be rendered substantially free of naturally associated components (or components associated with the cellular expression system used to produce the antibody) by isolation, using protein purification techniques well known in the art. Isolated cells are removed from their in vivo cellular milieu, for example, are no longer associated with the tissue they are associated with in vivo (e.g., blood, tumor) and may be partially or substantially isolated from or enriched with respect to other cell types present in the tissue they are associated with in vivo. In various embodiments, constructs of the disclosure thatencode chimeric antigen receptors, or host cells that include and / or express the disclosed constructs encoding the chimeric antigen receptors, are isolated.

[0068] The terms “nucleic acid”, "polynucleotide" and "oligonucleotide" and are used interchangeably and refers to polymers of nucleotides. Nucleic acids include naturally-occurring and recombinant forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecule can be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules of the disclosure comprise a contiguous open reading frame encoding an antibody, or a fragment or scFv, derivative, mutein, or variant thereof.

[0069] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides comprise natural and non-natural amino acids. Polypeptides can be naturally-occurring or recombinant forms. These terms encompass native and artificial proteins, protein fragments and polypeptide analogs (such as muteins, variants, chimeric proteins and fusion proteins) of a protein sequence as well as post-translationally, or otherwise covalently or non-covalently, modified proteins. A peptide, polypeptide, or protein may be monomeric or polymeric. Polypeptides includes antibodies, antibody chains, scFv and chimeric antigen receptor constructs.

[0070] The “percent identity” or “percent homology” refers to a quantitative measurement of the similarity between two polypeptide or between two polynucleotide sequences. The percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions that are shared between the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polypeptide sequences. In a similar manner, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions that are shared between the two polynucleotide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polynucleotide sequences. A comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between twopolynucleotide sequences, may be accomplished using a mathematical algorithm. For example, the "percent identity" or "percent homology" of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters.

[0071] The terms "Chimeric Antigen Receptor" or "CAR" describes a fusion protein comprising an extracellular antigen-binding protein, preferably a single chain variable fragment (scFv or sFv) derived from fusing the variable heavy and light regions of a monoclonal antibody, that is fused to an intracellular signaling domain capable of activating or stimulating an immune cell. Alternatively, scFvs may be used that are derived from Fab's (instead of from an antibody, e.g., obtained from Fab libraries). Disclosed herein are CD19 CARs that include targeting moieties that specifically bind CD19.

[0072] The term “antibody” describes an immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. The term antibody includes, for example, single chain variable fragment antibodies (scFvs).

[0073] The terms “anti-CD19 antibody” and “an antibody that binds to CD19” refer to an antibody that is capable of specifically binding CD19.

[0074] A “single-chain antibody” or “scFv” or “single chain variable fragment [antibody]” is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain. In one embodiment, the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).

[0075] A Fab fragment is a monovalent fragment having the VL, VH, CL and CHI domains; a F(ab')2fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge atthe hinge region; a Fd fragment has the VH and CHI domains; an Fv fragment has the VL and VH domains of a single arm of an antibody; and a dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain (U.S. Patents 6,846,634 and 6,696,245).

[0076] The term “hinge” refers to an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the overall construct and movement of one or both of the domains relative to one another. Structurally, a hinge region typically comprises from about 8 to about 100 amino acids, e.g., from about 9 to about 75 amino acids, from about 10 to about 70 amino acids, or from about 20 to about 65 amino acids. In various embodiments, a hinge region is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. The hinge region of a CAR can be derived from is a hinge region of a naturally-occurring protein, such as a hinge region of an antibody (e.g., an IgG, such as human IgG4). The hinge region can be derived from an immunoglobulin superfamily member or an antibody and may or may not comprise one or more constant regions of an antibody. A hinge region can comprise the hinge region of an antibody and the CH3 constant region of the antibody, or a hinge region can comprise the hinge region of an antibody and the CH2 and CH3 constant regions of the antibody. A hinge region can also be a non-naturally occurring peptide. A hinge region can be positioned between the C- terminus of the scFv moiety of a CAR and the N-terminus of the transmembrane domain of the CAR. In some embodiments, the hinge region comprises any one or any combination of two or more regions comprising an upper, core, or lower hinge sequence from IgG4 immunoglobulin molecule (e.g., human IgG4). In some embodiments, the hinge region comprises one, two, three or more cysteines that can form at least one, two, three or more interchain disulfide bonds.

[0077] A "vector" refers to a nucleic acid molecule (e.g., DNA or RNA) which can be operably linked to foreign genetic material (e.g., nucleic acid transgene). Vectors can be single-stranded or double-stranded nucleic acid molecules. Vectors can be linear or circular nucleic acid molecules. Vectors can be used as a vehicle to introduce foreign genetic material into a cell (e.g., host cell). One type of vector is a "plasmid," which refers to a linear or circular double stranded extrachromosomal DNA molecule which can be linked to a transgene, and is capable ofreplicating in a host cell, and transcribing and translating the transgene. A viral vector typically contains viral RNA or DNA backbone sequences which can be linked to the transgene. The viral backbone sequences can be modified to disable infection but retain insertion of the viral backbone and the co-linked transgene into a host cell genome. Examples of viral vectors include retroviral, lentiviral and adenoviral vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector" is a type of vector that can contain one or more regulatory sequences, such as inducible and / or constitutive promoters, or ribosomal binding sites, which directs transcription, or transcription and translation, of a transgene linked to the expression vector which is transduced into a host cell.

[0078] A transgene is “operably linked” to a vector when there is linkage between the transgene and the vector to permit functioning or expression of the vector sequences contained in the vector. Vector sequences can any one or any combination of an original-of-replication sequence, an inducible or constitutive promoter or enhancer sequence, at least one selectable marker sequence, 5’ and 3’ LTR sequences, and optionally viral env, pol and / or gag sequences.

[0079] A transgene is "operably linked" to a regulatory sequence when the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene. A "regulatory sequence" is a nucleic acid sequence that affects the expression (e.g., the level, timing, or location of expression) of a transgene to which it is operably linked. The regulatory sequence can, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or the nucleic acid). Regulatory sequences can be part of a vector. Examples of regulatory sequences include promoters, enhancers, ribosomal binding sites and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif, and Baron et al., 1995, Nucleic Acids Res.23:3605-3606.

[0080] A "host cell" or “or a population of host cells” refers to a cell (or a population thereof) into which foreign (exogenous) nucleic acids have been introduced. The foreign nucleic acids can include an expression vector operably linked to a transgene, and the host cell can be used to express the foreign nucleic acid (transgene). In one example, the host cell (or population thereof) can be introduced with an expression vector operably linked to a nucleic acid encoding the chimeric antigen receptors (CAR) described herein. A host cell (or a population thereof) can be a cultured cell or can be extracted from a subject. The host cell (or a population thereof) includes the primary subject cell and its progeny without any regard for the number of passages. Progeny cells may or may not harbor identical genetic material compared to the parent cell. Host cells encompass progeny cells.

[0081] A host cell can be a prokaryotic cell, for example, E. coli. or it can be a eukaryotic cell, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Examples of host cells include RPMI8226 (Gentry et al., 2004 Leuk. Res.28(3):307-313), and human chronic myelogenous leukemia cell line K562. Other examples include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media (Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX- B11, which is deficient in DHFR (Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1ZEBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293,293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. In one embodiment, a host cell is a mammalian host cell, for example a human host cell. Typically, a host cell is primary cell or a cultured cell that can be introduced with an exogenous polypeptide-encoding nucleic acid which can then be expressed in the host cell. It is understood that the term host cell refers to the particular subject cell and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeedinggenerations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0082] A host cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and / or manipulated in any way to express an a CAR construct, such as an anti-CD19-CAR construct, as disclosed herein. Preferably, the host cell is a human T cell, placenta cell or NK cell.

[0083] “Pan T cells” refers to a population of T cells that have not been selected by T cell type, but include all of the T cell types of the biological sample from which they are obtained, for example, pan T cells may include CD4+ “helper” T cells, CD4+CD25+ regulatory T cells, CD8+ cytotoxic T cells, gamma delta (y5 or gd) T cells, Natural Killer (NK) T cells, and “double negative” T cells. The biological source can be, as nonlimiting examples, peripheral blood, PBMCs, cord blood (CB), or placental blood, as nonlimiting examples.

[0084] As used herein, "allogeneic" refers to any material (such as cells) derived from a different individual of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.

[0085] The terms "transfected" or "transformed" or "transduced" refer to a process by which exogenous nucleic acid (e.g., transgene) is transferred or introduced into a host cell. The term “transduced” is used to refer to introduction of nucleic acid into a host cell by means of a viral vector, such as by viral infection of the host cell. A "transfected", "transformed" or "transduced" host cell is a cell which has been transfected, transformed, or transduced with exogenous nucleic acid or is the progeny of the directly transformed, transfected, or transduced cell.

[0086] Transgenes, such as the disclosed nucleic acid sequences encoding chimeric antigen receptors (CAR) constructs can be operably linked to a vector, including a viral vector, which is used as a vehicle to introduce a transgene into a host cell. Transgenes introduced (e.g., via transduction, transfection or transformation) into host cells can be transiently introduced or preferably stably integrated into the host cell’s genome. Transgenes introduced into host cellscan be propagated in progeny cells. Vectors can be single- or double-stranded DNA or RNA vectors. Vectors include expression vectors which direct expression of transgenes in a host cell. Suitable vectors include expression vectors which can contain an original of replication sequence, an inducible or constitutive promoter sequence, and at least one selectable marker sequence, where these sequences are functional in a packaging cell and / or host cell. Viral vectors used to introduce transgene into a host cell include vectors derived from the viral family Retroviridae which includes retroviral and lentiviral vectors. Retroviral vectors can be used to transduce dividing host cells, and lentiviral vectors can be used to transduce non-dividing host cells. Host cells transduced with the desired transgene linked to an expression vector include T cells, placental derived natural killer host cells, and cord blood derived natural killer host cells.

