Chimeric receptor therapy

Abstract

A non-naturally occurring polynucleotide encoding a miRNA that inhibits expression of an immune checkpoint protein. The polynucleotide may further encode a chimeric receptor, a cytokine, and / or a cell tag. A vector comprising said polynucleotide. A modified immune effector cell comprising said polynucleotide. Compositions and kits comprising said polynucleotide and / or cell. A method for treating a subject suffering from a disease or disorder, comprising administering said cell to a subject in need of such treatment. Use of said cell in the manufacture of a medicament for the treatment of a disease or disorder. A method for detecting a disease or disorder associated with overexpression of an antigen in a subject. A method for treating a disease or disorder comprising sequential administration of a polynucleotide encoding a chimeric antigen receptor or a cell comprising same.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on January 10, 2022, is named 75594-348568_SL.txt, and is 579,627 bytes in size. [Background technology]

[0002] Chimeric antigen receptor (CAR-T) cells and T cell receptor (TCR) therapy have undergone rapid development in recent years and have been shown to successfully direct the killing of tumor cells. Such therapy is useful, for example, in the treatment of autoimmune disorders and cancer. Indeed, several targets for such therapy have been identified so far, including, but not limited to, CD19, CD33, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, folate receptor alpha, mucins such as MUC1, MUC4 or MUC16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123, or VEGF-R2. Of these, CD19, CD33, MUC1, MUC16, and ROR1 have shown particular promise as targets for immunotherapy.

[0003] CD19 is an attractive target for immunotherapy due to several factors. It is expressed in various B cell lymphomas and leukemias, as well as in normal B cells, but is not found in hematopoietic stem cells, plasma cells, and other healthy tissues. In addition, CD19 has a broader expression profile than that of CD20, the target of monoclonal antibody therapy such as rituximab, and is considered to be a better target for antibody-drug conjugates (ADCs) compared to CD20, which suffers from inefficient internalization. CD19 has also been shown to be expressed when monoclonal antibody therapy (e.g., rituximab) is ineffective due to CD20 downregulation or other factors. In addition, CD19 targeting agents have a different mode of action than anti-CD20 antibodies, so they can complement existing monoclonal antibody regimens.

[0004] CD33 is an attractive target for immunotherapy due to its high expression in cancer and minimal expression in healthy adult tissues. CD33 is overexpressed on myeloid leukemias and leukemic stem cells. CD33 is overexpressed in acute myeloid leukemia (AML), the most common acute leukemia in adults. 85-90% of AML patients show expression of CD33 on blast cells. CD33 is also overexpressed in myelodysplastic syndromes (MDS), a cancerous condition of the bone marrow commonly found in adults in their seventh decade.

[0005] MUC1 is an attractive target for immunotherapy because it is overexpressed in breast cancer and is absent or expressed at low levels in normal mammary gland.In addition, MUC1 is mostly abnormally underglycosylated in cancer, and the antigen on the surface of cancer is different from that on normal cells.Therefore, targeting MUC1 for cancer immunotherapy can take advantage of the difference between cancer cells and normal cells.

[0006] MUC16 is an attractive target for immunotherapy due to its high expression in cancer and minimal expression in healthy adult tissues. MUC16 is abnormally expressed in ovarian, breast, pancreatic, endothermic, and lung cancers. For example, MUC16 is overexpressed in more than 80% of ovarian tumors, which are the most lethal of gynecological malignancies. Meanwhile, limited expression of MUC16 is found on healthy tissues. The current standard treatment for ovarian cancer is surgery, followed by chemotherapy with a combination of platinum agents and taxanes. However, recurrence of the disease occurs in most patients after initial treatment, resulting in repeated surgeries and additional rounds of chemotherapy cycles.

[0007] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an attractive target for immunotherapy due to its high expression in cancer and minimal expression in healthy adult tissues. ROR1 is aberrantly expressed in multiple hematological malignancies, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and diffuse B-cell lymphoma (DLBCL), as well as solid tumors, including breast adenocarcinoma, pancreatic cancer, ovarian cancer, and lung adenocarcinoma, including triple-negative breast cancer (TNBC).

[0008] Many patients have a sustained response to CAR-T and TCR therapy, but for some patients, the antitumor effects of such therapy are either short-lived or ineffective. Another immunotherapy that has shown promise is immune checkpoint inhibition, which can prevent T cells from switching off and promote the activity of these cells. Examples of checkpoint inhibitor targets include, but are not limited to, PD1, PD-L1, CTLA-4, TIGIT, 4-1BB, PIK3IP1, CD27, CD28, CD40, CD70, CD122, CD137, OX40 (CD134), GITR, ICOS, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, or VISTA. Of these, targeting CTLA4, PD-1, PD-L1, TIM3, TIGIT, LAG3, and / or PIK3IP1 has shown the most promise. One of the most studied checkpoint inhibitor pathways is the PD-1 / programmed death-ligand 1 (PD-L1) pathway, which plays a key role in how tumor cells evade immune responses. Immunotherapies utilizing PD-1 / PD-L1 blocking antibodies have been widely evaluated in the clinic and have been shown to improve tumor regression across multiple malignancies, especially when administered in conjunction with CAR-T cells. However, checkpoint inhibitor blocking antibodies are not consistently implemented across cancer types, may have limited access to the tumor microenvironment, require repeated dosing, and may lose efficacy over time. Genome editing is an alternative approach to eliminate PD-1-mediated CAR-T cell exhaustion, and has the advantage of restricting PD-1 blockade to only engineered CAR-T cells. However, gene editing complicates the manufacturing process and increases the turnaround time and cost of cell therapy. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a continuing need in the art for safer, more effective, and less expensive therapies for antigen-associated diseases and conditions, including those that combine CAR-T and / or TCR therapy with systemic checkpoint blockade.

[0010] There is also a need to devise ways to diversify therapeutic regimens to provide multifaceted targeting of antigens to address complex in vivo biological problems such as loss of immunosurveillance, genetic alterations in tumor antigen composition, and tumor heterogeneity (leading to phenotypic differences in cancer cells). [Means for solving the problem]

[0011] The present invention relates, in part, to a non-naturally occurring polynucleotide encoding an miRNA that inhibits expression of an immune checkpoint protein. In certain embodiments, the miRNA targets CTLA4, PD-1, PD-L1, TIM3, TIGIT, LAG3, GITR, or PIK3IP1. In certain embodiments, the miRNA targets PD-1.

[0012] In certain embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 72-87 or that can hybridize under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 72-87. In certain such embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 84, and 86 or that can hybridize under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 704, 705, 709, and 710. In certain embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 80% sequence identity to SEQ ID NOs: 179 or 180 or that can hybridize under stringent hybridization conditions to the complement of SEQ ID NOs: 179 or 180. In certain embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:267 or capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO:267.

[0013] In certain embodiments, the polynucleotide further comprises: a) a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:292 or capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO:291; and b) a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:292 or capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO:292.

[0014] In certain embodiments, the polynucleotide further encodes a chimeric receptor, hi certain embodiments, the chimeric receptor is a T cell receptor or a chimeric antigen receptor.

[0015] In certain embodiments, the chimeric antigen receptor comprises an antigen binding domain that binds to an epitope on CD19, CD33, MUC1, MUC16, or ROR1. In certain embodiments, the antigen binding domain binds to an epitope on ROR1.

[0016] In certain embodiments, the antigen binding domain comprises a variable light chain domain comprising the amino acid sequence of any one of SEQ ID NOs: 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, and 463, or a functional fragment or variant thereof. In certain such embodiments, the variable light chain domain comprises the amino acid sequence of SEQ ID NO: 387, or a functional fragment or variant thereof.

[0017] In certain embodiments, the antigen binding domain comprises a variable heavy domain comprising the amino acid sequence of any one of SEQ ID NOs: 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, and 461, or a functional fragment or variant thereof. In certain such embodiments, the variable heavy domain comprises the amino acid sequence of SEQ ID NO: 349, or a functional fragment or variant thereof.

[0018] In certain embodiments, the chimeric antigen receptor comprises a spacer. In certain such embodiments, the spacer comprises a stalk region that is a CD8α hinge domain, or a functional fragment or variant thereof. In certain such embodiments, the stalk region comprises the amino acid sequence of SEQ ID NO: 467, or a functional fragment or variant thereof. In certain such embodiments, the spacer comprises a stalk extension region. In certain such embodiments, the stalk extension region comprises the amino acid sequence of SEQ ID NO: 473, or a functional fragment or variant thereof.

[0019] In certain embodiments, the chimeric antigen receptor further comprises a transmembrane domain. In certain such embodiments, the transmembrane domain comprises the CD8α transmembrane domain, or a functional fragment or variant thereof. In certain such embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 475, or a functional fragment or variant thereof.

[0020] In certain embodiments, the chimeric antigen receptor further comprises an intracellular signaling domain. In certain such embodiments, the intracellular signaling domain comprises a CD3 zeta signaling domain, or a functional fragment or variant thereof. In certain such embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 479, or a functional fragment or variant thereof.

[0021] In certain embodiments, the intracellular signaling domain comprises a costimulatory domain. In certain such embodiments, the intracellular signaling domain comprises a CD28 signaling domain, or a functional fragment or variant thereof. In certain embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 481, or a functional fragment or variant thereof.

[0022] In certain embodiments, the cytokine further encodes a cell tag. In certain embodiments, the cytokine is IL-15, or a functional fragment or variant thereof. In certain such embodiments, the cytokine comprises the amino acid sequence of SEQ ID NO: 519, or a functional fragment or variant thereof.

[0023] In certain embodiments, IL-15, or a functional fragment or variant thereof, is membrane bound. In certain such embodiments, IL-15, or a functional fragment or variant thereof, forms part of a fusion protein that also includes IL-15Rα, or a functional fragment or variant thereof. In certain such embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 523, or a functional fragment or variant thereof. In certain embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 525, or a functional fragment or variant thereof.

[0024] In certain embodiments, the polynucleotide further encodes a cell tag. In certain embodiments, the cell tag comprises a truncated HER1, or a functional fragment or variant thereof. In certain embodiments, the truncated HER1 comprises HER1 domain III, or a functional fragment or variant thereof, and a truncated HER1 domain IV, or a functional fragment or variant thereof. In certain such embodiments, the truncated HER1 comprises the amino acid sequence of SEQ ID NO: 565, or a functional fragment or variant thereof, and the amino acid sequence of SEQ ID NO: 567, or a functional fragment or variant thereof.

[0025] In certain embodiments, the cell tag comprises the CD28 transmembrane domain, or a functional fragment or variant thereof. In certain such embodiments, the cell tag comprises the amino acid sequence of SEQ ID NO: 571, or a functional fragment or variant thereof.

[0026] The present invention also relates to a vector comprising the aforementioned polynucleotide. The vector may be viral or non-viral. In a particular embodiment, the vector comprises a Sleeping Beauty transposon.

[0027] The present invention further relates to a modified immune effector cell comprising the aforementioned polynucleotide. In a particular embodiment, the cell is a T cell.

[0028] The present invention further relates to compositions and kits comprising the aforementioned polynucleotides and / or cells.

[0029] A further aspect of the invention is a method for treating a subject suffering from a disease or disorder, comprising administering said cells to a subject in need of said treatment. The invention also relates to the use of said cells in the manufacture of a medicament for the treatment of a disease or disorder. The disease or disorder may be one associated with overexpression of an antigen, for example CD19, CD33, ROR1, MUC1, or MUC16. In a particular embodiment, the disease or disorder is one associated with overexpression of ROR1.

[0030] In certain embodiments, the disease or disorder is cancer. In certain embodiments, the disease or disorder is chronic lymphocytic leukemia, mantle cell lymphoma, acute lymphoblastic leukemia, or diffuse large B-cell lymphoma, breast adenocarcinoma, including triple-negative breast cancer, pancreatic cancer, ovarian cancer, or lung adenocarcinoma.

[0031] Another aspect of the invention is a method for detecting a disease or disorder associated with overexpression of an antigen in a subject, comprising: a) contacting a sample from the subject with one or more antibodies, or antigen-binding fragments thereof; and b) detecting an increased level of binding of the antibody or fragment thereof to the sample compared to such binding to a control sample lacking the disease, thereby detecting the disease in the subject.

