Co-expressed chimeric antigen receptors for cancer treatment

Abstract

Aspects of the present disclosure include compositions related to two chimeric antigen receptors (CARs), the first CAR comprising a CD4 or CD8 intracellular domain, and the second CAR comprising stimulatory and costimulatory domain(s) (e.g., 4-1BB or CD28 co-stimulatory domains and / or CD3ζ stimulatory domain), vectors encoding the CARs, and immune cells co-expressing the CARs. Also disclosed are methods for generating the CARs and immune cells co-expressing the CARs. Further aspects are related to methods of treating cancer with the compositions, stimulating an immune response with the compositions, and generating and expanding populations of immune cells co-expressing the CARs.

Description

CO-EXPRESSED CHIMERIC ANTIGEN RECEPTORS FOR CANCER TREATMENT CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the U.S. Provisional Application No. 63 / 661,416 filed June 18, 2024, the disclosure of which is herein incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AI144245 awarded by National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy, created on June 13, 2025, is named “ARCD0827 - Sequence Listing” and is 38,742 bytes in size. TECHNICAL FIELD

[0004] This disclosure relates at least to the fields of cancer biology, cell biology, immunology, and medicine. BACKGROUND

[0005] T cells are part of the adaptive immune system and recognize infected, malignant, or non-self cells. Although there are many subtypes with distinct immunological functions, most T cells recognize peptides presented on major histocompatibility complexes (pMHC) through their T cell receptor (TCR). The TCR when complexed with the CD4 or CD8 coreceptor is highly sensitive, capable of inducing activation by binding a single pMHC. Despite the sensitivity and diversity of the T cell repertoire, some cancer cells, transformed or infected cells, and autoreactive cells can evade T cell recognition. Therapies such as chimeric antigen receptor (CAR) T cells can be used to target these immune-evasive populations. CAR- T cells, genetically engineered to express an antigen recognition domain coupled to intracellular signaling domains borrowed from the endogenous TCR complex, provide an alternative, pMHC-independent pathway of T cell activation.

[0006] CAR-T therapy has seen great clinical success in the treatment of B cell malignancies, especially those targeting CD19, a common B cell surface marker expressed in298507734.1 - 1 -cell cancers. Use of tisa-cel in B-ALL, diffuse large B cell lymphoma (DLBCL), and follicular lymphoma (FL) show complete response rates of 90%, 46%, and 71%, respectively. Despite these impressive initial responses, however, long term remission is not achieved for all patients, with relapse observed in 26%, 73%, and 54% of patients.

[0007] Relapse can be caused by multiple factors, two of which are the CAR’s poor antigen sensitivity and loss of antigen expression. Other have published CAR-T cell designs addressing antigen loss in target cells, for example, by directly increasing sensitivity of CARs. Liu et al. 2021 designed the synthetic T cell receptor and antigen receptor (STAR) that fused the light and heavy chains of an scFv with the TCRA and TCRB constant regions, allowing the STAR to recruit CD3 complex components. However, while the STAR showed increased sensitivity to antigen, it did not recruit coreceptor-bound LCK and may in fact compete with endogenous TCRs for CD3 complex components. Mansilla-Soto et al.2022 generated an HLA-independent T cell (HIT) receptor, using TRAC locus editing to replace the TCRA and TCRB variable regions with light and heavy chains of an scFv. This design provided increased antigen sensitivity and phosphorylation of signaling molecules such as ITAMs and ZAP-70, but like the STAR, the HIT does not recruit coreceptor-bound LCK. Furthermore, editing the TRAC locus the HIT eliminates functionality of endogenous TCRs. Hartl et al. 2020 generated an eRKzCAR by inserting the CD3ε RK motif, capable of binding LCK, into a traditional CAR design. This design increased CD3ζ-associated LCK and antigen sensitivity without inducing induce tonic signaling. However, it undesirably induced CD3 ζ-associated LCK at rest, and improper sequestration of CD3ζ-associated LCK may interfere with endogenous TCR function.

[0008] A second strategy of preventing relapse due to antigen loss is by the use of dual- CAR systems targeting two unique antigens, so that if one antigen is downregulated the other can still be targeted. However, this approach also suffers flaws. It does not increase sensitivity of CARs, and if both targets are downregulated (as is the case with many B cell markers), it provides an avenue for relapse. Furthermore, while this approach is feasible for targeting B cells, not every tumor type has multiple antigens that can be safely targeted.

[0009] Comparative studies have shown that many clinically used CARs are limited by up to 1,000-fold reduced sensitivity for antigen relative to TCRs, in part due to poor recruitment of downstream signaling molecules to facilitate T-cell signaling. Upon recognition of pMHC through the TCR or an epitope through the CAR, both receptors undergo conformational changes and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) found in cytoplasmic domains, such as CD3ζ. In the case of the TCR, this signaling is further298507734.1 - 2 -amplified by co-receptor-associated LCK which further phosphorylates CD3 ITAMs as well as ZAP-70. Initial phosphorylation of ITAMs is amplified through kinase cascades and leads to major changes such as increased glycolytic metabolism, increased adhesion, and dramatic shifts in transcriptional expression. Furthermore, T cell activation induces differentiation and homing to secondary lymph nodes to aid in the immune response. Comparative studies have shown while CARs and TCRs engage their target molecules similarly, synaptic CD3ζ, CD3δ, CD3ε, CD3γ, LAT, LCK, and ZAP-70 are significantly less phosphorylated in CAR-mediated activation than with the TCR. As most clinically used CARs use similar intracellular signaling domains and differ mainly by choice of scFv, low sensitivity is common across designs.

[0010] Collectively, in the patients who relapsed after receiving a CD19 CAR-T therapy in B-ALL, DLBCL, and FL, ~40% of patients exhibited reduced CD19 expression. This association between low antigen expression and relapse has been observed for other antigen targets such as CD22, another B cell lineage marker expressed in >90% of B cell malignancies. Although many clinical trials are focused on repurposing older generations of CARs to target new surface antigens, it is clear there is still a great need for improving CAR-T signaling to reduce instances of relapse.

[0011] Given the importance of reducing instances of relapse in cancer patients receiving CAR-T therapy, pursuit of superior compositions and methods to reduce instances of relapse that can (1) improve CAR antigen sensitivity, (2) improve CAR avidity to stabilize CAR / antigen binding, (3) account for decreased antigen expression, (4) preserve endogenous TCR function, (5) avoid interference with assembly of CD3 complex components, and (6) recruit only LCK in the presence of antigen are warranted. BRIEF SUMMARY

[0012] A discovery has been made that provides a solution to at least one or more of the aforementioned problems associated with reducing instances of relapse in cancer patients receiving CAR-T therapy. In some aspects, the inventors discovered that targeting at least two tumor antigen epitopes using a system including co-expressed CAR molecules can induce CAR signaling that more closely mimics TCR signaling and can improve CAR sensitivity to target antigen, with possible clinical implications for cases of downregulation of target antigen. In some aspects, a system includes a novel, chimeric CD4, CD8, or CD28 coreceptor (mimicking CD8 or CD4) to enhance signaling and a stimulatory 41BB or CD28 and CD3ζ CAR (mimicking TCR). In some aspects, the inventors discovered that the co-expressed CAR molecules can interact with each other through the ITAM domain of CD3ζ and chimeric CD4-298507734.1 - 3 -or CD8-coreceptor-bound LCK. In some aspects, the inventors discovered that the system can increase in vitro functionality of CARs by, for example, increasing levels of TNF-α and IFNγ. In some aspects, the inventors discovered the system increases antigen sensitivity and / or improves CAR avidity to stabilize CAR / antigen binding while preserving endogenous TCR function, avoiding interference with assembly of CD3 complex components, recruiting only LCK in the presence of antigen, or a combination thereof. This specific strategy is novel and can provide support for colocalizing other CAR molecules.

[0013] In some aspects, the methods and compositions provided herein provide several advantages. In some aspects, the system including co-expressed CAR molecules can induce CAR signaling that more closely mimics TCR signaling and can improve CAR efficacy in cases of downregulation of target antigen. This activity can increase CAR sensitivity and / or improve CAR avidity to stabilize CAR / antigen binding to improve patient outcomes by reducing occurrences of relapse due to low antigen expression. In some aspects, the system employing the chimeric-coreceptor approach is useful for treating B cell and solid tumor cancers by targeting multiple epitopes of an antigen expressed by the cancer cells.

[0014] Aspects of the present disclosure include polynucleotides, polypeptides, vectors, expression constructs, engineered receptors, chimeric antigen receptors, immune cells, antibodies, antibody fragments, nanobodies, scFvs, pharmaceutical compositions, methods for generating a CAR, methods for generating a CAR immune cell, methods for treating a subject for cancer, methods of killing cancer cells in a subject, methods for stimulating an immune response to cancer cells in a subject, and methods of generating a persisting population of genetically engineered immune cells. Polypeptides and polynucleotides of the disclosure can encode at least or at most 1, 2, 3, or more of: an antigen-binding region, a variable heavy chain region, a variable light chain region, a transmembrane domain, an intracellular domain, a costimulatory domain, a hinge region, a signal peptide, a polypeptide linker, and an immune cell binding region. Any one of more of the preceding components may be excluded from polypeptides or polynucleotides of the disclosure in certain aspects.

[0015] Accordingly, provided herein, in some aspects, are compositions and methods relating to two CARs, wherein: the first CAR comprises: an antigen-binding domain that binds a first epitope on an antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; and an intracellular domain from CD4 or CD8, and the second CAR comprises: an antigen-binding domain that binds a second epitope on the antigen; a hinge from CD4, CD8,298507734.1 - 4 -or CD28; a transmembrane domain from CD4 or CD8; a 4-1BB or CD28 co-stimulatory domain; and a CD3ζ stimulatory domain.

[0016] Also provided herein, in some aspects, is an immune cell expressing two CARs, wherein: the first CAR comprises: an antigen-binding domain that binds a first epitope on an antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; and an intracellular domain from CD4 or CD8, and the second CAR comprises: an antigen-binding domain that binds a second epitope on the antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; a 4-1BB or CD28 co-stimulatory domain; and a CD3ζ stimulatory domain.

[0017] In some aspects, the antigen-binding domain of the first CAR comprises a first antibody or antigen-binding fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor- ligand pair that targets the antigen. In some aspects, the antigen-binding domain of the second CAR comprises a second antibody or antigen-binding fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally- occurring receptor-ligand pair that targets the antigen. In some aspects, the antigen is expressed by a cancer cell. In some aspects, the antigen is CD22, CD19, BCMA, HER2, or mesothelin.

[0018] In some aspects, the hinge of the first CAR and / or the second CAR is a CD4 hinge. In some aspects, the hinge of the first CAR and / or the second CAR is a CD8 hinge. In some aspects, the hinge of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5. In some aspects, the hinge of the first CAR and / or the second CAR comprises SEQ ID NO:5. In some aspects, the hinge of the first CAR and / or the second CAR consists of SEQ ID NO:5. In some aspects, the hinge of the first CAR and / or the second CAR is a CD28 hinge. In some aspects, the hinge of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6. In some aspects, the hinge of the first CAR and / or the second CAR comprises SEQ ID NO:6. In some aspects, the hinge of the first CAR and / or the second CAR consists of SEQ ID NO:6.

[0019] In some aspects, the transmembrane domain of the first CAR and / or the second CAR is a CD4 transmembrane domain. In some aspects, the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:7. In some aspects, the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:7. In some aspects, the298507734.1 - 5 -transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:7. In some aspects, the transmembrane domain of the first CAR and / or the second CAR is a CD8 transmembrane domain. In some aspects, the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:8. In some aspects, the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:8. In some aspects, the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:8. In some aspects, the transmembrane domain of the first CAR and / or the second CAR is a CD28 transmembrane domain. In some aspects, the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9. In some aspects, the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:9. In some aspects, the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:9.

[0020] In some aspects, the intracellular domain of the first CAR is a CD4 intracellular domain. In some aspects, the intracellular domain of the first CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:10. In some aspects, the intracellular domain of the first CAR comprises SEQ ID NO:10. In some aspects, the intracellular domain of the first CAR consists of SEQ ID NO:10. In some aspects, the intracellular domain of the first CAR is a CD8 intracellular domain. In some aspects, the intracellular domain comprises of the first CAR an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:11. In some aspects, the intracellular domain of the first CAR comprises SEQ ID NO:11. In some aspects, the intracellular domain of the first CAR consists of SEQ ID NO:11.

[0021] In some aspects, the 4-1BB co-stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12.

[0022] 39. The composition of any one of claims 1-37, wherein the 4-1BB co-stimulatory domain of the second CAR comprises SEQ ID NO:12. In some aspects, the 4-1BB co- stimulatory domain of the second CAR consists of SEQ ID NO:12. In some aspects, the CD28 co-stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:13. In some aspects, the CD28 co- stimulatory domain of the second CAR comprises SEQ ID NO:13. In some aspects, the CD28 co-stimulatory domain of the second CAR consists of SEQ ID NO:13. In some aspects, the298507734.1 - 6 -CD3ζ stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:14. In some aspects, the CD3ζ stimulatory domain of the second CAR comprises SEQ ID NO:14. In some aspects, the CD3ζ stimulatory domain of the second CAR consists of SEQ ID NO:14.

[0023] In some aspects, the first CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34. In some aspects, the first CAR comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34. In some aspects, the first CAR consists of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34. In some aspects, the second CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33. In some aspects, the second CAR comprises SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33. In some aspects, the second CAR consists of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33.

[0024] In some aspects, the first and / or the second CAR are encoded by one or more polynucleotides. In some aspects, the first and / or the second CAR are encoded by the same polynucleotide. In some aspects, the first and / or the second CAR are encoded by different polynucleotides. In some aspects, the one or more polynucleotides are comprised in one or more vectors. In some aspects, the one or more vectors are viral vectors. In some aspects, the viral vectors are adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, or a combination thereof. In some aspects, the one or more vectors are non-viral vectors. In some aspects, the non-viral vectors are plasmids. In some aspects, the vectors are multicistronic.

[0025] In some aspects, the one or more vectors are comprised in a cell or a population of cells. In some aspects, the cell or cells express the first and / or the second CAR encoded by one or more polynucleotides comprised in the one or more vectors. In some aspects, the cell or cells are immune cells. In some aspects, the cell or cells are T cells, natural killer (NK) cells, macrophages, microglial cells, or neutrophils. In some aspects, the cell or cells are T cells. In some aspects, the cell or cells are NK cells. In some aspects, the cells are human cells, including human cells that are T cell or NK cells.298507734.1 - 7 -

[0026] In some aspects, the composition further comprises a pharmaceutically-acceptable excipient. In some aspects, the composition further comprises an additional therapeutic. In some aspects, the additional therapeutic is a chemotherapeutic or an immunotherapeutic.

[0027] Disclosed herein, in some aspects, is a method for generating the first CAR and / or the second CAR of the composition of any one of claims 1-67, the method comprising: (a) providing one or more polynucleotides encoding the first CAR and / or the second CAR to a cell or cells; and (b) subjecting the cell or cells to conditions sufficient to express the first CAR and / or the second CAR from the one or more polynucleotides.

[0028] Disclosed herein, in some aspects, is a chimeric antigen receptor (CAR) comprising: an antigen-binding domain that binds a first epitope on an antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; and an intracellular domain from CD4 or CD8.

[0029] In some aspects, the antigen-binding domain comprises an antibody or antigen- binding fragment thereof, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen. In some aspects, the antigen-binding domain comprises an scFv or a nanobody. In some aspects, the antigen is expressed by a cancer cell. In some aspects, the antigen is CD22, CD19, BCMA, HER2, or mesothelin.

[0030] In some aspects, the hinge is a CD4 hinge. In some aspects, the hinge of the first CAR and / or the second CAR is a CD8 hinge. In some aspects, the hinge comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5. In some aspects, the hinge comprises SEQ ID NO:5. In some aspects, the hinge consists of SEQ ID NO:5. In some aspects, the hinge is a CD28 hinge. In some aspects, the hinge comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6. In some aspects, the hinge of comprises SEQ ID NO:6. In some aspects, the hinge consists of SEQ ID NO:6.

[0031] In some aspects, the transmembrane domain is a CD4 transmembrane domain. In some aspects, the transmembrane domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:7. In some aspects, the transmembrane domain comprises SEQ ID NO:7. In some aspects, the transmembrane domain consists of SEQ ID NO:7. In some aspects, the transmembrane domain is a CD8 transmembrane domain. In some aspects, the transmembrane domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:8. In some aspects, the transmembrane domain comprises SEQ ID NO:8. In some aspects, the298507734.1 - 8 -transmembrane domain consists of SEQ ID NO:8. In some aspects, the transmembrane domain is a CD28 transmembrane domain. In some aspects, the transmembrane domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9. In some aspects, the transmembrane domain comprises SEQ ID NO:9. In some aspects, the transmembrane domain consists of SEQ ID NO:9.

[0032] In some aspects, the intracellular domain is a CD4 intracellular domain. In some aspects, the intracellular domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:10. In some aspects, the intracellular domain comprises SEQ ID NO:10. In some aspects, the intracellular domain consists of SEQ ID NO:10. In some aspects, the intracellular domain is a CD8 intracellular domain. In some aspects, the intracellular domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:11. In some aspects, the intracellular domain comprises SEQ ID NO:11. In some aspects, the intracellular domain consists of SEQ ID NO:11.

[0033] In some aspects, the CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34. In some aspects, the CAR comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34. In some aspects, the CAR consists of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34.

[0034] In some aspects, the CAR is encoded by one or more polynucleotides. In some aspects, the CAR is a first CAR, and wherein the one or more polynucleotides further encode a second CAR. In some aspects, the first CAR and the second CAR are encoded by the same polynucleotide. In some aspects, the first CAR and the second CAR are encoded by different polynucleotides.

[0035] In some aspects, the second CAR comprises: an antigen-binding domain that binds a second epitope on the antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; a 4-1BB or CD28 co-stimulatory domain; and a CD3ζ stimulatory domain.

[0036] In some aspects, the antigen-binding domain of the second CAR comprises an antibody or antigen-binding fragment thereof, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen. In some aspects, the antigen-binding domain of the second CAR comprises an scFv or a nanobody.298507734.1 - 9 -

[0037] In some aspects, the hinge of the second CAR is a CD4 hinge. In some aspects, the hinge of the second CAR is a CD8 hinge. In some aspects, the hinge of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5. In some aspects, the hinge of the second CAR comprises SEQ ID NO:5. In some aspects, the hinge of the second CAR consists of SEQ ID NO:5. In some aspects, the hinge of the second CAR is a CD28 hinge. In some aspects, the hinge of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6. In some aspects, the hinge of the second CAR comprises SEQ ID NO:6. In some aspects, the hinge of the second CAR consists of SEQ ID NO:6.

[0038] In some aspects, the transmembrane domain of the second CAR is a CD4 transmembrane domain. In some aspects, the transmembrane domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:7. In some aspects, the transmembrane domain of the second CAR comprises SEQ ID NO:7. In some aspects, the transmembrane domain of the second CAR consists of SEQ ID NO:7. In some aspects, the transmembrane domain of the second CAR is a CD8 transmembrane domain. In some aspects, the transmembrane domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:8. In some aspects, the transmembrane domain of the second CAR comprises SEQ ID NO:8. In some aspects, the transmembrane domain of the second CAR consists of SEQ ID NO:8. In some aspects, the transmembrane domain of the second CAR is a CD28 transmembrane domain. In some aspects, the transmembrane domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9. In some aspects, the transmembrane domain of the second CAR comprises SEQ ID NO:9. In some aspects, the transmembrane domain of the second CAR consists of SEQ ID NO:9.

[0039] In some aspects, the 4-1BB co-stimulatory domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12. In some aspects, the 4-1BB co-stimulatory domain comprises SEQ ID NO:12. In some aspects, the 4- 1BB co-stimulatory domain consists of SEQ ID NO:12. In some aspects, the CD28 co- stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:13. In some aspects, the CD28 co- stimulatory domain of the second CAR comprises SEQ ID NO:13. In some aspects, the CD28 co-stimulatory domain of the second CAR consists of SEQ ID NO:13. In some aspects, the298507734.1 - 10 -CD3ζ stimulatory domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:14. In some aspects, the CD3ζ stimulatory domain comprises SEQ ID NO:14. In some aspects, the CD3ζ stimulatory domain consists of SEQ ID NO:14.

[0040] In some aspects, the second CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33. In some aspects, the second CAR comprises SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33. In some aspects, the second CAR consists of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33.

[0041] In some aspects, the one or more polynucleotides are comprised in a vector. In some aspects, the vector is a viral vector. In some aspects, the viral vector is an adenoviral vector, adeno-associated viral vector, lentiviral vector, or retroviral vector. In some aspects, the vector is a non-viral vector. In some aspects, the non-viral vector is a plasmid. In some aspects, the vector is multicistronic.

[0042] In some aspects, the one or more polynucleotides or the vector are comprised in an immune cell. In some aspects, the immune cell is a T cell, natural killer (NK) cell, macrophage, microglial cell, or neutrophil. In some aspects, the immune cell is a T cell. In some aspects, the immune cell is a NK cell. In some aspect, the immune cells comprise a population of immune cells.

[0043] In some aspects, the one or more polynucleotides, the vector, the immune cell, or the population of immune cells are comprised in a composition with a pharmaceutically acceptable excipient. In some aspects, the composition further comprises an additional therapeutic. In some aspects, the additional therapeutic is a chemotherapeutic or an immunotherapeutic.

[0044] Disclosed herein, in some aspects, is a method of killing cancer cells in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition; immune cell; CAR; one or more polynucleotides; vector; or population of immune cells according to any one or more aspects described herein.

[0045] Disclosed herein, in some aspects, is a method for treating a subject for cancer, the method comprising administering to the subject a therapeutically effective amount of a composition; immune cell; CAR; one or more polynucleotides; vector; or population of immune cells according to any one or more aspects described herein.298507734.1 - 11 -

[0046] Disclosed herein, in some aspects, is a method for stimulating an immune response to cancer cells in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of a composition; immune cell; CAR; one or more polynucleotides; vector; or population of immune cells according to any one or more aspects described herein.

[0047] In some aspects of the foregoing methods, the cancer is lymphoma, leukemia, multiple myeloma, glioblastoma, mesothelioma, head and neck cancers, osteosarcoma, melanoma, non-small cell lung cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer, germ cell tumors, or breast cancer. In some aspects, the cancer is a B-cell cancer. In some aspects, the cancer is a CD19-positive, CD22-positive, BCMA-positive cancer, HER2-positive cancer, or a mesothelin-positive cancer.

[0048] In some aspects, the method further comprises administering to the subject an additional therapy. In some aspects, the additional therapy is radiotherapy, chemotherapy, or immunotherapy.

[0049] Disclosed herein, in some aspects, is a method of generating a persisting population of genetically engineered immune cells in a subject, the method comprising administering to the subject an effective amount of a composition; immune cell; CAR; one or more polynucleotides; vector; or population of immune cells according to any one or more aspects described herein. In some aspects, the genetically engineered immune cells persist in the subject for at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days, weeks, months, or years after administration.

[0050] Disclosed herein, in some aspects, is a method of expanding a population of genetically engineered immune cells in a subject, the method comprising administering to the subject an effective amount of a composition; immune cell; CAR; one or more polynucleotides; vector; or population of immune cells according to any one or more aspects described herein.

[0051] Also disclosed herein are the following enumerated Aspects 1-273.