[0087] Retroviral vectors can be derived from any avian or mammalian source. Retroviral vectors can be capable of infecting host cells of several different species (e.g., amphotropic), including mice, rats and humans, or can have limited host range (e.g., ecotropic). Retroviral vectors can be derived from Moloney murine leukemia virus (MoMLV) (e.g., the MFG vector or a derivative thereof, Riviere et al. (1995) Proc. Nat ’I. Acad. ofSci. USA 92:6733-6737), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV).

[0088] In a typical first generation retroviral transfer vector (e.g., gammaretroviral vector) system, sequences that encode retroviral gag, pol and env can be replaced with a desired transgene, and the transgene can be flanked on both sides by cv.s-acting long terminal repeat (LTR) sequences. The gag and pol sequences can be carried on a packaging plasmid, the env sequence can be carried separately on an envelope plasmid, and expression of these three viral sequences act in-trans. The transfer vector (containing the transgene) along with the packaging and envelope plasmids, are reacted with packaging cells in the presence of a transfection reagent to transduce the vector and plasmids into the packaging cells. The transduced packaging cells produce cell culture supernatant containing infectious virions harboring the transfer vector carrying the transgene. Transduced host cells are generated by reacting the host cells with the virion supernatant. Upon transduction the retroviral transfer vector (carrying the transgene) integrates into the host cell’s genome (Morgan and Boyerinas 2016 Biomedicines 4(2): 9“Review: Genetic Modification of T Cells”). Retroviral transfer vectors can also contain a promoter that directs inducible or constitutive transcription of the transgene. A second generation retroviral vector system typically includes gag, pol and env sequence stably expressed in a packaging cell line which obviates the need for separate packaging and envelope plasmids. The packaging cell line is reacted with the packaging vector (carrying the transgene) to generate transduced packaging cells and virion supernatant. Phoenix helper-free retroviral packaging cell lines is an example of a second generation retroviral system. Retroviral vectors are used for host cell transduction (WO2014 / 055668).

[0089] Lentivirus vectors derived from HIV, SIV or FIV, can be used to introduce a transgene into a host cell. Several generations of lentivirus vectors have been developed. First generation lentiviral systems are similar to first generation retroviral systems in that they employ a transfer vector (carrying the transgene), packaging plasmid (carrying gag, pol, tat, rev and accessory sequences), and envelope plasmid (carrying a heterologous env sequence). Second generation lentiviral systems employ a transfer vector (transgene), packaging plasmid (gag, pol, tat and rev, and accessory sequences removed), and envelope plasmid (carrying a heterologous env sequence). Third generation lentiviral systems, sometimes called self-inactivating (SIN) systems, employ a transfer vector (transgene and 3’ LTR having tat removed), a first packaging plasmid (gag and pol), a second packaging plasmid (rev), and envelope plasmid (carrying a heterologous env sequence). Similar to retroviral systems, any of these lentiviral systems involves reacting the vector / plasmids with packaging cells and a transduction reagent to produce cell culture supernatant containing virions which is in turn used to transduce host cells. Lentivirus vectors are used to transduce host cells (WO2012 / 031744; U.S. Patent 8,802,374; and U.S. 2016 / 0152723).

[0090] Other viral vectors used to introduce transgenes into host cells include simian virus 40 (SV40), herpes simplex virus 1, adenovirus, adeno-associated virus (AAV) and Rous sarcoma virus (RSV) (Gross 1989 Proc. Natl. Acad. Sci. USA 86: 10024-10028).

[0091] Expression vectors typically include a promoter and / or enhancer sequence that directs inducible or constitutive expression (e.g., transcription) in packaging cells and / or host cells to be introduced with a transgene. Constitutive promoters include retroviral LTR, immediate earlycytomegalovirus (CMV) promoter, elongation growth factor 1 alpha (EF-la), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney murine leukemia virus (MoMuLV) LTR promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, PGK (phosphoglycerate kinase), UbC (Ubiquitin C), MLV (Moloney leukemia virus) and CAG (cytomegalovirus early enhancer element, promoter from first exon and intron of chicken beta-actin, and splice acceptor of rabbit betaglobin) enhancer sequence. Inducible promoter sequences include tetracycline operator (TetO) sites (Sakemura 2016 Cancer Immunology Research 4(8):658-668) and lac repressor system from E. coll. Promoters suitable for high expression from lentiviral vectors include human ubiquitin, MHC class I, MHC class II, and P2 microglobulin promoters (WO 2016 / 012623). Retroviral and lentiviral expression vectors are commercially available from several sources including Applied Biological Materials (ABM) (Vancouver, Canada) and Addgene (Watertown, Massachusetts).

[0092] The term “target cells” are cells expressing one or more target polypeptides which renders them recognizable by an antibody or antibody derivative. In one embodiment, target cells include cancer (tumor) target cells expressing CD19 polypeptides which are recognized for binding by chimeric antigen receptor (CAR) constructs of the present disclosure.

[0093] The patient or subject may be a human patient or human subject. Alternatively, the patient or subject may be a mammal such as a cat, dog, or horse. Chimeric Antigen Receptors (CARs)

[0094] The present disclosure describes a new CAR construct comprising an scFv (such as anti- CD19 antibody) onto a CAR construct scaffold. The present disclosure provides a nucleic acid sequence encoding a CAR for transduction into T cells, including pan T cells, T cells isolated from placental tissue or cord blood, or gamma delta (gd) T cells, in which the CAR directs the T cells (e.g., to CD19-expressing tumor cells).

[0095] CARs are generally constructed by joining the antigen recognition domains of an antibody with the signaling domains of receptors from T cells. A CAR construct will typically contain sequences encoding an extracellular region, e.g., a single chain variable fragment (scFv) of an antibody recognizing an antigen present on cancer cells (such as CD19), sequences encoding an intracellular region, e.g., a T-cell receptor such as (TCR) zeta chain that mimics TCR activation, and sequences encoding at least one signaling domain derived from CD28 to mimic co-stimulation. Modification of T cells with nucleic acid sequences encoding CARs equips T cells with retargeted antibody-type antitumor cytotoxicity. Because killing is MHC- unrestricted, the approach offers a general therapy for all patients bearing the same antigen. These T cells engineered with CARs are often called “CAR-T cells”, “designer T cells”, or “T- bodies” (Eshhar et al. Proc. Natl. Acad. Sci. USA 90(2): 720-724,1993; Ma et al., Cancer Chemother. Bio. I Response Modif.20: 315-341, 2002).

[0096] The anti-CD19 scFv can be any known in the art.

[0097] The CAR may further comprise a hinge region, that preferably includes a hIgG4 hinge region, a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the hIgG4 hinge region, or a functional fragment of the hIgG4 hinge region.

[0098] The CAR may further comprise a transmembrane domain, preferably a transmembrane domain from the transmembrane domain of human CD28, or a functional fragment thereof.

[0099] The CAR may further comprise an intracellular signaling domain comprising signaling domains from the group consisting of CD28 (such as human CD28), the CD3-zeta chain, and combination thereof. If there are two signaling domains, the second one is called a costimulatory signaling domain. Preferably, the co-stimulatory signaling domain comprises an intracellular domain, or fragment thereof, of hCD3-zeta. In some embodiments, the intracellular signaling domain comprises a hCD28 signaling domain. In some embodiments, the intracellular signaling domain comprises a hCD28 signaling domain and a hCD3-zeta signaling domain.

[0100] The present disclosure encompasses isolated nucleic acid molecules comprising sequences encoding the disclosed CAR construct. It should be noted that where an amino acidsequence is described, also included is a nucleic acid sequence that encodes the amino acid sequence. 1. Extracellular Anti-CD19 Binding Protein

[0101] The present disclosure provides a CAR comprising an antigen binding protein that binds to a target, such as CD19. VL and VH domains can be joined in an scFv by a linker, such as but not limited to a GS (G4S) linker, that can have, for example, between one and eight repeating units of G4S. In some examples, the linker has three G4S units.

[0102] Further, the present disclosure provides a CAR comprising an antigen binding protein that binds to CD19, where the antigen binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain.

[0103] Single chain antibodies may be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) have been prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VL and VH). The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et al., 1997, Prot. Eng.10:423; Kortt et al., 2001, Biomol. Eng.18:95-108). By combining different VL- and VH-comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al., 2001, Biomol. Eng. 18:31-40). Techniques developed for the production of single chain antibodies include those described in U.S. Patent 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, and de Graaf et al., 2002, Methods Mol. Biol.178:379-87. 2. Transmembrane Domains

[0104] A transmembrane domain of the CAR construct may be a polypeptide structure that is thermodynamically stable in a cell membrane, preferably a mammalian cell membrane. Transmembrane domains compatible for use in the CAR construct may be obtained from anynatural transmembrane protein, or a fragment thereof. Alternatively, the transmembrane domain can be a synthetic, non-naturally occurring transmembrane protein, or a fragment thereof, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane (e.g., a mammalian cell membrane).

[0105] Preferably, the transmembrane domain used in a CAR is derived from the transmembrane domain of human CD28. In various embodiments provided herein the transmembrane domain of a CAR comprises an amino acid sequence derived from CD28, for example, an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the transmembrane domain of human CD28. 3. Intracellular Domains

[0106] The CAR comprises at least one intracellular signaling domain. A signaling domain is generally responsible for activation of at least one of the normal effector functions of a cell. The term "effector function" describes a specialized function of a cell. For example, the effector function of a T cell or an NK cell can include a cytolytic activity or a helper activity. "Signaling domain" describes the portion of a protein which transduces the effector function signal and directs the cell to perform its specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use an entire chain or domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact domain as long as it transduces the effector function signal.