[0032] A still further aspect of the invention is a method for treating a disease or disorder, such as cancer and an autoimmune disease or disorder, comprising sequential administration of cells, nucleic acids, viral vectors, or non-viral vectors comprising a polynucleotide encoding a chimeric antigen receptor selected from a collection of chimeric antigen receptors having different structural compositions and binding specificities for an antigen target sequence. In certain such embodiments, the method comprises a first administration of cells expressing one or more chimeric antigen receptors from the collection, followed by a second administration of cells expressing one or more chimeric antigen receptors from the collection, with a period of time elapsed between the first and second administrations. [Brief description of the drawings]

[0033] [Figure 1A] FIG. 1 is an exemplary diagram of a vector comprising one or more checkpoint inhibitor miRNAs in combination with a chimeric receptor (SD=splice donor; SA=splice acceptor). [Figure 1B] FIG. 1 is an exemplary diagram of hairpin and loop designs of various pri-miRNAs, comprising target miRNAs and complementary sequences at various 5′ or 3′ positions. [Figure 1C] FIG. 1 is an exemplary diagram of the hairpin and loop designs of various pri-miRNAs, including their locations within the transgene cassette. [Diagram 2] 1 is a graph showing PD1 relative RNA expression after transfection of various combinations of miRNA constructs in the presence or absence of a MUC16-specific CAR. Constructs #1-8 shown on the X-axis are as presented generally in Table 10. [Figure 3A-C]3A-3C are graphs showing normalized absolute transcript counts obtained from gene analysis of over 700 genes using the Nanostring human gene panel code set with CD3 / CD28 bead stimulated CD33 CAR-T cells. In FIG. 3A, the Y-axis plots transcript counts from CAR-T cells containing introns encoding two checkpoint inhibitor miRNAs targeting PD-1 and TIGIT (CD33 CAR-mbIL15-HER1t+miRNA(PD-1+TIGIT)), and the X-axis plots transcripts only from CAR-T cells (not containing any checkpoint inhibitor miRNAs). Circles indicate genes of interest. In FIG. 3B, the Y-axis plots transcript counts from CAR-T cells containing PD-1 miRNA (CD33 CAR-mbIL15-HER1t+miRNA(PD-1+PD-1)), and the X-axis plots transcripts only from CAR-T cells. Figure 3C plots non-targeting miRNA control (CD33 CAR-mbIL15-HER1t+miRNA scramble*) on the Y-axis and miRNA-containing intronless CAR-T cells on the X-axis. Circles indicate genes of interest and are used to demonstrate on-target specificity of the checkpoint inhibitor miRNA design. All three graphs were derived from one donor. *Scramble control is non-targeting miRNA. [Figure 4A-C]4A-4D are graphs showing normalized absolute transcript counts obtained from gene minutes of over 700 genes using a Nanostring human gene panel code set in CD3 / CD28 bead-stimulated MUC16-specific CAR-T cells. In FIG. 4A, the Y-axis plots transcript counts from CAR-T cells containing introns encoding miRNAs targeting two different sequences in PD-1 and sequences for TIGIT (MUC16CAR-mbIL15-HER1t (also collectively referred to as "MUC16CAR") + miRNA (PD-1 / PD-1 / TIGIT)), and the X-axis plots transcripts from CAR-T cells without the miRNA-containing intron (MUC16CAR-mbIL15-HER1t). Filled circles indicate genes of interest. In Figure 4B, the X-axis is the same and the Y-axis plots the transcript counts from dual PD-1 targeting miRNA containing CAR-T cells (MUC16CAR-mbIL15-HER1t+miRNA(PD-1 / PD-1)). Figure 4C plots the non-targeting miRNA control (MUC16CAR-mbIL15-HER1t+miRNA(scrambled)) on the Y-axis and the miRNA containing intronless CAR-T cells on the X-axis. All three graphs are from one donor. [Figure 5A]Graph showing the number of GFP+ K562 cells expressing MUC16 over time. At each time point, the line with black circle filled points indicates the number of GFP+ target cells in wells without CAR-T cells. The line with square open points indicates target cell counts in wells with MUC16 CAR-mbIL15-HER1t CAR-T cells that do not contain a miRNA-containing intron ("with CAR-T cells"). The line with grey circle filled points indicates target cell counts in wells with CAR-T cells containing a synthetic intron with dual PD-1 targeting miRNAs (MUC16 CAR-mbIL15-HER1t+miRNA(PD-1 / PD-1) ("with CAR-T+miRNA cells"). Data are from one donor and plots are mean+SD of triplicate wells. ***P<0.001 based on 2-way ANOVA with Dunnett's multiple comparison post-hoc test. [Figure 5B] Graph showing the number of GFP+K562 / MUC16+ / PD-L1+ / CD155+ cells over time. At each time point, the line with square filled dots indicates the number of GFP+ target cells only in the wells. The line with open circle dots indicates the target cell count in wells with MUC16-specific CAR-T cells without miRNA-containing introns (MUC16 CAR-mbIL15-HER1t (with CAR-T cells)). The line with open circle filled dots indicates the target cell count in wells with CAR-T cells containing a synthetic intron with double PD-1 and TIGIT-targeting miRNAs (MUC16CAR-mbIL15-HER1t+miRNA(PD-1 / PD-1 / TIGIT) (with CAR-T+miRNA cells)). Data is from one donor and plots are the mean + SD of triplicate wells. [Figure 6A-B] Shows cytokine expression levels of IFN-gamma and GM-CSF in MUC16 CAR-T cells with one or more checkpoint inhibitor miRNA combinations after co-culture with tumor target cells (K562 / MUC16t). [Figure 6C-D]Figure 1 shows cytokine expression levels of IFN gamma and GM-CSF in MUC16 CAR-T cells with one or more checkpoint inhibitor miRNA combinations without co-culture with tumor target cells. Constructs #1-11 shown on the X-axis are as presented diagrammatically in Table 11. [Figure 7] 1 shows tumor burden in mice treated with MUC16 CAR+mbIL-15+HER1t (denoted as "MUC16 CAR") in combination with various miRNAs. [Figure 8A] PD-1 levels in cell populations after gating for hCD45 / CD3+ / HER1t+ expression in blood of MUC16CAR+mbIL15+HER1t (CAR only) and MUC16CAR+mbIL15+HER1t+miRNA(PD1 / PD-1) (CAR+miRNA(PD-1 / PD-1)) treated mice are shown. [Figure 8B] PD-1 levels as measured by median fluorescence intensity (MFI) in CAR and CAR+miRNA (PD-1 / PD-1) treated mice are shown. [Figure 9A-9B] PD-1 and TIGIT MFI levels in cell populations after gating for hCD45 / CD3+ / HER1t+ expression in blood of various CAR and CAR+miRNA treated mice. Groups #1-9 shown on the X-axis are as presented diagrammatically in Table 12. [Figure 10A] PD1 silencer module produces guide miRNA and corresponding reduction in PD1 mRNA expression in UltraCAR-T cells generated from five T cell donors. RT-qPCR results of PD1-targeting guide miRNA are shown. [Figure 10B] PD1 silencer module produces guide miRNA and corresponding reduction in PD1 mRNA expression in ultraCAR-T cells generated from five T cell donors. RT-qPCR results of PD1 mRNA are shown. [Figure 11]1 shows that the PD1 silencing module preferentially produced PD1-targeting guide miRNA over non-targeting passenger miRNA. [Figure 12A-E] We show that guide miRNAs are the predominant small RNA species derived from the PD1 silencer module. [Figure 13] Quantification of mature miRNAs mapping to the PD1 silencer module as a percentage of all small RNAseq reads is shown. [Figure 14A-B] Differential gene expression in ROR1+PD1 silenced cells compared to ROR1 UltraCAR-T control cells. [Figure 15A-D] A comparison of predicted miRNA binding strength against log fold change of transcript is shown. [Figure 16] 1 provides an exemplary scheme for a genetic construct of the present disclosure. [Figure 17] 1 provides a schematic diagram of pathways and elements of the present disclosure. [Figure 18] 1 provides examples of pathways along which the therapeutic regimens of the present disclosure may proceed. [Figure 19] 1 provides an indication of additional embodiments of the present disclosure. [Figure 20] 1 provides an indication of additional embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The following description and examples specifically illustrate embodiments of the present disclosure.

[0035] It should be understood that the present disclosure is not limited to the specific embodiments described herein, and as such may vary, and those skilled in the art will recognize that there are variations and modifications of the present disclosure that are encompassed within the scope of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0037] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0038] Although various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein for clarity in terms of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0039] The following definitions are complementary to those of the art and are directed to the current application and are not intended to be limiting in any related or unrelated case, such as any commonly owned patents or applications. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0040] I. Definition In this application, the use of the singular includes the plural unless specifically noted otherwise. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0041] In this application, the use of "or" means "and / or" unless otherwise specified. The terms "and / or" and "any combination thereof" as well as their grammatical equivalents used herein may be used interchangeably. These terms may convey that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "individually A, individually B, individually C, A and B, B and C, A and C, and A, B, and C." The term "or" may be used conjunctively or disjunctively unless the context specifically dictates disjunctive use.

[0042] Furthermore, use of the term "including" as well as other forms such as "include," "includes," and "included" is not limiting.

[0043] References herein to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0044] As used in the specification and claims, the terms "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or "containing" (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited, elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Additionally, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0045] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount "about 10" includes any amount between 10 and 9-11. In yet another example, the term "about" in connection with a reference numerical value can also include values ​​in a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where specific values ​​are recited in this application and in the claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable range of error for the particular value.

[0046] A "therapeutically effective amount" or "therapeutically effective dose" refers to an amount or dose effective, for a period of time necessary, to achieve the desired therapeutic result. This amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the nucleic acid sequence of the invention to elicit a desired response in the individual.

[0047] "Polynucleotide" or "oligonucleotide" refers to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, triple-stranded DNA, and double- and single-stranded RNA. It also includes modifications, for example, by methylation and / or capping, as well as unmodified forms of polynucleotides. The term is also meant to include molecules that contain non-natural or synthetic nucleotides and nucleotide analogs.

[0048] Unless otherwise indicated, nucleic acid sequences in the text of the specification are given in 5' to 3' orientation when read from left to right.

[0049] As used herein, the terms "transfection," "transformation," "nucleofection," or "transduction" refer to the introduction of one or more exogenous polynucleotides into a host cell or organism by using physical, chemical, and / or electrical methods. The nucleic acid sequences and vectors disclosed herein can be introduced into a cell or organism by any such method, including, for example, electroporation, calcium phosphate co-precipitation, strontium phosphate DNA co-precipitation, liposome-mediated transfection, DEAE-dextran-mediated transfection, polycation-mediated transfection, tungsten particle-facilitated microparticle bombardment, viral, and / or non-viral mediated transfection. In some cases, the method of introducing a nucleic acid into a cell or organism involves the use of a viral, retroviral, lentiviral, or transposon- or transposable element-mediated (e.g., Sleeping Beauty) vector.

[0050] As used herein, "polypeptide", "peptide" and their grammatical equivalents refer to a polymer of amino acid residues. A polypeptide can optionally include glycosylation or other modifications typical of a given protein in a given cellular environment. The polypeptides and proteins disclosed herein (including functional fragments and functional variants thereof) can include synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylser ... β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxy-lysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. The present disclosure further contemplates that expression of the polypeptides or proteins described herein in engineered cells may be associated with post-translational modification of one or more amino acids of the polypeptide or protein.Non-limiting examples of post-translational modifications include phosphorylation, acylation, including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation, including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0051] The term "conservative amino acid substitution" or "conservative mutation" refers to the substitution of one amino acid with another amino acid that has common properties. A functional method of defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such an analysis, groups of amino acids can be defined where the amino acids within the group preferentially exchange with each other and are therefore most similar to each other in their impact on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Examples of conservative mutations include amino acid substitutions of amino acids within the following subgroups, such as lysine for arginine to maintain a positive charge, glutamic acid for aspartic acid to maintain a negative charge, and vice versa, serine for threonine to maintain a free -OH, and glutamine for asparagine to maintain a free -NH2. Exemplary conservative amino acid substitutions are shown in the table below.

number

[0052] In some embodiments, a functional variant may include an amino acid sequence of a reference protein with at least one non-conservative amino acid substitution. The term "non-conservative mutation" involves an amino acid substitution between different groups, such as lysine for tryptophan or phenylalanine for serine. In this case, the non-conservative amino acid substitution preferably does not prevent or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the homologous parent protein. Amino acid exchangeability is discussed in detail, for example, by LY Yampolsky and A. Stoltzfus, "The Exchangeability of Amino acids in Proteins," Genetics 2005 Aug.;170(4):1459-1472.

[0053] As used herein, in the context of two nucleic acid or amino acid sequences of a polypeptide, the term "identical" and its grammatical equivalents, or "sequence identity," refers to residues in two sequences that are the same when aligned for maximum correspondence over a particular comparison window. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150, and a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art.Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970), by the similarity search method of Pearson and Lipman, Proc. Nat. Acad. Sci USA, 85:2444 (1988), by computerized implementations of these algorithms (including, but not limited to, CLUSTAL in the PC / Gene program by Intelligentics, Mountain View Calif, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA), the CLUSTAL program being a program developed by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989), Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988), Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992), and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is often performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein are at least 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference polypeptide (i.e., its full length), or a fragment thereof, as measured, for example, by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters.Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference nucleic acid (i.e., its entire length), or a fragment thereof, as measured, e.g., by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have a certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, the percentage of residues in the smaller molecule that find matching residues in the larger molecule will be those according to the order in which the two molecules are optimally aligned.

[0054] For purposes of this specification and claims, a reference to a reference sequence "having at least 50% sequence identity to," or any range therein (e.g., "at least 80% sequence identity to") is understood to include the reference sequence itself. Thus, for example, a claim reciting "a nucleic acid having at least 80% sequence identity to SEQ ID NO:0" includes SEQ ID NO:0 itself.