[0052] Aspect 1 is a composition comprising two CARs, wherein: the first CAR comprises: an antigen-binding domain that binds a first epitope on an antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; and an intracellular domain from CD4 or CD8, and the second CAR comprises: an antigen-binding domain that binds a second epitope on the antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; a 4-1BB or CD28 co-stimulatory domain; and a CD3ζ stimulatory domain.298507734.1 - 12 -

[0053] Aspect 2 is a composition comprising two CARs, wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

[0054] Aspect 3 is a composition comprising two CARs, wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD4; and an intracellular domain from CD4, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CDs22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

[0055] Aspect 4 is a composition comprising two CARs, wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD28; a transmembrane domain from CD28; and an intracellular domain from CD4, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

[0056] Aspect 5 is a composition comprising two CARs, wherein: the first CAR comprises: a BCMA-binding domain that binds a first epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8, and the second CAR comprises: a BCMA-binding domain that binds a second epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

[0057] Aspect 6 is the composition of any one of Aspects 1-5, wherein the antigen-binding domain of the first CAR comprises a first antibody or antigen-binding fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen. Aspect 7 is the composition of any one of Aspects 1-6, wherein the antigen-binding domain of the second CAR comprises a second antibody or antigen-binding fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally- occurring receptor-ligand pair that targets the antigen. Aspect 8 is the composition of any one of Aspects 1-7, wherein the antigen is expressed by a cancer cell. Aspect 9 is the composition298507734.1 - 13 -of any one of Aspects 1-8, wherein the antigen is CD22, CD19, BCMA, HER2, or mesothelin. Aspect 10 is the composition of any one of Aspects 1-9, wherein the hinge of the first CAR and / or the second CAR is a CD4 hinge. Aspect 11 is the composition of any one of Aspects 1- 9, wherein the hinge of the first CAR and / or the second CAR is a CD8 hinge. Aspect 12 is the composition of any one of Aspects 1-9, wherein the hinge of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5. Aspect 13 is the composition of any one of Aspects 1-9, wherein the hinge of the first CAR and / or the second CAR comprises SEQ ID NO:5. Aspect 14 is the composition of any one of Aspects 1-9, wherein the hinge of the first CAR and / or the second CAR consists of SEQ ID NO:5. Aspect 15 is the composition of any one of Aspects 1-9, wherein the hinge of the first CAR and / or the second CAR is a CD28 hinge. Aspect 16 is the composition of any one of Aspects 1-9, wherein the hinge of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6. Aspect 17 is the composition of any one of Aspects 1-9, wherein the hinge of the first CAR and / or the second CAR comprises SEQ ID NO:6. Aspect 18 is the composition of any one of Aspects 1-9, wherein the hinge of the first CAR and / or the second CAR consists of SEQ ID NO:6.

[0058] Aspect 19 is the composition of any one of Aspects 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD4 transmembrane domain. Aspect 20 is the composition of any one of Aspects 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:7. Aspect 21 is the composition of any one of Aspects 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:7. Aspect 22 is the composition of any one of Aspects 1- 18, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:7. Aspect 23 is the composition of any one of Aspects 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD8 transmembrane domain. Aspect 24 is the composition of any one of Aspects 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:8. Aspect 25 is the composition of any one of Aspects 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:8. Aspect 26 is the composition of any one of Aspects 1- 18, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:8. Aspect 27 is the composition of any one of Aspects 1-18, wherein the298507734.1 - 14 -transmembrane domain of the first CAR and / or the second CAR is a CD28 transmembrane domain. Aspect 28 is the composition of any one of Aspects 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9. Aspect 29 is the composition of any one of Aspects 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:9. Aspect 30 is the composition of any one of Aspects 1- 18, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:9.

[0059] Aspect 31 is the composition of any one of Aspects 1-30, wherein the intracellular domain of the first CAR is a CD4 intracellular domain. Aspect 32 is the composition of any one of Aspects 1-30, wherein the intracellular domain of the first CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:10. Aspect 33 is the composition of any one of Aspects 1-30, wherein the intracellular domain of the first CAR comprises SEQ ID NO:10. Aspect 34 is the composition of any one of Aspects 1-30, wherein the intracellular domain of the first CAR consists of SEQ ID NO:10. Aspect 35 is the composition of any one of Aspects 1-30, wherein the intracellular domain of the first CAR is a CD8 intracellular domain. Aspect 36 is the composition of any one of Aspects 1-30, wherein the intracellular domain comprises of the first CAR an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:11. Aspect 37 is the composition of any one of Aspects 1-30, wherein the intracellular domain of the first CAR comprises SEQ ID NO:11. Aspect 38 is the composition of any one of Aspects 1-30, wherein the intracellular domain of the first CAR consists of SEQ ID NO:11.

[0060] Aspect 39 is the composition of any one of Aspects 1-38, wherein the first CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34. Aspect 40 is the composition of any one of Aspects 1-38, wherein the first CAR comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34. Aspect 41 is the composition of any one of Aspects 1-38, wherein the first CAR consists of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34.

[0061] Aspect 42 is the composition of any one of Aspects 1-41, wherein the 4-1BB co- stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12. Aspect 43 is the composition of any one of Aspects 1-41, wherein the 4-1BB co-stimulatory domain of the second CAR comprises298507734.1 - 15 -SEQ ID NO:12. Aspect 44 is the composition of any one of Aspects 1-41, wherein the 4-1BB co-stimulatory domain of the second CAR consists of SEQ ID NO:12. Aspect 45 is the composition of any one of Aspects 1-44, wherein the CD28 co-stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:13. Aspect 46 is the composition of any one of Aspects 1-44, wherein the CD28 co-stimulatory domain of the second CAR comprises SEQ ID NO:13. Aspect 47 is the composition of any one of Aspects 1-44, wherein the CD28 co-stimulatory domain of the second CAR consists of SEQ ID NO:13.

[0062] Aspect 48 is the composition of any one of Aspects 1-47, wherein the CD3ζ stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:14. Aspect 49 is the composition of any one of Aspects 1-47, wherein the CD3ζ stimulatory domain of the second CAR comprises SEQ ID NO:14. Aspect 50 is the composition of any one of Aspects 1-47, wherein the CD3ζ stimulatory domain of the second CAR consists of SEQ ID NO:14.

[0063] Aspect 51 is the composition of any one of Aspects 1-50, wherein the second CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33. Aspect 52 is the composition of any one of Aspects 1-50, wherein the second CAR comprises SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33. Aspect 53 is the composition of any one of Aspects 1-50, wherein the second CAR consists of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33.

[0064] Aspect 54 is the composition of any one of Aspects 1-53, wherein the first and / or the second CAR are encoded by one or more polynucleotides. Aspect 55 is the composition of any one of Aspects 1-53, wherein the first and / or the second CAR are encoded by the same polynucleotide. Aspect 56 is the composition of any one of Aspects 1-53, wherein the first and / or the second CAR are encoded by different polynucleotides. Aspect 57 is the composition of any one of Aspects 54-56, wherein the one or more polynucleotides are comprised in one or more vectors. Aspect 58 is the composition of Aspect 57, wherein the one or more vectors are viral vectors. Aspect 59 is the composition of Aspect 58, wherein the viral vectors are adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, or a combination thereof. Aspect 60 is the composition of Aspect 57, wherein the one or more vectors are non-viral vectors. Aspect 61 is the composition of Aspect 60, wherein the non-viral vectors are plasmids. Aspect 62 is the composition of any one of Aspects 57-61, wherein the298507734.1 - 16 -vectors are multicistronic. Aspect 63 is the composition of any one of Aspects 57-62, wherein the one or more vectors are comprised in a cell or a population of cells. Aspect 64 is the composition of Aspect 63, wherein the cell or cells express the first and / or the second CAR encoded by one or more polynucleotides comprised in the one or more vectors. Aspect 65 is the composition of Aspect 63, wherein the cell or cells are immune cells. Aspect 66 is the composition of any one of Aspects 63-65, wherein the cell or cells are T cells, natural killer (NK) cells, macrophages, microglial cells, or neutrophils. Aspect 67 is the composition of any one of Aspects 63-66, wherein the cell or cells are T cells. Aspect 68 is the composition of any one of Aspects 63-66, wherein the cell or cells are NK cells. Aspect 69 is the composition of any one of Aspects 63-68, wherein the cell or cells are human cells. Aspect 70 is the composition of any one of Aspects 1-69, further comprising a pharmaceutically-acceptable excipient. Aspect 71 is the composition of any one of Aspects 1-70, further comprising an additional therapeutic. Aspect 72 is the composition of Aspect 71, wherein the additional therapeutic is a chemotherapeutic or an immunotherapeutic.

[0065] Aspect 73 is a method for generating the first CAR and / or the second CAR of the composition of any one of Aspects 1-72, the method comprising: (a) providing one or more polynucleotides encoding the first CAR and / or the second CAR to a cell or cells; and (b) subjecting the cell or cells to conditions sufficient to express the first CAR and / or the second CAR from the one or more polynucleotides.

[0066] Aspect 74 is an immune cell expressing two CARs, wherein: the first CAR comprises: an antigen-binding domain that binds a first epitope on an antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; and an intracellular domain from CD4 or CD8, and the second CAR comprises: an antigen-binding domain that binds a second epitope on the antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; a 4-1BB or CD28 co-stimulatory domain; and a CD3ζ stimulatory domain.

[0067] Aspect 75 is an immune cell expressing two CARs, wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

[0068] Aspect 76 is an immune cell expressing two CARs, wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a298507734.1 - 17 -transmembrane domain from CD4; and an intracellular domain from CD4, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

[0069] Aspect 77 is an immune cell expressing two CARs, wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD28; a transmembrane domain from CD28; and an intracellular domain from CD4, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

[0070] Aspect 78 is an immune cell expressing two CARs, wherein: the first CAR comprises: a BCMA-binding domain that binds a first epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8, and the second CAR comprises: a BCMA-binding domain that binds a second epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

[0071] Aspect 79 is the immune cell of any one of Aspects 74-78, wherein the antigen- binding domain of the first CAR comprises a first antibody or antigen-binding fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen. Aspect 80 is the immune cell of any one of Aspects 74-79, wherein the antigen-binding domain of the second CAR comprises a second antibody or antigen-binding fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen. Aspect 81 is the immune cell of any one of Aspects 74-80, wherein the antigen is expressed by a cancer cell. Aspect 82 is the immune cell of any one of Aspects 74-81, wherein the antigen is CD22, CD19, BCMA, HER2, or mesothelin.

[0072] Aspect 83 is the immune cell of any one of Aspects 74-82, wherein the hinge of the first CAR and / or the second CAR is a CD4 hinge. Aspect 84 is the immune cell of any one of Aspects 74-82, wherein the hinge of the first CAR and / or the second CAR is a CD8 hinge. Aspect 85 is the immune cell of any one of Aspects 74-82, wherein the hinge of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5. Aspect 86 is the immune cell of any one of Aspects298507734.1 - 18 -74-82, wherein the hinge of the first CAR and / or the second CAR comprises SEQ ID NO:5. Aspect 87 is the immune cell of any one of Aspects 74-82, wherein the hinge of the first CAR and / or the second CAR consists of SEQ ID NO:5. Aspect 88 is the immune cell of any one of Aspects 74-82, wherein the hinge of the first CAR and / or the second CAR is a CD28 hinge. Aspect 89 is the immune cell of any one of Aspects 74-82, wherein the hinge of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6. Aspect 90 is the immune cell of any one of Aspects 74-82, wherein the hinge of the first CAR and / or the second CAR comprises SEQ ID NO:6. Aspect 91 is the immune cell of any one of Aspects 74-82, wherein the hinge of the first CAR and / or the second CAR consists of SEQ ID NO:6.

[0073] Aspect 92 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD4 transmembrane domain. Aspect 93 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:7. Aspect 94 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:7. Aspect 95 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:7. Aspect 96 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD8 transmembrane domain. Aspect 97 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:8. Aspect 98 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:8. Aspect 99 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:8. Aspect 100 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD28 transmembrane domain. Aspect 101 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9. Aspect 102 is the immune cell of any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:9. Aspect 103 is the immune cell of298507734.1 - 19 -any one of Aspects 74-91, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:9.

[0074] Aspect 104 is the immune cell of any one of Aspects 74-103, wherein the intracellular domain of the first CAR is a CD4 intracellular domain. Aspect 105 is the immune cell of any one of Aspects 74-103, wherein the intracellular domain of the first CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:10. Aspect 106 is the immune cell of any one of Aspects 74-103, wherein the intracellular domain of the first CAR comprises SEQ ID NO:10. Aspect 107 is the immune cell of any one of Aspects 74-103, wherein the intracellular domain of the first CAR consists of SEQ ID NO:10. Aspect 108 is the immune cell of any one of Aspects 74-103, wherein the intracellular domain of the first CAR is a CD8 intracellular domain. Aspect 109 is the immune cell of any one of Aspects 74-103, wherein the intracellular domain comprises of the first CAR an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:11. Aspect 110 is the immune cell of any one of Aspects 74-103, wherein the intracellular domain of the first CAR comprises SEQ ID NO:11. Aspect 111 is the immune cell of any one of Aspects 74-103, wherein the intracellular domain of the first CAR consists of SEQ ID NO:11.

[0075] Aspect 112 is the immune cell of any one of Aspects 74-111, wherein the first CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34. Aspect 113 is the immune cell of any one of Aspects 74-111, wherein the first CAR comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34. Aspect 114 is the immune cell of any one of Aspects 74-111, wherein the first CAR consists of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34.

[0076] Aspect 115 is the immune cell of any one of Aspects 74-114, wherein the 4-1BB co-stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12. Aspect 116 is the immune cell of any one of Aspects 74-114, wherein the 4-1BB co-stimulatory domain of the second CAR comprises SEQ ID NO:12. Aspect 117 is the immune cell of any one of Aspects 74-114, wherein the 4-1BB co-stimulatory domain of the second CAR consists of SEQ ID NO:12. Aspect 118 is the immune cell of any one of Aspects 74-117, wherein the CD28 co-stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:13. Aspect 119 is the immune cell of any one of Aspects 74-117, wherein the CD28 co-stimulatory domain of the second CAR comprises SEQ298507734.1 - 20 -ID NO:13. Aspect 120 is the immune cell of any one of Aspects 74-117, wherein the CD28 co- stimulatory domain of the second CAR consists of SEQ ID NO:13.

[0077] Aspect 121 is the immune cell of any one of Aspects 74-120, wherein the CD3ζ stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:14. Aspect 122 is the immune cell of any one of Aspects 74-120, wherein the CD3ζ stimulatory domain of the second CAR comprises SEQ ID NO:14. Aspect 123 is the immune cell of any one of Aspects 74-120, wherein the CD3ζ stimulatory domain of the second CAR consists of SEQ ID NO:14.

[0078] Aspect 124 is the immune cell of any one of Aspects 74-123, wherein the second CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33. Aspect 125 is the immune cell of any one of Aspects 74-123, wherein the second CAR comprises SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33. Aspect 126 is the immune cell of any one of Aspects 74-123, wherein the second CAR consists of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33.

[0079] Aspect 127 is the immune cell of any one of Aspects 74-126, wherein the first and / or the second CAR are encoded by one or more polynucleotides. Aspect 128 is the immune cell of any one of Aspects 74-126, wherein the first CAR and the second CAR are encoded by the same polynucleotide. Aspect 129 is the immune cell of any one of Aspects 74-126, wherein the first CAR and the second CAR are encoded by different polynucleotides. Aspect 130 is the immune cell of any one of Aspects 127-129, wherein the one or more polynucleotides are comprised in one or more vectors. Aspect 131 is the immune cell of Aspect 130, wherein the one or more vectors are viral vectors. Aspect 132 is the immune cell of Aspect 131, wherein the viral vectors are adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, or a combination thereof. Aspect 133 is the immune cell of Aspect 130, wherein the one or more vectors are non-viral vectors. Aspect 134 is the immune cell of Aspect 133, wherein the non-viral vectors are plasmids. Aspect 135 is the immune cell of any one of Aspects 130-134, wherein the vectors are multicistronic. Aspect 136 is the immune cell of any one of Aspects 130-135, wherein the immune cell expresses the first and / or the second CAR encoded by one or more polynucleotides comprised in the one or more vectors.

[0080] Aspect 137 is the immune cell of any one of Aspects 74-136, wherein the immune cell is a T cell, natural killer (NK) cell, macrophage, microglial cell, or neutrophil. Aspect 138 is the immune cell of any one of Aspects 74-137, wherein the immune cell is a T cell. Aspect298507734.1 - 21 -139 is the immune cell of any one of Aspects 74-137, wherein the immune cell is a NK cell. Aspect 140 is the immune cell of any one of Aspects 74-139, wherein the immune cell is a human cell.

[0081] Aspect 141 is a composition comprising: (a) the immune cell of any one of Aspects 74-140; and (b) a pharmaceutically acceptable excipient. Aspect 142 is the composition of Aspect 141, wherein the pharmaceutical composition further comprises an additional therapeutic. Aspect 143 is the composition of Aspect 142, wherein the additional therapeutic is a chemotherapeutic or an immunotherapeutic.

[0082] Aspect 144 is a chimeric antigen receptor (CAR) comprising: an antigen-binding domain that binds a first epitope on an antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; and an intracellular domain from CD4 or CD8.

[0083] Aspect 145 is a chimeric antigen receptor (CAR) comprising: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8.

[0084] Aspect 146 is a chimeric antigen receptor (CAR) comprising: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD4; and an intracellular domain from CD4.

[0085] Aspect 147 is a chimeric antigen receptor (CAR) comprising: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD28; a transmembrane domain from CD28; and an intracellular domain from CD4.

[0086] Aspect 148 is a chimeric antigen receptor (CAR) comprising: a BCMA-binding domain that binds a first epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8.

[0087] Aspect 149 is the CAR of any one of Aspects 144-148, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment thereof, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor- ligand pair that targets the antigen. Aspect 150 is the CAR of any one of Aspects 144-149, wherein the antigen-binding domain comprises an scFv or a nanobody. Aspect 151 is the CAR of any one of Aspects 144-150, wherein the antigen is expressed by a cancer cell. Aspect 152 is the CAR of any one of Aspects 144-151, wherein the antigen is CD22, CD19, BCMA, HER2, or mesothelin.

[0088] Aspect 153 is the CAR of any one of Aspects 144-152, wherein the hinge is a CD4 hinge. Aspect 154 is the CAR of any one of Aspects 144-152, wherein the hinge is a CD8 hinge. Aspect 155 is the CAR of any one of Aspects 144-152, wherein the hinge comprises an298507734.1 - 22 -amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5. Aspect 156 is the CAR of any one of Aspects 144-152, wherein the hinge comprises SEQ ID NO:5. Aspect 157 is the CAR of any one of Aspects 144-152, wherein the hinge consists of SEQ ID NO:5. Aspect 158 is the CAR of any one of Aspects 144-152, wherein the hinge is a CD28 hinge. Aspect 159 is the CAR of any one of Aspects 144-152, wherein the comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6. Aspect 160 is the CAR of any one of Aspects 144-152, wherein the hinge comprises SEQ ID NO:6. Aspect 161 is the CAR of any one of Aspects 144-152, wherein the hinge consists of SEQ ID NO:6.

[0089] Aspect 162 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain is a CD4 transmembrane domain. Aspect 163 is the CAR of any one of Aspects 144- 161, wherein the transmembrane domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:7. Aspect 164 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain comprises SEQ ID NO:7. Aspect 165 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain consists of SEQ ID NO:7. Aspect 166 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain is a CD8 transmembrane domain. Aspect 167 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:8. Aspect 168 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain comprises SEQ ID NO:8. Aspect 169 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain consists of SEQ ID NO:8. Aspect 170 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain is a CD28 transmembrane domain. Aspect 171 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9. Aspect 172 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain comprises SEQ ID NO:9. Aspect 173 is the CAR of any one of Aspects 144-161, wherein the transmembrane domain consists of SEQ ID NO:9.

[0090] Aspect 174 is the CAR of any one of Aspects 144-173, wherein the intracellular domain is a CD4 intracellular domain. Aspect 175 is the CAR of any one of Aspects 144-173, wherein the intracellular domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:10. Aspect 176 is the CAR of any one of Aspects 144-173, wherein the intracellular domain comprises SEQ ID NO:10. Aspect 177 is the CAR of any one of Aspects 144-173, wherein the intracellular domain consists of SEQ ID NO:10.298507734.1 - 23 -Aspect 178 is the CAR of any one of Aspects 144-173, wherein the intracellular domain is a CD8 intracellular domain. Aspect 179 is the CAR of any one of Aspects 144-173, wherein the intracellular domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:11. Aspect 180 is the CAR of any one of Aspects 144-173, wherein the intracellular domain comprises SEQ ID NO:11. Aspect 181 is the CAR of any one of Aspects 144-173, wherein the intracellular domain consists of SEQ ID NO:11. Aspect 182 is the CAR of any one of Aspects 144-181, wherein the CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34.

[0091] Aspect 183 is the CAR of any one of Aspects 144-181, wherein the CAR comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34. Aspect 184 is the CAR of any one of Aspects 144-181, wherein the CAR consists of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:34.

[0092] Aspect 185 is one or more polynucleotides encoding the CAR of any one of Aspects 144-184. Aspect 186 is the one or more polynucleotides of Aspect 185, wherein the one or more polynucleotides further encode a second CAR. Aspect 187 is the one or more polynucleotides of Aspect 186, wherein the first CAR and the second CAR are encoded by the same polynucleotide. Aspect 188 is the one or more polynucleotides of Aspects 186 or 187, wherein the first CAR and the second CAR are encoded by different polynucleotides. Aspect 189 is the one or more polynucleotides of any one of Aspects 186-188, wherein the second CAR comprises: an antigen-binding domain that binds a second epitope on the antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; a 4-1BB or CD28 co- stimulatory domain; and a CD3ζ stimulatory domain. Aspect 190 is the one or more polynucleotides of any one of Aspects 186-188, wherein the second CAR comprises: a CD22- binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain. Aspect 191 is the one or more polynucleotides of any one of Aspects 186-188, wherein the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain. Aspect 192 is the one or more polynucleotides of any one of Aspects 186- 188, wherein the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory298507734.1 - 24 -domain; and a CD3ζ stimulatory domain. Aspect 193 is the one or more polynucleotides of any one of Aspects 186-188, wherein the second CAR comprises: a BCMA-binding domain that binds a second epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

[0093] Aspect 194 is the one or more polynucleotides of any one of Aspects 189-193, wherein the antigen-binding domain of the second CAR comprises an antibody or antigen- binding fragment thereof, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen. Aspect 195 is the one or more polynucleotides of any one of Aspects 189-194, wherein the antigen-binding domain of the second CAR comprises an scFv or a nanobody.

[0094] Aspect 196 is the one or more polynucleotides of any one of Aspects 189-195, wherein the hinge of the second CAR is a CD4 hinge. Aspect 197 is the one or more polynucleotides of any one of Aspects 189-195, wherein the hinge of the second CAR is a CD8 hinge. Aspect 198 is the one or more polynucleotides of any one of Aspects 189-195, wherein the hinge of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5. Aspect 199 is the one or more polynucleotides of any one of Aspects 189-195, wherein the hinge of the second CAR comprises SEQ ID NO:5. Aspect 200 is the one or more polynucleotides of any one of Aspects 189-195, wherein the hinge of the second CAR consists of SEQ ID NO:5. Aspect 201 is the one or more polynucleotides of any one of Aspects 189-195, wherein the hinge of the second CAR is a CD28 hinge. Aspect 202 is the one or more polynucleotides of any one of Aspects 189-195, wherein the hinge of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6. Aspect 203 is the one or more polynucleotides of any one of Aspects 189-195, wherein the hinge of the second CAR comprises SEQ ID NO:6. Aspect 204 is the one or more polynucleotides of any one of Aspects 189-195, wherein the hinge of the second CAR consists of SEQ ID NO:6.