[0107] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way or in an inhibitory way. Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (IT AMs). Primary signaling domains containing ITAMs for CARs include the signaling domains of TCR zeta (CD3 zeta) and FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Preferably, a primary signaling domain is CD3-zeta.

[0108] In various embodiments, a CAR may further comprise a co-stimulatory signaling domain. Examples of co-stimulatory signaling domains for use in the chimeric receptors are cytoplasmic signaling domain of co-stimulatory proteins selected from the group consisting of members of the B7 / CD28 family (B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7- DC, and PDCD6); members of the TNF superfamily (4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-alpha / TNF-beta, OX40 / TNFRSF4, 0X40 ligand / TNFSF4, RELT / TNFRSF19L, TACVTNFRSF13B, TL1A / TNFSF15, TNF-a, and TNF RII / TNFRSF1B); members of the interleukin-1 receptor / toll-like receptor (TLR) superfamily (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10); members of the SLAM family (2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB- A / SLAMF6, and SLAM / CD150); CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thyl, CD96, CD160, CD200, CD300a / LMIRl, HLA Class I, HLA-DR, ikaros, integrin alpha 4 / CD49d, integrin alpha 4 beta 1, integrin alpha 4 beta 7 / LPAM-l, LAG-3, TCL1A, TCL1B, CRTAM, DAP10, DAP12, MYD88, TRIF, TIRAP, TRAF, Dectin- 1 / CLEC7A, DPPIV / CD26, EphB6, TIM- 1 / KIM- 1 / HA VCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), and NKG2C. Preferably, the co-stimulatory domain comprises an intracellular domain of human CD28.

[0109] In various embodiments of a CAR provided herein, the includes a first signaling domain and a second signaling domain. For example, the first signaling domain of the CAR can comprise a signaling domain derived from human CD28. The first signaling domain can comprise an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of intracellular T-cell signaling domain of hCD28. The second signaling domain comprise a signaling domain derived from hCD3-zeta. The first signaling domain can comprise an amino acid sequence that is at least 80%, 90%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of intracellular T-cell signaling domain of hCD3-zeta. 4. Hinge Regions

[0110] The CAR can further comprise a hinge region. The hinge region is located between the scFv antibody region and the transmembrane domain. A hinge region is an amino acid segment that is generally found between two domains of a protein and allows for flexibility of the CAR and movement of one or both of the domains relative to one another. The hinge region can comprise from about 7 to about 120 amino acids, e.g., from about 8 to about 100 amino acids, from about 10 to about 90 amino acids, or from about 50 to about 90 amino acids. Prefereably the hinge region comprises at least 65 amino acids, for example, at least 65, at least 70, at least 75, at least 80, or at least 85 amino acids. A hinge region can be a hinge region of a naturally occurring protein or derived from a hinge region of a naturally occurring protein and can be derived from hinge regions of more than one polypeptide. In various embodiments, the hinge region comprises a hIgG4 hinge region. In various embodiment, the hinge region comprises an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the hIgG4 hinge region. Preferably, the hinge region is disposed between the C-terminus of the scFv and the N-terminus of the transmembrane domain of the CAR. 5. Signal Peptides

[0111] The CAR may include one or more signal peptides or signal sequences. Signal sequences are peptide sequences that target a polypeptide to the desired site in a cell, such as the secretory pathway of the cell, and will allow for integration and anchoring of the CAR into the lipid bilayer of the cellular membrane. A signal peptide encoded by a CAR construct can be positioned at the N-terminus of the encoded CAR and is operable in the cell type in which the CAR is to be expressed. Preferably, the signal peptide is cleaved during integration of the CAR into the cell membrane and is not present on the mature CAR expressed by the host cell.

[0112] In various examples the signal sequence of a CAR as provided herein is derived from a signal sequence of a protein of the immunoglobulin superfamily, and may be derived from, for example, an immunoglobulin heavy chain signal sequence, or a signal sequence of a molecule expressed on the cell membrane such as, for example, CD8a, CD28, or CD 16. Host Cells

[0113] Isolated host cells or populations of host cells are transduced with a CAR construct to express the CAR. The host cells may be transduced with a lipid nano-particle containing an mRNA encoding the CAR as described herein. The host cells may also be transduced with a lentivirus or retrovirus, for example, as disclosed herein. In the alternative, host cells can be generated by CRISPR / Cas methods, for example, as disclosed in US 2020 / 0224160 and WO 2020 / 185867, which are incorporated herein by reference.

[0114] In some embodiments the host cells are patient-derived isolated T cells, i.e., autologous T cells. Such populations of autologous T cells are obtained from a patient (e.g., from peripheral blood which is processed to provide peripheral blood mononuclear cells (PBMCs) from which T cells are isolated), isolated, and expanded. The isolated and expanded T cells originating from the patient are then transduced with a CAR construct as disclosed herein to achieve a population of transduced CAR-T cells that are typically expanded and returned (administered) to the individual patient.

[0115] In other embodiments the host cells are cultured T cells of derived from placenta or cord blood (CB) after pregnancy. Such populations of T cells are obtained from tissue not derived from the patient (allogeneic). The T cells are isolated and then expanded. The expanded placental or CB T cells are then transduced with a CAR construct as disclosed herein to achieve a population of transduced CAR-T cells. The transduced CAR-T cells are expanded and administered to a patient unrelated to the source of the T cells.

[0116] The present disclosure provides cultured T cells transduced with a CAR construct as provided herein that express the CAR. The T cells may be isolated from PBMCs or may be isolated from placental tissue or cord blood (CB).Therapeutic Methods and Uses of CARs

[0117] The present disclosure provides methods for treating a cancer or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an isolated host cell comprising a CAR (such as one targeting CD19), or a population of transduced host cells. Hematologic cancer can be treated using the CARs disclosed herein. Examples of hematologic cancer that can be treated using the methods of the disclosure include non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), B acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), T acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML). For example, a patient can be administered isolated CAR-T cells (e.g., CD19 CAR-T cells) to treat non-Hodgkin's lymphoma (NHL), B chronic lymphocytic leukemia (B-CLL), or B-cell acute lymphocytic leukemia (ALL). Other types of cancer that can be treated diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myeloid lymphoid leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

[0118] The disclosure provides a method of inhibiting growth of a tumor expressing a cancer associated antigen, comprising contacting a cancer cell of the tumor with a transduced host cell comprising a CAR, or a population of transduced host cells, wherein the host cell is an autologous T cell or a placenta-derived or CB-derived T cell.

[0119] The transduced host cells may also be used to treat other disorders, such as autoimmune disorders and inflammatory disorders. Autoimmune or inflammatory disorders include diseases or disorders arising from and directed against an individual's own tissues or organs or a manifestation thereof or a condition resulting therefrom. In one embodiment, it refers to a condition that results from, or is aggravated by, the production of T cells that are reactive with normal body tissues and antigens. In one embodiment, it refers to a condition that results from, or is aggravated by, the production by antibodies that are reactive with normal body tissues and antigens. In some embodiments, autoimmune or inflammatory disorders include, but are notlimited to arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis (such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails), atopy (including atopic diseases such as hay fever and Job's syndrome), dermatitis (including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, exfoliative psoriatic dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis), x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria (such as chronic allergic urticaria, chronic idiopathic urticaria, chronic autoimmune urticaria), myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis (such as systemic sclerosis; multiple sclerosis (MS), MS associated with EBV infection, spino-optical MS, primary progressive MS (PPMS), relapsing-remitting MS (RRMS), progressive relapsing MS, secondary progressive MS (SPMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis), neuromyelitis optica spectrum disorder (NMO, also known as Devic's Disease or Devic's Syndrome), inflammatory bowel disease (IBD) including Crohn's disease; autoimmune-mediated gastrointestinal diseases; colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis; and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome (including adult or acute respiratory distress syndrome (ARDS)), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such asRasmussen's encephalitis and limbic and / or brainstem encephalitis, uveitis (such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis), glomerulonephritis (GN) with and without nephrotic syndrome (such as chronic or acute glomerulonephritis, primary GN, immunemediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, or proliferative nephritis), autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema (including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema), asthma (such as asthma bronchiale,bronchial asthma, and auto-immune asthma), conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus (including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE), cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, Type I diabetes, Type II diabetes, and latent autoimmune diabetes in adults (or Type 1.5 diabetes), juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), idiopathic diabetes, insipidus, diabetic retinopathy, diabetic nephropathy, and diabetic large-artery disorder; immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis (including lymphomatoid granulomatosis, Wegener's granulomatosis, or agranulocytosis), vasculitides (including vasculitis, large-vessel vasculitis, polymyalgia rheumatica and giant cell (Takayasu's) arteritis, medium-vessel vasculitis, Kawasaki's disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNSvasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis (such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis)), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA); Factor VIII deficiency; hemophilia A; autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome (such as those secondary to septicemia, trauma, or hemorrhage), antigen-antibody complex-mediated diseases, anti- glomerular basement membrane disease, anti-phospholipid antibody syndrome, antiphospholipid syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury, preeclampsia, an immune complex disorder such as immune complex nephritis, antibodymediated nephritis, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgMmediated neuropathy, thrombocytopenia (as developed by myocardial infarction patients, for example), including thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin- induced thrombocytopenia, autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, acquired thrombocytopenic purpura, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases, including thyroiditis autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis), autoimmune thyroid disease, idiopathic hypothyroidism, or Grave's disease), polyglandular syndromes, autoimmune polyglandular syndromes ( or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-manor stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, hepatitis, including autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia areata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility (e.g., due to antispermatozoan antibodies) mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, postmyocardial infarction cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Samter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis ( acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemiareperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome,antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, emphysema, alopecia areata, adipose tissue inflammation / diabetes type II, obesity associated adipose tissue inflammation / insulin resistance, endometriosis, multiple sclerosis, and pulmonary hemosiderosis.