[0055] The term "substantially identical" and its grammatical equivalents as applied to a nucleic acid or amino acid sequence means that the nucleic acid or amino acid sequence includes sequences having at least 95% sequence identity to a reference sequence using the above programs, e.g., BLAST, using standard parameters.

[0056] "Homology" is generally inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The exact percentage of identity between sequences useful for establishing homology varies depending on the nucleic acid and protein in question, but sequence identity is typically no more than 25% used to establish homology. Higher levels of sequence identity, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining percentages of sequence identity (e.g., BLASTP and BLASTN using default parameters) are described herein and are publicly available. Nucleic acids and / or nucleic acid sequences are "homologous" if they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and / or protein sequences are "homologous" if their coding DNA is derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homologous molecules may be referred to as homologs. For example, any naturally occurring protein can be modified by any available mutagenesis method, and when expressed, the mutagenized nucleic acid will encode a polypeptide that is homologous to the protein encoded by the original nucleic acid.

[0057] Nucleic acid molecules that hybridize to the disclosed sequences are also contemplated and included herein. Hybridization conditions may be mild, moderate, or stringent, as warranted.

[0058] Suitable stringency conditions that promote DNA hybridization, such as 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by a 2x SSC wash at 50°C, are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. "Stringent hybridization conditions" include a salt concentration of 1.0 M NaCl in 50% formamide at a temperature of 37°C for 4-12 hours, followed by a wash in 0.1x SSC at 60-65°C.

[0059] As will be understood by those skilled in the art, slight changes in a nucleic acid sequence do not necessarily change the amino acid sequence of the encoded polypeptide. The present disclosure encompasses the degeneracy of codon usage, as will be understood by those skilled in the art. For example, as is known in the art, different codons code for the same amino acid, as shown in the table below.

number

[0060] In addition, those skilled in the art will understand that partial sequences often work as effectively as full-length versions. The manner in which nucleotide sequences can be altered or shortened is well known to those skilled in the art, as are the manners for testing the suitability or effectiveness of modified genes. In certain embodiments, the suitability and / or effectiveness of modified genes can be easily tested, for example, by conventional gas chromatography. Thus, all such alterations of genes are included as part of the present disclosure.

[0061] As used herein, the term "isolated" and its grammatical equivalents refer to the removal of a nucleic acid from its natural environment. However, it is understood that nucleic acids and proteins can be formulated with a diluent or adjuvant and still be isolated for practical purposes.

[0062] As used herein, the term "purified" and its grammatical equivalents refer to a molecule or composition that has been removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and / or amplified under laboratory conditions, and "purity" is a relative term, not "absolute purity". For example, when a nucleic acid is used for introduction into a cell, it is typically mixed with an acceptable carrier or diluent. As used herein, the term "substantially purified" and its grammatical equivalents refer to a nucleic acid sequence, polypeptide, protein, or other compound that is essentially free, i.e., more than about 50% free, more than about 70% free, more than about 90% free, of polynucleotides, proteins, polypeptides, and other molecules with which the nucleic acid, polypeptide, protein, or other compound is naturally associated.

[0063] As used herein, a "T cell" or "T lymphocyte" is a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface.

[0064] "Transposon", "transposable element", or "TE" refers to a vector DNA sequence that can change its position within a genome, sometimes creating or reversing mutations and changing the genome size of a cell. Transposition often results in duplication of the transposon. Class I transposons are copied in two steps: first, they are transcribed from DNA to RNA, and then the RNA produced is reverse transcribed into DNA. This copied DNA is then inserted into the genome at a new location. The reverse transcription step is catalyzed by a reverse transcriptase enzyme that may be encoded by the transposon itself. The characteristics of retrotransposons are similar to retroviruses such as HIV. The cut-and-paste transposition mechanism of class II transposons does not involve an RNA intermediate. Transposition is catalyzed by several transposase enzymes. Some transposases bind nonspecifically to any target site in DNA, while others bind to specific DNA sequence targets. The transposases cut with a stagger at the target site, resulting in single-stranded 5' or 3' DNA overhangs (sticky ends). This step cuts out the DNA transposon and then ligates it to a new target site, a process that involves the activity of DNA polymerase to fill the gap and DNA ligase to close the sugar-phosphate backbone. This results in a duplication of the target site. The insertion site of the DNA transposon can be identified by a short direct repeat that can be created by staggered cuts in the target DNA and filled in by DNA polymerase, followed by a series of inverted repeats that are important for transposon excision by the transposase. Cut-and-paste transposons can be replicated if transposition occurs during the S phase of the cell cycle, when the donor site has already been replicated but the target site has not yet been replicated. Transposition can be classified as either "autonomous" or "non-autonomous" for both class I and class II transposons. Autonomous transposons can move on their own, while non-autonomous transposons require the presence of another transposon to move.This is because non-autonomous transposons often lack a transposase (in the case of class II) or a reverse transcriptase (in the case of class I).

[0065] "Transposase" refers to an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut-and-paste mechanism or a replicative transposition mechanism. In some embodiments, the catalytic activity of a transposase can be used to move a gene from a vector into the genome.

[0066] An "expression vector" or "vector" is any genetic element, e.g., plasmid, minicircle, nanoplasmid, chromosome, virus, transposon, that functions either as an autonomous unit of polynucleotide replication within a cell (i.e., capable of replicating under its own control) or capable of replicating by insertion into a host cell chromosome to which it is attached another polynucleotide segment, resulting in replication and / or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages, and cosmids. A vector can include polynucleotide sequences necessary to effect ligation or insertion of the vector into a desired host cell and to effect expression of the attached segment. Such sequences vary depending on the host organism, and they include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosome binding site sequences, and transcription and translation termination sequences. Alternatively, an expression vector may be capable of directly expressing a nucleic acid sequence product encoded therein, without ligation or integration of the vector into a host cell DNA sequence. In some embodiments, the vector is an "episomal expression vector" or "episome" that can replicate in a host cell and persist as an extrachromosomal segment of DNA in the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize the Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.), and pBK-CMV from Stratagene (La Jolla, Calif.) represent non-limiting examples of episomal vectors that use T antigen and the SV40 origin of replication instead of EBNA1 and oriP. The vector may also include a selectable marker gene.In certain embodiments in which a nanoplasmid is utilized, a strain such as R6K that utilizes an antisense RNA selection marker (e.g., sucrose resistance) can be used.

[0067] The term "selectable marker gene" refers to a nucleic acid sequence that allows for the specific selection of, or for, cells expressing the nucleic acid sequence in the presence of a corresponding selective agent. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Applications WO 1992 / 08796 and WO 1994 / 28143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981); Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981); Santerre et al., Gene, 30:147 (1984); Kent et al., Science, 237:901-903 (1987); Wigler et al. al., Cell, 11:223 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026 (1962); Lowy et al., Cell, 22:817 (1980); and U.S. Patent Nos. 5,122,464 and 5,770,359.

[0068] The term "coding sequence" refers to a segment of a polynucleotide that encodes a protein or polypeptide. The region or sequence is bounded proximal to the 5' end by a start codon and proximal to the 3' end by a stop codon. A coding sequence may also be referred to as an open reading frame.

[0069] The term "operably linked" refers to the physical and / or functional linkage of a DNA segment to another DNA segment in such a manner as to allow the segment to function in an intended manner. A DNA sequence encoding a gene product is operably linked to a regulatory sequence in a manner that allows for the regulation of transcription of the DNA sequence, directly or indirectly, when it is linked to a regulatory sequence, such as a promoter, enhancer, and / or silencer. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream of the promoter with respect to the transcription start site of the promoter and in the correct reading frame with respect to the transcription start site, so as to allow transcription elongation to proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence encoding a gene product when it is ligated to the DNA sequence in such a manner as to increase or decrease the transcription of the DNA sequence, respectively. Enhancers and silencers can be located upstream or downstream of the coding region of the DNA sequence, or can be integrated within the coding region of the DNA sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if the signal sequence is expressed as a preprotein that participates in the secretion of the polypeptide. Linking of a DNA sequence to a regulatory sequence is typically accomplished by ligation at suitable restriction sites or via adapters or linkers inserted into the sequence using restriction endonucleases known to those skilled in the art.

[0070] As used herein, the terms "induce" and "induction," and their grammatical synonyms, refer to an increase in nucleic acid sequence transcription, promoter activity and / or expression brought about by a transcriptional regulator as compared to some basal level of transcription.

[0071] The term "transcriptional regulator" refers to a biochemical element that acts to block or inhibit transcription of a DNA sequence driven by a promoter under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein) or to permit or stimulate transcription of a DNA sequence driven by a promoter under certain environmental conditions (e.g., an inducer or enhancer).

[0072] As used herein, the term "enhancer" refers to a DNA sequence that, for example, increases the transcription of a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, the pattern of DNA methylation, or changes in DNA structure. Numerous enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (e.g., from depositories such as ATCC, as well as other commercial or individual sources). Some polynucleotides that contain a promoter (such as the commonly used CMV promoter) also contain an enhancer sequence. Enhancers can be located upstream or downstream of the coding sequence, or within the coding sequence. The term "Ig enhancer" refers to an enhancer element derived from enhancer regions mapped within the immunoglobulin (Ig) locus (such enhancers include, for example, the heavy chain (mu) 5' enhancer, the light chain (kappa) 5' enhancer, the kappa and mu intronic enhancers, and the 3' enhancers (see generally, Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363; and U.S. Patent No. 5,885,827).

[0073] The term "promoter" refers to a region of a polynucleotide that initiates transcription of a coding sequence. Promoters are located near the transcription start site of a gene, on the same strand, and upstream of the DNA (towards the 5' region of the sense strand). Some promoters are constitutive, as they are active in all contexts within a cell, while other promoters are regulated to become active in response to a particular stimulus, e.g., inducible promoters. As used herein, the term "promoter activity" and its grammatical equivalents refer to the degree of expression of a nucleotide sequence operably linked to a promoter whose activity is being measured. Promoter activity can be measured directly by determining the amount of RNA transcript produced, for example, by Northern blot analysis, or indirectly by determining the amount of product encoded by a linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

[0074] "Inducible promoter" refers to a promoter that is induced to activity by the presence or absence of a transcriptional regulator, e.g., a biological or non-biological factor. Inducible promoters are useful because the expression of a gene operably linked to them can be turned on or off at a particular developmental stage or in a particular tissue of an organism. Non-limiting examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature-regulated promoters, and light-regulated promoters. An inducible promoter can be a gene switch or part of a gene switch.

[0075] As used herein, a "T cell" or "T lymphocyte" is a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface.

[0076] As used herein, the phrase "functional fragment" when used in reference to a polypeptide refers to a fragment of such a polypeptide that has the primary function of the reference polypeptide. For example, a functional fragment of a polypeptide that functions as a transmembrane domain is a fragment of that polypeptide that also functions as a transmembrane domain. In certain embodiments, a functional fragment of a polypeptide is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the reference polypeptide. When used in reference to a nucleic acid, the phrase "functional fragment" refers to a fragment of a reference nucleic acid that encodes a polypeptide that has the same primary function as the polypeptide encoded by the reference nucleic acid.

[0077] As used herein, the phrase "functional variant" when used in reference to a polypeptide refers to a polypeptide that differs from a reference polypeptide but has the primary function of the reference polypeptide. For example, a functional variant of a polypeptide that functions as a transmembrane domain is a fragment of that polypeptide that also functions as a transmembrane domain. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the reference amino acid sequence and / or is a conservatively substituted variant of the reference sequence. When used in reference to a nucleic acid, the phrase "functional variant" refers to a nucleic acid that encodes a polypeptide that differs from the reference nucleic acid but has the same primary function as the polypeptide encoded by the reference nucleic acid. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference nucleic acid sequence, hybridizes under stringent hybridization conditions to the complement of a reference nucleic acid sequence, or is a codon-degenerate variant of a nucleic acid sequence.

[0078] As used herein, the term "antibody", also known as immunoglobulin (Ig), can refer to a monoclonal or polyclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B cells and that binds to the same epitope. In contrast, a "polyclonal antibody" refers to a population of antibodies produced by different B cells and that bind to different epitopes of the same antigen. The antibody can be from any animal. The antibody can be IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. In some embodiments, the antibody can be a single-chain whole antibody. An antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (V H ) domain and three C-terminal constant (CH1, CH2 and CH3) domains, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. H and V LThe regions have a similar general structure, and each region contains four framework regions whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody that is responsible for antigen binding. These particular regions are described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977), and Kabat et al., Sequences of proteins of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and the definitions include overlapping or subsets of amino acid residues when compared to each other. Preferably, the term "CDR" refers to the CDRs defined by Kabat, based on sequence comparisons: CDRH1, CDRH2 and CDRH3 represent the heavy chain CDRs, and CDRL1, CDRL2 and CDRL3 represent the light chain CDRs.