[0095] Aspect 205 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR is a CD4 transmembrane domain. Aspect 206 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:7. Aspect 207 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR comprises SEQ ID NO:7. Aspect 208 is the one or more polynucleotides of any298507734.1 - 25 -one of Aspects 189-204, wherein the transmembrane domain of the second CAR consists of SEQ ID NO:7. Aspect 209 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR is a CD8 transmembrane domain. Aspect 210 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:8. Aspect 211 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR comprises SEQ ID NO:8. Aspect 212 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR consists of SEQ ID NO:8. Aspect 213 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR is a CD28 transmembrane domain. Aspect 214 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9. Aspect 215 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR comprises SEQ ID NO:9. Aspect 216 is the one or more polynucleotides of any one of Aspects 189-204, wherein the transmembrane domain of the second CAR consists of SEQ ID NO:9.

[0096] Aspect 217 is the one or more polynucleotides of any one of Aspects 189-216, wherein the 4-1BB co-stimulatory domain comprises an amino 218 sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12. Aspect 214 is the one or more polynucleotides of any one of Aspects 189-216, wherein the 4-1BB co-stimulatory domain comprises SEQ ID NO:12. Aspect 219 is the one or more polynucleotides of any one of Aspects 189-216, wherein the 4-1BB co-stimulatory domain consists of SEQ ID NO:12. Aspect 220 is the one or more polynucleotides of any one of Aspects 189-219, wherein the CD28 co- stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:13. Aspect 221 is the one or more polynucleotides of any one of Aspects 189-219, wherein the CD28 co-stimulatory domain of the second CAR comprises SEQ ID NO:13. Aspect 222 is the one or more polynucleotides of any one of Aspects 189-219, wherein the CD28 co-stimulatory domain of the second CAR consists of SEQ ID NO:13.

[0097] Aspect 223 is the one or more polynucleotides of any one of Aspects 189-222, wherein the CD3ζ stimulatory domain comprises an amino acid sequence having at least 80%,298507734.1 - 26 -90%, 95%, or 99% sequence identity to SEQ ID NO:14. Aspect 224 is the one or more polynucleotides of any one of Aspects 189-222, wherein the CD3ζ stimulatory domain comprises SEQ ID NO:14. Aspect 225 is the one or more polynucleotides of any one of Aspects 189-222, wherein the CD3ζ stimulatory domain consists of SEQ ID NO:14.

[0098] Aspect 226 is the one or more polynucleotides of any one of Aspects 189-225, wherein the second CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33. Aspect 227 is the one or more polynucleotides of any one of Aspects 189-225, wherein the second CAR comprises SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33. Aspect 228 is the one or more polynucleotides of any one of Aspects 189-225, wherein the second CAR consists of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:33.

[0099] Aspect 229 is a vector comprising the one or more polynucleotides of any one of claims 185-228. Aspect 230 is the vector of Aspect 229, wherein the vector is a viral vector. Aspect 231 is the vector of Aspect 230, wherein the viral vector is an adenoviral vector, adeno- associated viral vector, lentiviral vector, or retroviral vector. Aspect 232 is the vector of Aspect 229, wherein the vector is a non-viral vector. Aspect 233 is the vector of Aspect 232, wherein the non-viral vector is a plasmid. Aspect 234 is the vector of any one of Aspects 229-233, wherein the vector is multicistronic.

[0100] Aspect 235 is an immune cell comprising the one or more polynucleotides of any one of Aspects 185-228 or the vector of any one of Aspects 229-233. Aspect 236 is the immune cell of Aspect 235, wherein the immune cell is a T cell, natural killer (NK) cell, macrophage, microglial cell, or neutrophil. Aspect 237 is the immune cell of Aspect 235 or 236, wherein the immune cell is a T cell. Aspect 238 is the immune cell of Aspect 235 or 236, wherein the immune cell is a NK cell. Aspect 239 is the immune cell of any one of Aspects 235-238, wherein the immune cell is a human cell.

[0101] Aspect 240 is a population of the immune cells of any one of Aspects 235-239.

[0102] Aspect 241 is a composition comprising: (a) the polynucleotide of any one of Aspects 185-228, the vector of any one of Aspects 229-234, the immune cell of any of Aspects 235-239, or the population of immune cells of Aspect 240; and (b) a pharmaceutically acceptable excipient. Aspect 242 is the composition of Aspect 241, further comprising an additional therapeutic. Aspect 243 is the composition of Aspect 242, wherein the additional therapeutic is a chemotherapeutic or an immunotherapeutic.298507734.1 - 27 -

[0103] Aspect 244 is a method of killing cancer cells in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235- 239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240.

[0104] Aspect 245 is a method for treating a subject for cancer, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235- 239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240.

[0105] Aspect 246 is a method for stimulating an immune response to cancer cells in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144- 184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240.

[0106] Aspect 247 is the method of any one of Aspects 244-246, wherein the cancer is lymphoma, leukemia, multiple myeloma, glioblastoma, mesothelioma, head and neck cancer, osteosarcoma, melanoma, non-small cell lung cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer, germ cell tumors, or breast cancer. Aspect 248 is the method of any one of Aspects 244-247, wherein the cancer is a B-cell cancer. Aspect 249 is the method of any one of Aspects 244-248, wherein the cancer is a CD19-positive, CD22-positive, BCMA-positive cancer, HER2-positive cancer, or a mesothelin-positive cancer. Aspect 250 is the method of any one of Aspects 244-249, further comprising administering to the subject an additional therapy. Aspect 251 is the method of Aspect 250, wherein the additional therapy is radiotherapy, chemotherapy, or immunotherapy.

[0107] Aspect 252 is a method of generating a persisting population of genetically engineered immune cells in a subject, the method comprising administering to the subject an effective amount of the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144- 184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240. Aspect 253 is the method298507734.1 - 28 -of Aspect 252, wherein the genetically engineered immune cells persist in the subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days, weeks, months, or years after administration.

[0108] Aspect 254 is a method of expanding a population of genetically engineered immune cells in a subject, the method comprising administering to the subject an effective amount of the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229- 234; or the population of immune cells of Aspect 240.

[0109] Aspect 255 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144- 184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first CAR and / or the second CAR further comprises a signal peptide. Aspect 256 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first CAR and / or the second CAR further comprises a signal peptide comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:30. Aspect 257 is the composition of any one of Aspects 1-72, 141- 143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first CAR and / or the second CAR further comprises a signal peptide comprising SEQ ID NO:30. Aspect 258 is the composition of any one of Aspects 1-72, 141-143, or 241- 243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first CAR and / or the second CAR further comprises a signal peptide consisting of SEQ ID NO:30.

[0110] Aspect 259 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144- 184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first CAR and / or the second CAR further comprises an epitope tag. Aspect 260 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or298507734.1 - 29 -235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first CAR and / or the second CAR further comprises an epitope tag comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:31 or SEQ ID NO:32. Aspect 261 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first CAR and / or the second CAR further comprises an epitope tag comprising SEQ ID NO:31 or SEQ ID NO:32. Aspect 262 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first CAR and / or the second CAR further comprises an epitope tag consisting of SEQ ID NO:31 or SEQ ID NO:32.

[0111] Aspect 263 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144- 184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a 2A sequence. Aspect 264 is the composition of any one of Aspects 1- 72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185- 228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence. Aspect 265 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:29. Aspect 266 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR298507734.1 - 30 -of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185- 228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence comprising SEQ ID NO:29. Aspect 267 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144- 184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence consisting of SEQ ID NO:29.

[0112] Aspect 268 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144- 184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first and the second CAR together comprise an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:35. Aspect 269 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185- 228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first and the second CAR together comprise SEQ ID NO:35. Aspect 270 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first and the second CAR together consist of SEQ ID NO:35. Aspect 271 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:35. Aspect 272 is the composition of any one of Aspects 1- 72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185- 228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect298507734.1 - 31 -240, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence comprising SEQ ID NO:35. Aspect 273 is the composition of any one of Aspects 1-72, 141-143, or 241-243; the immune cell of any one of Aspects 74-140 or 235-239; the CAR of any one of Aspects 144-184; the one or more polynucleotides of any one of Aspects 185-228; the vector of any one of Aspects 229-234; or the population of immune cells of Aspect 240, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence consisting of SEQ ID NO:35.

[0113] The references to the methods of treatment by therapy or surgery or in vivo diagnosis methods in Examples of this description and in the claims and disclosure of this description are to be interpreted as references to compounds, pharmaceutical compositions and medicaments of the present invention for use in those methods.

[0114] It is specifically contemplated that any limitation discussed with respect to one aspect of the invention may apply to any other aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of the disclosure set forth in the Examples are also aspects that may be implemented in the context of and / or as discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, Drawings, and description of the Drawings.

[0115] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0117] FIGS. 1A-1E show CAR-CoCAR expressing cells display increased in vitro sensitivity and functionality to CD22+ cells. FIG. 1A. Schematics showing components of298507734.1 - 32 -both CAR and CoCAR molecules. FIG.1B. Representative flow cytometry plots of primary T cells transduced with CAR or both CAR and CoCAR. Untransduced cells were used as a gating control. Summary graphs of FIG. 1C, intracellular IL-2, TNFα, and IFNγ after a 6-hour coculture with Nalm6 cells and FIG.1D, percent of triple-positive, polyfunctional CAR-T cells within the entire T cell population. FIG.1E. Intracellular cytokine staining after coculture with Nalm6 expressing different CD22 surface levels.

[0118] FIGS. 2A-2E. FIG. 2A. Staining of CAR or CoCAR expressing primary T cells with AF647-conjugated CD22-Fc. FIG. 2B. Representative flow cytometry plots of intracellular IL-2, TNFα, and IFNγ after a 6-hour coculture with Nalm6 cells. FIG. 2C, Summary graphs of intracellular IL-2, TNFα, and IFNγ after a 6-hour coculture with Nalm6 cells and FIG.2D, percent of triple-positive, polyfunctional CAR-T cells within the entire T cell population from two alternative PBMC donors. FIG.2E. Flow histograms of CD22 from Nalm6 cells expressing different levels of surface antigen and antigen-binding capacity quantified by AF647 calibration beads.

[0119] FIGS.3A-3E show CoCAR-bound LCK acts to supplement CAR function. FIG. 3A. Summary graphs of intracellular IL-2, TNFα, and IFNγ of cells transduced with CAR, CoCAR, or both CAR and CoCAR. FIG.3B. Schematic of CoCAR variants (AXAP and Δ8a) to assess the importance of CoCAR-bound signaling molecules. FIG.3C. Representative flow cytometry plots of primary T cells transduced with CAR or both CAR and CoCAR (or CoCAR variant). Untransduced cells were used as a gating control. Summary graphs of FIG. 3D, intracellular IL-2, TNFα, and IFNγ after a 6-hour coculture with Nalm6 cells and FIG. 3E, percent of triple-positive, polyfunctional CAR-T cells within the entire T cell population.

[0120] FIGS. 4A-4C. FIG. 4A, IFNγ measured from the supernatant and FIG. 4B, representative flow cytometry plots of intracellular IFNγ after coculture with Nalm6 cells (+) or CAR-T cells cultured in the absence of target cells. FIG.4C, Representative flow cytometry plots intracellular IL-2, TNFα, and IFNγ after a 6-hour coculture with Nalm6 cells, gated into CD4+ or CD8+ T cells.

[0121] FIGS.5A-5B show increased control of tumor in vivo by CAR-CoCAR expressing cells. FIG. 5A. Schematic showing mouse injection strategy. FIG. 5B. First experiment showing survival of mice in different experimental groups over time.

[0122] FIGS.6A-6D show redirection of the CoCAR system to target alternative antigens. FIG. 6A. Schematics showing components of both BCMA-CAR and BCMA-CoCAR molecules. FIG.6B. Representative flow cytometry plots of primary T cells transduced with CAR or both CAR and CoCAR. Untransduced cells were used as a gating control. Summary298507734.1 - 33 -graphs of FIG.6C, intracellular IL-2 and TNFα after a 6-hour coculture with MM.1s cells and FIG. 6D, percent of double-positive, polyfunctional CAR-T cells within the entire T cell population.

[0123] FIGS. 7A-7E. Epitope tags allow effective detection of bicistronically-expressed CAR and CoCAR molecules. FIG.7A. Schematic illustrations depicting components found in CAR and CAR-CoCAR lentiviral constructs. FIG.7B. Expression of CAR and CAR-CoCAR constructs in primary human T cells. FIG.7C. Cytotoxicity of bicistronically-expressed CAR and CoCAR molecules. FIG. 7D. Survival of mice in different experimental groups over treatment time course illustrated in FIG.5A. FIG.7E. In vivo bioluminescent imaging of mice tracking tumor size / intensity over the treatment time course illustrated in FIG.5A.

[0124] FIG.8. Confocal images showing the co-localization of CARs and CoCARs in the immune synapse. DETAILED DESCRIPTION

[0125] The present disclosure relates to compositions including a novel system including co-expressed CAR molecules to induce CAR signaling that more closely mimics TCR signaling and to improve CAR efficacy in cases of downregulation of target antigen, for example, upon relapse in a cancer patient. The compositions can be useful in methods for treating a subject for cancer, killing cancer cells in a subject, stimulating an immune response to cancer cells in a subject, and generating a persisting population of genetically engineered immune cells. The compositions provided herein can provide greater beneficial effects for treating cancer as compared to other commercially or clinically available CAR therapies. For example, the co-expressed CAR molecule system increases antigen sensitivity and / or improves CAR avidity to stabilize CAR / antigen binding while preserving endogenous TCR function, avoiding interference with assembly of CD3 complex components, recruiting only LCK in the presence of antigen, or a combination thereof. The co-expressed CAR molecule system also showed superior antitumor functionality of the compared to CAR alone, due in part to LCK binding. Furthermore, the system can be redirected to target a wide range of antigens. Thus, the methods and compositions described in this disclosure, including the methods and compositions directed to co-expressed CAR molecules to induce CAR signaling that more closely mimics TCR signaling and to improve CAR efficacy in cases of downregulation of target antigen, may provide more favorable conditions to support the cancer treatment in comparison to existing methods and compositions.298507734.1 - 34 -I. Examples of Definitions

[0126] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” As used herein “another” may mean at least a second or more.

[0127] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an aspect.

[0128] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0129] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of” excludes any element, step, or ingredient not specified. The phrase “consisting essentially of” limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of” or “consisting essentially of.”

[0130] The terms “optional” or “optionally” as used herein mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0131] The terms “about” or “approximately” as used herein are used according to their plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In one non-limiting aspect the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5% of an associated disclosed value. The terms may be removed from the associated disclosed value and the exact value may be used instead.298507734.1 - 35 -

[0132] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated aspect that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these aspects are explicitly disclosed.

[0133] When the lower limit value of a given percentage range does not include the % symbol and / or the percentage type (e.g., sequence identity), then the percentage type for the lower limit value is the same as for the upper limit value of the given percentage range. For example, the percentage range of “80 to 90% sequence identity” means “80% sequence identity to 90% sequence identity.”

[0134] For purposes of this application, a number value with one or more decimal places can be rounded to the nearest whole number using standard rounding guidelines, i.e., round up if the number being rounded is 5, 6, 7, 8, or 9; and round down if the number being rounded is 0, 1, 2, 3, or 4. For example, 0.42 can be rounded to 0.4.

[0135] The term “amino acid sequence” refers to a list of abbreviations, letters, characters, or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0136] The term “antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be298507734.1 - 36 -monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class from any species, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In exemplary aspects, antibodies used with the methods and compositions described herein are derivatives of the IgG class. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen. In some aspects, antibodies or fragments thereof may be substituted with an antigen-binding moiety described in Hanssens et al., Med Res Rev. (2022) 42 (1): 306–342 or Liu et al., Front Bioeng Biotechnol. 2022; 10: 928169, both of which are incorporated by reference herein in their entirety. For example, the antibody or fragment thereof antigen-targeting moiety may be replaced by a peptide ligand that interacts with tumor-overexpressed receptors (e.g., NKG2D receptors, IL-13-cytokine receptors, integrin v6-binding receptors, heregulin-chimaera receptors) or a receptor or ligand portion of a naturally associating receptor‐ligand pair.

[0137] The term “antibody fragment” refers to any derivative of an antibody which is less than full-length. In exemplary aspects, the antibody fragment retains at least a significant portion of the full-length antibody’s specific binding ability.

[0138] Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, scFv, Fv, dsFv diabody, Fc, Fd fragments, and nanobodies. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.

[0139] The term “antigen” refers to any substance that causes an immune system to produce antibodies against it, or to which a T cell responds. In some aspects, an antigen is a peptide that is 5-50 amino acids in length or is, is at least, or is at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, or 300 amino acids, or any derivable range therein. The term “antigen-binding site” refers to a region of an antibody that specifically binds an epitope on an antigen.

[0140] The term “cell” is herein used in its broadest sense in the art and refers to a living body which is a structural unit of tissue of a multicellular organism, is surrounded by a298507734.1 - 37 -membrane structure which isolates it from the outside, has the capability of self-replicating, and has genetic information and a mechanism for expressing it. Cells used herein may be naturally-occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.).

[0141] The term “chimeric molecule” refers to a single molecule created by joining two or more molecules that exist separately in their native state. The single, chimeric molecule has the desired functionality of all of its constituent molecules. One type of chimeric molecule is a fusion protein. Another type of chimeric molecule is a chimeric antigen receptor (CAR).

[0142] The term “engineered antibody” refers to a recombinant molecule that comprises at least an antibody fragment comprising an antigen-binding site derived from the variable domain of the heavy chain and / or light chain of an antibody and may optionally comprise the entire or part of the variable and / or constant domains of an antibody from any of the Ig classes (for example IgA, IgD, IgE, IgG, IgM and IgY). As a nonlimiting example, nanobodies are one class of engineered antibodies contemplated to be useful according to the compositions and methods disclosed herein.

[0143] The term “epitope” refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody binds preferentially to a single specific epitope of a molecule that can be molecularly defined. In the present disclosure, multiple epitopes can be recognized by a multispecific antibody.

[0144] The term “exogenous,” when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial means, or in relation a cell refers to a cell which was isolated and subsequently introduced to other cells or to an organism by artificial means. An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell. An exogenous cell may be from a different organism, or it may be from the same organism. By way of a non-limiting example, an exogenous nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.

[0145] The term “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein.298507734.1 - 38 -A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

[0146] The term “Fab fragment” refers to a fragment of an antibody comprising an antigen- binding site generated by cleavage of the antibody with the enzyme papain, which cuts at the hinge region N-terminally to the inter-H-chain disulfide bond and generates two Fab fragments from one antibody molecule.

[0147] The term “F(ab’)2 fragment” refers to a fragment of an antibody containing two antigen-binding sites, generated by cleavage of the antibody molecule with the enzyme pepsin which cuts at the hinge region C-terminally to the inter-H-chain disulfide bond.

[0148] The term “fragment” refers to the fragment of an antibody comprising the constant domain of its heavy chain. The term ”Fv fragment” refers to the fragment of an antibody comprising the variable domains of its heavy chain and light chain.

[0149] A “gene,” “polynucleotide,” “coding region,” “sequence,” “segment,” “fragment,” or “transgene” which “encodes” a particular protein, is a nucleic acid molecule which is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences. The coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3’ to the gene sequence.

[0150] “Gene construct” refers to a nucleic acid, such as a vector, plasmid, viral genome or the like which includes a “coding sequence” for a polypeptide or which is otherwise transcribable to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), may be transfected into cells, e.g. in certain aspects mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct. The gene construct may include one or more regulatory elements operably linked to the coding sequence, as well as intronic sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0151] The term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which298507734.1 - 39 -may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.

[0152] As used herein, “isolated” for example, with respect to cells and / or nucleic acids means altered or removed from the natural state through human intervention.

[0153] The term “linker” is art-recognized and refers to a molecule or group of molecules connecting two compounds, such as two polypeptides. The linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a compound by a specific distance.

[0154] The terms “nanobody” or “variable domain of the heavy chain of heavy chain antibodies” (“VHH”) refer to an antibody molecule that retains the antigen binding region of the heavy chain domain of a conventional antibody, but does not have the other light variable chain domains. Single domain nanobodies or VHH antibodies may have a small molecular weight (i.e., about 15 kDa). One or more nanobodies or VHH antibodies may be linked to other antibody fragments (such as one or more other nanobodies or VHH antibodies) to form antibody constructs having one or more antigen recognition sites.

[0155] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0156] By “operably linked” with reference to nucleic acid molecules is meant that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter, and298507734.1 - 40 -an enhancer element) are connected in such a way as to permit transcription of the nucleic acid molecule. “Operably linked” with reference to peptide and / or polypeptide molecules is meant that two or more peptide and / or polypeptide molecules are connected in such a way as to yield a single polypeptide chain, i.e., a fusion polypeptide, having at least one property of each peptide and / or polypeptide component of the fusion. The fusion polypeptide is particularly chimeric, i.e., composed of heterologous molecules.

[0157] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0158] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0159] The terms “polypeptide fragment” or “fragment”, when used in reference to a particular polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to that of the reference polypeptide. Such deletions may occur at the amino- terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least about 5, 6, 8 or 10 amino acids long, at least about 14 amino acids long, at least about 20, 30, 40 or 50 amino acids long, at least about 75 amino acids long, or at least about 100, 150, 200, 300, 500 or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. In various aspects, a fragment may comprise an enzymatic activity and / or an interaction site of the reference polypeptide. In another aspect, a fragment may have immunogenic properties.

[0160] The term “protein domain” refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.

[0161] The term “single chain variable fragment” or “scFv” refers to an Fv fragment in which the heavy chain domain and the light chain domain are linked. One or more scFv fragments may be linked to other antibody fragments (such as the constant domain of a heavy chain or a light chain) to form antibody constructs having one or more antigen recognition sites.

[0162] A “spacer” as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally, a spacer has no specific biological activity other than to join the298507734.1 - 41 -proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.

[0163] The term “specifically binds”, as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologies. Thus, under designated conditions (e.g., immunoassay conditions in the case of an antibody), a specified ligand or antibody “specifically binds” to its particular “target” (e.g., an antibody specifically binds to a tumor cell antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 105M_1(e.g., 106M-\ 107M-\ 108M-\ 109M-\ 101° M_1, 1011M_1, and 1012M_1or more) with that second molecule.

[0164] The terms “subject” and “individual” are used interchangeably here and refer to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician, and the term “patient” may be used interchangeably with the term “subject”; treatment may or may not include correspondence through the internet. The subject may be of any gender, age, or race.

[0165] As used herein, the term “therapeutically effective amount” is synonymous with “effective amount”, “therapeutically effective dose”, and / or “effective dose” refers to an amount of an agent sufficient to produce a desired result or exert a desired influence on the particular condition being treated. In some aspects, a therapeutically effective amount is an amount sufficient to inhibit or ameliorate at least one symptom, behavior, or event, associated with a pathological, abnormal, or otherwise undesirable condition, or an amount sufficient to prevent or lessen the probability that such a condition will occur or re-occur, or an amount sufficient to delay worsening of such a condition. Effective amount can also mean the amount of a compound, material, or composition comprising a compound of the present disclosure that is effective for producing some desired effect, e.g., treating or preventing cancer. The effective amount may vary depending on the organism or individual treated.