[0120] Yet another embodiment is a method for treating a mammal (e.g., a human) having an autoimmune disease, wherein the method comprises administering to the mammal identified as having an autoimmune disease an effective amount of a population of T cells comprising a CARas described herein. In one embodiment, the T cells express a first chimeric antigen receptor polypeptide having a first antigen binding domain that binds a first antigen on a CD11c+Tbet+B cell with low affinity, where the binding activates the T cell, and where the T cell expresses a second chimeric antigen receptor polypeptide having a second antigen binding domain that binds a second antigen on a CD11c+Tbet+B cell and stimulates the T cell. In one embodiment, the method reduces the number of age-associated B-cells.

[0121] In one embodiment, the autoimmune disease results from production of autoantibodies by age-associated B cells. The autoimmune disease may be lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, Crohn's disease, Celiac disease, or polyarteritis nodosa.

[0122] The transduced host cells may be administered at a dosage of about 101to about 109cells / kg body weight. Ranges intermediate to the above recited dosage, e.g., about 102to about 108cells / kg body weight, about 104to about 107cells / kg body weight, about 105to about 106cells / kg body weight, are also intended to be part of this disclosure.

[0123] The host cells administered to a subject or patient can be allogeneic with respect to the subject or patient.

[0124] The transduced host cells may be administered as a single dose or multiple doses, and may be administered daily or preferably less frequently.Lipid Nanoparticles

[0125] In some embodiments, the CAR nucleic acid construct (and preferably an mRNA encoding a CAR) is encapsulated by a lipid nanoparticle (LNP). In one embodiment, the lipid nanoparticle includes a CAR nucleic acid construction (such as an mRNA encoding a CAR), a cationic or other ionizable lipid, a structural lipid (such as a sterol), a non-cationic (helper) lipid (or phospholipid), and optionally a PEGylated lipid. In another embodiment, the lipid nanoparticle includes a CAR nucleic acid construction (such as an mRNA encoding a CAR), and a lipid blend. The lipid blend may comprise an ionizable cationic lipid and / or lipid-immune cell targeting group conjugate (e.g., a lipid-T-cell targeting group conjugate). In some embodiments, the lipid blend comprises one or more of a structural lipid (e.g., a sterol), a neutral phospholipid, and a free PEG-lipid.

[0126] The lipid nano-particle may include a lipid (e.g., an ionizable cationic lipid) to enable intracellular delivery of a nucleic acid, e.g., mRNA, to the cytosolic compartment of a target cell type and optionally rapidly degrade into non-toxic components. The lipid may be an ionizable cationic lipid and / or a lipid-immune cell targeting group conjugate (e.g., a lipid-T-cell targeting group conjugate). The lipid nano-particle and lipid may be any of those described in International Application No. PCT / US2025 / 015513, filed February 12, 2025, which is hereby incorporated by reference.

[0127] In some embodiments, the cationic lipid is present in the lipid nanoparticle at a molar percentage of about 30% to about 80%.

[0128] In some embodiments, the sterol is present in the lipid nanoparticle at a molar percentage of about 20% to about 50%.

[0129] In some embodiments, the non-cationic lipid is present in the lipid nanoparticle at a molar percentage of about 2% to about 20%.

[0130] In some embodiments, the at least one PEGylated lipid is present in the lipid nanoparticle at a molar percentage of about 2.1% to about 10%, or about 1% to about 2%.

[0131] In some embodiments, the lipid nanoparticle comprises an ionizable lipid (such as one that targets certain cells), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), optionally at a molar ratio of about 48.5:10:40:1.5, respectively.

[0132] In some embodiments, the lipid nanoparticle comprises an ionizable lipid (such as one that targets certain cells), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), optionally at a molar ratio of about 48.5:10:40:1.5, respectively.

[0133] In some embodiments, the lipid nanoparticle comprises an ionizable lipid (such as one that targets certain cells), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), optionally at a molar ratio of about 48.5:10:39:2.5, respectively.

[0134] The amount of cationic lipid may be selected taking the amount of the nucleic acid cargo into account. In one embodiment, these amounts are selected such as to result in an N / P ratio of the nanoparticle(s) in the range from about 0.1 to about 30, or (i) at an amount such as to achieve an N / P ratio in the range of about 1 to about 20, about 2 to about 15, about 3 to about 10, about 4 to about 9, about 6, about 5 to about 20, about 10 to about 18, about 12 to about 16, about 14, or (ii) at an amount such as to achieve a lipid : mRNA weight ratio in the range of 20 to 60, about 3 to about 15, about 5 to about 13, about 4 to about 8, or from about 7 to about 11.

[0135] In other preferred embodiments, the N / P ratio can be in the range of about 1 to about 50. In other embodiments, the range is about 1 to about 20, and preferably about 1 to about 15. In one embodiment, the N / P (lipid to RNA mol ratio) is about 14 or about 17. In another embodiment, the N / P, i.e., lipid to RNA mol ratio is about 6. In yet another embodiment, the N / P ratio is about 4.85 or 5 (lipid to RNA mol ratio).

[0136] In some embodiments, the amount of the ionizable cationic lipid is selected taking the amount of the nucleic acid cargo into account, at an amount such as to achieve an N / P ratio in therange of about 12 to about 16, such as about 14. In this context, the N / P ratio is defined as the mole ratio of the nitrogen atoms (“N”) of the basic nitrogen-containing groups of the lipid or lipidoid to the phosphate groups (“P”) of the nucleic acid which is used as cargo. The N / P ratio may be calculated on the basis that, for example, 1 pg RNA typically contains about 3 nmol phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The “N”- value of the cationic lipid or lipidoid may be calculated on the basis of its molecular weight and the relative content of permanently cationic and - if present -ionizable groups. If more than one cationic lipid is present, the N-value should be calculated on the basis of all cationic lipids comprised in the lipid nanoparticles.

[0137] In some embodiments, the LNP comprises a cationic lipid, DOPC, cholesterol and DMG- PEG. In some embodiments, the LNP comprises a cationic lipid, DOPC, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide. In some embodiments, the LNP comprises a cationic lipid, DOPE, cholesterol and DMG-PEG. In some embodiments, the LNP comprises a cationic lipid, DOPE, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide. In some embodiments, the LNP comprises a cationic lipid, DSPC, cholesterol and DMG-PEG. In some embodiments, the LNP comprises a cationic lipid, DSPC, cholesterol, DMG-PEG, and DSPE-PEG or DSPE-PEG- azide. In some embodiments, the LNP comprises a cationic lipid, DOPC, beta-sitosterol and DMG-PEG. In some embodiments, the LNP comprises a cationic lipid, DOPC, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide. In some embodiments, the LNP comprises a cationic lipid, DOPE, beta-sitosterol and DMG-PEG. In some embodiments, the LNP comprises a cationic lipid, DOPE, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide. In some embodiments, the LNP comprises a cationic lipid, DSPC, beta-sitosterol and DMG-PEG. In some embodiments, the LNP comprises a cationic lipid, DSPC, beta-sitosterol, DMG-PEG, and DSPE-PEG or DSPE-PEG-azide.

[0138] In some embodiments, the DMG-PEG is DMG-PEG2000. In some embodiments, the DSPE-PEG is DSPE-PEG2000 or DSPE-PEG5000. In some embodiments, the DSPE-PEG-azide is DSPE-PEG2000-azide or DSPE-PEG5000-azide. In some embodiments, the LNP comprises a cationic lipid, DOPC, sterol, DMG-PEG and DSPE-PEG or DSPE-PEG-azide at molar ratios of about 51: 7.3 : 38.3 : 2.9 : 0.5.

[0139] In some embodiments, the lipid component of the nanoparticle composition includes about 30 mol % to about 60 mol % cationic (or other ionizable) lipid, about 0 mol % to about 30 mol % non-cationic lipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol% to about 10 mol% of PEGylated lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition includes about 35 mol % to about 55 mol % cationic lipid, about 5 mol % to about 25 mol % non-cationic lipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEGylated lipid. In a particular embodiment, the lipid component includes about 50 mol % cationic lipid, about 10 mol % non-cationic lipid, about 38.5 mol % structural lipid, and about 1.5 mol% of PEGylated lipid. In another particular embodiment, the lipid component includes about 40 mol % cationic lipid, about 20 mol % non-cationic lipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEGylated lipid. In another particular embodiment, the lipid component includes about 48.5 mol % cationic lipid, about 10 mol % non-cationic lipid, about 40 mol % structural lipid, and about 1.5 mol % of PEGylated lipid. In another particular embodiment, the lipid component includes about 48.5 mol % cationic lipid, about 10 mol % non- cationic lipid, about 39 mol % structural lipid, and about 2.5 mol % of PEGylated lipid. In some embodiments, the non-cationic lipid may be DOPE or DSPC. In other embodiments, the PEGylated lipid may be PEG-DMG and / or the structural lipid may be cholesterol. The amount of nucleic acid in a nanoparticle composition may depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition as well as on the properties of the nucleic acid. For example, the amount of nucleic acid useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the nucleic acid. The relative amounts of nucleic acid and other elements (e.g., lipids) in a nanoparticle composition may also vary.

[0140] In some embodiments, a nanoparticle composition comprising a CAR nucleic acid construct of the present disclosure is formulated to provide a specific E:P ratio. The E:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA. In general, a lower E:P ratio is preferred. The amounts of these components may be selected to provide an E:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1.In certain embodiments, the E:P ratio may be from about 2:1 to about 8:1. In other embodiments, the E:P ratio is from about 5:1 to about 8:1. For example, the E:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. Ionizable cationic lipids or ionizable lipids.