[0079] The terms "antibody fragment," "antibody fragment," "antibody fragment," "antigen-binding portion," and their grammatical equivalents are used interchangeably herein to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). Antibody fragments desirably contain, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations thereof. Non-limiting examples of antibody fragments include: (1) V L , V H , C L (2) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the stalk region; and (3) a V(ab')2 fragment, which is a bivalent fragment consisting of the V(ab')2 domain of a single arm of an antibody. L and V H(4) an Fv fragment consisting of two domains of an Fv fragment (i.e., V L and V H ), which are monovalent molecules consisting of a single Fv domain joined by a linker that allows the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16:778 (1998)); and (5) diabodies, which are dimers of polypeptide chains, each of which is composed of a V domain on the same polypeptide chain. H and V L V is bound by a peptide linker that is too short to allow pairing between L V connected to H and therefore different V H -V L Diabodies, which drive pairing between complementary domains on polypeptide chains to produce dimeric molecules with two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent No. 8,603,950.

[0080] The terms "antigen recognition moiety," "antigen recognition domain," "antigen binding domain," and "antigen binding region" refer to a molecule or a portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, an antibody-like molecule, or a fragment thereof.

[0081] The term "proliferative disorder" refers to the unifying concept in which excessive cell proliferation and / or cell-matrix turnover contributes significantly to the pathogenesis of disease, including cancer. In some embodiments, the proliferative disorder is cancer.

[0082] "Patient" or "subject" refers to a mammalian subject who has been diagnosed as having or suspected of having, or is developing, a proliferative disorder, such as cancer. In some embodiments, the term "patient" refers to a mammalian subject who has a higher than average likelihood of developing a proliferative disorder, such as cancer. Exemplary patients can be humans, apes, dogs, pigs, cows, cats, horses, goats, sheep, rodents, and other mammals that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female. "Patient in need thereof" or "subject in need thereof" refers to a patient who has been diagnosed as having or suspected of having a disease or disorder, such as, for example, but not limited to, cancer.

[0083] "Administering" as used herein refers to providing one or more compositions described herein to a patient or subject. By way of example and not limitation, composition administration, e.g., injection, can be by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be used. Parenteral administration can be by, for example, bolus injection or by gradual perfusion over time. Alternatively, or simultaneously, administration can be by oral route. In addition, administration can be by surgical deposition of a bolus or pellet of cells, or by positioning of a medical device.

[0084] As used herein, the terms "treatment", "treating", and their grammatical equivalents refer to obtaining a desired pharmacological and / or physiological effect. In some embodiments, the effect is therapeutic, i.e., the effect partially or completely cures a disease and / or adverse symptoms resulting from a disease. In some embodiments, the term "treating" can include "preventing" a disease or condition.

[0085] As used herein, a "treatment interval" refers to a treatment cycle, e.g., the course of administration of a therapeutic agent, which may be repeated on a regular schedule. In some embodiments, the dosing regimen may have one or more periods during which no administration of a therapeutic agent occurs during the treatment interval.

[0086] As used herein, the terms "co-administration," "co-administration," "co-administering," and "co-providing" refer to two (or more) different therapies being delivered to a subject during the course of the subject's illness, e.g., two or more therapies being delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated or the treatments are discontinued for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so there is an overlap in terms of administration. This may be referred to herein as "simultaneous" or "co-delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments in either case, the treatments are more effective due to the co-administration. For example, the second treatment is more effective than if the second treatment was administered in the absence of the first treatment, or a similar situation would be seen with the first treatment, e.g., a comparable effect is seen with less of the second treatment, or the second treatment relieves symptoms to a greater extent. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed with one treatment delivered in the absence of the other treatment. The effect of the two treatments can be partially additive, fully additive, or greater than additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0087] In some embodiments, the first and second treatments can be administered simultaneously (e.g., at the same time), in the same or separate compositions, or sequentially. Sequential administration refers to administration of one treatment prior to (e.g., immediately before, less than 5, 10, 15, 30, 45, 60 minutes, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96 hours or more, 4, 5, 6, 7, 8, 9 days or more, 1, 2, 3, 4, 5, 6, 7, 8 weeks or more) administration of an additional, e.g., secondary, treatment. The order of administration of the first and second treatments can also be reversed.

[0088] The terms "therapeutically effective amount", "therapeutic amount", "immunologically effective amount", "antitumor effective amount", "tumor inhibiting effective amount", and their grammatical equivalents refer to an amount effective at dosages and for periods of time necessary to achieve a desired therapeutic result. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the compositions described herein to induce a desired response in one or more subjects. The exact amount of the composition of the present disclosure to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0089] Alternatively, the pharmacological and / or physiological effect of administering one or more compositions described herein to a patient or subject can be "prophylactic," i.e., the effect can prevent, completely or partially, a disease or its symptoms. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., prevention of disease onset).

[0090] As used herein, the term "immune checkpoint protein" refers to a molecule that transmits inhibitory signals or has an immunosuppressive function. Examples of such immune checkpoint proteins include, but are not limited to, CTLA-4, PD-1, PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), LAG-3 (lymphocyte activation gene 3), TIM3 (T cell immunoglobulin and mucin-3), BTLA (B and T lymphocyte attenuator), B7H3, B7H4, CD160, CD39, CD70, CD73, A2aR (adenosine A2a receptor), KIR (killer inhibitory receptor), VISTA (V domain Ig-containing inhibitor of T cell activation), IDO1 (indoleamine 2,3-dioxygenase), arginase I, TIGIT (T cell immunoglobulin and ITIM domain), CD115, and the like (Nature Reviews Cancer, 12, p. 252-264, 2012, and Cancer See Cell, 27, p.450-461, 2015).

[0091] As used herein, the terms used in the identification of biological moieties may or may not include a dash "-" within the term. The presence or absence of the dash does not change the intended meaning or identification of the biological moiety. For purposes of illustration only, but not limited to these biological moieties, each of the following paired terms (shown with / without a dash) denotes and identifies the same biological entity: CCR-4 / CCR4, CD-3 / CD3, CD-4 / CD4, CD-33 / CD33, EGFR-2 / EGFR2, FLT-1 / FLT1, HER-1 / HER1, HER-1t / HER1t, IL-12 / IL12, IL-15 / IL15, IL-15Rα / IL15Rα MUC-1 / MUC1, MUC-16 / MUC16, ROR-1 / ROR1, ROR-1R / ROR1R, TGF-beta / TGF-beta, VEGF-1 / VEGF1, VEGF-R2 / VEGFR2.”

[0092] II. Combinations In certain embodiments, the miRNAs or polynucleotides encoding the miRNAs described herein may be used in combination with or may further comprise a nucleic acid sequence encoding a chimeric receptor, respectively. In some cases, the chimeric receptor is a chimeric antigen receptor (CAR). In some examples, the CAR comprises a pattern recognition receptor. In other cases, the chimeric receptor comprises an engineered T cell receptor (TCR). The miRNAs may be used in combination with a specific CAR, cytokine, and cell tag, for example, a MUC16-specific CAR with a fusion protein comprising IL15 and IL-15Rα (mbIL15) and truncated HER1 (HER1t), a MUC1-specific CAR with mbIL15 and HER1t, or a CD33-specific CAR with mbIL15 and HER1t.

[0093] In certain embodiments, the genetic construct can include a 5' untranslated region (5'UTR) and / or a 3' untranslated region (3'UTR). In some embodiments, the sequence encoding the miRNA can be located in the 5'UTR and / or the 3'UTR. In some embodiments, the 5'UTR and / or the 3'UTR do not include an intron.

[0094] In certain embodiments, a nucleic acid sequence encoding a synthetic intron with a checkpoint inhibitor miRNA is inserted into the same genetic construct as that encoding the chimeric receptor (e.g., in the portion of the construct corresponding to the 5'UTR of the mRNA encoding the chimeric receptor). An exemplary depiction of the order of the polynucleotide sequences can be found in FIG. 1A. In one embodiment, the 5'UTR contains one or more mature miRNAs. In some embodiments, one or more mature miRNAs may be directed to the same immune checkpoint protein target, e.g., PD-1. In certain other embodiments, a mature miRNA may be directed to multiple immune checkpoint protein targets, e.g., PD-1 and TIGIT, PD1 and CTLA4, or TIGIT and CTLA4.

[0095] III. miRNA As used herein, the terms "miR," "mir," and "miRNA" are used to refer to microRNAs, which are a class of small non-coding RNA molecules that can affect the expression of genes ("target genes") by regulating the translation of messenger RNA transcribed from those genes ("target genes") (either increasing or decreasing expression of the gene) and / or by destabilizing such messenger RNA.

[0096] miRNAs may not be naturally occurring. As used herein to describe miRNAs, the terms "non-naturally occurring," "synthetic," and "artificial" are used interchangeably to refer to miRNAs that have sequences that do not occur in nature. In some embodiments, non-naturally occurring miRNAs effectively mimic naturally occurring miRNAs. Non-naturally occurring miRNAs can be designed such that the desired hairpin or loop of the corresponding naturally occurring miRNA is maintained when the naturally occurring mature miRNA sequence is replaced with a synthetic sequence designed to target a specific transcript. For example, non-naturally occurring miRNAs can have at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity with a naturally occurring miRNA and / or can hybridize under stringent hybridization conditions with a naturally occurring miRNA. Unless otherwise indicated, the term "miRNA" generally refers to the mature, pri, and preforms of a particular microRNA, as well as functional fragments and variants thereof.

[0097] The term "pri-miRNA" refers to a primary miRNA transcript that contains at least one RNA hairpin. An exemplary depiction of a pri-miRNA is shown in FIG. 1B. The RNA hairpin is cleaved from the pri-miRNA in the cell nucleus to form one or more precursor miRNAs ("pre-miRNAs"). The pre-miRNA is exported to the cytoplasm where the stem-loop structure is cleaved to generate a double-stranded miRNA that contains a miRNA-5p strand from the original 5' arm of the hairpin loop and a miRNA-3p strand from the original 3' arm of the hairpin loop. Argonaute proteins then bind to the double-stranded miRNA and one of the strands (either the miRNA-5p sequence or the miRNA-3p sequence) is released. The remaining bound strand becomes the "guide strand", while the released strand is known as the "passenger strand" and is preferably degraded. The guide strand then interacts with the messenger RNA derived from the target gene, thus affecting its translation.

[0098] Both the miRNA-5p and miRNA-3p strand sequences are referred to herein as the "mature miRNA" sequence. The remaining portions of the pri-miRNA (its portion 5' to the miRNA-5p sequence, its portion 3' to the miRNA-3p sequence, and the stem-loop sequence between the miRNA-5p and miRNA-3p sequences) are collectively referred to as the miRNA scaffold sequence. The term "5' scaffold sequence" is used herein to refer to the scaffold sequence that is 5' to the miRNA-5p sequence in the pri-miRNA. The term "3' scaffold sequence" is used herein to refer to the scaffold sequence that is 3' to the miRNA-3p sequence in the pri-miRNA. The term "stem-loop sequence" refers to the scaffold sequence that is between the miRNA-5p and miRNA-3p sequences in the pri-miRNA.

[0099] The pri-miRNA of the present invention can be produced from naturally occurring pri-miRNA by removing the natural mature miRNA sequences and replacing them with non-natural mature miRNA sequences, one of which can function as a guide miRNA to target a gene of interest. In designing non-naturally occurring pri-miRNAs, the backbone miRNA nucleic acid sequence can be derived from mouse, rat, or human miRNA sequences. As a non-limiting example, the backbone miRNA sequence can be derived from miR150, miR206, miR204, miR17, miR16, miR30a, miR126, miR122, miR213, miR29b1, or miR133a1.

[0100] Examples of scaffold sequences that can be used in the practice of the invention include, but are not limited to, those encoded by the DNA sequences listed in Table 1 below. The symbols "X" and "Y" in Table 1 indicate the nucleic acid sequences encoding the guide miRNA (which may be either miRNA-5p or miRNA-3p) and passenger miRNA (which may be either miRNA-5p or miRNA-3p), respectively, and the symbol "n" indicates the number of nucleotides in such sequences, e.g., 16-30, preferably 18-25. In some embodiments, n can be 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, or 35 nucleotides. In certain embodiments, the scaffold miRNA sequence is one that is encoded by a sequence that hybridizes under stringent hybridization conditions to the complement of any one of the sequences listed in Table 1. [Table 1]

[0101] Although miRNA-5p and miRNA-3p sequences hybridize with each other, they are not necessarily exactly complementary. In designing non-naturally occurring miRNAs, compensatory mutations can be made in miRNA-5p and / or miRNA-3p sequences to maintain the RNA folding and free energy of natural miRNAs. In certain embodiments, the sequence encoding the miRNA-3p sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the complement of the sequence encoding the miRNA-5p sequence, or can hybridize with the sequence encoding the miRNA-5p sequence under stringent hybridization conditions.