[0166] The appropriate effective amount to be administered for a particular application of the disclosed methods can be determined by those skilled in the art, using the guidance provided herein. For example, an effective amount can be determined experimentally using various298507734.1 - 42 -techniques and / or extrapolated from in vitro and in vivo assays including dose escalation studies. Various concentrations of an agent may be used in preparing compositions incorporating the agent to provide for variations in the age of the patient to be treated, the severity of the condition, and / or the duration of the treatment and the mode of administration. One skilled in the art will recognize that the condition of the individual can be monitored throughout the course of therapy and that the effective amount of a compound or composition disclosed herein that is administered can be adjusted accordingly. Further, one of skill in the art recognizes that an amount may be considered effective even if the medical condition is not totally eradicated but improved partially. For example, the medical condition may be halted or reduced or its onset delayed, a side effect from the medical condition may be inhibited or partially reduced or completed eliminated, and so forth.

[0167] The terms “transformation” and “transfection” and “transduction” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said cell.

[0168] As used herein, the terms “treatment,” “treat,” or “treating” refers to intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of pathology of a disease or condition, such as for example B cell and solid tumor cancers. Treatment may serve to accomplish one or more of various desired outcomes, including, for example, preventing occurrence or recurrence of disease, alleviation of symptoms, and diminishment of any direct or indirect pathological consequences of the disease, preventing disease spread, lowering the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis, and / or producing some desired effect.

[0169] The term “variant” refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e., a degenerate variant), substitutions within the wobble position of each codon (i.e., DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0170] A “vector” or “construct” (sometimes referred to as gene delivery or gene transfer “vehicle”) refers to a macromolecule, complex of molecules, or viral particle, comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. The polynucleotide can be a linear or a circular molecule. A “plasmid”, a common type of a vector, is an extra- chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In certain cases, it is circular and double-298507734.1 - 43 -stranded. By “expression construct” or “expression cassette” or “expression vector” is meant a nucleic acid molecule that is capable of directing transcription. An expression construct includes, at the least, a promoter or a structure functionally equivalent to a promoter. Additional elements, such as an enhancer, and / or a transcription termination signal, may also be included. II. Chimeric Antigen Receptors

[0171] Disclosed herein are methods and compositions which utilize chimeric antigen receptors (CARs) encoded by polypeptides and polynucleotides, including CARs comprising an antigen-binding domain that bind to one or more epitopes or binding regions of cancer- associated antigens. These CARs can induce CAR signaling that more closely mimics TCR signaling and can improve CAR efficacy in cases of downregulation of target antigen.

[0172] Thus, some aspects comprise methods and compositions for cell-based immunotherapies that increase in vitro functionality of CARs by mimicking TCR signaling and when target antigen is downregulated. For example, a novel, chimeric CD4, CD8, or CD28 coreceptor (mimicking CD8 or CD4) to enhance signaling and a stimulatory 41BB or CD28 and CD3ζ CAR (mimicking TCR) can interact with each other through the ITAM domain of CD3ζ and chimeric CD4- or CD8-coreceptor-bound LCK. This can increase in vitro functionality of CARs by, for example, increasing levels of TNF-α and IFNγ. These co- expressed CARs can increase antigen sensitivity while preserving endogenous TCR function, avoiding interference with assembly of CD3 complex components, recruiting only LCK in the presence of antigen, or a combination thereof. Together, this activity can increase CAR sensitivity and / or improve CAR avidity to stabilize CAR / antigen binding to improve patient outcomes by reducing occurrences of relapse due to low antigen expression. In some aspects, the system employing the chimeric-coreceptor approach is useful for treating B cell and solid tumor cancers by targeting multiple antigens expressed by the cancer cells.

[0173] Immune cells, such as T cells and Natural Killer (NK) cells, may be engineered to express these CARs. Therefore, also disclosed are methods for providing an immunotherapy in a subject using the disclosed immune cells. In some aspects, expression of CARs by immune cells increases the likelihood of immune cell activation at tumor sites, thereby increasing specificity and safety of the composition. In some aspects, the immune cell promotes an immune response against a cancer when the antibody of a CAR binds a tumor antigen or a binding region thereof on a target cell.

[0174] The term “chimeric antigen receptor” or “CAR” refers to engineered receptors, which graft an arbitrary specificity onto an immune cell. For example, these receptors can be298507734.1 - 44 -used to graft the specificity of an antibody or fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen onto a NK cell, a T cell, or other immune cell, with transfer of the coding sequence facilitated by a vector (e.g., a retroviral or lentiviral vector). The receptors are called chimeric because they are composed of parts from different sources. Such molecules result in the transmission of a signal in response to recognition by the antibody or fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen of its target. To target malignant B cells, for example, T cell specificity can be redirected using a chimeric immunoreceptor specific for the B-lineage molecule, CD19.

[0175] The variable portions of an immunoglobulin heavy and light chain are fused by a flexible linker to form a scFv. The single domain variable region of the heavy chain makes up a nanobody molecule (VHH). These nanobodies and scFvs can be preceded by a signal peptide, e.g., a GM-CSF signal peptide, to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (this is cleaved). Additionally, or alternatively, these nanobodies and scFvs can be preceded or followed by an epitope tag, e.g., a FLAG epitope tag, a Myc epitope tag, a His-tag, an HA epitope tag, a V5 epitope tag, etc. Any one or more of the foregoing tags may be excluded from the constructs disclosed herein. A flexible spacer allows the nanobodies and scFvs to orient in different directions to enable antigen-binding. The transmembrane domain can be a typical hydrophobic alpha helix usually derived from the original molecule of the signaling endodomain which protrudes into the cell and transmits the desired signal.

[0176] In certain aspects, the CAR comprises at least one extracellular and at least one intracellular domain. An extracellular domain can comprise a target-specific binding element, otherwise referred to as an antigen-binding moiety, that specifically binds to any particular antigen of interest. In specific aspects, the extracellular domain comprises an antigen-binding moiety that binds one or more epitopes of a tumor or cancer cell antigen or a fragment thereof. In various aspects, the target-specific binding element comprises an anti-CD22, CD19, BCMA, HER2, or mesothelin antibody or a fragment thereof, an anti-CD22, CD19, BCMA, HER2, or mesothelin nanobody, or an anti-CD22, CD19, BCMA, HER2 scFv, or mesothelin. In various aspects, the target-specific binding element does not comprise an anti-CD22, CD19, BCMA, HER2, or mesothelin antibody or a fragment thereof, an anti-CD22, CD19, BCMA, HER2, or mesothelin nanobody, or an anti-CD22, CD19, BCMA, HER2 scFv, or mesothelin.298507734.1 - 45 -

[0177] In various aspects the intracellular domain or otherwise the cytoplasmic domain comprises, one or more costimulatory signaling region(s), and in various aspects, a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. In various aspects, costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of immune cells to antigen.

[0178] Between the extracellular domain and the transmembrane domain of the CAR, and / or between the cytoplasmic domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. As used herein, the term “spacer domain” generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain. In various aspects the spacer domain may comprise up to 300 amino acids, or in various aspects about 10 to about 100 amino acids, and in certain aspects about 25 to about 50 amino acids.

[0179] In various aspects a DNA construct comprising sequences of the one or more CARs disclosed herein are provided. In some aspects, the one or more CARs are comprised in or encoded by the same construct, or isolated nucleic acid, while in others, the one or more CARs are comprised in separate constructs or isolated nucleic acids. In some aspects, the one or more CARs comprise at least two CARs and the at least two CARs are encoded by the same construct, or isolated nucleic acid, and are bicistronically expressed. In some aspects, a polynucleotide (e.g., an mRNA) encoding the CARs contains two open reading frames (ORFs), each with its own start and stop codon, allowing for the translation of two separate proteins. In some aspects, an IRES element separates the two ORFs, which can allow ribosomes to initiate translation at the second ORF independently of the first. Exemplary IRES sequences that may be used in the constructs disclosed herein may be derived from, e.g., EMCV, CrPV, HCV, Poliovirus, FMDV, c-Myc, VEGF, IGF1R, etc. Any one or more of the foregoing IRES sequences may be excluded from a construct disclosed herein. In some aspects, a 2A sequence separates the two ORFs. Exemplary 2A sequences that may be used in the constructs disclosed herein include but are not limited to P2A, T2A, E2A, F2A, etc. Any one or more of the foregoing 2A sequences may be excluded from a construct disclosed herein. Unlike IRES elements, which rely on ribosome re-initiation, 2A sequences cause a “ribosomal skip” during translation, resulting in the production of separate proteins. In some aspects, the CARs comprising an antigen-binding domain that binds to one or more cancer or tumor cell- associated antigens, epitopes, or binding regions thereof can comprise an exemplary P2A having, having at least, having at most, or having between any two of (inclusive of end values)298507734.1 - 46 -75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:29:GSGATNFSLLKQAGDVEENPGP.

[0180] In some aspects, the CAR comprises one or more antigen-binding domains that bind to one or more cancer-associated antigens, epitopes, or binding regions thereof comprise an anti-cancer or tumor cell antigen antibody or fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen, or other antigen-binding domain that binds to the cancer or tumor cell antigens, epitopes, or binding regions thereof, one or more signal peptides, one or more epitope tags, one or more hinges or spacers, a transmembrane domain, one or more cytoplasmic signaling domains, and optionally, one or more detection domains and / or molecules. In some aspects, a CAR comprising an antigen-binding domain that binds to one or more cancer or tumor cell-associated antigens, epitopes, or binding regions thereof comprises an antigen-binding domain that binds a first epitope of the antigen, a hinge from CD4, CD8, or CD28, a transmembrane domain from CD4 or CD8, an intracellular domain from CD4 or CD8, or a combination of the foregoing. In some aspects, a CAR comprising an antigen-binding domain that binds to one or more cancer or tumor cell-associated antigens, epitopes, or binding regions thereof comprises an antigen-binding domain that binds a second epitope of the antigen, a hinge from CD4, CD8, or CD28, a transmembrane domain from CD4 or CD8, a 4- 1BB or CD28 co-stimulatory domain, a CD3ζ stimulatory domain, or a combination of the foregoing. In some aspects, a CAR comprising an antigen-binding domain that binds to one or more cancer or tumor cell-associated antigens, epitopes, or binding regions thereof comprises an antigen-binding domain that binds a first epitope of the antigen, does not comprise a hinge from any one or more of CD4, CD8, or CD28, does not comprise a transmembrane domain from any one or more of CD4 or CD8, does not comprise an intracellular domain from any one or more of CD4 or CD8, or any combination of the foregoing. In some aspects, a CAR comprising an antigen-binding domain that binds to one or more cancer or tumor cell- associated antigens, epitopes, or binding regions thereof comprises an antigen-binding domain that binds a second epitope of the antigen, does not comprise a hinge from any one or more of CD4, CD8, or CD28, does not comprise a transmembrane domain from any one or more of CD4 or CD8, does not comprise a co-stimulatory domain from one or more of 4-1BB or CD28 co-stimulatory domain, does not comprise a CD3ζ stimulatory domain, or any combination of the foregoing.298507734.1 - 47 -

[0181] In some aspects, the CARs comprising an antigen-binding domain that binds to one or more cancer or tumor cell-associated antigens, epitopes, or binding regions thereof comprise one or more of the following sequences:

[0182] An exemplary signal peptide sequence from GM-CSF having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:30: MLLLVTSLLLCELPHPAFLLIP (UniProt Accession No. P15509), or another functionally equivalent art-recognized signal peptide sequence that ensures the protein is properly localized.

[0183] Exemplary epitope tag sequences having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to the following SEQ ID NOs:31 and 32, or another functionally equivalent art-recognized epitope tag sequence that provides for detection, purification, and / or localization of the protein:

[0184] FLAG epitope tag:DYKDDDDK (SEQ ID NO:31)

[0185] Myc epitope tag:EQKLISEEDL (SEQ ID NO:32)

[0186] Exemplary antigen-binding domain sequences having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to the following SEQ ID NOs:1-4:

[0187] M971, also known as CD22 scFv 1: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYND YAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSS GGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSG VPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO:1), as described in U.S. Pat. App. Ser. No. 18 / 503,107, incorporated by reference herein in its entirety, or another functionally equivalent CD22-binding nucleotide and / or amino acid sequence of a CD22-binding domain (e.g., an antibody or antigen-binding fragment thereof, a nanobody, or an scFv) that binds to cancer or tumor cell-associated CD22 antigens, epitopes, or fragments thereof.

[0188] HA22, also known as CD22 scFv 2: EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKRLEWVAYISSGGGTYYPDT VKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTLVTVSAGG GGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIY YTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGGGTKLEIK (SEQ298507734.1 - 48 -ID NO:2), as described in Haso et al., Blood (2013) 121 (7): 1165–1174, and Ho et al., JBC (2005) 280 (1): 607–671, both of which are incorporated by reference herein in their entirety, or another functionally equivalent CD22-binding nucleotide and / or amino acid sequence of a CD22-binding domain (e.g., an antibody or antigen-binding fragment thereof, a nanobody, or an scFv) that binds to cancer or tumor cell-associated CD22 antigens, epitopes, or fragments thereof.

[0189] Cilta VHH1, also known as BCMA VHH 1: QVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYAD SVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSS (SEQ ID NO:3), as described in U.S. Pat. App. Ser. No.17 / 540,736, incorporated by reference herein in its entirety, or another functionally equivalent BCMA-binding nucleotide and / or amino acid sequence of a BCMA-binding domain (e.g., an antibody or antigen-binding fragment thereof, a nanobody, or an scFv) that binds to cancer or tumor cell-associated BCMA antigens, epitopes, or fragments thereof.

[0190] Cilta VHH2, also known as BCMA VHH 2: EVQLVESGGGLVQAGGSLRLSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAYYAESVK GRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMSIRPDYWGQGTQVTVSS (SEQ ID NO:4), as described in U.S. Pat. App. Ser. No.17 / 540,736, incorporated by reference herein in its entirety, or another functionally equivalent BCMA-binding nucleotide and / or amino acid sequence of a BCMA-binding domain (e.g., an antibody or antigen-binding fragment thereof, a nanobody, or an scFv) that binds to cancer or tumor cell-associated BCMA antigens, epitopes, or fragments thereof.

[0191] FRP5, also known as HER2 scFv 1: QVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFAD DFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGS GGGGSGGGGSDIQLTQSHKFLSTSVGDRVSITCKASQDVYNAVAWYQQKPGQSPKLLIYSAS SRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIK (SEQ ID NO:27), as described in Wels et al., Cancer Res. (1992) 52 (22): 6310-7, incorporated by reference herein in its entirety, or another functionally equivalent HER2-binding nucleotide and / or amino acid sequence of a HER2-binding domain (e.g., an antibody or antigen-binding fragment thereof, a nanobody, or an scFv) that binds to cancer or tumor cell-associated HER2 antigens, epitopes, or fragments thereof.

[0192] 4D5, also known as HER2 scFv 2:298507734.1 - 49 -DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEV QLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSV KGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS (SEQ ID NO:28), as described in Carter et al., Proc Natl Acad Sci U S A. (1992) 89 (10): 4285–4289, and Zhao et al., J Immunol. (2009) 183 (9): 5563–5574, both of which are incorporated by reference herein in their entirety, or another functionally equivalent HER2-binding nucleotide and / or amino acid sequence of a HER2-binding domain (e.g., an antibody or antigen-binding fragment thereof, a nanobody, or an scFv) that binds to cancer or tumor cell-associated HER2 antigens, epitopes, or fragments thereof.

[0193] CD4 hinge sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to one or more art-recognized CD4 hinge sequences that provide sufficient structural flexibility and spacing to flanking polypeptide regions.

[0194] CD8 hinge sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:5: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD, as described by UniProt Accession No. P01732, incorporated by reference herein in its entirety, or another functionally equivalent art-recognized CD8 hinge sequence that provides sufficient structural flexibility and spacing to flanking polypeptide regions.

[0195] CD28 hinge sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:6: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP, as described by UniProt Accession No. P10747, incorporated by reference herein in its entirety, or another functionally equivalent art-recognized CD28 hinge sequence that provides sufficient structural flexibility and spacing to flanking polypeptide regions.

[0196] CD4 transmembrane sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:7:MALIVLGGVAGLLLFIGLGIFF, as described by UniProt Accession No. P01730, incorporated by reference herein in its entirety, or another298507734.1 - 50 -functionally equivalent art-recognized CD4 transmembrane sequence that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell.

[0197] CD8 transmembrane sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:8: IYIWAPLAGTCGVLLLSLVITLYC, as described by UniProt Accession No. P01732, incorporated by reference herein in its entirety, or another functionally equivalent art-recognized CD8 transmembrane sequence that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell.

[0198] CD28 transmembrane sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:9: FWVLVVVGGVLACYSLLVTVAFIIFWV, as described by UniProt Accession No. P10747, incorporated by reference herein in its entirety, or another functionally equivalent art-recognized CD28 transmembrane sequence that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell.

[0199] CD4 intracellular sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:10: CVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI, as described by UniProt Accession No. P01730, incorporated by reference herein in its entirety, or another functionally equivalent art-recognized CD4 intracellular sequence that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen-binding domain.

[0200] CD8 intracellular sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:11: NHRNRRRVCKCPRPVVKSGDKPSLSARYV, as described by UniProt Accession No. P01732, incorporated by reference herein in its entirety, or another functionally equivalent art-recognized CD8 intracellular sequence that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of298507734.1 - 51 -cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen-binding domain.

[0201] 4-1BB co-stimulatory domain sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:12: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, as described by UniProt Accession No. Q07011, incorporated by reference herein in its entirety, or another functionally equivalent art-recognized co-stimulatory domain sequence that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen-binding domain. CD28 co-stimulatory domain sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:13: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS, or another functionally equivalent art-recognized co-stimulatory domain sequence that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen-binding domain.

[0202] CD3ζ stimulatory domain sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:14: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, or another functionally equivalent art-recognized stimulatory domain sequence that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular298507734.1 - 52 -signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen-binding domain.

[0203] In some aspects, a CAR comprising an antigen-binding domain that binds to one or more cancer or tumor cell-associated antigens, epitopes, or binding regions thereof, including the novel, chimeric CD4, CD8, or CD28 coreceptors disclosed herein to enhance signaling, comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NOs:15-19.

[0204] GM-CSF Signal Peptide, a CD22-binding (HA22) domain with CD8 hinge, transmembrane, and intracellular components: MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQT PEKRLEWVAYISSGGGTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYG THWGVLFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQ DISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQ QGNTLPWTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO:15).

[0205] GM-CSF Signal Peptide, a CD22-binding (HA22) domain with CD8 hinge, CD4 transmembrane, and CD4 intracellular components: MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQT PEKRLEWVAYISSGGGTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYG THWGVLFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQ DISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQ QGNTLPWTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKT CSPI (SEQ ID NO:16).

[0206] GM-CSF Signal Peptide, a CD22-binding domain (HA22) with CD28 hinge, CD28 transmembrane domain, and CD4 intracellular components: MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQT PEKRLEWVAYISSGGGTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYG THWGVLFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQ DISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQ QGNTLPWTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW298507734.1 - 53 -VLVVVGGVLACYSLLVTVAFIIFWVCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTC SPI (SEQ ID NO:17).

[0207] GM-CSF Signal Peptide, a BCMA-binding domain (Cilta VHH1) with CD8a hinge, transmembrane, and intracellular components: MLLLVTSLLLCELPHPAFLLIPQVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQA PGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRID AADFDSWGQGTQVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO:18).

[0208] GM-CSF Signal Peptide, a BCMA-binding domain (Cilta VHH2) with CD8 hinge, transmembrane, and intracellular components: MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQAGGSLRLSCAASGRTFTMGWFRQAPGK EREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMS IRPDYWGQGTQVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO:19).

[0209] In some aspects, a CAR comprising an antigen-binding domain comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NOs:18 and 19:

[0210] GM-CSF Signal Peptide, a CD22-binding domain (m971) with CD8a hinge, CD8a transmembrane, 4-1BB costimulatory, and CD3z stimulatory components: MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIR QSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARE VTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNW YQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQT FGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:20).

[0211] GM-CSF Signal Peptide, a BCMA-binding domain (Cilta VHH1) with CD8a hinge, CD8a transmembrane, 4-1BB costimulatory, and CD3z stimulatory components: MLLLVTSLLLCELPHPAFLLIPQVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQA PGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRID298507734.1 - 54 -AADFDSWGQGTQVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:21).

[0212] GM-CSF Signal Peptide, a BCMA-binding domain (Cilta VHH2) with CD8a hinge, CD8a transmembrane, 4-1BB costimulatory, and CD3z stimulatory components: MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQAGGSLRLSCAASGRTFTMGWFRQAPGK EREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMS IRPDYWGQGTQVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:22).

[0213] GM-CSF Signal Peptide, a FLAG epitope tag, a CD22-binding domain (m971) with CD8a hinge, CD8a transmembrane, 4-1BB costimulatory, and CD3z stimulatory components: MLLLVTSLLLCELPHPAFLLIPDYKDDDDKQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSN SAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDT AVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ TIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQ QSYSIPQTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC IYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:33)

[0214] GM-CSF Signal Peptide, a CD22-binding (HA22) domain with a Myc epitope tag, a CD8 hinge, transmembrane, and intracellular components: MVLLVTSLLLCELPHPAFLLIPDTEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVR QTPEKRLEWVAYISSGGGTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSG YGTHWGVLFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRA SQDISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYF CQQGNTLPWTFGGGTKLEIKEQKLISEEDLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSA RYV (SEQ ID NO:34).298507734.1 - 55 -

[0215] GM-CSF Signal Peptide, a FLAG epitope tag, a CD22-binding domain (m971) with CD8a hinge, CD8a transmembrane, 4-1BB costimulatory, and CD3z stimulatory components follwed by a P2A sequence for bicistronic expression of GM-CSF Signal Peptide, a CD22- binding (HA22) domain with a Myc epitope tag, a CD8 hinge, transmembrane, and intracellular components: MLLLVTSLLLCELPHPAFLLIPDYKDDDDKQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSN SAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDT AVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ TIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQ QSYSIPQTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC IYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLL KQAGDVEENPGPMVLLVTSLLLCELPHPAFLLIPDTEVQLVESGGGLVKPGGSLKLSCAASG FAFSIYDMSWVRQTPEKRLEWVAYISSGGGTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSE DTAMYYCARHSGYGTHWGVLFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQTTSSLSA SLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTI SNLEQEDFATYFCQQGNTLPWTFGGGTKLEIKEQKLISEEDLTTTPAPRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRP VVKSGDKPSLSARYV (SEQ ID NO:35)

[0216] Also disclosed are dual CAR immune cells containing the CAR aspects disclosed above, which bind at least two different cancer or tumor cell-associated antigens, epitopes, or binding regions thereof. In these aspects, one CAR can include a CD4 or CD8 intracellular domain, while the other CAR can include stimulatory and costimulatory domain(s). The co- expressed CARs can increase antigen sensitivity while preserving endogenous TCR function, avoiding interference with assembly of CD3 complex components, recruiting only LCK in the presence of antigen, or a combination thereof. In these aspects, dual CAR immune cell activation uses co-expression of both targets on the target cell. These CAR immune cells can be useful in methods for treating a subject for cancer, killing cancer cells in a subject, stimulating an immune response to cancer cells in a subject, and generating a persisting population of genetically engineered immune cells. Furthermore, the immune cells can be redirected to target a wide range of cancer or tumor cell-associated antigens, epitopes, or binding regions thereof. Thus, the immune cells co-expressing the CAR molecules can induce CAR signaling that more closely mimics TCR signaling and can improve CAR efficacy in298507734.1 - 56 -cases of downregulation of target antigen, which may provide more favorable conditions to support cancer treatment in comparison to existing methods and compositions.