[0141] Ionizable lipids are ionizable such that they can dissociate to exist in a positively charged form depending on pH. The ionization of an ionizable lipid affects the surface charge of a lipid nanoparticle comprising the ionizable lipid under different pH conditions. The surface charge of the lipid nanoparticle in turn can influence its plasma protein absorption, blood clearance, and tissue distribution (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as its ability to form endosomolytic non-bilayer structures (Hafez, I.M., et al., Gene Ther 8: 1188-1196 (2001)) that can influence the intracellular delivery of nucleic acids. Ionizable lipids containing at least one group (e.g., a head group) that can be ionized, e.g., dissociated to produce one or more electrically charged species, under a given condition (e.g., pH). The ionizable lipid may be a permanent cationic lipid.

[0142] The term “ionizable” as used herein means that a compound, or group or atom, is positively charged at a lower pH and uncharged at a higher pH of its environment. Also in non- aqueous environments where no pH value can be determined, a ionizable compound, group or atom is positively charged at a high hydrogen ion concentration and uncharged at a low concentration or activity of hydrogen ions. It depends on the individual properties of the ionizable or polyionizable compound, in particular the pKa of the respective ionizable group or atom, at which pH or hydrogen ion concentration it is charged or uncharged. In diluted aqueous environments, the fraction of ionizable compounds, groups or atoms bearing a positive charge may be estimated using the so-called Henderson-Hasselbalch equation which is well-known to a person skilled in the art. E.g., if a compound or moiety is ionizable, it is preferred that it is positively charged at a pH value of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferably of a pH value of or below 9, of or below 8, of or below 7, most preferably at physiological pH values, e.g. about 7.3 to 7.4, i.e. under physiological conditions, particularly under physiological salt conditions of the cell in vivo. In embodiments, it is preferred that the ionizable compound or moiety is predominantly neutral at physiological pH values, e.g. about7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, the preferred range of pKa for the ionizable compound or moiety is about 5 to about 7. In some embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.

[0143] The permanent cationic lipids means that the respective lipid, or group or atom, is positively charged at any pH value or hydrogen ion activity of its environment. Very often, the positive charge results from the presence of a quaternary nitrogen atom. Where a compound carries a plurality of such positive charges, it may be referred to as permanently polycationic, which is a subcategory of permanently cationic.

[0144] In some embodiments, the cationic lipid is N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (“DOTMA”). (Feigner et al. (Proc. Nat’l Acad. Sci.84, 7413 (1987); U.S. Pat. No.4,897,355, which is incorporated herein by reference). Other cationic lipids suitable for the lipid nanoparticles include, for example, 5- carboxyspermylglycinedioctadecylamide (“DOGS”); 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (“DOSPA”) (Behr et al. Proc. Nat.’l Acad. Sci.86, 6982 (1989), U.S. Pat. No.5,171,678; U.S. Pat. No.5,334,761); 1,2-Dioleoyl-3- Dimethylammonium-Propane (“DODAP”); l,2-Dioleoyl-3-Trimethylammonium- Propane (“DOTAP”).

[0145] Additional exemplary cationic lipids suitable for the lipid nano-particles also include: l,2- distearyloxy-N,N-dimethyl-3-aminopropane ( “DSDMA”); l,2-dioleyloxy-N,N-dimethyl-3- aminopropane (“DODMA”); 1 ,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (“DLinDMA”); l,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (“DLenDMA”); N-dioleyl-N,N- dimethylammonium chloride (“DODAC”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”); 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12- octadecadienoxy)propane (“CLinDMA”); 2-[5’-(cholest-5-en-3-beta-oxy)-3’-oxapentoxy)-3- dimethy l-l-(cis,cis-9’, l-2’-octadecadienoxy)propane (“CpLinDMA”); N,N-dimethyl-3,4- dioleyloxybenzylamine (“DMOBA”); 1 ,2-N,N’-dioleylcarbamyl-3-dimethylaminopropane (“DOcarbDAP”); 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (“DLinDAP”); 1,2-N,N’-Dilinoleylcarbamyl-3-dimethylaminopropane (“DLincarbDAP”); 1 ,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (“DLinCDAP”); 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (“DLin-K-DMA”); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, N-dimethyl-3-[(9Z, 12Z)- octadeca-9, 12-dien-l -yloxy]propane-l -amine (“Octyl-CLinDMA”); (2R)-2-((8-[(3beta)-cholest- 5-en-3-yloxy]octyl)oxy)-N, N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]propan-l - amine (“Octyl-CLinDMA (2R)”); (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, fsl- dimethyh3-[(9Z, 12Z)-octadeca-9, 12-dien-l -yloxy]propan-l -amine (“Octyl-CLinDMA (2S)”); 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (“DLin-K-XTC2-DMA”); and 2-(2,2- di((9Z,12Z)-octadeca-9,l 2-dien- l-yl)-l ,3-dioxolan-4-yl)-N,N-dimethylethanamine (“DLin-KC2- DMA”) (see, WO 2010 / 042877, which is incorporated herein by reference; Semple et al. , Nature Biotech.28: 172-176 (2010)). (Heyes, J., et al. , J Controlled Release 107: 276-287 (2005); Morrissey, DV., et al. , Nat. Biotechnol.23(8): 1003-1007 (2005); International Patent Publication WO 2005 / 121348). In some embodiments, one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.

[0146] Additional cationic lipids of the invention can be selected from the non-limiting group consisting of 3-{didodecylamino)-Nl,Nl,4-tridodecyl-l-piperazineethanamine (KL10), N 1 -[2- (didodecylamino)ethyl]-N 1 ,N4,N4-tridodecyl- 1 ,4-piperazinediethanamine (KL22), 14,25- ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin- MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), l,2- dioleyloxy-N,N-dimethylaminopropane (DODMA), (13Z,165Z)-N,N-dimethyl-3-nonydocosa- 13-16-dien-l-amine (L608), 2-({8-[(3b)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yl oxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-({8-[(3b)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-die n-l-yloxy]propan- l -amine (Octyl-CLinDMA (2R)), and (2S)-2-({8-[(3b)-cholest-5-en-3-yloxy]octyl}oxy)-N,N- dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien -1 -yloxy]propan-l-amine (Octyl-CLinDMA (2S)). In addition to these, an ionizable amino lipid can also be a lipid including a cyclic amine group.

[0147] In some embodiments, the cationic lipids are one or more of the compounds described in PCT Publication Nos. WO 2015 / 074805, WO 2015 / 199952, WO 2017 / 112865, WO 2017 / 075531, and WO 2021 / 026358 hereby incorporated by reference in their entireties. Other cationic lipids suitable for inclusion in the lipid nano-particles include those described in US 2015 / 0239834, hereby incorporated by reference in its entirety.

[0148] In some embodiments, one or more cationic lipids suitable for the lipid nano-particles include 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (“XTC”); (3aR,5s,6aS)-N,N- dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [1 ,3]dioxol-5- amine (“ALNY-100”) and / or 4,7 , 13 -tris(3 -oxo-3 -(undecylamino)propyl)-N 1 ,N 16- diundecyl-4,7 ,10,13-tetraazahexadecane- 1,16-diamide (“NC98-5”).

[0149] In some embodiments, the cationic lipid is selected from the group consisting of 1 ,1 ’- ((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl)piperazin-1- yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (CKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01 , 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315. In one embodiment, the cationic lipid is C12-200, TT3 or heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA).

[0150] C12-200 is described, e.g., in Whitehead et al. , Nature Communications, 5: Article No. 4277, 2014. MD1 (cKK-E12), TT3, LP01, and Lipid 5 are described, e.g., in Miao et al., Molecular Cancer, 20(41), 2021. OF2 (OF-02) is described, e.g., in Han et al., Nature Communications, 12: Article No.7233, 2021. EPC is available from AVANTI® Polar Lipids. ZA3-Ep10 is described, e.g., in Miller et al., Angew Chem Int Ed Engl, 56(4): 1059-1063, 2017. 5A2-SC8 is described, e.g., in Zhou et al., Proc Natl Acad Sci USA, 113(3): 520-525, 2016. 98N12-5 is described, e.g., in Akinc et al., Mol Ther, 17(5): 872-879, 2009.In some embodiments, the ionizable lipid is In one aspect, the present invention provides a compound represented by Formula (A-I) or Formula (A-II): or(A-I) (A-II) or its N-oxide, isomer, or a pharmaceutically acceptable salt thereof, wherein L is optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl; L1is optionally substituted C1-6alkylenyl, or C2-6heteroalkylenyl; each L2 is independently C1-6 alkylenyl;L3 is optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl; L4is absent, optionally substituted C1-10alkylenyl, or optionally substituted C2-10heteroalkylenyl; L5 is optionally substituted C1-10alkyl, optionally substituted C1-10alkenyl, optionally substituted C1-10alkynyl, or optionally substituted C2-10heteroalkylenyl; X is absent, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -OC(O)O-; each X2 is absent, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -OC(O)O-; each of R is independently hydrogen, OR6, or an optionally substituted group selected from C6-20aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; R1is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered cycloaliphatic, optionally substituted 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6- membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR2, - C(O)OR2, -C(O)SR2, -OC(O)R2, -OC(O)OR2, -CN, -N(R2)2, -C(O)N(R2)2, -S(O)2N(R2)2, - NR2C(O)R2, -OC(O)N(R2)2, -N(R2)C(O)OR2, -NR2S(O)2R2, -NR2C(O)N(R2)2, -NR2C(S)N(R2)2, -NR2C(NR2)N(R2)2, -NR2C(CHR2)N(R2)2, -N(OR2)C(O)R2, -N(OR2)S(O)2R2, - N(OR2)C(O)OR2, -N(OR2)C(O)N(R2)2, -N(OR2)C(S)N(R2)2, -N(OR2)C(NR2)N(R2)2, - N(OR2)C(CHR2)N(R2)2, -C(NR2)N(R2)2, -C(NR2)R2, -C(O)N(R2)OR2, -C(R2)N(R2)2C(O)OR2, - CR2(R3)2, -OP(O)(OR2)2, or -P(O)(OR2)2; or R1 is a ring selected from 3- to 7-membered cycloaliphatic and 3- to 7- membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R2or R3groups;each R2 is independently hydrogen, oxo, -CN, -NO2, -OR4, -S(O)2R4, -S(O)2N(R4)2, -(CH2)n-R4, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 3- to 7-membered cycloaliphatic, 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two occurrences of R2, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R3 is independently -(CH2)n-R4; or two occurrences of R3, taken together with the atom(s) to which they are attached, form optionally substituted 5- to 6- membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R4 is independently hydrogen, -OR5, -N(R5)2, -OC(O)R5, -OC(O)OR5, -CN, - C(O)N(R5)2, -NR5C(O)R5, -OC(O)N(R5)2, -N(R5)C(O)OR5, -NR5S(O)2R5, -NR5C(O)N(R5)2, - NR5C(S)N(R5)2, or -NR5C(NR5)N(R5)2; and each R5is independently hydrogen, or optionally substituted C1-6 aliphatic; or two occurrences of R5, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R6is independently C4-12aliphatic; and each n is independently 0 to 4.