[0102] In certain embodiments, the target gene encodes a checkpoint inhibitor protein. Thus, the present invention relates, in part, to a polynucleotide encoding a non-naturally occurring miRNA that inhibits the expression of an immune checkpoint protein. In certain such embodiments, the target gene encodes CTLA4, PD-1, PD-L1, TIGIT, TIM3, LAG3, GITR, or PIK3IP1. Non-limiting examples of nucleic acid sequences encoding guide miRNA targeting genes encoding such checkpoint inhibitors are listed in Table 2. Table 2 also lists sequences encoding passenger strands. As previously discussed, the guide and passenger strands are not necessarily complementary. It is contemplated that the passenger strand may also serve to target messenger RNA associated with the target gene. It is also contemplated that sequences may be used that are sequences that hybridize to the complement of the sequences listed in Table 2 under stringent hybridization conditions. The mature miRNA sequence used may be combined with a particular pri-miRNA scaffold. Table 2 also lists scaffolds that can be combined with the mature guide and passenger miRNAs listed therein. [Table 2] TIFF2024502479000006.tif228170

[0103] In certain embodiments, the present invention relates to polynucleotides comprising a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 64-83, 85, 87-171, and 704-713, or capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 64-83, 85, 87-171, and 704-713. In certain such embodiments, the present invention relates to polynucleotides comprising a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 64-83, 85, 87-171, and 704-713, or capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 139, 140, 142, 144, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 170, 171 6, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 704, 705, 709, and 710. or can hybridize under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 704, 705, 709, and 710.

[0104] In certain such embodiments, the miRNA targets PD-1.In certain such embodiments, the invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 72-83, 85, 87, and 704-713, or capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 72-83, 85, 87, and 704-713. In certain embodiments, the invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 704, 705, 709, and 710, or capable of hybridizing to the complement of any one of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 704, 705, 709, and 710 under stringent hybridization conditions.

[0105] In certain embodiments, the invention relates to polynucleotides comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 72-75, or capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 72-75. In certain such embodiments, the invention relates to polynucleotides comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 72 or 74, or capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NOs: 72 or 74.

[0106] In a particular embodiment, the present invention relates to a polynucleotide comprising: a) a sequence encoding the 5' miRNA scaffold sequence; b) a sequence encoding a guide miRNA sequence; c) a sequence encoding a stem-loop sequence; d) a sequence encoding a passenger miRNA sequence, and e) A sequence encoding the 3' backbone sequence.

[0107] In certain embodiments, the sequence encoding the guide miRNA sequence is selected from the group consisting of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 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, 2 , 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 704, 705, 709, and 710, or can hybridize under stringent hybridization conditions to the complement of any one of such sequences.

[0108] In certain embodiments, the sequence encoding the passenger miRNA sequence is selected from the group consisting of SEQ ID NOs: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 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, 198, 199, 200, 201, 202, 203, 204 9, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 706-708, and 711-713, or can hybridize under stringent hybridization conditions to the complement of any one of such sequences.

[0109] In certain embodiments, the polynucleotide encoding the pri-miRNA encodes: a) SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160 , 162, 164, 166, 168, 170, 704, 705, 709, and 710, or a guide miRNA sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of such sequences or that can hybridize to the complement of any one of such sequences under stringent hybridization conditions; b) SEQ ID NOs: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 1 61, 163, 165, 167, 169, 171, 706-708, and 711-713, respectively, or a passenger sequence that can hybridize to the complement of any one of such sequences under stringent hybridization conditions.

[0110] In any of the foregoing embodiments, the sequences encoding the 5' miRNA scaffold sequence, the stem-loop sequence, and the 3' miRNA scaffold sequence are each SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively. SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively. SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively. SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively. SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively. SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively. SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively. SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively. SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively. SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively. SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively. SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively. SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively. SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively. SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively. SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, respectively; or SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, respectively. Or it may include a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to such a sequence, or a sequence capable of hybridizing under stringent hybridization conditions to the complement of such a sequence.

[0111] Nucleic acids encoding exemplary non-naturally occurring pri-miRNA sequences that target specific checkpoint inhibitors are set forth in Table 3. In certain embodiments, the non-naturally occurring pri-miRNA sequence can be a sequence that can hybridize under stringent hybridization conditions to the complement of any one of the sequences listed in Table 3. [Table 3] TIFF2024502479000008.tif254170TIFF2024502479000009.tif23170

[0112] In certain such embodiments, the invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 178-263, or capable of hybridizing to the complement of any one of SEQ ID NOs: 178-263 under stringent hybridization conditions.

[0113] In certain embodiments, the miRNA targets PD-1. In certain such embodiments, the invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 179, 180, and 241-249, or capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 179, 180, and 241-249. In certain embodiments, the invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 179 or 180, or capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 179 or 180.

[0114] Certain embodiments of the present invention relate to polynucleotides comprising nucleic acid sequences encoding at least two pri-miRNAs. Two or more pri-miRNAs may comprise guide miRNA sequences targeting the same target gene, or different guide miRNAs may target different genes. In the case of multiple pri-miRNAs, each design may be based on a different natural miRNA backbone to reduce the possibility of one miRNA misfolding with another miRNA. Table 4 provides examples of nucleic acid sequences encoding one or more pri-miRNAs. [Table 4-1]

[0115] In certain such embodiments, the invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 267-290, or capable of hybridizing to the complement of any one of SEQ ID NOs: 267-290 under stringent hybridization conditions.

[0116] In certain such embodiments, the pri-miRNA comprises at least two pre-miRNAs that target PD-1.In certain embodiments, the invention relates to a polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 267, or capable of hybridizing to the complement of any one of SEQ ID NOs: 267 under stringent hybridization conditions.

[0117] In certain embodiments, the nucleic acid encoding the pri-miRNA is included in the same genetic construct as one that includes one or more genes encoding a protein of interest (e.g., a chimeric antigen receptor, a cytokine, or a cell tag). In certain embodiments, such genetic constructs include a nucleic acid sequence encoding a 5' untranslated region (5'UTR) directly upstream of the gene of interest, and the sequence encoding the miRNA is included in the 5'UTR. In certain embodiments, such genetic constructs include a nucleic acid sequence encoding a 3' untranslated region (3'UTR) directly downstream of the gene of interest, and the sequence encoding the miRNA is included in the 3'UTR.

[0118] In embodiments in which the sequence encoding the pri-miRNA is included in the sequence corresponding to the 5'UTR, the transcribed RNA may include additional sequences such as splice donor, branch point, and / or acceptor site sequences. The inclusion of splice donor, branch point, and acceptor sites is important for the splicing of the miRNA from the transcribed RNA. Without splicing, the highly structured miRNA sequence would likely prevent ribosome scanning to the translation initiation sequence associated with the gene of interest. Examples of sequences encoding such splice donor / acceptor sites include SEQ ID NOs: 291 and 292, sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with such sequences, and sequences capable of hybridizing to the complement of such sequences under stringent hybridization conditions.

[0119] Thus, in certain embodiments, the polynucleotides of the invention further comprise a) a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:291 or capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO:291, and b) a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:292 or capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO:292.

[0120] IV. Chimeric Antigen Receptors (CARs) In any of the foregoing embodiments, the polynucleotide of the present disclosure can further encode a chimeric receptor, such as a chimeric antigen receptor (CAR). Thus, the polynucleotide of the present disclosure can encode a miRNA and a CAR, as described herein. In any of the embodiments of the present disclosure, the modified immune effector cell can include a chimeric receptor, as described herein.

[0121] CARs are engineered receptors that transfer exogenous specificity to immune effector cells. In some instances, CARs comprise an extracellular domain (ectodomain) that comprises an antigen-binding domain, a transmembrane domain, and an intracellular (endodomain) domain. The intracellular domain comprises an intracellular signaling domain. In certain embodiments, the extracellular domain further comprises a spacer between the antigen-binding domain and the transmembrane domain.

[0122] A. Antigen-binding Domains The antigen-binding domain may comprise the complementarity determining region of a monoclonal antibody and / or its antigen-binding fragment. Complementarity determining regions (CDRs) are short amino acid sequences found in the variable domains of antigen receptor (e.g., immunoglobulin and T-cell receptor) proteins that bind to an antigen, thus providing the receptor with its specificity for that particular antigen. Each polypeptide chain of an antigen receptor may contain three CDRs (CDR1, CDR2, and CDR3).

[0123] In certain embodiments, the antigen-binding domain comprises an antibody, or a functional fragment or variant thereof, that binds to a target antigen. The functional fragment or variant may comprise the variable domain of the heavy chain of the antibody (VH) and / or the variable domain of the light chain of the antibody (VL), or a functional fragment or variant thereof. In certain embodiments, the antigen-binding domain comprises the Fv, Fab, Fab of an antibody. 2 , Fab', F(ab') 2 or F(ab') 3In certain embodiments, the antigen-binding domain comprises an scFv, sc(Fv) 2 , dsFv, diabody, minibody, nanobody, or binding fragments thereof. In certain embodiments, the antigen-binding domain further comprises an Fc fragment of an antibody, for example, it may comprise an scFv linked to an Fc fragment.

[0124] In some embodiments, the CAR targets an antigen elevated in cancer cells, autoimmune cells, or cells infected with a virus, bacteria, or parasite. Pathogens that can be targeted include, but are not limited to, Plasmodium, trypanosome, Aspergillus, Candida, Hepatitis A, Hepatitis B, Hepatitis C, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus, or Ebola pathogens. Autoimmune diseases can include graft-versus-host disease, rheumatoid arthritis, lupus, celiac disease, Crohn's disease, Sjogren's syndrome, polymyalgic rheumatoid arthritis, multiple sclerosis, neuromyelitis optica, ankylosing spondylitis, type 1 diabetes, patchy alopecia, vasculitis, temporal arteritis, bullous pemphigus, psoriasis, pemphigus vulgaris, and autoimmune uveitis. The pathogen recognized by the CAR can be essentially any type of pathogen, but in some embodiments, the pathogen is a fungus, a bacterium, or a virus. Exemplary viral pathogens include Adenoviridae, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV), JC virus, BK virus, HPV, HSV, HHV family of viruses, Hepatitis family of viruses, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae. Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, chickenpox, Ebola, and rubella. Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella. In some embodiments, the pathogen receptor Dectin-1 may be used to generate CARs that recognize carbohydrate structures on the cell walls of fungi such as Aspergillus. In another embodiment, CARs can be made based on antibodies that recognize viral determinants (e.g., glycoproteins from CMV and Ebola) to interrupt viral infection and pathology.

[0125] In some embodiments, the CAR described herein is selected from the group consisting of B7H4, BCMA, BTLA, CAIX, CA125, CCR4, CD3, CD4, CD5, CD7, CD16, CD19, CD20, CD22, CD24, CD25, CD28, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / v8, CD47, CD52, CD56, CD70, CD79b, CD80, CD81, CD86, CD123, CD133, CD137, CD138, CD151, CD171, CD174, CD276, CEA, CE ACAM6, CLL-1, c-MET, CS1, CSPG4, CTLA-4, DLL3, EDB-F, EGFR, EGFR2, EGFRvIII, EGP-2, EGP-40, EphA2, FAP, FLT1, FLT4, folate binding protein, folate receptor, folate receptor alpha, alpha-folate receptor, frizzled, GD2, GD3, GHR, GHRHR, GITR, GPC3, Gp100, gp130, HBV antigen, HER1, HER2, HER3, HER4, HER1 / HER3, h5T4, HPV antigen, HVEM, IGF1R, Ig KAppa, IL-1-RAP, IL-2R, IL6R, IL-11Rα, IL-13R-a2, KDR, KRASG12V, LewisA, LewisY, L1-CAM, LIFRP, LRP5, LTPR, MAGE-A, MAGE-A1, MAGE-A10, MAGE-A3, MAGEA3 / A6, MAGE-A4, MAGE-A6, MART-1, MCAM, mesothelin, PSCA, mucins such as MUC1, MUC-4 or MUC16, NGFR, NKG2D, Notch-1-4, NY-ESO-1, O-acetyl GD2, O- The antigen-binding domain comprises an antigen-binding domain that binds to an epitope on acetyl-GD3, OX40, P53, PD1, PDE10A, PD-L1, PD-L2, PMSA, PRAME, PSCA, PSMA, PTCH1, RANK, Robol, ROR1, ROR1R, ROR-2, TACI, TAG-72, TCRa, TCRp, TGF, TGF beta, TGF beta-II, TGFBR1, TGFBR2, Titin, TLR7, TLR9, TNFR1, TNFR2, TNFRSF4, TRBC1, TWEAK-R, VEGF, VEGF-R2, or WT-1.

[0126] In some embodiments, the CARs described herein comprise an antigen binding domain that binds to an epitope on CD19, CD33, MUC1, MUC16, or ROR1. In some examples, the CARs described herein comprise an antigen binding domain that binds to an epitope on CD19. In some cases, the CARs described herein comprise an antigen binding domain that binds to an epitope on CD33. In some examples, the CARs described herein comprise an antigen binding domain that binds to an epitope on MUC1. In some examples, the CARs described herein comprise an antigen binding domain that binds to an epitope on MUC16. In some examples, the CARs described herein comprise an antigen binding domain that binds to an epitope on ROR1. In further embodiments, the CARs described herein comprise an autoantigen or antigen binding domain that binds to an epitope on HLA-A2, myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAdCAM1, SDF1, or type II collagen.