[0217] Exemplary CD22-targeting CAR constructs include but are not limited to:

[0218] CD22-targeting CAR comprising a M971 scFv, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z stimulatory domain.

[0219] CD22-targeting CoCARs comprising (1) a HA22 scFv, a CD8 hinge, a CD8 transmembrane domain, and a CD8 intracellular domain; (2) a HA22 scFv, a CD8 hinge, a CD4 transmembrane domain, and a CD4 intracellular domain; and / or (3) a HA22 scFv, a CD28 hinge, a CD28 transmembrane domain, and a CD4 intracellular domain.

[0220] Exemplary BCMA-targeting CAR constructs include but are not limited to:

[0221] BCMA-targeting CAR comprising a Cilta VHH1, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z stimulatory domain; and / or a Cilta VHH2, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z stimulatory domain.

[0222] BCMA-targeting CoCARs comprising: a Cilta VHH1, a CD8 hinge, a CD8 transmembrane domain, and a CD8 intracellular domain; and / or a Cilta VHH2, a CD8 hinge, a CD8 transmembrane domain, and a CD8 intracellular domain.

[0223] In some aspects, CAR systems disclosed herein include one CAR molecule and one CoCAR molecule expressed at the same time. Exemplary CAR / CoCAR combinations include but are not limited to:

[0224] A CD22-targeting CAR comprising a M971 scFv, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z stimulatory domain and a CD22-targeting CoCAR comprising HA22 scFv, a CD8 hinge, a CD8 transmembrane domain, and a CD8 intracellular domain.

[0225] A CD22-targeting CAR comprising a M971 scFv, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z stimulatory domain and a CD22-targeting CoCAR comprising HA22 scFv, a CD8 hinge, a CD4 transmembrane domain, and a CD4 intracellular domain.

[0226] A CD22-targeting CAR comprising a M971 scFv, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z stimulatory domain and a CD22-targeting CoCAR comprising HA22 scFv, a CD28 hinge, a CD28 transmembrane domain, and a CD4 intracellular domain.

[0227] A BCMA-targeting CAR comprising a Cilta VHH1, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z stimulatory domain and298507734.1 - 57 -a BCMA-targeting CoCAR comprising: a Cilta VHH2, a CD8 hinge, a CD8 transmembrane domain, and a CD8 intracellular domain.

[0228] A BCMA-targeting CAR comprising a Cilta VHH2, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z stimulatory domain and a BCMA-targeting CoCAR comprising: a Cilta VHH1, a CD8 hinge, a CD8 transmembrane domain, and a CD8 intracellular domain. A. Antigen-Binding Domains

[0229] In some aspects, the CARs comprise an antigen-binding domain that binds to one or more cancer or tumor cell-associated antigens, epitopes, or binding regions thereof. Cancer or tumor cell-associated antigens are proteins that are produced by cancer or tumor cells that elicit an immune response. The selection of the antigen-binding domain will depend on the particular type of cancer to be treated. In some aspects, the CARs comprise at least two antigen- binding domains that bind to one or more cancer or tumor cell-associated antigens at different, distinct, and / or non-overlapping epitopes. In some aspects, the CARs comprise at least two antigen-binding domains that bind to one or more cancer or tumor cell-associated antigens at different but overlapping epitopes. In some aspects, the CARs comprise at least two antigen- binding domains that bind to one or more cancer or tumor cell-associated antigens at overlapping epitopes. In some aspects, the CARs do not comprise two antigen-binding domains that bind to the same epitope.

[0230] Non-limiting examples of cancer or tumor cell-associated antigens that may be targeted include at least the following: Differentiation antigens such as tyrosinase, TRP-1 , TRP-2 and tumor-specific multilineage antigens such as MAGE-1 , MAGE-3, BAGE, GAGE- 1 , GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor- suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH- IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm- 23H1 , PSA, CA 19-9, CA 72-4, CAM 17.1 , NuMa, K-ras, beta-Catenin, CDK4, Mum-1 , p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha- fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1 , CO-029, FGF-5, G250, Ga733\EpCAM, HTgp- 175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1 , RCASI, SDCCAG1 6, TA- 90\Mac-2 binding protein\cyclophilm C-associated protein,298507734.1 - 58 -TAAL6, TAG72, TLP, TPS, GPC3, MUC16, MUC18, LMP1 , EBMA-1 , BARF-1 , CS1 , CD319, HER1 , B7H6, L1 CAM, IL6, and MET. Tumor antigens include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvlll, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, b-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD19, cyclin Bl, lectin-reactive AFP, Fos-related antigen 1 , ADRB3, thyroglobulin, EphA2, RAGE-1 , RUI, RU2, SSX2, AKAP-4, LCK, OY- TESI, PAX5, SART3, CLL-1 , fucosyl GM1 , GloboH, MN-CA IX, EPCAM, EVT6- AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1 , RUI, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP- 2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1 , LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51 E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1 , VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1 , prostate-carcinoma tumor antigen- 1 (PCTA-1), ML-IAP, MAGE, MAGE-A1.MAD-CT-1 , MAD-CT-2, MelanA / MART 1 , XAGE1 , ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1 , ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171 , CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61 , folate receptor (FRa), folate receptor beta, ROR1 , Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, mesothelin, and any combination thereof. In specific aspects, cancer or tumor cell-associated antigens comprise CD22, CD19, BCMA, HER2, or mesothelin. In some aspects, any one or more of the antigens describe herein may be expressly excluded from the list of antigens targeted by the one or more CARs.

[0231] In some aspects of the disclosure, there are compositions comprising CARs which encompass a nucleotide and / or amino acid sequence of an antigen-binding domain (e.g., an antibody or antigen-binding fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor- ligand pair that targets the antigen) that binds to the cancer or tumor cell-associated antigens, epitopes, or fragments thereof. As a reference sequence, CD22, CD19, BCMA, HER2, and mesothelin polypeptide sequences are in the UniProtKB database at Accession Numbers P20273-1, P15391-1, Q02223-1, P04626-1, and Q13421, respectively:

[0232] Human CD22:298507734.1 - 59 -MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFHNP EYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNKNCTLSIHPVHLNDSGQLGLRMESK TEKWMERIHLNVSERPFPPHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQA AVTSTSLTIKSVFTRSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKV TPSDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVTKDQSGKYCCQ VSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVEFLCMSLANPLPTNYTWYHNGKE MQGRTEEKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPKKVTTVIQNPMPIRE GDTVTLSCNYNSSNPSVTRYEWKPHGAWEEPSLGVLKIQNVGWDNTTIACAACNSWCSWASP VALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQFFWEKNGRLLGKESQLNF DSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSMSPGDQVMEGKSATLTCESDA NPPVSHYTWFDWNNQSLPYHSQKLRLEPVKVQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPE TIGRRVAVGLGSCLAILILAICGLKLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSE GPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALHKRQVGD YENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH (SEQ ID NO:23).

[0233] Human CD19: MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFL KLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRW NVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQ DLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLL PRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGI LHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLG GTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQ DGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLG SQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR (SEQ ID NO:24).

[0234] Human BCMA: MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGL SLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVE ECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR (SEQ ID NO:25).

[0235] Human HER2: MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLEL TYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLN NTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDT298507734.1 - 60 -NRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPK HSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGS CTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFG SLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGR ILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHT ANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNAR HCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEE GACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKI RKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGE NVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCL LDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLAR LLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPARE IPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNE DLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRS GGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDP TVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKN GVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGT PTAENPEYLGLDVPV (SEQ ID NO:26).

[0236] Human mesothelin: MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLS PRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLL FLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALG GLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDAL RGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKARE IDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHL GYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLD KDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMN GSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGL KAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTV LALLLASTLA (SEQ ID NO:36).

[0237] The term “antibody” refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes chimeric, humanized, fully human, and bispecific antibodies. As used herein, the terms “antibody” or “immunoglobulin” are used interchangeably and refer to any298507734.1 - 61 -of several classes of structurally related proteins that function as part of the immune response of an animal, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen-binding activity.

[0238] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody. An antigen may possess one or more epitopes that are capable of interacting with different antibodies.

[0239] The term “epitope” includes any region or portion of molecule capable eliciting an immune response by binding to an immunoglobulin or to a T-cell receptor. Epitope determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen within a complex mixture.

[0240] The epitope regions of a given polypeptide can be identified using many different epitope mapping techniques are well known in the art, including: x-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, see, e.g., Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, N.Y. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986). Additionally, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydropathy plots.

[0241] The term “immunogenic sequence” means a molecule that includes an amino acid sequence of at least one epitope such that the molecule is capable of stimulating the production of antibodies in an appropriate host. The term “immunogenic composition” means a composition that comprises at least one immunogenic molecule (e.g., an antigen or carbohydrate).

[0242] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains, such as antibodies naturally occurring in camelids that may comprise only heavy chains. Antibodies as disclosed herein may be derived solely from a single source or may be “chimeric,” that is, different portions of the antibody may be derived from two different antibodies. For example, the variable or CDR regions may be derived from a rat or murine source, while the constant region is derived from a different animal source, such as a human. The antibodies or binding fragments298507734.1 - 62 -may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes derivatives, variants, fragments, and muteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol.2013; 4: 302; 2013).

[0243] The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain has a molecular weight of around 25,000 Daltons and includes a variable region domain (abbreviated herein as VL), and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, identified as kappa (κ) and lambda (λ). The term “VL fragment” means a fragment of the light chain of a monoclonal antibody that includes all or part of the light chain variable region, including CDRs. A VL fragment can further include light chain constant region sequences. The variable region domain of the light chain is at the amino-terminus of the polypeptide.

[0244] The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of around 50,000 Daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as CH1, CH2, and CH3). The term “VH fragment” means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the heavy chain variable region, including CDRs. A VH fragment can further include heavy chain constant region sequences. The number of heavy chain constant region domains will depend on the isotype. The VH domain is at the amino- terminus of the polypeptide, and the CH domains are at the carboxy-terminus, with the CH3 being closest to the —COOH end. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE and is defined by the heavy chains present of which there are five classifications: mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chains, respectively. IgG has several subtypes, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0245] In some aspects, the antigen-binding domains that bind to cancer or tumor cell- associated antigens, epitopes, or fragments thereof comprise a single-chain variable fragment (scFv) derived from an antibody. “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0246] The variable regions of the antigen-binding domains of the polypeptides of the disclosure can be modified by mutating amino acid residues within the VH and / or VL CDR 1,298507734.1 - 63 -CDR 2 and / or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term “CDR” refers to a complementarity-determining region that is based on a part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B cells and T cells respectively, where these molecules bind to their specific antigen. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions, or deletions.

[0247] Framework modifications can be made to the antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to the corresponding germline sequence.

[0248] The binding affinity of the antigen-binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, or 10-13M. In some aspects, the KDof the antigen-binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, or 10-13M (or any derivable range therein).

[0249] Binding affinity, KA, or KD can be determined by methods known in the art such as by surface plasmon resonance (SRP)-based biosensors, by kinetic exclusion assay (KinExA), by optical scanner for microarray detection based on polarization-modulated oblique-incidence reflectivity difference (OI-RD), or by ELISA.

[0250] In some aspects, the framework regions, such as FR1, FR2, FR3, and / or FR4 of a human framework can each or collectively have, have at least, have at most, or have about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,298507734.1 - 64 -180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) amino acid substitutions, contiguous amino acid additions, or contiguous amino acid deletions with respect to a mouse framework.

[0251] In some aspects, the framework regions, such as FR1, FR2, FR3, and / or FR4 of a mouse framework can each or collectively have, have at least, have at most, or have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) amino acid substitutions, contiguous amino acid additions, or contiguous amino acid deletions with respect to a human framework.

[0252] The substitution may be at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 of FR1, FR2, FR3, or FR4 of a heavy or light chain variable region. B. Extracellular Spacer

[0253] An extracellular spacer may link the antigen-binding domain to the transmembrane domain. It should be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen-binding. In some aspects, the spacer is the hinge region from IgG. In some aspects, the spacer is an IgG4 hinge. In some aspects, the spacer is an IgG1 spacer. In some aspects, the spacer comprises both an IgG4 hinge and an IgG1 spacer.

[0254] As used herein, the term “hinge” refers to a flexible polypeptide connector region (also referred to herein as “hinge region” or “spacer”) providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides. A “spacer” derived from an immunoglobulin (e.g., IgGl) is generally defined as stretching from Glu216 to Pro230 of human IgGl, for example (Burton (1985) Molec. Immunol., 22: 161- 206).298507734.1 - 65 -Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions. The hinge region may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region as described in U.S. Pat. No.5,677,425. The hinge region can include a complete hinge region derived from an antibody of a different class or subclass from that of the CH1 domain. The term “hinge” can also include regions derived from CD8 and other receptors that provide a similar function in providing flexibility and spacing to flanking regions. Other alternatives include the CH2CH3 region of immunoglobulin and portions of CD3.

[0255] The extracellular spacer can have a length of, of at least, of at most, or of about 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or 400 amino acids (or any derivable range therein). In some aspects, the extracellular spacer consists of or comprises a hinge region from an immunoglobulin (e.g. IgG). Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al. (1986) Nucl. Acids Res.

[0256] The length of an extracellular spacer may have effects on the CAR’s signaling activity and / or the CAR-T cells’ expansion properties in response to antigen-stimulated CAR signaling. In some aspects, a shorter spacer such as less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids is used. In some aspects, a longer spacer, such as one that is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, or 290 amino acids may have the advantage of increased expansion in vivo or in vitro.

[0257] In some aspects, the hinge region is derived from CD4 and comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to one or more art-recognized CD4 hinge sequences that provide sufficient structural flexibility and spacing to flanking polypeptide regions. In some aspects, the hinge region is not derived from CD4. In some aspects, the hinge region is derived from CD8 and comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of298507734.1 - 66 -end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:5, or another functionally equivalent art-recognized CD8 hinge sequence that provides sufficient structural flexibility and spacing to flanking polypeptide regions. In some aspects, the hinge region is not derived from CD8. In some aspects, the hinge region is derived from CD28 and comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:6, or another functionally equivalent art-recognized CD28 hinge sequence that provides sufficient structural flexibility and spacing to flanking polypeptide regions. In some aspects, the hinge region is not derived from CD28.

[0258] The extracellular spacer can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4 hinge region. In some aspects, the extracellular spacer does not comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4 hinge region. The extracellular spacer may also include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally-occurring) hinge region. C. Transmembrane Domain

[0259] With respect to the transmembrane domain, the CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR, optionally, via the hinge or spacer. The transmembrane domain can be a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different receptor stability. In one aspect, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0260] In various aspects the transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Illustrative, but non-limiting, examples of transmembrane regions of particular use in the CAR constructs contemplated here can be derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain can be synthetic, in which case it can comprise predominantly hydrophobic residues such as leucine and valine. In certain aspects, a triplet of phenylalanine, tryptophan and valine298507734.1 - 67 -will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, e.g., between 2 and about 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In certain aspects a glycine-serine doublet provides a particularly suitable linker. In certain aspects, any one or more of the aforementioned transmembrane domains and / or linkers may be expressly excluded from a CAR.

[0261] The transmembrane domain is interposed between the extracellular spacer and the cytoplasmic region. In some aspects, the transmembrane domain is interposed between the extracellular spacer and one or more costimulatory regions. In some aspects, a linker is between the transmembrane domain and the one or more costimulatory regions.

[0262] Any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. As one non-limiting example, the transmembrane domain is derived from CD4 and comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:7, or another functionally equivalent art-recognized CD4 transmembrane sequence that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell. In some aspects, a transmembrane domain is not from CD4. As another example, the transmembrane domain is derived from CD8 and comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:8, or another functionally equivalent art-recognized CD8 transmembrane sequence that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell. In some aspects, a transmembrane domain is not from CD8. As another example, the transmembrane domain is derived from CD28 and comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:9, or another functionally equivalent art-recognized CD28 transmembrane sequence that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell. In some aspects, a transmembrane domain is not from CD28. D. Cytoplasmic Region

[0263] The cytoplasmic or intracellular domain or otherwise the intracellular signaling domain of the CAR is responsible for activation of at least one of the normal effector functions298507734.1 - 68 -of the immune cell in which the CAR has been placed. After antigen and / or ligand recognition, receptors cluster and a signal is transmitted to the cell through the cytoplasmic region. In some aspects, the stimulatory and costimulatory domains described herein are part of the cytoplasmic or intracellular region.

[0264] The term “effector function” refers to a specialized function of a cell. An effector function of a T cell, for example, may be cytolytic activity, or helper activity including the secretion of cytokines. Thus, the term “intracellular signaling domain” refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0265] Cytoplasmic or intracellular stimulatory and / or costimulatory regions or domains suitable for use in the polypeptides of the disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen-binding domain. By the term “stimulation,” it is meant a primary response induced by binding of a stimulatory molecule with its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction. Stimulation can mediate altered expression of certain molecules. A “stimulatory molecule,” as the term is used herein, means a molecule on an immune cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell. A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an APC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on an immune cell, thereby mediating a primary response by the immune cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. The term “costimulatory ligand,” as the term is used herein, includes a molecule on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, and the like) that specifically binds a cognate costimulatory molecule on an immune cell, thereby providing a signal which, in addition to the primary signal to mediate the immune cell298507734.1 - 69 -response, including, but not limited to, proliferation, activation, differentiation, and the like. A costimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a costimulatory molecule present on an immune cell. A “costimulatory molecule” refers to the cognate binding partner on an immune cell that specifically binds with a costimulatory ligand, thereby mediating a co- stimulatory response by the immune effector, such as, but not limited to, proliferation and / or activation. A “costimulatory signal”, as used herein, refers to a signal, that in combination with a primary signal, leads to immune cell activation, proliferation, and / or upregulation or downregulation of key molecules.

[0266] In some aspects, the cytoplasmic region includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motifs as described herein. In some aspects, the cytoplasmic region includes at most one, two, three, four, five, or six ITAM motifs as described herein. Cytoplasmic regions suitable for use in the polypeptides of the disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. An ITAM motif is YX1X2(L / I), where X1 and X2 are independently any amino acid. In some cases, the cytoplasmic region comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twice in an endodomain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids, e.g., (YX1X2(L / I))(X3)n(YX1X2(L / I)), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid.

[0267] A suitable cytoplasmic region may be an ITAM motif-containing a portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable endodomain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, CD3ε; CD3γ, and CD3ζ. In some aspects, an ITAM motif-containing peptide is not CD3ε, CD3γ, and / or CD3ζ.

[0268] In some cases, an intracellular domain is derived from CD4 and comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:10, or another functionally equivalent art-recognized CD4 intracellular sequence that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way298507734.1 - 70 -of binding of the antigen to the antigen-binding domain. In some aspects, an intracellular domain is from CD4. In some aspects, an intracellular domain is not from CD4.

[0269] In some cases, an intracellular domain is derived from CD8 and comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:11, or another functionally equivalent art-recognized CD8 intracellular sequence that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen-binding domain. In some aspects, an intracellular domain is from CD8. In some aspects, an intracellular domain is not from CD8.

[0270] In some aspects, a cytoplasmic stimulatory domain is derived from 4-1BB and comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:12, or another functionally equivalent art-recognized 4- 1BB intracellular sequence that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen-binding domain. In some aspects, an intracellular domain is from 41BB. In some aspects, an intracellular domain is not from 41BB.

[0271] In some aspects, a cytoplasmic stimulatory domain is derived from CD28 and comprises an amino acid sequence having, having at least, having at most, or having between any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:13, or another functionally equivalent art-recognized CD28 intracellular sequence that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen-binding domain. In some aspects, an intracellular domain is from CD28. In some aspects, an intracellular domain is not from CD28.

[0272] In some aspects, a cytoplasmic stimulatory domain is derived from CD3ζ and comprises an amino acid sequence having, having at least, having at most, or having between298507734.1 - 71 -any two of (inclusive of end values) 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, amino acid sequence identity to SEQ ID NO:14, or another functionally equivalent art-recognized CD3ζ intracellular sequence that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen-binding domain. In some aspects, an intracellular domain is from CD3ζ. In some aspects, an intracellular domain is not from CD3ζ. E. Detection peptides

[0273] In some aspects, the CARs described herein may further comprise a detection peptide or molecule. Suitable detection peptides include hemagglutinin (HA; e.g., YPYDVPDYA; FLAG (e.g., DYKDDDDK (SEQ ID NO:31); c-myc (e.g., EQKLISEEDL; SEQ ID NO: 32), and the like. Other suitable detection peptides are known in the art. In some aspects, a CAR described herein does not comprise a detection peptide or molecule. F. Therapeutic Controls

[0274] In some aspects of the methods and compositions described herein, the CAR is co- expressed with a therapeutic control.

[0275] Therapeutic controls can regulate cell proliferation, facilitate cell selection (for example selecting cells which express the chimeric antigen receptors of the disclosure) or a combination thereof. In one aspect, regulating cell proliferation comprises up-regulating cell proliferation to promote cell propagation. In another aspect, regulating cell proliferation comprises down-regulating cell proliferation so as to reduce or inhibit cell propagation. In some aspects, the agents that serve as therapeutic controls may promote enrichment of cells which express the chimeric antigen receptors which may result in a therapeutic advantage. In some aspects, agents which serve as therapeutic controls may biochemically interact with additional compositions so as to regulate the functioning of the therapeutic controls.

[0276] Further aspects of the therapeutic controls, CAR molecules, and methods of use for the compositions of the disclosure can be found in U.S. Patent No.9,447,194, which is herein incorporated by reference for all purposes. G. Additional Modifications and Polypeptides

[0277] As used herein, a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version298507734.1 - 72 -of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity.

[0278] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0279] In certain aspects the size of a protein or polypeptide (wild-type or modified) may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein.

[0280] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,298507734.1 - 73 -44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids or nucleic acids, or any range derivable therein, of SEQ ID NOs:1-28.

[0281] It is contemplated that a region or fragment of a polypeptide of the disclosure may have an amino acid sequence that has, has at least or has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 or more amino acid substitutions, contiguous amino acid additions, or contiguous amino acid deletions with respect to any of SEQ ID NOs:1-28. Alternatively, a region or fragment of a polypeptide of the disclosure may have an amino acid sequence that comprises or consists of an amino acid sequence that is, is at least, or is at most 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,298507734.1 - 74 -64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% (or any range derivable therein) identical to any of SEQ ID NOs:1-28. Moreover, in some aspects, a region or fragment comprises an amino acid region of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 or more contiguous amino acids starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,298507734.1 - 75 -130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 in any of SEQ ID NOs:1-28 (where position 1 is at the N-terminus of the SEQ ID NOs:1-28). The polypeptides of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous with at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,298507734.1 - 76 -162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000, 1500, or 2000 or more contiguous amino acids or nucleic acids, or any range derivable therein, of any of SEQ ID NOs:1-28.

[0282] The polypeptides of the disclosure may include, include at least, or include at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,298507734.1 - 77 -521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 substitutions (or any range derivable therein).

[0283] The substitution may be at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,298507734.1 - 78 -547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 650, 700, 750, 800, 850, 900, 1000, 1500, or 2000 of any of SEQ ID NOs:1-28 (or any derivable range therein).