[0151] In some embodiments, R1 is -N(R2)2.

[0152] In some embodiments, L1-R1is selected from the group consisting of.

[0153] In some embodiments, is selected from the group consisting of: ,

[0154] In some embodiments, is .

[0155] In some embodiments, is where R1000is optionally substituted C1-10alkyl, optionally substituted C2-10alkenyl, optionally substituted C2-10alkynyl, optionally substituted C3-10cyclooalkyl or optionally substituted C5-10 heteroalkyl.

[0156] In some embodiments, is .

[0157] In some embodiments, is .

[0158] In some embodiments, x is -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -OC(O)O-.

[0159] In some embodiments, x is -OC(O)-.

[0160] In some embodiments, when at least one is not , then is where R1000is optionally substituted C1-10alkyl, optionally substituted C2-10alkenyl, optionally substituted C2- 10 alkynyl, optionally substituted C3-10cyclooalkyl or optionally substituted C5-10 heteroalkyl.

[0161] In some embodiments, when both are not , then is where R1000 is optionally substituted C1-10alkyl, optionally substituted C2-10alkenyl, optionally substituted C2- 10 alkynyl, optionally substituted C3-10cyclooalkyl or optionally substituted C5-10 heteroalkyl.

[0162] In some embodiments, a compound of formula (A-III)(A-III); where L1-L5, X2, R1, R” and R1000are as previously defined.

[0163] In some embodiments, X2is OC(O) or C(O)O; and -L3-R” is independently selected from:

[0164] In some embodiments, a compound of formula (A-IV)(A-IV): where each X100 is independently OC(O), C(O)O, OC(O)O, OC(O)NH, or NHC(O)O; L100 is C1-C6 akylene; and L1-L5, X2, R1, R” and R1000are as previously defined.

[0165] In some embodiments, L100 is CH2and X100 is OC(O).

[0166] In some embodiments, L4 is absent, and L100 is CH2and X100 is OC(O).

[0167] In some embodiments, L2is CH2, X2is OC(O), and L3is CH2.

[0168] In some embodiments, L2is CH2, X2is OC(O), L3is CH2, L4is absent, and L100is CH2and X100 is OC(O).

[0169] In some embodiments, L1 is C2-C6 alkylene.

[0170] In some embodiments. -L1-R1is selected from:

[0171] In some embodiments, a compound of formula (B-I): (B-I) or its N-oxide, isomer, or a pharmaceutically acceptable salt thereof, wherein each R’ is independently absent, optionally substituted C1-6 alkyl, or optionally substituted C1-6 heteroalkyl; each of L is independently optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl;each of X is independently O, S, N(R”), S-S, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH, or NHC(O)O; each Z is independently , , or ; provided that at least one or two of Z is or ; each of m is independently 1-9; t is 1, 2 or 3; X10 is absent, O, S, N(R”), S-S, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH, or NHC(O)O; R” is H, , , or ; L10 is optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl; each of R11 is independently C2-20 aliphatic, 3- to 12-membered cycloaliphatic, C2-C10 aliphatic substituted with 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, or phenyl; and each of R10 is independently hydrogen, OR6, or an optionally substituted group selected from C6- 20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2- adamantyl, sterolyl, or phenyl.

[0172] In some embodiments, Z is .

[0173] In some embodiments, Z is .

[0174] In some embodiments, R’ is H or CH3.

[0175] In some embodiments, a compound represented by formula (B-II): (B-II) wherein Z and R” are each independently , , or ; provided at least two or three of Z and R” are or .

[0176] In some embodiments, is selected from the group consisting of:

[0177] In some embodiments, is selected from the group consisting of: , , , and .

[0178] In some embodiments, a compound of formula (C-I):(C-I) or its N-oxide, isomer, or a pharmaceutically acceptable salt thereof, wherein each of X20is independently absent, O, S, N(R”), S-S, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH, or NHC(O)O; each of L20is independently optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl; each of Z20is independently , , or ; provided that at least one of Z20 is or ; each of m is independently 1-9; t is 1, 2 or 3; L10is optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl;each of R11 is independently C2-20 aliphatic, 3- to 12-membered cycloaliphatic, C2-C10 aliphatic substituted with 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, or phenyl; and each of R10 is independently hydrogen, OR6, or an optionally substituted group selected from C6- 20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2- adamantyl, sterolyl, or phenyl; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. is 4- to 8- membered cyclic or heterocyclic ring; L1 is absent, C1-6 alkylenyl, or C2-6 heteroalkylenyl; R1 is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered cycloaliphatic, optionally substituted 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6- membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR2, -C(O)OR2, -C(O)SR2, -OC(O)R2, -OC(O)OR2, -CN, -N(R2)2, -C(O)N(R2)2, -S(O)2N(R2)2, -NR2C(O)R2, -OC(O)N(R2)2, -N(R2)C(O)OR2, -NR2S(O)2R2, -NR2C(O)N(R2)2, - NR2C(S)N(R2)2, -NR2C(NR2)N(R2)2, -NR2C(CHR2)N(R2)2, -N(OR2)C(O)R2, -N(OR2)S(O)2R2, - N(OR2)C(O)OR2, -N(OR2)C(O)N(R2)2, -N(OR2)C(S)N(R2)2, -N(OR2)C(NR2)N(R2)2, - N(OR2)C(CHR2)N(R2)2, -C(NR2)N(R2)2, -C(NR2)R2, -C(O)N(R2)OR2, -C(R2)N(R2)2C(O)OR2, - CR2(R3)2, -OP(O)(OR2)2, or -P(O)(OR2)2; or R1is a ring selected from 3- to 7-membered cycloaliphatic and 3- to 7- membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R2or R3groups;each R2 is independently hydrogen, oxo, -CN, -NO2, -OR4, -S(O)2R4, -S(O)2N(R4)2, -(CH2)n-R4, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 3- to 7-membered cycloaliphatic, 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two occurrences of R2, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R3 is independently -(CH2)n-R4; or two occurrences of R3, taken together with the atom(s) to which they are attached, form optionally substituted 5- to 6- membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R4 is independently hydrogen, -OR5, -N(R5)2, -OC(O)R5, -OC(O)OR5, -CN, - C(O)N(R5)2, -NR5C(O)R5, -OC(O)N(R5)2, -N(R5)C(O)OR5, -NR5S(O)2R5, -NR5C(O)N(R5)2, - NR5C(S)N(R5)2, or -NR5C(NR5)N(R5)2; and each R5is independently hydrogen, or optionally substituted C1-6 aliphatic; or two occurrences of R5, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R6is independently C4-12aliphatic; and each n is independently 0 to 4.

[0179] In some embodiments, the is selected from the group consisting of

[0180] In some embodiments, the is selected from the group consisting of

[0181] In some embodiments, the is or .

[0182] In some embodiments, L1is absent, and R1is -OR2, -C(O)OR2, -C(O)SR2, -OC(O)R2, - OC(O)OR2, -N(R2)2, -C(O)N(R2)2, -S(O)2N(R2)2, -NR2C(O)R2, -OC(O)N(R2)2, -N(R2)C(O)OR2, -NR2S(O)2R2, or -NR2C(O)N(R2)2; and R2 is as defined above.

[0183] In some embodiments, R1is -OR2or -C(O)OR2.

[0184] In some embodiments, L1-R1is selected from the group consisting of

[0185] In some embodiments. -L1-R1 is selected from:

[0186] In some embodiments, the compound represented by formula (C-II): (C-II) wherein L1 is absent, R1 is OR2, q is 2-9, X20 is OC(O) or C(O)O, R2, L20 and Z20 are as defined above.

[0187] In some embodiments, a compound represented by formula (C-III):(C-III) wherein L1is absent, R1is OR2, q is 2-9, X20is OC(O) or C(O)O, R2, L20and Z20are as defined above.

[0188] In some embodiments, -L20-Z20 is selected from the group consisting of:

[0189] In some embodiments, a compound represented by formula (C-IV): (C-IV) wherein L1 is absent, R1 is OR2, q is 2-9, X20 is OC(O) or C(O)O, R2, L20 and Z20 are as defined above.

[0190] In some embodiments, is selected from the group consisting of:

[0191] In some embodiments, is selected from the group consisting of: , , , and .