[0127] Antigen binding can be evaluated by flow cytometry or cell-based assays, or any other equivalent assay. Cell-based assays can utilize cell types expressing the antigen of interest on the surface to evaluate antigen binding. Antigens expressed as soluble proteins or fragments thereof can be used to evaluate antigen binding using flow cytometry or similar assays. Improved antigen binding can be indirectly evaluated by functional measurements of the antigen-binding domain or chimeric receptor. For example, improved antigen binding of the chimeric receptor or CAR described herein can be measured by increased specific cytotoxicity against target cells expressing the antigen.

[0128] The cell surface expression level of the polypeptides of the present disclosure can be assessed, for example, using a flow cytometry-based assay. Improved expression of an antigen-binding polypeptide can be measured as the percentage of analyzed cells expressing the antigen-binding polypeptide, or alternatively, as the average density of the antigen-binding polypeptide on the surface of the cells. Additional suitable methods that can be used to assess cell surface expression of the antigen-binding polypeptides described herein include Western blotting or any other equivalent assay.

[0129] 1. CD19-specific antigen-binding domain CD19 is a cell surface glycoprotein of the immunoglobulin superfamily that is found primarily on malignant B-lineage cells. In some instances, CD19 has also been detected in solid tumors, such as pancreatic, liver, and prostate cancers.

[0130] In some embodiments, the CAR comprises a CD19-specific antigen-binding domain, and the antigen-binding domain is F(ab') 2 , Fab', Fab, Fv, or scFv. In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by FMC63. In some embodiments, the scFv and / or VH / VL domains are derived from FMC63. FMC63 generally refers to a murine monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, NR, el al. (1987). Leucocyte typing III. 302).

[0131] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics).

[0132] In some embodiments, the antigen binding domain comprises an scFv antigen binding domain that recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics).

[0133] In some embodiments, the antigen binding domain comprises an anti-CD19 antibody described in U.S. Patent Application Publication No. 2016 / 0152723. In some embodiments, the antigen binding domain comprises an anti-CD19 antibody described in WO 2015 / 123642. In some embodiments, the antigen binding domain comprises an anti-CD19 scFv derived from clone FMC63 (Nicholson et al., Construction and characterization of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukemia and lymphoma., Mol. Immunol. 34:1157-1165 (1997)).

[0134] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19 (Kite Pharma, Inc.).

[0135] In some embodiments, the antigen binding domain comprises an scFv antigen binding domain, wherein the antigen binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19.

[0136] In some embodiments, the antigen binding domain comprises an anti-CD19 antibody described in WO 2015 / 187528, or a fragment or variant thereof.

[0137] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by CTL019 (Novartis).

[0138] In some embodiments, the antigen binding domain comprises an scFv antigen binding domain, wherein the antigen binding domain recognizes an epitope on CD19 that is also recognized by CTL019.

[0139] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by UCART19 (Cellectis).

[0140] In some embodiments, the antigen binding domain comprises an scFv antigen binding domain, and the antigen binding domain recognizes an epitope on CD19 that is also recognized by UCART19.

[0141] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by BPX-401 (Bellicum).

[0142] In some embodiments, the antigen binding domain comprises an scFv antigen binding domain, wherein the antigen binding domain recognizes an epitope on CD19 that is also recognized by BPX-401.

[0143] In some cases, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), cortuximab ravtansine (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-551 (Medimmune), denintuzumab mafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomab paptox (National Cancer Institute), XmAb 5871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex), or AFM11 (Affimed).

[0144] In some embodiments, the antigen binding domain is an F(ab') 2, Fab', Fab, Fv, or scFv, wherein the antigen-binding domain recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), cortuximab ravtansine (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-551 (Medimmune), denintuzumab mafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomab paptox (National Cancer Institute), XmAb 5871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex) or AFM11 (Affimed).

[0145] In some cases, the antigen binding domain comprises an scFv antigen binding domain, wherein the antigen binding domain recognizes an epitope on CD19 that is also recognized by FMC63, blinatumomab (Amgen), cortuximab ravtansine (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-551 (Medimmune), denintuzumab mafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomab paptox (National Cancer Institute), XmAb 5871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex), or AFM11 (Affimed).

[0146] 2. CD33-specific antigen-binding domain "CD33," a 67 kDa single-pass transmembrane glycoprotein, is a member of the sialic acid-binding immunoglobulin-like lectin (Siglecs) superfamily. CD33 is characterized by a V-set Ig-like domain involved in sialic acid binding and a C2-set Ig-like domain in its extracellular domain. CD33 is expressed in myeloid cells and has also been detected in lymphoid cells. Alternative splicing of CD33 mRNA results in a shorter isoform (CD33m) that lacks the V-set Ig-like domain and the disulfide bond linking the V- and C2-set Ig-like domains. In healthy subjects, CD33 is expressed primarily as a myeloid differentiation antigen found on normal multipotent myeloid precursors, unipotent colony-forming cells, monocytes, and mature granulocytes. CD33 is expressed on more than 80% of myeloid leukemia cells, but not on normal hematopoietic stem cells or mature granulocytes. (Andrews, R. et al., The L4F3 antigen is expressed by unipotent and multipotent colony-forming cells but not by their precursors, Blood, 68(5): 1030-5 (1986)). CD33 has been reported to be expressed on malignant myeloid cells, activated T cells, and activated NK cells, and is found on at least a subset of blasts in the majority of AML patients (Pollard, J. et al., Correlation of CD33 expression level with disease characteristics and response to gemtuzumab ozogamicin containing chemotherapy in childhood AML, Blood, 119(16): 3705-11 (2012)). In addition to widespread expression on AML blasts, CD33 can be expressed on the stem cells that underlie AML.

[0147] In some embodiments, the antigen binding domain of the CAR described herein is specific for CD33 (CD33 CAR). CD33-specific CARs, when expressed on the cell surface, redirect the specificity of T cells to human CD33. In some embodiments, the antigen binding domain comprises a single-chain antibody fragment (scFv) comprising a variable domain light chain (VL) and a variable domain heavy chain (VH) of a target antigen-specific monoclonal anti-CD33 antibody linked by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In some embodiments, the scFv is M195, m2H12, DRB2, and / or My9-6. In some embodiments, the scFv is humanized, e.g., hM195. In some embodiments, the antigen binding domain can comprise a VH and a VL directionally linked, e.g., from N-terminus to C-terminus, VH-linker-VL or VL-linker-VH. In some embodiments, the antigen binding domain comprises a F(ab')2, Fab', Fab, Fv, or scFv that binds to CD33. In some embodiments, the antigen binding region recognizes an epitope on CD33 that is also recognized by Lintuzumab (Seattle Genetics), BI 836858 (Boehringer Ingelheim).

[0148] In some embodiments, a CAR described herein comprises an antigen binding domain comprising a VL polypeptide.

[0149] In certain embodiments, the antigen binding domain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 293 (hM195 VL domain), SEQ ID NO: 296 (M2H12 VL domain), SEQ ID NO: 298 (DRB2 VL domain), SEQ ID NO: 300 (My9-6 VL domain), or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than any one of the foregoing sequences. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 293, 296, 298, and 300, and / or is a conservatively substituted variant of any one of such sequences.

[0150] In certain embodiments, the antigen binding domain comprises a VL domain encoded by SEQ ID NO: 301 (nucleic acid encoding the VL domain for hM195). In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 301, hybridizes under stringent hybridization conditions to the complement of the nucleic acid sequence of SEQ ID NO: 301, or is a codon-degenerate variant of SEQ ID NO: 301.

[0151] In some embodiments, a CAR described herein comprises an antigen-binding domain comprising a VH polypeptide.

[0152] In certain embodiments, the antigen binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 294 (hM195 VH domain), SEQ ID NO: 295 (M2H12 VH domain), SEQ ID NO: 297 (DRB2 VH domain), SEQ ID NO: 299 (My9-6 VH domain), or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than any one of the foregoing sequences. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 294, 295, 297, or 299, and / or is a conservatively substituted variant of the amino acid sequence of any one of SEQ ID NOs: 294, 295, 297, or 299.

[0153] In certain embodiments, the antigen binding domain comprises a VH domain encoded by SEQ ID NO: 302 (nucleic acid encoding a VH domain for hM195), or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 302, hybridizes under stringent hybridization conditions to the complement of the nucleic acid sequence of SEQ ID NO: 302, or is a codon-degenerate variant of SEQ ID NO: 302.

[0154] In some embodiments, an antigen binding domain can comprise a VH and a VL directionally linked, for example, from N-terminus to C-terminus, VH-linker-VL, or VL-linker-VH. Any linker described herein can be used to link the VH and VL domains.

[0155] In certain embodiments, the antigen binding domain comprises an scFv. In certain such embodiments, the domain comprises the amino acid sequence of SEQ ID NO: 303, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than any one of the foregoing sequences. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 303 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 303.

[0156] In certain embodiments, the antigen binding domain is encoded by SEQ ID NO: 304, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 304, hybridizes under stringent hybridization conditions to the complement of the nucleic acid sequence of SEQ ID NO: 304, or is a codon-degenerate variant of SEQ ID NO: 304.

[0157] 3. MUC1-specific antigen-binding domain In one embodiment, the antigen binding domain binds to an epitope on MUC1. In some embodiments, the anti-MUC1 antibody fragment comprises an anti-MUC1 Fab, Fab 2 , (Fab') 2 , Fv, (Fv) 2 , scFv, scFv-FC, FC, diabody, triabody, or minibody. In some embodiments, the anti-MUC1 antibody fragment is a single domain antibody of an anti-MUC1 antibody. In some embodiments, the single domain antibody is a V of an anti-MUC1 antibody. NAR Or V HH It is a fragment.

[0158] In some embodiments, a CAR described herein comprises an antigen-binding domain comprising a VL polypeptide of MUC1.

[0159] In certain embodiments, the antigen-binding domain comprises a VL domain comprising the amino acid sequence of any one of SEQ ID NOs: 310-314, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of any one of SEQ ID NOs: 310-314. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 310-314 and / or is a conservatively substituted variant of the amino acid sequence of any one of SEQ ID NOs: 310-314.

[0160] In some embodiments, a CAR described herein comprises an antigen-binding domain comprising a VH polypeptide of MUC1.

[0161] In certain embodiments, the antigen-binding domain comprises a VH domain comprising the amino acid sequence of any one of SEQ ID NOs: 305-309, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of any one of SEQ ID NOs: 305-309. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 305-309 and / or is a conservatively substituted variant of the amino acid sequence of any one of SEQ ID NOs: 305-309.

[0162] In some embodiments, an antigen-binding portion may comprise a VH and a VL directionally linked, for example, from N-terminus to C-terminus, VH-linker-VL, or VL-linker-VH. Any linker described herein may be used to link the VH and VL domains.

[0163] 4. MUC16-specific antigen-binding domain In some embodiments, the antigen-binding portion of the CAR described herein is specific for MUC16 (MUC16 CAR). A MUC16-specific CAR, when expressed on the cell surface, redirects the specificity of T cells to human MUC16.

[0164] In certain embodiments, the antigen binding domain comprises a VL domain comprising the amino acid sequence of any one of SEQ ID NOs: 329, 331, 333, 335, 337, 339, 341, 662, 664, 666, 668, 670, 688, 690, 692, 694, 696, 698, and 700, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of any one of the foregoing sequences. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 329, 331, 333, 335, 337, 339, 341, 662, 664, 666, 668, 670, 688, 690, 692, 694, 696, 698, and 700, and / or is a conservatively substituted variant of the amino acid sequence of any one of SEQ ID NOs: 329, 331, 333, 335, 337, 339, 341, 662, 664, 666, 668, 670, 688, 690, 692, 694, 696, 698, and 700.

[0165] In certain embodiments, the antigen binding domain comprises a VL domain encoded by any one of SEQ ID NOs: 330, 332, 334, 336, 338, 340, 342, 663, 665, 667, 669, 671, 689, 691, 693, 695, 697, 699, and 701, or a functional fragment or variant thereof. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 330, 332, 334, 336, 338, 340, 342, 663, 665, 667, 669, 671, 689, 691, 693, 695, 697, 699, and 701, or hybridizes under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 330, 332, 334, 336, 338, 340, 342, 663, 665, 667, 669, 671, 689, 691, 693, 695, 697, 699, and 701.

[0166] In certain embodiments, the antigen binding domain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 692, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 692. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 692 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 692.

[0167] In certain embodiments, the antigen binding domain comprises a VL domain encoded by SEQ ID NO: 693, or a functional fragment or variant thereof. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 693, or hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 693.

[0168] In certain embodiments, the antigen binding domain comprises a VH domain comprising the amino acid sequence of any one of SEQ ID NOs: 315, 317, 319, 321, 323, 325, 327, 648, 650, 652, 654, 656, 658, 660, 672, 674, 676, 678, 680, 682, 684, and 686, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of any one of the foregoing sequences. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 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%, 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, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 178, 179, 180, 182, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198 8% or 99% sequence identity and / or are conservatively substituted variants of any one of the amino acid sequences of SEQ ID NOs: 315, 317, 319, 321, 323, 325, 327, 648, 650, 652, 654, 656, 658, 660, 672, 674, 676, 678, 680, 682, 684, and 686.