[0284] The polypeptides described herein may be of a fixed length of, of at least, or of at most, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more amino acids (or any derivable range therein).

[0285] It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.

[0286] Additionally, the polypeptides of the disclosure may be chemically modified. Glycosylation of the polypeptides can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos.5,714,350 and 6,350,861).

[0287] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge.298507734.1 - 79 -Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Non- conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.

[0288] Proteins may be recombinant, or synthesized in vitro. Alternatively, a non- recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that bacteria containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.

[0289] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.

[0290] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5’ or 3’ sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non- coding sequences flanking either of the 5’ or 3’ portions of the coding region.

[0291] The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity. Structures such as, for example, an enzymatic catalytic domain or interaction components may have amino acid substituted to maintain such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties.298507734.1 - 80 -It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.

[0292] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0293] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.

[0294] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5ʹ or 3ʹ sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5ʹ or 3ʹ portions of the coding region.

[0295] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.

[0296] Insertional mutants typically involve the addition of amino acid residues at a non- terminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein

[0297] In other aspects, alteration of the function of a polypeptide is intended by introducing one or more substitutions. For example, certain amino acids may be substituted for other amino acids in a protein structure with the intent to modify the interactive binding298507734.1 - 81 -capacity of interaction components. Structures such as, for example, protein interaction domains, nucleic acid interaction domains, and catalytic sites may have amino acids substituted to alter such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with different properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes with appreciable alteration of their biological utility or activity.

[0298] In making such changes, the hydropathic index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain aspects, the substitution of amino acids whose hydropathy indices are within ±2 is included. In some aspects of the disclosure, those that are within ±1 are included, and in other aspects of the disclosure, those within ±0.5 are included.

[0299] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain aspects, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen- binding, that is, as a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate298507734.1 - 82 -(+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4). In making changes based upon similar hydrophilicity values, in certain aspects, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other aspects, those which are within ±1 are included, and in still other aspects, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0300] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0301] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy.org / proteomics / protein_structure.

[0302] In some aspects of the disclosure, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen-binding affinities, and / or (5) confer or modify other physicochemical or functional properties on such polypeptides. For example,298507734.1 - 83 -single or multiple amino acid substitutions (in certain aspects, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. In such aspects, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).

[0303] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0304] In specific aspects, all or part of proteins described herein can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence that encodes a peptide or polypeptide is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultivated under conditions suitable for expression.

[0305] Aspects can include the use of gene transfer to cells, including microorganisms, for the production and / or presentation of proteins. The gene for the protein of interest may be transferred into appropriate host cells followed by culture of cells under the appropriate conditions. A nucleic acid encoding virtually any polypeptide may be employed. The generation of recombinant expression vectors, and the elements included therein, are discussed herein. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell used for protein production.

[0306] The nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.298507734.1 - 84 -III. Immune Cells

[0307] Disclosed herein are methods and compositions which utilize immune cells. In some aspects, the immune cells are αβ–T cells, γδ–T cell, Natural Killer (NK) cells, Natural Killer T (NKT) cells, B cells, macrophages, microglial cells, neutrophils, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, cytotoxic T lymphocytes (CTL), lymphokine activated killer (LAK) cells, and / or regulatory T cells.

[0308] The immune cells described herein may be engineered to express the disclosed CARs. These cells are preferably obtained from the subject to be treated (i.e., are autologous). However, in some aspects, immune cell lines or donor effector cells (i.e., allogeneic) are used. Immune cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some aspects, immune cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune cells can be further isolated by positive or negative selection techniques. For example, immune cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune cells. Alternatively, enrichment of immune cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0309] Following genetic modification with the vector(s), the immune cells may be immediately infused or may be stored. In certain aspects, following genetic modification, the immune cells may be propagated for days, weeks, or months ex vivo as a bulk population within about 1, 2, 3, 4, 5 days or more following gene transfer into cells. In a further aspect, the transfectants are cloned and a clone demonstrating presence of a single integrated or episomally maintained expression cassette or plasmid, and expression of the viral protein polypeptide(s) and / or antigen-targeting polypeptide(s) (e.g., an antibody and / or CAR) is expanded ex vivo. The clone selected for expansion demonstrates the capacity to specifically recognize and lyse antigen-expressing target cells. The recombinant immune cells may be expanded by stimulation with IL-2, or other cytokines that bind the common gamma-chain (e.g., IL-7, IL-298507734.1 - 85 -12, IL-15, IL-21, IL-23, and others). The recombinant immune cells may be expanded by stimulation with artificial antigen presenting cells. In a further aspect, the genetically modified cells may be cryopreserved.

[0310] Aspects of the disclosure encompass cells that express antigen-targeting CARs as encompassed herein. The immune cell comprises a recombinant nucleic acid that encodes one or more antigen-targeting CARs, in specific aspects. In specific aspects, in addition to expressing one or more antigen-targeting CARs, the cell also comprises a nucleic acid that encodes one or more therapeutic gene products.

[0311] The immune cells may be from an individual in need of therapy for a medical condition, and following their manipulation to express the CARs (using standard techniques for transduction and expansion for adoptive cell therapy, for example), they may be provided back to the individual from which they were originally sourced. In some cases, the cells are stored for later use for the individual or another individual.

[0312] The immune cells may be comprised in a population of cells, and that population may have a majority that are transduced with one or more antigen-targeting CARs. A cell population may comprise 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of immune cells that are transduced with one or more antigen- targeting CARs. The one or more antigen-targeting CARs may be separate polypeptides encoded by separate polynucleotides, or may be on the same polypeptide and encoded by the same polynucleotide. IV. Formulations and Culture of Cells

[0313] In particular aspects, the immune cells and / or precursors thereto may be specifically formulated and / or they may be cultured in a particular medium at any stage of a process of generating the immune cells which express one or more of the CARs disclosed herein. The cells may be formulated in such a manner as to be suitable for delivery to a recipient without deleterious effects.

[0314] The medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer’s media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as298507734.1 - 86 -it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined.

[0315] The medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the stem cell(s). The serum- free medium refers to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

[0316] The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid- rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3’- thiolglycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98 / 30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0317] In further aspects, the medium may be a serum-free medium that is suitable for cell development. For example, the medium may comprise B-27®supplement, xeno-free B-27®supplement (available at world wide web at thermofisher.com / us / en / home / technical- resources / media-formulation.250.html), NS21 supplement (Chen et al., J Neurosci Methods, 2008 Jun 30; 171(2): 239–247, incorporated herein in its entirety), GS21TMsupplement (available at world wide web at amsbio.com / B-27.aspx), or a combination thereof at a concentration effective for producing T cells from the 3D cell aggregate.

[0318] In certain aspects, the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha-Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HCl; Glutathione (reduced); L-Carnitine HCl; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HCl; Sodium Selenite; and / or T3 (triodo-I-thyronine).298507734.1 - 87 -

[0319] In some aspects, the medium further comprises vitamins. In some aspects, the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof. In some aspects, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some aspects, the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some aspects, the medium further comprises proteins. In some aspects, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some aspects, the medium further comprises one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some aspects, the medium comprises one or more of the following: a B-27®supplement, xeno-free B-27®supplement, GS21TMsupplement, or combinations thereof. In some aspects, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some aspects, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some aspects, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some aspects, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain aspects, the medium comprises or consists essentially of one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, a B-27®supplement, xeno-free B-27®supplement, GS21TMsupplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese.298507734.1 - 88 -

[0320] In further aspects, the medium may comprise externally added ascorbic acid. The medium can also contain one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2- mercaptoethanol, pyruvic acid, buffering agents, and / or inorganic salts.

[0321] One or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, µg / ml, mg / ml, or any range derivable therein.

[0322] The medium used may be supplemented with at least one externally added cytokine at a concentration from about 0.1 ng / mL to about 500 ng / mL, more particularly 1 ng / mL to 100 ng / mL, or at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, µg / ml, mg / ml, or any range derivable therein. Suitable cytokines, include but are not limited to, FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL- 21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, and / or midkine. Particularly, the culture medium may include at least or at most one of FLT3L and IL-7. More particularly, the culture may include both FLT3L and IL-7.

[0323] Other culturing conditions can be appropriately defined. For example, the culturing temperature can be about 20 to 40°C, such as at least, at most, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C (or any range derivable therein), though the temperature may be above or below these values. The CO2concentration can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (or any range derivable therein), such as about 2% to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen tension can be at least, at most, or about 1, 5, 8, 10, 20%, or any range derivable therein.

[0324] In specific aspects, immune cells which express one or more of the CARs disclosed herein are specifically formulated. They may or may not be formulated as a cell suspension. In specific cases they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases the cells are formulated for storage prior to use, and the cell formulation may comprise one or more cryopreservation agents, such as DMSO (for example, in 5% DMSO). The cell formulation may comprise albumin, including human albumin, with a specific formulation comprising 2.5% human albumin. The cells may be formulated specifically for intravenous administration; for example, they are formulated for intravenous administration over less than one hour. In particular aspects the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from time of thawing.298507734.1 - 89 -

[0325] In some aspects, the immune cells or population thereof comprise a one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens and epitopes thereof to which a CAR may be directed include at least 5T4, 8H9, αvβ6integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CD133, CEA, c-Met, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRα, GD2, G250 / CAIX, GD3, Glypican-3 (GPC3), GUCY2C, HER1, HER2, ICAM-1, IL-13Rα2, IL-11Rα, Kras, Kras G12D, L1CAM, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, MET, Mesothelin, Muc1, Muc16, MUC18, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, laminin receptor, HPV E6, E7, BING-4, Calcium-activated chloride channel 2, Cyclin-B1, 9D7, EphA3, Telomerase, SAP-1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, Prostate-specific antigen, β-catenin, BRCA1 / 2, CML66, Fibronectin, MART-2, TGF-βRII, WT-1, or VEGF receptors (e.g., VEGFR2), for example. In specific aspects, the CARs are directed to one or more epitopes of CD22, CD19, BCMA, HER2, or mesothelin. The CAR may be a first, second, third, or more generation CAR. The CAR may be bispecific for any two nonidentical antigens, or it may be specific for more than two nonidentical antigens.

[0326] The therapeutic compositions and treatments disclosed herein may comprise administration of a combination of therapeutic agents, such as a first therapeutic or pharmaceutical composition or treatment and a second therapeutic or pharmaceutical composition or treatment. The therapies may be administered in any suitable manner known in the art. For example, the therapeutic or pharmaceutical compositions or treatments may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, the therapeutic or pharmaceutical compositions or treatments are administered in a separate composition. In some aspects, the therapeutic or pharmaceutical compositions or treatments are in the same composition.

[0327] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapeutic or pharmaceutical compositions or treatments may be administered in one composition or in more than one composition, such as298507734.1 - 90 -2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0328] The therapeutic or pharmaceutical compositions and treatments disclosed herein may precede, be co-current with and / or follow another treatment or agent by intervals ranging from minutes to weeks. In aspects where agents are applied separately to a cell, tissue, or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapeutic or pharmaceutical agents would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more agents or treatments substantially simultaneously (i.e., within less than about a minute). In other aspects, one or more therapeutic agents or treatments may be administered or provided within 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks or more, and any range derivable therein, prior to and / or after administering another therapeutic agent or treatment.

[0329] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0330] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain aspects, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120,298507734.1 - 91 -125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 µg / kg, mg / kg, µg / day, or mg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0331] In some aspects, the therapeutically effective or sufficient amount of the therapeutic composition or treatment administered to a human will be in the range of about 102up to about 1010cells per kg of patient body weight whether by one or more administrations. In some aspects, the therapy used is about 102cells to about 109cells / kg, about 102cells to about 108cells / kg, about 102cells to about 107cells / kg, about 102cells to about 106cells / kg, about 102cells to about 105cells / kg, about 102cells to about 104cells / kg, or about 102cells to about 103cells / kg administered daily, for example. In one aspect, a therapy described herein is administered to a subject at a dose of about 102cells, about 103cells, about 104cells, about 105cells, about 106cells, about 107cells, about 108cells, about 109cells, or about 1010cells. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The progress of this therapy is easily monitored by conventional techniques.

[0332] In some aspects, the therapeutically effective or sufficient amount of the therapeutic composition or treatment administered to a human will be in the range of about 0.01 to about 50 mg / kg of patient body weight whether by one or more administrations. In some aspects, the therapy used is about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg administered daily, for example. In one aspect, a therapy described herein is administered to a subject at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21-day cycles. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The progress of this therapy is easily monitored by conventional techniques.

[0333] In certain aspects, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 µM to 150 µM. In another aspect, the effective dose provides a blood level of about 4 µM to 100 µM.; or about 1 µM to 100 µM; or about 1 µM to 50 µM; or about 1 µM to 40 µM; or about 1 µM to 30 µM; or about 1 µM to 20 µM; or about 1 µM to 10 µM; or about 10 µM to 150 µM; or about 10 µM to 100 µM; or about 10 µM to 50 µM; or about 25 µM to 150 µM; or about 25 µM to 100 µM; or about 25 µM to 50 µM; or298507734.1 - 92 -about 50 µM to 150 µM; or about 50 µM to 100 µM (or any range derivable therein). In other aspects, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM or any range derivable therein. In certain aspects, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0334] In various aspects the CAR-modified cells described herein can be administered either as a therapeutic or pharmaceutical composition alone, or as a therapeutic or pharmaceutical composition in combination with diluents and / or with other components such other cytokines or cell populations. Briefly, in certain aspects, pharmaceutical compositions can comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants ( e.g., aluminum hydroxide); and preservatives.

[0335] Pharmaceutical or therapeutic compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the subject’s disease, although appropriate dosages may be determined by clinical trials. Precise amounts of the therapeutic or pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0336] When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise298507734.1 - 93 -amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0337] It will be understood by those skilled in the art and made aware that dosage units of µg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of µg / ml or mM (blood levels), such as 4 µM to 100 µM. It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein. V. Methods of Producing CAR-Expressing Immune Cells

[0338] Immune cells that express one or more of the CARs disclosed herein may be produced by any suitable method(s), including those described herein. The method(s) may utilize one or more successive steps for one or more modifications to cells and / or utilize one or more simultaneous steps for one or more modifications to cells.

[0339] Cells produced by the preparation methods may be frozen. The produced cells may be in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and DMSO. The solution may be sterile, nonpyrogenic, and isotonic.

[0340] Preparation methods of the disclosure may produce a population of immune cells comprising at least or at most about 102-106clonal cells. The method may produce a cell population comprising at least or at most about 106-1012total cells. The produced cell population may be frozen and then thawed. In some cases of the preparation method, the method further comprises introducing one or more additional nucleic acids into the frozen and thawed cell population, such as the one or more additional nucleic acids encoding one or more therapeutic gene products, for example.

[0341] Genetic modification may also be introduced to certain components to generate antigen- and / or ligand-specific immune cells and to model positive and negative selection. These modifications include, for example, transduction of immune cells with a vector encoding an antigen- and / or ligand-specific chimeric antigen receptor (CAR) for the generation of antigen- and / or ligand-specific cells and / or transduction of the immune cell lines with an antigen and / or ligand plus costimulatory molecules or cytokines to enhance the positive selection of CAR immune cells. In specific aspects, the nucleic acid encoding the CARs is298507734.1 - 94 -introduced into the cell using a recombinant vector such as a viral vector including at least a lentivirus, a retrovirus, gamma-retroviruses, an adeno-associated virus (AAV), a herpesvirus, or adenovirus, for example.

[0342] CARs can be achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0343] The expression constructs described herein can also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art (see, e.g., U.S. Pat. Nos.5,399,346, 5,580,859, and 5,589,466). In certain aspects gene therapy vectors are provided.

[0344] The nucleic acid encoding the CAR can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0345] In certain aspects the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.

[0346] Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses (including self- inactivating lentivirus vectors). In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No.6,326,193).

[0347] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some aspects, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one aspect, lentivirus vectors are used.298507734.1 - 95 -

[0348] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements can function either cooperatively or independently to activate transcription.

[0349] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-lalpha (EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Moreover, the constructs are not be limited to the use of constitutive promoters and inducible and / or tissue-specific promoters are also contemplated. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0350] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0351] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the298507734.1 - 96 -expression vector can be transferred into a host cell by physical, chemical, or biological means. Examples of viral vectors include at least retroviral, lentiviral, adenoviral, or adeno-associated viral vectors. Examples of non-viral vectors include at least plasmids, transposons, lipids, nanoparticles, and so forth. One of skill in the art would be well-equipped to construct a vector through standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference) for the expression of the CARs of the present disclosure.

[0352] Biological methods for introducing a polynucleotide of interest into a host cell can include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like (see, e.g. U.S. Pat. Nos.5,350,674 and 5,585,362, and the like).

[0353] In cases wherein the immune cell is transduced with more than one antigen-specific CARs, the CARs may or may not be comprised on or with the same vector. In some cases, the CARs are expressed from the same vector molecule, such as the same viral vector molecule. In such cases, the expression of the CARs may or may not be regulated by the same regulatory element(s). When the CARs are on the same vector, they may or may not be expressed as separate polypeptides. In cases wherein they are expressed as separate polypeptides, they may be separated on the vector by a 2A element or IRES element (or both kinds may be used on the same vector once or more than once), for example.

[0354] In particular aspects, the CARs are expressed from a multicistronic vector (the term “cistron” as used herein refers to a nucleic acid sequence from which a gene product may be produced). In specific aspects, the multicistronic vector encodes the a CAR and a CoCAR. In certain aspects, the present disclosure provides a flexible, modular system (the term “modular” as used herein refers to a cistron or component of a cistron that allows for interchangeability thereof, such as by removal and replacement of an entire cistron or of a component of a cistron, respectively, for example by using standard recombination techniques) utilizing a polycistronic vector having the ability to express multiple cistrons at substantially identical levels. The system may be used for cell engineering allowing for combinatorial expression (including overexpression) of multiple genes. In specific aspects, one or more of the genes expressed by the vector includes one, two, or more CARs or CoCARs. The vector also has aspects wherein multiple of the cistrons are translated and processed into polypeptides, thereby imparting an advantage for the vector to express separate gene products in substantially equimolar298507734.1 - 97 -concentrations. In some cases, cistrons are translated into a separate polypeptides, whereas in other cases multiple of the cistrons are translated into a single polypeptide and cleaved into separate polypeptides. Adjacent cistrons on the vector may be separated by a self cleavage site, such as a 2A self cleavage site. When 2A cleavages sites are utilized in the vector, the 2A cleavage site may comprise a P2A, T2A, E2A and / or F2A site. In some cases each of the cistrons express separate polypeptides from the vector. In some cases, adjacent cistrons on the vector are separated by an IRES element. This system may be applied to viral and non-viral vectors, including but not limited lentivirus, adenovirus AAV, as well as non-viral plasmids.

[0355] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An illustrative colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0356] In the case where a non-viral delivery system is utilized, one illustrative delivery vehicle is a lipid and / or a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro , ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0357] In various aspects lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl298507734.1 - 98 -phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform can be used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al. (1991) Glycobiology 5: 505-510). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0358] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.

[0359] The methods may further comprise culturing cells in media prior to introducing one or more nucleic acids into the cells. The culturing may comprise incubating the selected cells with medium comprising one or more growth factors, in some cases, and the one or more growth factors may comprise c-kit ligand, flt-3 ligand, and / or human thrombopoietin (TPO), for example. The growth factors may or may not be at a certain concentration, such as between about 5 ng / ml to about 500 ng / ml.

[0360] In manufacturing the engineered immune cells of the present disclosure, the cells may be present in a particular serum-free medium. In specific aspects, the serum-free medium further comprises externally added FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, midkine, or combinations thereof. The serum-free medium may298507734.1 - 99 -further comprise vitamins, including biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or combinations thereof or salts thereof. The serum-free medium may further comprise one or more externally added (or not) proteins, such as albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. The serum-free medium may further comprise corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. The serum-free medium may comprise a B-27®supplement, xeno-free B-27®supplement, GS21TMsupplement, or combinations thereof. Amino acids (including arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof), monosaccharides, and / or inorganic ions (including sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof, for example) may be present in the serum-free medium. The serum-free medium may further comprise molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof.

[0361] Cell culture conditions may be provided for the culture of immune cells described herein and for the production of immune cells, for example, T cells and NK cells, expressing one or more CARs disclosed herein and / or positive / negative selection thereof. In certain aspects, starting cells of a selected population may comprise at least, at most, or about 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013cells or any range derivable therein. The starting cell population may have a seeding density of at least, at most, or about 10, 101, 102, 103, 104, 105, 106, 107, 108cells / ml, or any range derivable therein.

[0362] A culture vessel used for culturing the 3D cell aggregates or progeny cells thereof can include, but is particularly not limited to: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK® Chambers, culture bag, and roller bottle, as long as it is capable of culturing the stem cells therein. The stem cells may be cultured in a volume of at least, at most, or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture. In a certain aspect, the culture vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment. The bioreactor may have a volume of at least, at most, or about 2, 4, 5, 6,298507734.1 - 100 -8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.

[0363] The culture vessel can be cellular adhesive or non-adhesive and selected depending on the purpose. The cellular adhesive culture vessel can be coated with any of substrates for cell adhesion such as extracellular matrix (ECM) to improve the adhesiveness of the vessel surface to the cells. The substrate for cell adhesion can be any material intended to attach stem cells or feeder cells (if used). The substrate for cell adhesion includes collagen, gelatin, poly- L-lysine, poly-D-lysine, laminin, and fibronectin and mixtures thereof for example MatrigelTM, and lysed cell membrane preparations.

[0364] Various defined matrix components may be used in the culturing methods or compositions. For example, recombinant collagen IV, fibronectin, laminin, and vitronectin in combination may be used to coat a culturing surface as a means of providing a solid support for pluripotent cell growth, as described in Ludwig et al. (2006a; 2006b), which are incorporated by reference in its entirety.

[0365] A matrix composition may be immobilized on a surface to provide support for cells. The matrix composition may include one or more extracellular matrix (ECM) proteins and an aqueous solvent. The term “extracellular matrix” is recognized in the art. Its components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. Other extracellular matrix proteins are described in Kleinman et al., (1993), herein incorporated by reference. It is intended that the term “extracellular matrix” encompass a presently unknown extracellular matrix that may be discovered in the future, since its characterization as an extracellular matrix will be readily determinable by persons skilled in the art.

[0366] In some aspects, the total protein concentration in the matrix composition may be about 1 ng / mL to about 1 mg / mL. In some aspects, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In more preferred aspects, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0367] The extracellular matrix (ECM) proteins may be of natural origin and purified from human or animal tissues. Alternatively, the ECM proteins may be genetically engineered recombinant proteins or synthetic in nature. The ECM proteins may be a whole protein or in the form of peptide fragments, native or engineered. Examples of ECM protein that may be useful in the matrix for cell culture include laminin, collagen I, collagen IV, fibronectin and298507734.1 - 101 -vitronectin. In some aspects, the matrix composition includes synthetically generated peptide fragments of fibronectin or recombinant fibronectin.

[0368] In still further aspects, the matrix composition includes a mixture of at least fibronectin and vitronectin. In some other aspects, the matrix composition preferably includes laminin.