[0192] In some embodiments. -L1-R1 is selected from:

[0193] In some embodiments, a compound of formula (D-I):or its N-oxide, isomer, or a pharmaceutically acceptable salt thereof, wherein L101 and L102 are each independently C1-6 alkylenyl; L103and L104are each independently optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl; X101 and X102 are each independently absent, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or - OC(O)O-; L105, L106, L107 and L108 are each independently optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl; R100, R101, R102, and R103 are each independently hydrogen, OC1-3 aliphatic, OR6, or an optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; L109is C1-6alkylenyl; X103 is absent, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -OC(O)O-, or -NR105-; R105 is hydrogen, C1-10alkane, C2-10alkene, or C3-12 cycloalkane; L110is C1-6alkylenyl; X104 is absent or -NR106-; L111 is C1-6 alkylenyl; R106is hydrogen, C1-10alkane, C2-10alkene, or C3-12cycloalkane; R104is hydrogen, OR107, C1-10alkane, C2-10alkene, C3-12cycloalkane, or an optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; and R107 is H or C1-12 aliphatic.

[0194] In some embodiments, a compound of formula (D-II):, wherein R100, R101, R102, and R103 are each independently hydrogen, OC1-3 aliphatic, OR6, or an optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, or phenyl; L105, L106, L107 and L108 are each independently optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl; X103 is absent, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -OC(O)O-, or -NR105-;R105 is hydrogen, C1-10alkane, C2-10alkene, or C3-12 cycloalkane; L110 is C1-6 alkylenyl; X104is absent or -NR106-;L111 is C1-6 alkylenyl; R106 is hydrogen, C1-10alkane, C2-10alkene, or C3-12 cycloalkane; and R104is hydrogen, OR107, C1-10alkane, C2-10alkene, C3-12cycloalkane, or an optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, or phenyl; and R107is H or C1-12aliphatic.

[0195] In some embodiments, and are each independently selected from the group consisting of:O O O OL105L106L107L108

[0196] In some embodiments,R100 R101andR102R103are each independently selected from the group consisting of: O O,O O,, , and .

[0197] In some embodiments, is selected from the group consisting of:.

[0198] In some embodiments. -L1-R1is selected from:

[0199] In some embodiments, a compound of formula (E-I): (E-I) or its N-oxide, isomer, or a pharmaceutically acceptable salt thereof, whereineach of X30 is independently absent, O, S, N(R”), S-S, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH, or NHC(O)O; each of L30is independently optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl; each of X31is independently NR’’; wherein R” is H, , , or ; provided that at least one of Z30 is or ; each of Z30 is independently , , or ; provided that at least one of Z30is or ; each of m is independently 1 to 9; t is 1, 2 or 3;L10 is optionally substituted C1-10alkylenyl, optionally substituted C1-10alkenylenyl, optionally substituted C1-10alkynylenyl, or optionally substituted C2-10heteroalkylenyl; each of R11is independently C2-20aliphatic, 3- to 12-membered cycloaliphatic, C2-C10aliphatic substituted with 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, or phenyl; and each of R10is an optionally substituted group selected from C6-20aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, or phenyl.

[0200] In some embodiments, a compound represented by formula (E-II) (E-II) wherein X30, L30, X31 and Z30 are as defined above.

[0201] In some embodiments, X30is C(O)O, OC(O), C(O)NH, NHC(O) or O.

[0202] In some embodiments, is selected from the group consisting of:

[0203] In some embodiments, is selected from the group consisting of: , , , and .

[0204] In some embodiments, X30is C(O)NH.

[0205] In some embodiments, X30is C(O)O.

[0206] In some embodiments, X31 is N-R”.

[0207] In some embodiments, Z30 is selected from the group consisting of:

[0208] In some embodiments. -L1-R1is selected from:

[0209] In some embodiments, the pKa of the protonated form of the compound is from about 4.5 to about 8.0.

[0210] In some embodiments, the cationic lipid can be selected from an ionizable cationic lipid set forth in tables below, or an isomer or a salt thereof. Table A

[0211] In some embodiments, the one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured by weight, of the total lipid content in the lipid nanoparticle. In some embodiments, the one or more cationic lipids constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured as a mol %, of the total lipid content in the nanoparticle. In some embodiments, the one or more cationic lipids constitute about 30-70 % (e.g., about 30-65%, about 30-60%, about30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured by weight, of the total lipid content in the lipid nanoparticle. In some embodiments, the one or more cationic lipids constitute about 30-70 % (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured as mol %, of the total lipid content in the lipid nanoparticle. Structural Lipid

[0212] In one embodiment, the structural lipid is a sterol or a derivative thereof.

[0213] In some embodiments, the sterol or a derivative thereof is a cholesterol.

[0214] In some embodiments, the sterol or a derivative thereof is beta-sitosterol. Non-cationic helper lipid (or phospholipid)

[0215] In some embodiments, the non-cationic lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2- oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3 -pho sphoethanolamine (DLPE); 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. In some embodiments, the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl- phosphatidylethanolamine (DOPE). Pegylated Lipid

[0216] In some embodiments, the PEGylated lipid is selected from the group consisting of PEG- dilauryloxypropyl; PEG-dimyristyloxypropyl; PEG-dipalmityloxypropyl, PEG- distearyloxypropyl; l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol], and distearoyl- rac-glycerol-poly(ethylene glycol) (DSG-PEG); PEG-dilaurylglycerol; PEG-dipalmitoylglycerol; PEG-disterylglycerol; PEG-dilaurylglycamide; PEG-dimyristylglycamide; PEG- dipalmitoylglycamide; PEG-disterylglycamide; (l-[8’-(Cholest-5-en-3[beta]-oxy)carboxamido- 3’,6’-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol) (PEG-cholesterol); 3,4- ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol) ether (PEG-DMB), and l,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol) (DSPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl (DSPE- PEG-OH); and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-azide (DSPE-PEG-azide).

[0217] In some embodiments, the PEGylated lipid is DMG-PEG, DSPE-PEG, DSPE-PEG-OH, DSPE-PEG-azide, DSG-PEG, or a combination thereof. In some embodiments, the at least one PEGylated lipid is DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-OH, DSPE-PEG2000- azide, DSG-PEG2000, or a combination thereof. Additional Lipid Components

[0218] In some embodiments, the LNP may further comprise one or more additional lipid components capable of influencing the tropism of the LNP. In some embodiments, the LNPfurther comprises at least one lipid selected from DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200 (see Cheng, et al. Nat Nanotechnol.2020 April; 15(4): 313–320.; Dillard, et al. PNAS 2021 Vol.118 No.52.).

[0219] The lipid nanoparticles may be in a composition. A lipid nanoparticle composition may include any substance useful in pharmaceutical compositions. For example, the lipid nanoparticle composition may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art (see for example Remington’s The Science and Practice of Pharmacy, 21stEdition, A. R. Gennaro: Lippincott, Williams & Wilkins, Baltimore, Md., 2006).

[0220] The characteristics of a nanoparticle composition may depend on the components thereof. For example, a nanoparticle composition including cholesterol as a structural lipid may have different characteristics than a nanoparticle composition that includes a different structural lipid. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For instance, a nanoparticle composition including a higher molar fraction of a phospholipid may have different characteristics than a nanoparticle composition including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition. Nanoparticle compositions may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure Zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) mayalso be used to measure multiple characteristics of a nanoparticle composition, Such as particle size, polydispersity index, and Zeta potential.

[0221] The mean size of a nanoparticle composition may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the mean size of a nanoparticle composition may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the mean size of a nanoparticle composition may be from about 70 nm to about 100 nm. In a particular embodiment, the mean size may be about 80 nm. In other embodiments, the mean size may be about 100 nm.

[0222] A nanoparticle composition may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.

[0223] The Zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the Zeta potential may describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the Zeta potential of a nanoparticle composition may be from about -10 mV to about +20 mV,from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV, to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV, to about +15 mV, or from about +5 mV to about +10 mV.

[0224] Lipids and their method of preparation are disclosed in, e.g., U.S. Patent Nos.8,569,256, 5,965,542 and U.S. Patent Publication Nos.2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2013 / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, 2013 / 0022649, 2013 / 0017223, 2012 / 0295832, 2012 / 0183581, 2012 / 0172411, 2012 / 0027803, 2012 / 0058188, 2011 / 0311583, 2011 / 0311582, 2011 / 0262527, 2011 / 0216622, 2011 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 0060032, 2010 / 0130588, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076 and PCT Pub. Nos. WO 99 / 39741, WO 2017 / 117528, WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2014 / 008334, WO 2013 / 086373, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, WO2011 / 141705, and WO 2001 / 07548 and Semple et. al, Nature Biotechnology, 2010, 28, 172-176, the full disclosures of which are herein incorporated by reference in their entirety for all purposes. Examples

[0225] The following examples are meant to be illustrative and can be used to further understand embodiments of the present disclosure and should not be construed as limiting the scope of the present teachings in any way. Example 1: CD19 CAR Construct

[0226] mRNA CAR constructs having SEQ ID NOs: 68-77 (CARS01 – CARS10), 80-81 (CAR11 and CAR12), and 202 (CAR13) were constructed. CAR11 = CAR03 with mCherry tag / CAR12 = CAR03 without CD3z domain / CAR13= UTR1 with mCherry tag (no CAR)

[0227] Preactivated T-cells were electroporated with the mRNA. Table 2 shows the results 24 hours post-RNA delivery. Cells were stained with the respective linkers (CAR01 and CAR04 with G4S linker antibody, and the others with Whitlow / 218 linker antibody. MFI values were calculated on CAR+ cells. The results are shown in Table 1 below. Table 1

[0228] Pre-activated T cells were electroporated with mRNA and rested overnight. Electroporated T cells were co-cultured with wildtype (WT) or CD19- / -Nalm6 cells at a 1:1 ratio for 5 hours. Cells were stained for respective linkers (CAR01 and CAR04 with G4S linker antibody, and all the others with Whitlow / 218 linker antibody), and TNFα. The results are shown in Table 2 below. Percentages are expressed amongst CD25+cells.Table 2