[0169] In certain embodiments, the antigen binding domain comprises a VH domain encoded by any one of SEQ ID NOs: 316, 318, 320, 322, 324, 326, 328, 649, 651, 653, 655, 657, 659, 661, 673, 675, 677, 679, 681, 683, 685, and 687, or a functional fragment or variant thereof. In certain embodiments, a functional variant has a sequence similar to that of any one of SEQ ID NOs: 316, 318, 320, 322, 324, 326, 328, 649, 651, 653, 655, 657, 659, 661, 673, 675, 677, 679, 681, 683, 685, and 687, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more identical to any one of SEQ ID NOs: 316, 318, 320, 322, 324, 326, 328, 649, 651, 653, 655, 657, 659, 661, 673, 675, 677, 679, 681, 683, 685, and 687. has 99% sequence identity or hybridizes under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 316, 318, 320, 322, 324, 326, 328, 649, 651, 653, 655, 657, 659, 661, 673, 675, 677, 679, 681, 683, 685, and 687.

[0170] In certain embodiments, the antigen binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 676, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 676. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 676 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 676.

[0171] In certain embodiments, the antigen binding domain comprises a VH domain encoded by SEQ ID NO: 677, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 677, or hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 677.

[0172] In some embodiments, an antigen-binding portion may comprise a VH and a VL directionally linked, for example, from N-terminus to C-terminus, VH-linker-VL, or VL-linker-VH. Any linker described herein may be used to link the VH and VL domains.

[0173] In some embodiments, the antigen-binding domain comprises a single chain antibody fragment (scFv) comprising the variable domain light chain (VL) and variable domain heavy chain (VH) of a target antigen-specific monoclonal anti-MUC16 antibody linked by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In certain embodiments, the scFv comprises the VH and VL from MUC16-3 and a linker. In certain such embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 343, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 343. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:343 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO:343.

[0174] In certain embodiments, the antigen binding domain comprises an scFv encoded by SEQ ID NO: 344, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 344, or hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 344.

[0175] 5. ROR1-specific antigen-binding domain In some embodiments, the CAR described herein comprises an antigen-binding domain comprising the VL domain of an anti-ROR1 antibody.

[0176] In certain such embodiments, the antigen binding domain may comprise the amino acid sequence of any one of SEQ ID NOs: 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, and 463, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than any one of the foregoing sequences. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 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 8% or 99% sequence identity and / or are conservatively substituted variants of any one of the amino acid sequences of SEQ ID NOs: 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, and 463.

[0177] In certain embodiments, the antigen binding domain comprises a V sequence encoded by any one of SEQ ID NOs: 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, and 464, or a functional fragment or variant thereof. LIn certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, and 464, or a domain similar to SEQ ID NOs: 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400 , 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, and 464, or a codon-degenerate variant of any one of SEQ ID NOs: 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, and 464.

[0178] In some embodiments, the CAR described herein comprises the VH domain of an anti-ROR1 antibody.

[0179] In certain such embodiments, the antigen binding domain may comprise the amino acid sequence of any one of SEQ ID NOs: 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, and 461, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than any one of the foregoing sequences. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 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, 1 8% or 99% sequence identity and / or are conservatively substituted variants of any one of the amino acid sequences of SEQ ID NOs: 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, and 461.

[0180] In certain embodiments, the antigen binding domain comprises a V sequence encoded by any one of SEQ ID NOs: 350, 354, 358, 362, 366, 370, 374, 378, 382, ​​386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, 450, 454, 458, and 462, or a functional fragment or variant thereof. HIn certain embodiments, the functional variant comprises a domain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 350, 354, 358, 362, 366, 370, 374, 378, 382, ​​386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, 450, 454, 458, and 462, or ... 02, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, 450, 454, 458, and 462, or a codon-degenerate variant of any one of SEQ ID NOs: 350, 354, 358, 362, 366, 370, 374, 378, 382, ​​386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, 450, 454, 458, and 462.

[0181] In certain embodiments, the antigen binding domain comprises at least one CDR selected from those comprising the amino acid sequence of any one of the CDRs that binds to ROR1, or a functional fragment or variant thereof.

[0182] In certain embodiments, the antigen-binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 715-725, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than any one of the above sequences. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 715-725, and / or is a conservatively substituted variant of any one of SEQ ID NOs: 715-725.

[0183] In certain embodiments, the antigen binding domain comprises the amino acid sequences of SEQ ID NO:715, SEQ ID NO:716, and SEQ ID NO:717. H Includes the domain.

[0184] In certain embodiments, the antigen binding domain comprises the amino acid sequences of SEQ ID NO:718, SEQ ID NO:719, and SEQ ID NO:720. H Includes the domain.

[0185] In certain embodiments, the antigen binding domain comprises the amino acid sequences of SEQ ID NO:721, SEQ ID NO:722, and SEQ ID NO:723. L Includes the domain.

[0186] In certain embodiments, the antigen binding domain comprises the amino acid sequences of SEQ ID NO:724, SEQ ID NO:725, and SEQ ID NO:723. L Includes the domain.

[0187] In certain embodiments, the antigen binding domain comprises one or more of the aforementioned V H Domain and V L Includes both the domain.

[0188] In certain embodiments, the antigen binding domain comprises a variable heavy domain comprising the amino acid sequence of SEQ ID NO: 349, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 349. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 349 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 349.

[0189] In certain embodiments, the antigen binding domain comprises a VH domain encoded by SEQ ID NO: 350, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 350, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 350, or is a codon-degenerate variant of SEQ ID NO: 350.

[0190] In certain embodiments, the antigen binding domain comprises a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 387, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 387. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 387 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 387.

[0191] In certain embodiments, the antigen binding domain comprises the V L In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:388, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO:388, or is a codon-degenerate variant of SEQ ID NO:388.

[0192] In certain embodiments, the antigen binding domain comprises a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 349, or a functional fragment or variant thereof, wherein the variable heavy chain domain comprises, in order from N-terminus to C-terminus, the sequences of SEQ ID NO: 715, SEQ ID NO: 716, and SEQ ID NO: 717. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 349. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 349 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 349.

[0193] In certain embodiments, the antigen binding domain comprises a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 349, or a functional fragment or variant thereof, wherein the variable heavy chain domain comprises, in order from N-terminus to C-terminus, the sequences of SEQ ID NO: 718, SEQ ID NO: 719, and SEQ ID NO: 720. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 349. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 349 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 349.

[0194] In certain embodiments, the antigen binding domain comprises a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 387, or a functional fragment or variant thereof, and the variable heavy chain domain comprises, in order from N-terminus to C-terminus, the sequences of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 387. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 387 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 387.

[0195] In certain embodiments, the antigen binding domain comprises a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 387, or a functional fragment or variant thereof, and the variable heavy chain domain comprises, in order from N-terminus to C-terminus, the sequences of SEQ ID NO: 724, SEQ ID NO: 725, and SEQ ID NO: 723. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 387. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 387 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 387.

[0196] In certain embodiments, the antigen binding domain comprises a variable heavy domain comprising the amino acid sequence of SEQ ID NO: 726, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 726. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 726 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 726.

[0197] In certain embodiments, the antigen binding domain comprises a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 727, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 727. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 727 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 727.

[0198] In certain embodiments, the antigen binding domain comprises a variable heavy domain comprising the amino acid sequence of SEQ ID NO: 728, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 728. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 728 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 728.

[0199] In certain embodiments, the antigen binding domain comprises a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 729, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 729. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 729 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 729.

[0200] In certain embodiments, the antigen binding domain comprises H Domain and V L In certain such embodiments, the linker comprises a linker having the amino acid sequence of SEQ ID NO: 424 ((G 4 S) 3 ) or a conservatively substituted variant thereof, or (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 424. In certain such embodiments, the linker is encoded by SEQ ID NO: 425, hybridizes under stringent conditions to the complement of SEQ ID NO: 425, or is a codon-degenerate version of SEQ ID NO: 425. Any of the linkers described herein can be used to link the VH and VL domains.

[0201] In certain embodiments, the antigen binding domain comprises an scFv. In certain such embodiments, the domain comprises the amino acid sequence of SEQ ID NO: 465, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 465. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 465 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 465.

[0202] In certain embodiments, the antigen binding domain comprises an scFv encoded by SEQ ID NO: 466, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 466, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 466, or is a codon-degenerate variant of SEQ ID NO: 466.

[0203] 6. EGFRvIII-specific antigen-binding domain In another embodiment, the CAR described herein is an EGFRvIII-specific CAR. "EGFRvIII", "EGFR variant III", "EGFR type III mutant", "EGFR.D2-7" or "de2-7EGFR" is a mutant form of the epidermal growth factor receptor (EGFR; ErbB-1; HER1), a transmembrane protein that is a receptor for members of the epidermal growth factor (EGF) family of extracellular protein ligands in human and non-human subjects. EGFRvIII is characterized by a deletion of exons 2-7 of the wild-type EGFR gene, resulting in an in-frame deletion of 267 amino acids in the extracellular domain of the full-length wild-type EGFR protein. EGFRvIII also contains a novel glycine residue inserted at the fusion junction compared to wild-type EGFR. Although the truncated receptor EGFRvIII cannot bind any known EGFR ligand, it does exhibit constitutive tyrosine kinase activity. This constitutive activation is critical for its pro-oncogenic effects. Kinase-deficient EGFRvIII fails to confer a similar oncogenic advantage. EGFRvIII is highly expressed in glioblastoma (GBM) and can be detected in several other solid tumor types, but not in normal tissues.

[0204] In some embodiments, the antigen-binding portion of the CAR described herein is specific for EGFRvIII (EGFRvIII CAR). EGFRvIII-specific CARs, when expressed on the cell surface, redirect the specificity of T cells to human EGFRvIII. In embodiments, the antigen-binding domain comprises a single-chain antibody fragment (scFv) comprising a variable domain light chain (VL) and a variable domain heavy chain (VH) of a target antigen-specific monoclonal anti-EGFRvIII antibody linked by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In some embodiments, the antigen-binding portion may comprise a VH and a VL directionally linked, for example, from the N-terminus to the C-terminus, VH-linker-VL or VL-linker-VH.

[0205] B. Spacer In some embodiments, the chimeric antigen receptor of the present disclosure further comprises a spacer that is used to link the antigen binding domain to the transmembrane domain, hi some embodiments, the spacer is sufficiently flexible to allow the antigen binding domain to be oriented in different directions to facilitate antigen recognition.

[0206] In certain embodiments, a chimeric antigen receptor comprising a spacer has improved functional activity compared to an otherwise identical antigen-binding polypeptide lacking the spacer. In certain embodiments, a chimeric antigen receptor comprising a spacer has increased expression on the cell surface compared to an otherwise identical polypeptide lacking the spacer. In one embodiment, a chimeric antigen receptor comprising a spacer is a polypeptide that, without the spacer, is not expressed on the cell membrane surface and / or is unable to bind its target due to lack of proximity or steric hindrance.

[0207] In certain embodiments, the spacer comprises a stalk region, e.g., a hinge region from an antibody. In some examples, the stalk region comprises a hinge region from an IgG, e.g., an IgG1. In alternative examples, the stalk region comprises the CH of an immunoglobulin. 2 CH 3 In some cases, the stalk region comprises a CD8α hinge region (SEQ ID NO: 426), an IgG4-Fc12 amino acid hinge region (SEQ ID NO: 631), or an IgG4 hinge region, as described in WO2016 / 073755. The stalk region may be the extracellular portion of the CAR that links the antigen binding domain to the cell surface and / or transmembrane region.

[0208] In some embodiments, the stalk region can be about 20 to about 300 amino acids in length. In some cases, the stalk region can be about 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 or more amino acids in length. In other cases, the stalk region can be about 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids in length. In some cases, the stalk region can be less than 20 amino acids in length.

[0209] In some embodiments, the stalk region comprises a CD8α hinge domain, a CD28 hinge domain, or a CTLA-4 hinge domain, or a functional fragment or variant thereof.

[0210] In certain embodiments, the stalk region comprises a CD8α hinge region, or a functional fragment or variant thereof. In certain such embodiments, the spacer comprises the amino acid sequence of SEQ ID NO: 426, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 467. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 467, and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 467.

[0211] In certain embodiments, the CD8 alpha hinge region, or a functional fragment or variant thereof, is encoded by SEQ ID NO: 468, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 468, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 468, or is a codon-degenerate variant of SEQ ID NO: 468.

[0212] In some embodiments, the stalk region may be capable of dimerizing with a homologous stalk region of a second CAR.

[0213] In certain embodiments, in addition to the stalk region, the spacer may include one or more stalk extension regions. In certain embodiments, the stalk extension region is a polypeptide that is homologous to the stalk region. For example, it may include a substitution of at least one amino acid residue compared to the stalk region. In some embodiments, the stalk region includes a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the stalk region, to which is attached, for example, a CD8α hinge domain, a CD28 hinge domain, or a CTLA-4 hinge domain.

[0214] In some embodiments, the spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 stalk extension regions.

[0215] In certain embodiments, the stalk region may be linked to the stalk extension region via a linker.

[0216] In certain embodiments, the stalk extension region may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the length of the stalk region as measured by the number of amino acids.