[0369] The matrix composition preferably includes a single type of extracellular matrix protein. In some aspects, the matrix composition includes fibronectin, particularly for use with culturing progenitor cells. For example, a suitable matrix composition may be prepared by diluting human fibronectin, such as human fibronectin sold by Becton, Dickinson & Co. of Franklin Lakes, N.J. (BD) (Cat#354008), in Dulbecco’s phosphate buffered saline (DPBS) to a protein concentration of 5 μg / mL to about 200 μg / mL. In a particular example, the matrix composition includes a fibronectin fragment, such as RetroNectin®. RetroNectin® is a ~63 kDa protein of (574 amino acids) that contains a central cell-binding domain (type III repeat, 8,9,10), a high affinity heparin-binding domain II (type III repeat, 12,13,14), and CS1 site within the alternatively spliced IIICS region of human fibronectin.

[0370] In some other aspects, the matrix composition may include laminin. For example, a suitable matrix composition may be prepared by diluting laminin (Sigma-Aldrich (St. Louis, Mo.); Cat#L6274 and L2020) in Dulbecco’s phosphate buffered saline (DPBS) to a protein concentration of 5 μg / ml to about 200 μg / ml.

[0371] In some aspects, the matrix composition is xeno-free, in that the matrix is or its component proteins are only of human origin. This may be desired for certain research applications. For example, in the xeno-free matrix to culture human cells, matrix components of human origin may be used, wherein any non-human animal components may be excluded. In certain aspects, MatrigelTMmay be excluded as a substrate from the culturing composition. MatrigelTMis a gelatinous protein mixture secreted by mouse tumor cells and is commercially available from BD Biosciences (New Jersey, USA). This mixture resembles the complex extracellular environment found in many tissues and is used frequently by cell biologists as a substrate for cell culture, but it may introduce undesired xeno antigens or contaminants.

[0372] Isolation of immune cells include any selection methods, including cell sorters, magnetic separation using antibody-coated magnetic beads, packed columns; affinity chromatography; cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including but not limited to, complement and cytotoxins; and “panning” with antibody attached to a solid matrix, e.g., plate, or any other convenient technique.298507734.1 - 102 -

[0373] The use of separation or isolation techniques include, but are not limited to, those based on differences in physical (density gradient centrifugation and counter-flow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondria-binding dye rho123 and DNA-binding dye Hoechst 33342). Techniques providing accurate separation include but are not limited to, FACS (Fluorescence-activated cell sorting) or MACS (Magnetic-activated cell sorting), which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.

[0374] The antibodies utilized in the preceding techniques or techniques used to assess cell type purity (such as flow cytometry) can be conjugated to identifiable agents including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorochromes, metal compounds, radioactive compounds, drugs or haptens. The enzymes that can be conjugated to the antibodies include, but are not limited to, alkaline phosphatase, peroxidase, urease, and β- galactosidase. The fluorochromes that can be conjugated to the antibodies include, but are not limited to, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin, allophycocyanins and Texas Red. For additional fluorochromes that can be conjugated to antibodies, see Haugland, Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals (1992-1994). The metal compounds that can be conjugated to the antibodies include, but are not limited to, ferritin, colloidal gold, and particularly, colloidal superparamagnetic beads. The haptens that can be conjugated to the antibodies include, but are not limited to, biotin, digoxygenin, oxazalone, and nitrophenol. The radioactive compounds that can be conjugated or incorporated into the antibodies are known to the art, and include but are not limited to technetium 99m (99TC), 125I and amino acids comprising any radionuclides, including, but not limited to, 14C, 3H and 35S.

[0375] Other techniques for positive selection may be employed, which permit accurate separation, such as affinity columns, and the like. The method should permit the removal to a residual amount of less than about 20%, preferably less than about 5%, of the non-target cell populations.

[0376] Cells may be selected based on light-scatter properties as well as their expression of various cell surface antigens. The purified stem cells have low side scatter and low to medium forward scatter profiles by FACS analysis. Cytospin preparations show the enriched stem cells to have a size between mature lymphoid cells and mature granulocytes.

[0377] Various techniques may be employed to separate the cells by initially removing cells of dedicated lineage. Monoclonal antibodies are particularly useful for identifying298507734.1 - 103 -markers associated with particular cell lineages and / or stages of differentiation. The antibodies may be attached to a solid support to allow for crude separation. The separation techniques employed should maximize the retention of viability of the fraction to be collected. Various techniques of different efficacy may be employed to obtain “relatively crude” separations. Such separations are where up to 10%, usually not more than about 5%, preferably not more than about 1%, of the total cells present are undesired cells that remain with the cell population to be retained. The particular technique employed will depend upon efficiency of separation, associated cytotoxicity, ease and speed of performance, and necessity for sophisticated equipment and / or technical skill.

[0378] Selection of the progenitor cells need not be achieved solely with a marker specific for the cells. By using a combination of negative selection and positive selection, enriched cell populations can be obtained.

[0379] In certain aspects, cells containing an exogenous nucleic acid may be identified in vitro or in vivo by including a marker in the expression vector or the exogenous nucleic acid. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selection marker may be one that confers a property that allows for selection. A positive selection marker may be one in which the presence of the marker allows for its selection, while a negative selection marker is one in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0380] Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes as negative selection markers such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selection and screenable markers are well known to one of skill in the art.

[0381] Selectable markers may include a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a298507734.1 - 104 -transfection or other procedure meant to introduce foreign DNA into a cell. Selectable markers are often antibiotic resistance genes; cells that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those cells that can grow have successfully taken up and expressed the introduced genetic material. Examples of selectable markers include: the Abicr gene or Neo gene from Tn5, which confers antibiotic resistance to geneticin.

[0382] A screenable marker may comprise a reporter gene, which allows the researcher to distinguish between wanted and unwanted cells. Certain aspects of the present disclosure utilize reporter genes to indicate specific cell lineages. For example, the reporter gene can be located within expression elements and under the control of the ventricular- or atrial-selective regulatory elements normally associated with the coding region of a ventricular- or atrial- selective gene for simultaneous expression. A reporter allows the cells of a specific lineage to be isolated without placing them under drug or other selective pressures or otherwise risking cell viability.

[0383] Examples of such reporters include genes encoding cell surface proteins (e.g., CD4, HA epitope), fluorescent proteins, antigenic determinants, and enzymes (e.g., β-galactosidase). The vector containing cells may be isolated, e.g., by FACS using fluorescently-tagged antibodies to the cell surface protein or substrates that can be converted to fluorescent products by a vector encoded enzyme.

[0384] In specific aspects, the reporter gene is a fluorescent protein. A broad range of fluorescent protein genetic variants have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum. Mutagenesis efforts in the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes that range in color from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone, Discosoma striata, and reef corals belonging to the class Anthozoa. Still other species have been mined to produce similar proteins having cyan, green, yellow, orange, and deep red fluorescence emission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness.

[0385] The cells in certain aspects can be made to contain one or more genetic alterations by genetic engineering of the cells either before or after differentiation (US 2002 / 0168766). A cell is said to be “genetically altered”, “genetically modified” or “transgenic” when an exogenous nucleic acid or polynucleotide has been transferred into the cell by any suitable298507734.1 - 105 -means of artificial manipulation, or where the cell is a progeny of the originally altered cell that has inherited the polynucleotide. For example, the cells can be processed to increase their replication potential by genetically altering the cells to express telomerase reverse transcriptase, either before or after they progress to restricted developmental lineage cells or terminally differentiated cells (U.S. Patent Application Publication 2003 / 0022367).

[0386] In aspects wherein cells are genetically modified, such as to add or reduce one or more features, the genetic modification may occur by any suitable method. For example, any genetic modification compositions or methods may be used to introduce exogenous nucleic acids into cells or to edit the genomic DNA, such as gene editing, homologous recombination or non-homologous recombination, RNA-mediated genetic delivery, or any conventional nucleic acid delivery methods. Non-limiting examples of the genetic modification methods may include gene editing methods such as by CRISPR / CAS9, zinc finger nuclease, or TALEN technology.

[0387] Genetic modification may also include the introduction of a selectable or screenable marker that aid selection or screen or imaging in vitro or in vivo. Particularly, in vivo imaging agents or suicide genes may be expressed exogenously or added to starting cells or progeny cells. In further aspects, the methods may involve image-guided adoptive cell therapy. VI. Methods of Using the Cells

[0388] The immune cells of the disclosure may or may not be utilized directly after production. In some cases they are stored for later purpose. In any event, they may be utilized in therapeutic or preventative applications for a mammalian subject (human, dog, cat, horse, etc.) such as a patient. The individual may be in need of cell therapy for a medical condition of any kind, including allogeneic cell therapy.

[0389] Methods of treating an individual with a therapeutically effective amount of immune cells of the disclosure comprise administering the cells or clonal populations thereof to the patient. The cells or cell populations may be allogeneic with respect to the patient. The individual does not exhibit signs of depletion of the cells or cell population, in particular aspects. The individual may or may not have cancer. In specific aspects wherein the individual has cancer, tumor cells of the cancer patient are killed after administering the cells or cell population to the individual.

[0390] One example of a therapeutic method for treating a subject for cancer comprises administering to a subject a therapeutically effective amount of an immune cell or population thereof disclosed herein. In some aspects, a polynucleotide encoding two CARs, the first CAR298507734.1 - 106 -comprising a CD4 or CD8 intracellular domain, and the second CAR comprising stimulatory and costimulatory domain(s) (e.g., 4-1BB or CD28 co-stimulatory domains and / or CD3ζ stimulatory domain), has been introduced into the immune cell(s), and administration of a therapeutically effective amount of the immune cell(s) decreases cancer or tumor burden or increases survival of the subject.

[0391] The present immune cells including co-expressed CAR molecules or compositions thereof can induce CAR signaling that more closely mimics TCR signaling and can improve CAR efficacy in cases of downregulation of target antigen, for example, upon relapse in a cancer patient. The compositions can be useful in methods for treating a subject for cancer, killing cancer cells in a subject, stimulating an immune response to cancer cells in a subject, and generating a persisting population of genetically engineered immune cells. The immune cells including co-expressed CAR molecules or compositions thereof provided herein can provide greater beneficial effects for treating cancer as compared to other commercially or clinically available CAR therapies. For example, the immune cells including co-expressed CAR molecules or compositions thereof can i...

Claims

CLAIMS 1. A composition comprising two chimeric antigen receptors (CARs), wherein: the first CAR comprises: an antigen-binding domain that binds a first epitope on an antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; and an intracellular domain from CD4 or CD8, and the second CAR comprises: an antigen-binding domain that binds a second epitope on the antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; a 4-1BB or CD28 co-stimulatory domain; and a CD3ζ stimulatory domain.

2. A composition comprising two chimeric antigen receptors (CARs), wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

3. A composition comprising two chimeric antigen receptors (CARs), wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD4; and an intracellular domain from CD4, and the second CAR comprises:298507734.1 - 135 -a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

4. A composition comprising two chimeric antigen receptors (CARs), wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD28; a transmembrane domain from CD28; and an intracellular domain from CD4, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

5. A composition comprising two chimeric antigen receptors (CARs), wherein: the first CAR comprises: a BCMA-binding domain that binds a first epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8, and the second CAR comprises: a BCMA-binding domain that binds a second epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

6. The composition of any one of claims 1-5, wherein the antigen-binding domain of the first CAR comprises a first antibody or antigen-binding fragment thereof, a nanobody, an scFv,298507734.1 - 136 -a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally- occurring receptor-ligand pair that targets the antigen.

7. The composition of any one of claims 1-6, wherein the antigen-binding domain of the second CAR comprises a second antibody or antigen-binding fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen.

8. The composition of any one of claims 1-7, wherein the antigen is expressed by a cancer cell.

9. The composition of any one of claims 1-8, wherein the antigen is CD22, CD19, BCMA, HER2, or mesothelin.

10. The composition of any one of claims 1-9, wherein the hinge of the first CAR and / or the second CAR is a CD4 hinge.

11. The composition of any one of claims 1-9, wherein the hinge of the first CAR and / or the second CAR is a CD8 hinge.

12. The composition of any one of claims 1-9, wherein the hinge of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

5.

13. The composition of any one of claims 1-9, wherein the hinge of the first CAR and / or the second CAR comprises SEQ ID NO:

5.

14. The composition of any one of claims 1-9, wherein the hinge of the first CAR and / or the second CAR consists of SEQ ID NO:

5.

15. The composition of any one of claims 1-9, wherein the hinge of the first CAR and / or the second CAR is a CD28 hinge.

16. The composition of any one of claims 1-9, wherein the hinge of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6.298507734.1 - 137 -17. The composition of any one of claims 1-9, wherein the hinge of the first CAR and / or the second CAR comprises SEQ ID NO:

6.

18. The composition of any one of claims 1-9, wherein the hinge of the first CAR and / or the second CAR consists of SEQ ID NO:

6.

19. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD4 transmembrane domain.

20. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

7.

21. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:

7.

22. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:

7.

23. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD8 transmembrane domain.

24. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

8.

25. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:

8.

26. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:

8.

27. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD28 transmembrane domain.

28. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:9.298507734.1 - 138 -29. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:

9.

30. The composition of any one of claims 1-18, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:

9.

31. The composition of any one of claims 1-30, wherein the intracellular domain of the first CAR is a CD4 intracellular domain.

32. The composition of any one of claims 1-30, wherein the intracellular domain of the first CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

10.

33. The composition of any one of claims 1-30, wherein the intracellular domain of the first CAR comprises SEQ ID NO:

10.

34. The composition of any one of claims 1-30, wherein the intracellular domain of the first CAR consists of SEQ ID NO:

10.

35. The composition of any one of claims 1-30, wherein the intracellular domain of the first CAR is a CD8 intracellular domain.

36. The composition of any one of claims 1-30, wherein the intracellular domain comprises of the first CAR an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

11.

37. The composition of any one of claims 1-30, wherein the intracellular domain of the first CAR comprises SEQ ID NO:

11.

38. The composition of any one of claims 1-30, wherein the intracellular domain of the first CAR consists of SEQ ID NO:

11.

39. The composition of any one of claims 1-38, wherein the first CAR further comprises a signal peptide.

40. The composition of any one of claims 1-39, wherein the first CAR further comprises a signal peptide comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:30.298507734.1 - 139 -41. The composition of any one of claims 1-40, wherein the first CAR further comprises a signal peptide comprising SEQ ID NO:

30.

42. The composition of any one of claims 1-41, wherein the first CAR further comprises a signal peptide consisting of SEQ ID NO:

30.

43. The composition of any one of claims 1-42, wherein the first CAR further comprises an epitope tag.

44. The composition of any one of claims 1-43, wherein the first CAR further comprises an epitope tag comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:31 or SEQ ID NO:

32.

45. The composition of any one of claims 1-44, wherein the first CAR further comprises an epitope tag comprising SEQ ID NO:31 or SEQ ID NO:

32.

46. The composition of any one of claims 1-45, wherein the first CAR further comprises an epitope tag consisting of SEQ ID NO:31 or SEQ ID NO:

32.

47. The composition of any one of claims 1-45, wherein the first CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:

34.

48. The composition of any one of claims 1-45, wherein the first CAR comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:

34.

49. The composition of any one of claims 1-45, wherein the first CAR consists of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:

34.

50. The composition of any one of claims 1-49, wherein the 4-1BB co-stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

12.

51. The composition of any one of claims 1-49, wherein the 4-1BB co-stimulatory domain of the second CAR comprises SEQ ID NO:

12.

52. The composition of any one of claims 1-49, wherein the 4-1BB co-stimulatory domain of the second CAR consists of SEQ ID NO:12.298507734.1 - 140 -53. The composition of any one of claims 1-49, wherein the CD28 co-stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

13.

54. The composition of any one of claims 1-49, wherein the CD28 co-stimulatory domain of the second CAR comprises SEQ ID NO:

13.

55. The composition of any one of claims 1-49, wherein the CD28 co-stimulatory domain of the second CAR consists of SEQ ID NO:

13.

56. The composition of any one of claims 1-55, wherein the CD3ζ stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

14.

57. The composition of any one of claims 1-55, wherein the CD3ζ stimulatory domain of the second CAR comprises SEQ ID NO:

14.

58. The composition of any one of claims 1-55, wherein the CD3ζ stimulatory domain of the second CAR consists of SEQ ID NO:

14.

59. The composition of any one of claims 1-58, wherein the second CAR further comprises a signal peptide.

60. The composition of any one of claims 1-59, wherein the second CAR further comprises a signal peptide comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

30.

61. The composition of any one of claims 1-60, wherein the second CAR further comprises a signal peptide comprising SEQ ID NO:

30.

62. The composition of any one of claims 1-61, wherein the second CAR further comprises a signal peptide consisting of SEQ ID NO:

30.

63. The composition of any one of claims 1-62, wherein the second CAR further comprises an epitope tag.298507734.1 - 141 -64. The composition of any one of claims 1-63, wherein the second CAR further comprises an epitope tag comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:31 or SEQ ID NO:

32.

65. The composition of any one of claims 1-64, wherein the second CAR further comprises an epitope tag comprising SEQ ID NO:31 or SEQ ID NO:

32.

66. The composition of any one of claims 1-65, wherein the second CAR further comprises an epitope tag consisting of SEQ ID NO:31 or SEQ ID NO:

32.

67. The composition of any one of claims 1-66, wherein the second CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:

33.

68. The composition of any one of claims 1-66, wherein the second CAR comprises SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:

33.

69. The composition of any one of claims 1-66, wherein the second CAR consists of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:

33.

70. The composition of any one of claims 1-69, wherein the first and the second CAR together comprise an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

35.

71. The composition of any one of claims 1-69, wherein the first and the second CAR together comprise SEQ ID NO:

35.

72. The composition of any one of claims 1-69, wherein the first and the second CAR together consist of SEQ ID NO:

35.

73. The composition as in any one of claims 1-72, wherein the first and / or the second CAR are encoded by one or more polynucleotides.

74. The composition as in any one of claims 1-73, wherein the first and the second CAR are encoded by the same polynucleotide.298507734.1 - 142 -75. The composition of any one of claims 1-74, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a 2A sequence.

76. The composition of any one of claims 1-74, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence.

77. The composition of any one of claims 1-74, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

29.

78. The composition of any one of claims 1-74, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence comprising SEQ ID NO:

29.

79. The composition of any one of claims 1-74, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence consisting of SEQ ID NO:

29.

80. The composition of any one of claims 1-79, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

35.

81. The composition of any one of claims 1-79, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence comprising SEQ ID NO:

35.

82. The composition of any one of claims 1-79, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence consisting of SEQ ID NO:

35.

83. The composition as in any one of claims 1-69, wherein the first and / or the second CAR are encoded by different polynucleotides.298507734.1 - 143 -84. The composition of any one of claims 73-83, wherein the one or more polynucleotides are comprised in one or more vectors.

85. The composition of claim 84, wherein the one or more vectors are viral vectors.

86. The composition of claim 85, wherein the viral vectors are adenoviral vectors, adeno- associated viral vectors, lentiviral vectors, retroviral vectors, or a combination thereof.

87. The composition of claim 84, wherein the one or more vectors are non-viral vectors.

88. The composition of claim 87, wherein the non-viral vectors are plasmids.

89. The composition of any one of claims 84-88, wherein the vectors are multicistronic.

90. The composition of any one of claims 84-89, wherein the one or more vectors are comprised in a cell or a population of cells.

91. The composition of claim 90, wherein the cell or cells express the first and / or the second CAR encoded by one or more polynucleotides comprised in the one or more vectors.

92. The composition of claim 90 or 91, wherein the cell or cells are immune cells.

93. The composition of any one of claims 90-92, wherein the cell or cells are T cells, natural killer (NK) cells, macrophages, microglial cells, or neutrophils.

94. The composition of any one of claims 90-93, wherein the cell or cells are T cells.

95. The composition of any one of claims 90-93, wherein the cell or cells are NK cells.

96. The composition of any one of claims 90-95, wherein the cell or cells are human cells.

97. The composition of any one of claims 1-96, further comprising a pharmaceutically- acceptable excipient.

98. The composition of any one of claims 1-97, further comprising an additional therapeutic.

99. The composition of claim 98, wherein the additional therapeutic is a chemotherapeutic or an immunotherapeutic.298507734.1 - 144 -100. A method for generating the first CAR and / or the second CAR of the composition of any one of claims 1-99, the method comprising: (a) providing one or more polynucleotides encoding the first CAR and / or the second CAR to a cell or cells; and (b) subjecting the cell or cells to conditions sufficient to express the first CAR and / or the second CAR from the one or more polynucleotides.

101. An immune cell expressing two chimeric antigen receptors (CARs), wherein: the first CAR comprises: an antigen-binding domain that binds a first epitope on an antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; and an intracellular domain from CD4 or CD8, and the second CAR comprises: an antigen-binding domain that binds a second epitope on the antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; a 4-1BB or CD28 co-stimulatory domain; and a CD3ζ stimulatory domain.

102. An immune cell expressing two chimeric antigen receptors (CARs), wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

103. An immune cell expressing two chimeric antigen receptors (CARs), wherein: the first CAR comprises:298507734.1 - 145 -a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD4; and an intracellular domain from CD4, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

104. An immune cell expressing two chimeric antigen receptors (CARs), wherein: the first CAR comprises: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD28; a transmembrane domain from CD28; and an intracellular domain from CD4, and the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

105. An immune cell expressing two chimeric antigen receptors (CARs), wherein: the first CAR comprises: a BCMA-binding domain that binds a first epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8, and the second CAR comprises: a BCMA-binding domain that binds a second epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8;298507734.1 - 146 -a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

106. The immune cell of any one of claims 101-105, wherein the antigen-binding domain of the first CAR comprises a first antibody or antigen-binding fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen.

107. The immune cell of any one of claims 101-106, wherein the antigen-binding domain of the second CAR comprises a second antibody or antigen-binding fragment thereof, a nanobody, an scFv, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen.

108. The immune cell of any one of claims 101-107, wherein the antigen is expressed by a cancer cell.

109. The immune cell of any one of claims 101-108, wherein the antigen is CD22, CD19, BCMA, HER2, or mesothelin.

110. The immune cell of any one of claims 101-109, wherein the hinge of the first CAR and / or the second CAR is a CD4 hinge.

111. The immune cell of any one of claims 101-109, wherein the hinge of the first CAR and / or the second CAR is a CD8 hinge.

112. The immune cell of any one of claims 101-109, wherein the hinge of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

5.

113. The immune cell of any one of claims 101-109, wherein the hinge of the first CAR and / or the second CAR comprises SEQ ID NO:

5.

114. The immune cell of any one of claims 101-109, wherein the hinge of the first CAR and / or the second CAR consists of SEQ ID NO:

5.

115. The immune cell of any one of claims 101-109, wherein the hinge of the first CAR and / or the second CAR is a CD28 hinge.298507734.1 - 147 -116. The immune cell of any one of claims 101-109, wherein the hinge of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

6.

117. The immune cell of any one of claims 101-109, wherein the hinge of the first CAR and / or the second CAR comprises SEQ ID NO:

6.

118. The immune cell of any one of claims 101-109, wherein the hinge of the first CAR and / or the second CAR consists of SEQ ID NO:

6.

119. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD4 transmembrane domain.

120. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

7.

121. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:

7.

122. The immune cell of claim 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:

7.

123. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD8 transmembrane domain.

124. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

8.

125. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:

8.

126. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:

8.

127. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR is a CD28 transmembrane domain.298507734.1 - 148 -128. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

9.

129. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR comprises SEQ ID NO:

9.

130. The immune cell of any one of claims 101-118, wherein the transmembrane domain of the first CAR and / or the second CAR consists of SEQ ID NO:

9.

131. The immune cell of any one of claims 101-130, wherein the intracellular domain of the first CAR is a CD4 intracellular domain.

132. The immune cell of any one of claims 101-130, wherein the intracellular domain of the first CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

10.