[0229] Pre-activated T cells were electroporated with mRNA and rested overnight. Electroporated T cells were co-cultured with WT or CD19- / - Nalm6 cells at a 1:1 ratio for 24 hours. Cells were stained for respective linkers (CAR01 and CAR04 with G4S linker antibody, and all the others with Whitlow / 218 linker antibody), and IFNγ. The results are shown in Table 3 below. Percentages are expressed amongst CD8+T cells. Table 3

[0230] Purified T cells were pre-activated for 24 hours, then electroporated with CAR mRNA. CAR expression was measured 24 hours after. The results are shown in Table 4 below. CAR percentages amongst total T cells are shown as Mean % ± SD. CAR geometric MFI was determined on total T cells and expressed as Mean gMFI ± SD. Table 4

[0231] Purified T cells were dosed with the indicated amount of lipid nanoparticles containing CAR mRNA (CAR-LNPs). The LNPs were washed away after an overnight incubation.48 hours after CAR-LNP delivery, transfected T cells were analyzed for CAR expression via flow cytometry. The results are shown in Table 5 below. Table 5

[0232] Purified T cells were dosed with the amount of CAR-LNPs indicated in Table 6. LNPs were washed away after an overnight incubation.24 hours after CAR-LNP delivery, transfected T cells were co-cultured with firefly luciferase-expressing Nalm6 cells at an effector to target ratio of 1:3. Luminescence was assessed 48 hours later. The results are shown in Table 6 below. Raw RLU are shown and expressed as Mean ± SD. Table 6

[0233] Purified T cells were dosed with the amount of CAR-LNPs indicated in Table 7 below. LNPs were washed away after an overnight incubation.24 hours after CAR-LNP delivery, transfected T cells were co-cultured with WT or CD19- / -Nalm6 cells at an effector to target ratio of 1:3. Culture supernatants were collected 48 hours later, and cytokine content measured. The results are shown in Table 7 below. Data are shown as Mean concentration (pg / mL) ± SD. Table 7

[0234] An in vivo comparison of CAR03 and CAR10 efficacy was performed. Immunodeficient NSG mice were humanized with human PBMCs. Mice were randomized at day 18 based off human PBMC engraftment levels, and treated with CAR-LNPs at a dose of 0.1 mg / kg on day 19. T cell characterization and B cell depletion were assessed 24 hours post-LNP delivery in selected tissues. The results are shown in Table 8 below. All data expressed as Mean ± SD. Table 8Table I - Sequences

Claims

1. We claim:

1. A chimeric antigen receptor (CAR) nucleic acid construct comprising a nucleic acid sequence encoding: (a) a single-chain variable fragment (scFv), (b) a hinge region comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the hinge region of hIgG4, (c) a transmembrane domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the transmembrane domain of hCD28, (d) a first intracellular domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the intracellular T-cell signaling domain of hCD28, 4-1BB, or CD4, and (e) a second intracellular domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the intracellular T-cell signaling domain of hCD3-zeta, wherein optionally a double leucine within the CAR (such as within transmembrane domain) is replaced with any other amino acid(s) that are not leucine (such as double alanine) in order to maintain the scFv on the surface of the cell.

2. A chimeric antigen receptor (CAR) nucleic acid construct comprising a nucleic acid sequence encoding: (a) a single-chain variable fragment (scFv), (b) a hinge region comprising the hinge region of hIgG4, (c) a transmembrane domain comprising the transmembrane domain of hCD28, (d) a first intracellular domain comprising the intracellular T-cell signaling domain of hCD28, 4-1BB, or CD4, and (e) a second intracellular domain comprising the intracellular T-cell signaling domain of hCD3-zeta,wherein optionally a double leucine within the CAR (such as within transmembrane domain) is replaced with any other amino acid(s) that are not leucine (such as double alanine) in order to maintain the scFv on the surface of the cell.

3. The CAR nucleic acid construct of claim 1 or 2, wherein the scFV is an scFv that binds CD19.

4. A chimeric antigen receptor (CAR) nucleic acid construct comprising a nucleic acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 71-134, 204, 207, and 210.

5. A chimeric antigen receptor (CAR) nucleic acid construct comprising a nucleic acid sequence that is any one of SEQ ID NOs: 71-134, 204, 207, and 210.

6. The CAR nucleic acid construct of any one of the preceding claims, wherein the CAR nucleic acid construct is an mRNA and optionally, the mRNA includes a poly(A) tail.

7. A lipid nano-particle comprising the CAR nucleic acid construct of claim 6.

8. A chimeric antigen receptor (CAR) comprising: (a) a single-chain variable fragment (scFv), (b) a hinge region comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the hinge region of hIgG4, (c) a transmembrane domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the transmembrane domain of hCD28, (d) a first intracellular domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the intracellular T-cell signaling domain of hCD28, 4-1BB, or CD4, and (e) a second intracellular domain comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of the intracellular T-cell signaling domain of hCD3-zeta,wherein optionally a double leucine within the CAR (such as within transmembrane domain) is replaced with any other amino acid(s) that are not leucine (such as double alanine) in order to maintain the scFv on the surface of the cell.

9. A chimeric antigen receptor (CAR) comprising: (a) a single-chain variable fragment (scFv), (b) a hinge region comprising the hinge region of hIgG4, (c) a transmembrane domain comprising the transmembrane domain of hCD28, (d) a first intracellular domain comprising the intracellular T-cell signaling domain of hCD28, 4-1BB, or CD4, and (e) a second intracellular domain comprising the intracellular T-cell signaling domain of hCD3-zeta, wherein optionally a double leucine within the CAR (such as within transmembrane domain) is replaced with any other amino acid(s) that are not leucine (such as double alanine) in order to maintain the scFv on the surface of the cell.

10. The CAR of claim 8 or 9, wherein the scFV is an scFv that binds CD19.

11. A chimeric antigen receptor (CAR) comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 138-201, 205, 208 and 211.

12. A chimeric antigen receptor (CAR) comprising an amino acid sequence that is any one of SEQ ID NOs: 138-201, 205, 208 and 211.

13. A host cell, or a population of host cells, which express the CAR of any one of claims 8- 12.

14. A host cell, or a population of host cells, comprising the CAR nucleic acid construct of claim 6.

15. A host cell, or a population of host cells, transfected with the lipid nano-particle of claim 7.

16. The host cell or population of host cells of any one of claims 13-15, which are selected from the group consisting of a PBMC-derived T cell (or population thereof), a placental derived T cell (or a population thereof), and a cord blood derived T cell (or population thereof).

17. The host cell or population of host cells of claim 16, wherein the host cell or cells are pan T cells or gd T cells.

18. A method of treating a cancer in a patient, comprising administering to the patient a population of host cells of any one of claims 13-17.

19. A method of inhibiting growth of a tumor expressing CD19 in a patient having cancer, comprising administering to the patient a population of T cells comprising a CAR of any one of claims 8-12, wherein the scFv binds CD19.

20. The method of claim 18 or 19, wherein the population of T cells are isolated from peripheral blood mononuclear cells (PBMCs).

21. The method of claim 98 or 16, wherein the population of T cells are isolated from placental tissue or cord blood.

22. The method of claim 18 or 19, wherein the population of T cells are allogeneic with respect to the patient.

23. A method of any one of claims 18-22, wherein the cancer is a hematological cancer.

24. The method of claim 23, wherein the cancer is non-Hodgkin's lymphoma (NHL), B chronic lymphocytic leukemia (B-CLL), or B acute lymphocytic leukemia (ALL).

25. A method for treating a mammal having an autoimmune disease, wherein the method comprises administering to the mammal identified as having an autoimmune disease an effective amount of a population of T cells comprising a CAR of any one of claims 8-12.

26. The method of claim 25, wherein T cells express a first chimeric antigen receptor polypeptide having a first antigen binding domain that binds a first antigen on a CD11c+Tbet+B cell with low affinity, wherein the binding activates the T cell, and wherein the T cell expresses a second chimeric antigen receptor polypeptide having a second antigen binding domain that binds a second antigen on a CD11c+Tbet+B cell and stimulates the T cell.

27. The method of claim 25 or 26, wherein the mammal is a human.

28. The method of any one of claims 25-27, wherein the autoimmune disease results from production of autoantibodies by age-associated B cells.

29. The method of any one of claims 25-28, wherein the autoimmune disease is lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Grave’s disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture’s syndrome, pemphigus vulgaris, acute rheumatic fever, post- streptococcal glomerulonephritis, Crohn’s disease, Celiac disease, or polyarteritis nodosa.

30. The method of any one of claims 25-29, wherein the method reduces the number of age- associated B-cells.

31. A method of decreasing the internalization of a receptor protein into the cell, the method comprising replacing any double leucine (LL) within the receptor protein with any other amino acid(s) that is not leucine, thereby maintaining the receptor protein on the surface of the cell.

32. The method of claim 31, wherein the receptor protein is a CAR protein.

33. The method of claim 31 or 32, wherein the double leucine (LL) is replaced with a double alanine (AA).

34. The method of any one of claims 31-33, wherein the double leucine (LL) is located within the transmembrane domain of the receptor protein.

35. The method of any of claims 31-34, where the receptor protein includes a cytosolic domain.

36. The method of claim 35, wherein the cytosolic domain is selected from a co-stimulatory domain and a signaling domain.

37. The method of claim 36, wherein the co-stimulatory domain is 4-1BB and / or CD4.

38. The method of claim 36, wherein the signaling domain is CD3 zeta.

39. The method of any one of claims 31-38, wherein the receptor protein has the amino acid sequence of any one of SEQ ID NOs: 157-159, 163, 166, and 169 (2-CAR-10-hCD28- LL>AA, 3-CAR-10-hCD28-LL>AA-LH, 4-CAR-10-hCD28-LL>AA-YMNM, 8-CAR- 10-CD4-LL>AA, 11-CAR-10-LL>AA-CD4LL>AA, and 14-CAR-10-4-1BB-LL>AA).

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