133. The immune cell of any one of claims 101-130, wherein the intracellular domain of the first CAR comprises SEQ ID NO:

10.

134. The immune cell of any one of claims 101-130, wherein the intracellular domain of the first CAR consists of SEQ ID NO:

10.

135. The immune cell of any one of claims 101-130, wherein the intracellular domain of the first CAR is a CD8 intracellular domain.

136. The immune cell of any one of claims 101-130, wherein the intracellular domain comprises of the first CAR an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

11.

137. The immune cell of any one of claims 101-130, wherein the intracellular domain of the first CAR comprises SEQ ID NO:

11.

138. The immune cell of any one of claims 101-130, wherein the intracellular domain of the first CAR consists of SEQ ID NO:

11.

139. The immune cell of any one of claims 101-138, wherein the first CAR further comprises a signal peptide.298507734.1 - 149 -140. The immune cell of any one of claims 101-139, wherein the first CAR further comprises a signal peptide comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

30.

141. The immune cell of any one of claims 101-140, wherein the first CAR further comprises a signal peptide comprising SEQ ID NO:

30.

142. The immune cell of any one of claims 101-141, wherein the first CAR further comprises a signal peptide consisting of SEQ ID NO:

30.

143. The immune cell of any one of claims 101-142, wherein the first CAR further comprises an epitope tag.

144. The immune cell of any one of claims 101-143, wherein the first CAR further comprises an epitope tag comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:31 or SEQ ID NO:

32.

145. The immune cell of any one of claims 101-144, wherein the first CAR further comprises an epitope tag comprising SEQ ID NO:31 or SEQ ID NO:

32.

146. The immune cell of any one of claims 101-145, wherein the first CAR further comprises an epitope tag consisting of SEQ ID NO:31 or SEQ ID NO:

32.

147. The immune cell of any one of claims 101-146, wherein the first CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:

34.

148. The immune cell of any one of claims 101-146, wherein the first CAR comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:

34.

149. The immune cell of any one of claims 101-146, wherein the first CAR consists of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:

34.

150. The immune cell of any one of claims 101-149, wherein the 4-1BB co-stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12.298507734.1 - 150 -151. The immune cell of any one of claims 101-149, wherein the 4-1BB co-stimulatory domain of the second CAR comprises SEQ ID NO:

12.

152. The immune cell of any one of claims 101-149, wherein the 4-1BB co-stimulatory domain of the second CAR consists of SEQ ID NO:

12.

153. The immune cell of any one of claims 101-149, wherein the CD28 co-stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

13.

154. The immune cell of any one of claims 101-149, wherein the CD28 co-stimulatory domain of the second CAR comprises SEQ ID NO:

13.

155. The immune cell of any one of claims 101-149, wherein the CD28 co-stimulatory domain of the second CAR consists of SEQ ID NO:

13.

156. The immune cell of any one of claims 101-155, wherein the CD3ζ stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

14.

157. The immune cell of any one of claims 101-155, wherein the CD3ζ stimulatory domain of the second CAR comprises SEQ ID NO:

14.

158. The immune cell of any one of claims 101-155, wherein the CD3ζ stimulatory domain of the second CAR consists of SEQ ID NO:

14.

159. The immune cell of any one of claims 101-158, wherein the second CAR further comprises a signal peptide.

160. The immune cell of any one of claims 101-159, wherein the second CAR further comprises a signal peptide comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

30.

161. The immune cell of any one of claims 101-160, wherein the second CAR further comprises a signal peptide comprising SEQ ID NO:

30.

162. The immune cell of any one of claims 101-161, wherein the second CAR further comprises a signal peptide consisting of SEQ ID NO:30.298507734.1 - 151 -163. The immune cell of any one of claims 101-162, wherein the second CAR further comprises an epitope tag.

164. The immune cell of any one of claims 101-163, wherein the second CAR further comprises an epitope tag comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:31 or SEQ ID NO:

32.

165. The immune cell of any one of claims 101-164, wherein the second CAR further comprises an epitope tag comprising SEQ ID NO:31 or SEQ ID NO:

32.

166. The immune cell of any one of claims 101-165, wherein the second CAR further comprises an epitope tag consisting of SEQ ID NO:31 or SEQ ID NO:

32.

167. The immune cell of any one of claims 101-166, wherein the second CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:

33.

168. The immune cell of any one of claims 101-166, wherein the second CAR comprises SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:

33.

169. The immune cell of any one of claims 101-166, wherein the second CAR consists of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:

33.

170. The immune cell of any one of claims 101-169, wherein the first and the second CAR together comprise an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

35.

171. The immune cell of any one of claims 101-169, wherein the first and the second CAR together comprise SEQ ID NO:

35.

172. The immune cell of any one of claims 101-169, wherein the first and the second CAR together consist of SEQ ID NO:

35.

173. The immune cell as in any one of claims 101-172, wherein the first and / or the second CAR are encoded by one or more polynucleotides.

174. The immune cell as in any one of claims 101-173, wherein the first CAR and the second CAR are encoded by the same polynucleotide.298507734.1 - 152 -175. The immune cell of any one of claims 101-174, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a 2A sequence.

176. The immune cell of any one of claims 101-174, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence.

177. The immune cell of any one of claims 101-174, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

29.

178. The immune cell of any one of claims 101-174, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence comprising SEQ ID NO:

29.

179. The immune cell of any one of claims 101-174, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence consisting of SEQ ID NO:

29.

180. The immune cell of any one of claims 101-179, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

35.

181. The immune cell of any one of claims 101-179, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence comprising SEQ ID NO:

35.

182. The immune cell of any one of claims 101-182, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence consisting of SEQ ID NO:

35.

183. The immune cell as in any one of claims 101-169, wherein the first CAR and the second CAR are encoded by different polynucleotides.298507734.1 - 153 -184. The immune cell of any one of claims 173-183, wherein the one or more polynucleotides are comprised in one or more vectors.

185. The immune cell of claim 184, wherein the one or more vectors are viral vectors.

186. The immune cell of claim 185, wherein the viral vectors are adenoviral vectors, adeno- associated viral vectors, lentiviral vectors, retroviral vectors, or a combination thereof.

187. The immune cell of claim 184, wherein the one or more vectors are non-viral vectors.

188. The immune cell of claim 187, wherein the non-viral vectors are plasmids.

189. The immune cell of any one of claims 184-188, wherein the vectors are multicistronic.

190. The immune cell of any one of claims 184-189, wherein the immune cell expresses the first and / or the second CAR encoded by one or more polynucleotides comprised in the one or more vectors.

191. The immune cell of any one of claims 101-190, wherein the immune cell is a T cell, natural killer (NK) cell, macrophage, microglial cell, or neutrophil.

192. The immune cell of any one of claims 101-191, wherein the immune cell is a T cell.

193. The immune cell of any one of claims 101-191, wherein the immune cell is a NK cell.

194. The composition of any one of claims 101-193, wherein the immune cell is a human cell.

195. A composition comprising: (a) the immune cell of any one of claims 101-194; and (b) a pharmaceutically acceptable excipient.

196. The composition of claim 195, wherein the pharmaceutical composition further comprises an additional therapeutic.

197. The composition of claim 196, wherein the additional therapeutic is a chemotherapeutic or an immunotherapeutic.

198. A chimeric antigen receptor (CAR) comprising:298507734.1 - 154 -an antigen-binding domain that binds a first epitope on an antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; and an intracellular domain from CD4 or CD8.

199. A chimeric antigen receptor (CAR) comprising: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8.

200. A chimeric antigen receptor (CAR) comprising: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD8; a transmembrane domain from CD4; and an intracellular domain from CD4.

201. A chimeric antigen receptor (CAR) comprising: a CD22-binding domain that binds a first epitope on CD22; a hinge from CD28; a transmembrane domain from CD28; and an intracellular domain from CD4.

202. A chimeric antigen receptor (CAR) comprising: a BCMA-binding domain that binds a first epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; and an intracellular domain from CD8.

203. The CAR of any one of claims 198-202, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment thereof, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally-occurring receptor-ligand pair that targets the antigen.

204. The CAR of any one of claims 198-203, wherein the antigen-binding domain comprises an scFv or a nanobody.298507734.1 - 155 -205. The CAR of any one of claims 198-204, wherein the antigen is expressed by a cancer cell.

206. The CAR of any one of claims 198-205, wherein the antigen is CD22, CD19, BCMA, HER2, or mesothelin.

207. The CAR of any one of claims 198-206, wherein the hinge is a CD4 hinge.

208. The CAR of any one of claims 198-206, wherein the hinge of the first CAR and / or the second CAR is a CD8 hinge.

209. The CAR of any one of claims 198-206, wherein the hinge comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

5.

210. The CAR of any one of claims 198-206, wherein the comprises SEQ ID NO:

5.

211. The CAR of any one of claims 198-206, wherein the hinge consists of SEQ ID NO:

5.

212. The CAR of any one of claims 198-206, wherein the hinge is a CD28 hinge.

213. The CAR of any one of claims 198-206, wherein the hinge comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

6.

214. The CAR of any one of claims 198-206, wherein the hinge comprises SEQ ID NO:

6.

215. The CAR of any one of claims 198-206, wherein the hinge consists of SEQ ID NO:

6.

216. The CAR of any one of claims 198-215, wherein the transmembrane domain is a CD4 transmembrane domain.

217. The CAR of any one of claims 198-215, wherein the transmembrane domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

7.

218. The CAR of any one of claims 198-215, wherein the transmembrane domain comprises SEQ ID NO:

7.

219. The CAR of any one of claims 198-215, wherein the transmembrane domain consists of SEQ ID NO:7.298507734.1 - 156 -220. The CAR of any one of claims 198-215, wherein the transmembrane domain is a CD8 transmembrane domain.

221. The CAR of any one of claims 198-215, wherein the transmembrane domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

8.

222. The CAR of any one of claims 198-215, wherein the transmembrane domain comprises SEQ ID NO:

8.

223. The CAR of any one of claims 198-215, wherein the transmembrane domain consists of SEQ ID NO:

8.

224. The CAR of any one of claims 198-215, wherein the transmembrane domain is a CD28 transmembrane domain.

225. The CAR of any one of claims 198-215, wherein the transmembrane domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

9.

226. The CAR of any one of claims 198-215, wherein the transmembrane domain comprises SEQ ID NO:

9.

227. The CAR of any one of claims 198-215, wherein the transmembrane domain consists of SEQ ID NO:

9.

228. The CAR of any one of claims 198-227, wherein the intracellular domain is a CD4 intracellular domain.

229. The CAR of any one of claims 198-227, wherein the intracellular domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

10.

230. The CAR of any one of claims 198-227, wherein the intracellular domain comprises SEQ ID NO:

10.

231. The CAR of any one of claims 198-227, wherein the intracellular domain consists of SEQ ID NO:10.298507734.1 - 157 -232. The CAR of any one of claims 198-227, wherein the intracellular domain is a CD8 intracellular domain.

233. The CAR of any one of claims 198-227, wherein the intracellular domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

11.

234. The CAR of any one of claims 198-227, wherein the intracellular domain comprises SEQ ID NO:

11.

235. The CAR of anyone of claims 198-227, wherein the intracellular domain consists of SEQ ID NO:

11.

236. The CAR of any one of claims 198-235, wherein the CAR further comprises a signal peptide.

237. The CAR of any one of claims 198-236, wherein the CAR further comprises a signal peptide comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

30.

238. The CAR of any one of claims 198-237, wherein the CAR further comprises a signal peptide comprising SEQ ID NO:

30.

239. The CAR of any one of claims 198-238, wherein the CAR further comprises a signal peptide consisting of SEQ ID NO:

30.

240. The CAR of any one of claims 198-239, wherein the CAR further comprises an epitope tag.

241. The CAR of any one of claims 198-240, wherein the CAR further comprises an epitope tag comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:31 or SEQ ID NO:

32.

242. The CAR of any one of claims 198-241, wherein the CAR further comprises an epitope tag comprising SEQ ID NO:31 or SEQ ID NO:

32.

243. The CAR of any one of claims 198-242, wherein the CAR further comprises an epitope tag consisting of SEQ ID NO:31 or SEQ ID NO:32.298507734.1 - 158 -244. The CAR of any one of claims 198-243, wherein the CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:

34.

245. The CAR of any one of claims 198-243, wherein the CAR comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:

34.

246. The CAR of any one of claims 198-243, wherein the CAR consists of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:

34.

247. One or more polynucleotides encoding the CAR of any one of claims 198-246.

248. The one or more polynucleotides of claim 247, wherein the one or more polynucleotides further encode a second CAR.

249. The one or more polynucleotides of claim 248, wherein the first CAR and the second CAR are encoded by the same polynucleotide.

250. The one or more polynucleotides of claim 249, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a 2A sequence.

251. The one or more polynucleotides of claim 249 or 250, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence.

252. The one or more polynucleotides of any one of claims 249-251, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

29.

253. The one or more polynucleotides of any one of claims 249-252, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence comprising SEQ ID NO:

29.

254. The one or more polynucleotides of any one of claims 249-253, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the first and the second CAR are separated by a P2A sequence consisting of SEQ ID NO:29.298507734.1 - 159 -255. The one or more polynucleotides of any one of claims 249-254, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

35.

256. The one or more polynucleotides of any one of claims 249-254, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence comprising SEQ ID NO:

35.

257. The one or more polynucleotides of any one of claims 249-254, wherein the first and the second CAR are encoded by the same polynucleotide, and wherein the polynucleotide encodes an amnio acid sequence consisting of SEQ ID NO:

35.

258. The one or more polynucleotides of claim 248, wherein the first CAR and the second CAR are encoded by different polynucleotides.

259. The one or more polynucleotides of any one of claims 248-258, wherein the second CAR comprises: an antigen-binding domain that binds a second epitope on the antigen; a hinge from CD4, CD8, or CD28; a transmembrane domain from CD4 or CD8; a 4-1BB or CD28 co-stimulatory domain; and a CD3ζ stimulatory domain.

260. The one or more polynucleotides of any one of claims 248-258, wherein the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

261. The one or more polynucleotides of any one of claims 248-258, wherein the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8;298507734.1 - 160 -a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

262. The one or more polynucleotides of any one of claims 248-258, wherein the second CAR comprises: a CD22-binding domain that binds a second epitope on CD22; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

263. The one or more polynucleotides of any one of claims 248-258, wherein the second CAR comprises: a BCMA-binding domain that binds a second epitope on BCMA; a hinge from CD8; a transmembrane domain from CD8; a 4-1BB co-stimulatory domain; and a CD3ζ stimulatory domain.

264. The one or more polynucleotides of any one of claims 259-263, wherein the antigen- binding domain of the second CAR comprises an antibody or antigen-binding fragment thereof, a peptide ligand that interacts with the antigen, or a receptor or ligand portion of a naturally- occurring receptor-ligand pair that targets the antigen.

265. The one or more polynucleotides of any one of claims 259-264, wherein the antigen- binding domain of the second CAR comprises an scFv or a nanobody.

266. The one or more polynucleotides of any one of claims 259-265, wherein the hinge of the second CAR is a CD4 hinge.

267. The one or more polynucleotides of any one of claims 259-265, wherein the hinge of the second CAR is a CD8 hinge.298507734.1 - 161 -268. The one or more polynucleotides of any one of claims 259-265, wherein the hinge of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

5.

269. The one or more polynucleotides of any one of claims 259-265, wherein the hinge of the second CAR comprises SEQ ID NO:

5.

270. The one or more polynucleotides of any one of claims 259-265, wherein the hinge of the second CAR consists of SEQ ID NO:

5.

271. The one or more polynucleotides of any one of claims 259-265, wherein the hinge of the second CAR is a CD28 hinge.

272. The one or more polynucleotides of any one of claims 259-265, wherein the hinge of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

6.

273. The one or more polynucleotides of any one of claims 259-265, wherein the hinge of the second CAR comprises SEQ ID NO:

6.

274. The one or more polynucleotides of any one of claims 259-265, wherein the hinge of the second CAR consists of SEQ ID NO:

6.

275. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR is a CD4 transmembrane domain.

276. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

7.

277. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR comprises SEQ ID NO:

7.

278. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR consists of SEQ ID NO:

7.

279. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR is a CD8 transmembrane domain.298507734.1 - 162 -280. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

8.

281. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR comprises SEQ ID NO:

8.

282. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR consists of SEQ ID NO:

8.

283. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR is a CD28 transmembrane domain.

284. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

9.

285. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR comprises SEQ ID NO:

9.

286. The one or more polynucleotides of any one of claims 259-274, wherein the transmembrane domain of the second CAR consists of SEQ ID NO:

9.

287. The one or more polynucleotides of any one of claims 259-286, wherein the 4-1BB co- stimulatory domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

12.

288. The one or more polynucleotides of any one of claims 259-286, wherein the 4-1BB co- stimulatory domain comprises SEQ ID NO:

12.

289. The one or more polynucleotides of any one of claims 259-286, wherein the 4-1BB co- stimulatory domain consists of SEQ ID NO:

12.

290. The one or more polynucleotides of any one of claims 259-286, wherein the CD28 co- stimulatory domain of the second CAR comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:13.298507734.1 - 163 -291. The one or more polynucleotides of any one of claims 259-286, wherein the CD28 co- stimulatory domain of the second CAR comprises SEQ ID NO:

13.

292. The one or more polynucleotides of any one of claims 259-286, wherein the CD28 co- stimulatory domain of the second CAR consists of SEQ ID NO:

13.

293. The one or more polynucleotides of any one of claims 259-292, wherein the CD3ζ stimulatory domain comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

14.

294. The one or more polynucleotides of any one of claims 259-292, wherein the CD3ζ stimulatory domain comprises SEQ ID NO:

14.

295. The one or more polynucleotides of any one of claims 259-292, wherein the CD3ζ stimulatory domain consists of SEQ ID NO:

14.

296. The one or more polynucleotides of any one of claims 259-295, wherein the second CAR further comprises a signal peptide.

297. The one or more polynucleotides of any one of claims 259-296, wherein the second CAR further comprises a signal peptide comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

30.

298. The one or more polynucleotides of any one of claims 259-297, wherein the second CAR further comprises a signal peptide comprising SEQ ID NO:

30.

299. The one or more polynucleotides of any one of claims 259-298, wherein the second CAR further comprises a signal peptide consisting of SEQ ID NO:

30.

300. The one or more polynucleotides of any one of claims 259-299, wherein the second CAR further comprises an epitope tag.

301. The one or more polynucleotides of any one of claims 259-300, wherein the second CAR further comprises an epitope tag comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:31 or SEQ ID NO:

32.

302. The one or more polynucleotides of any one of claims 259-301, wherein the second CAR further comprises an epitope tag comprising SEQ ID NO:31 or SEQ ID NO:32.298507734.1 - 164 -303. The one or more polynucleotides of any one of claims 259-302, wherein the second CAR further comprises an epitope tag consisting of SEQ ID NO:31 or SEQ ID NO:

32.

304. The one or more polynucleotides of any one of claims 259-303, wherein the second CAR comprises an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:

33.

305. The polynucleotide of any one of claims 259-303, wherein the second CAR comprises SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:

33.

306. The one or more polynucleotides of any one of claims 259-303, wherein the second CAR consists of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:

33.

307. The one or more polynucleotides of any one of claims 259-306, wherein the first and the second CAR together comprise an amnio acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

35.

308. The one or more polynucleotides of any one of claims 259-306, wherein the first and the second CAR together comprise SEQ ID NO:

35.

309. The one or more polynucleotides of any one of claims 259-306, wherein the first and the second CAR together consist of SEQ ID NO:

35.

310. A vector comprising the one or more polynucleotides of any one of claims 247-306.

311. The vector of claim 310, wherein the vector is a viral vector.

312. The vector of claim 311, wherein the viral vector is an adenoviral vector, adeno- associated viral vector, lentiviral vector, or retroviral vector.

313. The vector of claim 310, wherein the vector is a non-viral vector.

314. The vector of claim 313, wherein the non-viral vector is a plasmid.

315. The composition of any one of claims 310-314, wherein the vector is multicistronic.

316. An immune cell comprising the one or more polynucleotides of any one of claims 247- 306 or the vector of any one of claims 310-314.298507734.1 - 165 -317. The immune cell of claim 316, wherein the immune cell is a T cell, natural killer (NK) cell, macrophage, microglial cell, or neutrophil.

318. The immune cell of claim 316 or 317, wherein the immune cell is a T cell.

319. The immune cell of claim 316 or 317, wherein the immune cell is a NK cell.

320. The composition of any one of claims 316-319, wherein the immune cell is a human cell.

321. A population of the immune cells of any one of claims 316-320.

322. A composition comprising: (a) the polynucleotide of any one of claims 247-306, the vector of any one of claims 310-315, the immune cell of any of claims 316-320, or the population of immune cells of claim 321; and (b) a pharmaceutically acceptable excipient.

323. The composition of claim 322, further comprising an additional therapeutic.

324. The composition of claim 323, wherein the additional therapeutic is a chemotherapeutic or an immunotherapeutic.

325. A method of killing cancer cells in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-99, 195-197, or 322-324; the immune cell of any one of claims 101-194 or 316-320; the CAR of any one of claims 198-246; the one or more polynucleotides of any one of claims 247-306; the vector of any one of claims 310-315; or the population of immune cells of claim 321.

326. A method for treating a subject for cancer, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-99, 195- 197, or 322-324; the immune cell of any one of claims 101-194 or 316-320; the CAR of any one of claims 198-246; the one or more polynucleotides of any one of claims 247-306; the vector of any one of claims 310-315; or the population of immune cells of claim 321.

327. A method for stimulating an immune response to cancer cells in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of the composition of any one of claims 1-99, 195-197, or 322-324; the immune cell of any one298507734.1 - 166 -of claims 101-194 or 316-320; the CAR of any one of claims 198-246; the one or more polynucleotides of any one of claims 247-306; the vector of any one of claims 310-315; or the population of immune cells of claim 321.

328. The method of any one of claims 325-327, wherein the cancer is lymphoma, leukemia, multiple myeloma, glioblastoma, mesothelioma, head and neck cancer, osteosarcoma, melanoma, non-small cell lung cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer, germ cell tumors, or breast cancer.

329. The method of any one of claims 325-328, wherein the cancer is a B-cell cancer.

330. The method of any one of claims 325-329, wherein the cancer is a CD19-positive, CD22-positive, BCMA-positive cancer, HER2-positive cancer, or a mesothelin-positive cancer.

331. The method of any one of claims 325-330, further comprising administering to the subject an additional therapy.

332. The method of claim 331, wherein the additional therapy is radiotherapy, chemotherapy, or immunotherapy.

333. A method of generating a persisting population of genetically engineered immune cells in a subject, the method comprising administering to the subject an effective amount of the composition of any one of claims 1-99, 195-197, or 322-324; the immune cell of any one of claims 101-194 or 316-320; the CAR of any one of claims 198-246; the one or more polynucleotides of any one of claims 247-306; the vector of any one of claims 310-315; or the population of immune cells of claim 321.

334. The method of claim 333, wherein the genetically engineered immune cells persist in the subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days, weeks, months, or years after administration.

335. A method of expanding a population of genetically engineered immune cells in a subject, the method comprising administering to the subject an effective amount of the composition of any one of claims 1-99, 195-197, or 322-324; the immune cell of any one of claims 101-194 or 316-320; the CAR of any one of claims 198-246; the one or more298507734.1 - 167 -polynucleotides of any one of claims 247-306; the vector of any one of claims 310-315; or the population of immune cells of claim 321.298507734.1 - 168 -

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