[0217] In some embodiments, the stalk region comprises at least one dimerization site. In certain embodiments, the stalk region may comprise one or more dimerization sites for forming homo- or heterodimerized chimeric polypeptides. In other embodiments, the stalk region or one or more stalk extension regions may comprise a mutation that completely eliminates the dimerization site.

[0218] In certain embodiments, the stalk extension region has at least one less dimerization site compared to the stalk region. For example, if the stalk region contains two dimerization sites, the stalk extension region may contain one or zero dimerization sites. As another example, if the stalk region contains one dimerization site, the stalk extension region may contain zero dimerization sites. In some examples, the stalk extension region lacks dimerization sites. In some cases, one or more dimerization sites may be membrane proximal. In other cases, one or more dimerization sites may be membrane distal.

[0219] In certain embodiments, the dimerization site is a cysteine ​​residue capable of forming a disulfide bond. In certain embodiments, the stalk extension region can form fewer disulfide bonds compared to the stalk region. For example, if the stalk region can form two disulfide bonds, the stalk extension region may be capable of forming one disulfide bond or may not be capable of forming a disulfide bond. As another example, if the stalk region can form one disulfide bond, the stalk extension region may not be capable of forming such a bond.

[0220] Each of the stalk extension regions can be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or more amino acids in length.

[0221] In certain embodiments, the stalk extension region is homologous to the CD8α hinge region. In certain such embodiments, the stalk extension region comprises the amino acid sequence of SEQ ID NO: 469, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 469. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 469 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 469.

[0222] In certain embodiments, the stalk extension region is encoded by any one of SEQ ID NOs: 470-472, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 470-472, hybridizes under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 470-472, or is a codon-degenerate variant of any one of SEQ ID NOs: 470-472.

[0223] In certain embodiments, the spacer comprises a stalk region and one to three stalk extension regions. In certain such embodiments, the spacer comprises a stalk region and two stalk extension regions, e.g., a CD8α hinge region and two stalk extension regions, each stalk extension region being homologous to a CD8α hinge region.

[0224] In some embodiments, each of the stalk region and the stalk extension region may be derived from at least one of the CD8α hinge domain, the CD28 hinge domain, the CTLA-4 hinge domain, the LNGFR extracellular domain, the IgG1 hinge, the IgG4 hinge, and the CH2-CH3 domain. The stalk and the stalk extension region may be derived separately from any combination of the CD8α hinge domain, the CD28 hinge domain, the CTLA-4 hinge domain, the LNGFR extracellular domain, the IgG1 hinge, the IgG4 hinge, or the CH2-CH3 domain. As an example, the stalk region may be derived from the CD8α hinge domain and at least one stalk extension region may be derived from the CD28 hinge domain, thus creating a hybrid spacer. As another example, the stalk region may be derived from an IgG1 hinge or an IgG4 hinge and at least one stalk extension region may be derived from the CH2-CH3 domain of an IgG.

[0225] In certain such embodiments, the spacer comprises the amino acid sequence of SEQ ID NO: 473, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 473. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 473 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 473.

[0226] In certain embodiments, the spacer is encoded by SEQ ID NO: 474, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 474, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 474, or is a codon-degenerate variant of SEQ ID NO: 474.

[0227] C. Transmembrane domain The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from, for example, any membrane-bound or transmembrane protein. Suitable transmembrane domains include those derived from TCR-alpha chain, TCR-beta chain, TCR-gamma1 chain, TCR-delta chain, TCR-zeta chain, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, GITR, CD152 (CTLA-4), or CD154, or functional fragments or variants thereof. Alternatively, the transmembrane domain may be synthetic and may contain hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine are found at one or both ends of the synthetic transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain, a CD152 (CTLA-4), a TCRγ1, a TCRδ, or a CD3ζ transmembrane domain.

[0228] Optionally, in some embodiments, a short oligonucleotide or polypeptide linker, 2-10 amino acids in length, can link the transmembrane domain to the intracellular signaling domain of the CAR. In some embodiments, the linker is a glycine-serine linker.

[0229] In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain or a CD3ζ transmembrane domain, or a functional fragment or variant thereof.

[0230] In certain embodiments, the transmembrane domain comprises a CD8α transmembrane domain, or a functional fragment or variant thereof. In certain such embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 475, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 475. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 475 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 475.

[0231] In certain embodiments, the CD8α transmembrane domain, or a functional fragment or variant thereof, is encoded by SEQ ID NO: 476, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 476, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 476, or is a codon-degenerate variant of SEQ ID NO: 476.

[0232] In certain embodiments, the transmembrane domain comprises the CD28 transmembrane domain, or a functional fragment or variant thereof. In certain such embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 477, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 477. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 477 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 477.

[0233] In certain embodiments, the CD28 transmembrane domain, or a functional fragment or variant thereof, is encoded by SEQ ID NO: 478, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 478, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 478, or is a codon-degenerate variant of SEQ ID NO: 438.

[0234] D. Intracellular signaling domains The intracellular signaling domain of the CAR may be responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to the specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including secretion of cytokines. Usually, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the intact chain, so long as it transduces an effector function signal. In some embodiments, the intracellular domain further comprises a signaling domain for T cell activation.

[0235] In some embodiments, the intracellular cell signaling domain interacts with a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell.

[0236] The intracellular domains are FCER1G, CD19, CD40, KIR3DL1, KIR3DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, SIRPA, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, FCGR1A, FCGR2A, FCGR2B, FCGR3A, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, PILRB, NCR1, NCR2, NCR3, NKG2A, NKG2C, NKG2D, DAP12, FCER1G, DAP10, CD84, CD19, KIR3DL1, KIR3DL2, KIR2DL2, KIR2DL3, KI The antibody may comprise an amino acid sequence derived from R2DL4, KIR2DL5, KIR3DL2, KIR3DL3, SIRPA, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, CD4, CD8A, CD8B, LAT, FCGR1A, FCGR2A, FCGR2B, FCGR3A, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, NCR1, NCR2, NCR3, LY9, NKG2C, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b or CD66d, or a functional fragment or variant thereof. In some cases, the signaling domain for T cell activation comprises a domain derived from CD3ζ, or a functional fragment or variant thereof.

[0237] In certain embodiments, the intracellular signaling domain comprises a CD3 zeta domain, or a functional fragment or variant thereof. In certain such embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 479, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 479. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 479 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 479.

[0238] In certain embodiments, the CD3 zeta domain, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 480, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 480, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 480, or is a codon-degenerate variant of SEQ ID NO: 480.

[0239] The intracellular signaling domain can further comprise one or more costimulatory domains. Exemplary costimulatory domains include, but are not limited to, CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), and CD3-zeta costimulatory domains, and functional fragments or variants thereof. In some examples, the CARs described herein comprise one or more, or two or more, costimulatory domains selected from CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, and OX40 (CD134) costimulatory domains, and functional fragments or variants thereof. In some examples, the CARs described herein comprise one or more, or two or more, costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, and OX40 (CD134) costimulatory domains, and functional fragments or variants thereof. In some examples, the CARs described herein comprise one or more, or two or more, of costimulatory domains selected from CD8, CD28, 4-1BB (CD137), DAP10, and DAP12 costimulatory domains, and functional fragments or variants thereof. In some examples, the CARs described herein comprise one or more, or two or more, of costimulatory domains selected from CD28 and 4-1BB (CD137) costimulatory domains, and functional fragments or variants thereof. In some examples, the CARs described herein comprise CD28 and 4-1BB (CD137) costimulatory domains, or their respective functional fragments or variants. In some examples, the CARs described herein comprise CD28 and OX40 (CD134) costimulatory domains, or their respective functional fragments and variants. In some examples, the CARs described herein comprise CD8 and CD28 costimulatory domains, or their respective functional fragments and variants. In some examples, the CARs described herein comprise a CD28 costimulatory domain, or its functional fragments or variants. In some examples, the CAR described herein comprises a 4-1BB (CD137) costimulatory domain, or a functional fragment or variant thereof.In some examples, the CARs described herein comprise an OX40 (CD134) costimulatory domain, or a functional fragment or variant thereof. In some examples, the CARs described herein comprise a CD8 costimulatory domain, or a functional fragment or variant thereof. In some examples, the CARs described herein comprise a DAP10 costimulatory domain, or a functional fragment or variant thereof. In some examples, the CARs described herein comprise a DAP12 costimulatory domain, or a functional fragment or variant thereof.

[0240] In certain embodiments, the intracellular signaling domain comprises a CD28 costimulatory domain, or a functional fragment or variant thereof. In certain such embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 481, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 481. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 481 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 481.

[0241] In certain embodiments, the CD28 costimulatory domain, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 482, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 482, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 482, or is a codon-degenerate variant of SEQ ID NO: 442.

[0242] In certain embodiments, the intracellular signaling domain comprises a 4-1BB costimulatory domain, or a functional fragment or variant thereof. In certain such embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 483, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 483. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 483 and / or is a conservatively substituted variant of SEQ ID NO: 483.

[0243] In certain embodiments, the 4-1BB costimulatory domain, or a functional fragment or variant thereof, is encoded by SEQ ID NO: 484, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 484, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 484, or is a codon-degenerate variant of SEQ ID NO: 484.

[0244] In certain embodiments, the intracellular signaling domain comprises a DAP10 costimulatory domain, or a functional fragment or variant thereof. In certain embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 485, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 485. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 485 and / or is a conservatively substituted variant of SEQ ID NO: 485.

[0245] In certain embodiments, the DAP10 costimulatory domain, or a functional fragment or variant thereof, is encoded by the sequence of SEQ ID NO: 486, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 486, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 486, or is a codon-degenerate variant of SEQ ID NO: 486.

[0246] In certain embodiments, the intracellular signaling domain comprises a DAP12 costimulatory domain, or a functional fragment or variant thereof. In certain embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 487, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 487. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 487 and / or is a conservatively substituted variant of SEQ ID NO: 487.

[0247] In certain embodiments, the DAP12 costimulatory domain, or a functional fragment or variant thereof, is encoded by the sequence of SEQ ID NO: 488, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 488, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 488, or is a codon-degenerate variant of SEQ ID NO: 488.

[0248] In certain embodiments, the intracellular signaling domain comprises both a CD28 co-signaling domain and a 4-1BB co-signaling domain, or a functional fragment or variant thereof, as described above.

[0249] E. Signal Peptides In one embodiment, the signal peptide directs the nascent CAR protein to the endoplasmic reticulum. This is the case, for example, when the receptor is glycosylated and anchored to the cell membrane. Any eukaryotic signal peptide sequence is assumed to be functional. Generally, the signal peptide naturally associated with the protein or, in the case of a fusion protein, the component closest to the N-terminus is used (e.g., V L For scFvs where the moiety is closest to the N-terminus, the native signal of the light chain is used). In some embodiments, the signal peptide is native to GM-CSFRa (SEQ ID NO: 489) or IgK (SEQ ID NO: 491), IgE (SEQ ID NO: 493), or a functional fragment or variant thereof. Other signal peptides that may be used include those native to CD8α (SEQ ID NO: 495) and CD28. In some embodiments, the signal peptide is native to mouse Ig V H Native to region 3 (SEQ ID NO:497), β2M signal peptide (SEQ ID NO:499), azurocidin (SEQ ID NO:501), human serum albumin signal peptide (SEQ ID NO:503), A2M receptor-associated protein signal peptide (SEQ ID NO:505), IGHV3-23 (SEQ ID NO:507), IGKV1-D33(HuL1) (SEQ ID NO:509), IGKV1-D33(L14F)(HuH7) (SEQ ID NO:511), or a functional fragment or variant thereof.

[0250] In certain embodiments, the CAR is linked to a GM-CSFRa signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the GM-CSFRa signal peptide has the amino acid sequence of SEQ ID NO: 489, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 489. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 489 and / or is a conservatively substituted variant of SEQ ID NO: 489.

[0251] In certain embodiments, the GM-CSFRa signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 490, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 490, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 490, or is a codon-degenerate variant of SEQ ID NO: 490.

[0252] In certain embodiments, the CAR is linked to an Igκ signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the Igκ signal peptide has the amino acid sequence of SEQ ID NO: 491, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 491. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 491 and / or is a conservatively substituted variant of SEQ ID NO: 491.

[0253] In certain embodiments, the Igκ signal peptide, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 492, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 492, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 492, or is a codon-degenerate variant of SEQ ID NO: 492.

[0254] In certain embodiments, the CAR is linked to an IgE signal peptide ("IgE signal peptide") native to IgE, or a functional fragment or variant thereof. In certain such embodiments, the IgE signal peptide is the amino acid sequence of SEQ ID NO: 493, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 493. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 493 and / or is a conservatively substituted variant of SEQ ID NO: 493.

[0255] In certain embodiments, the IgE signal peptide, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 494, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 494, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 494, or is a codon-degenerate variant of SEQ ID NO: 494.

[0256] In certain embodiments, the CAR is linked to a CD8α signal peptide native to CD8α, or a functional fragment or variant thereof. In certain such embodiments, the CD8α signal peptide comprises the amino acid sequence of SEQ ID NO: 495, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 495. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 495 and / or is a conservatively substituted variant of SEQ ID NO: 495.

[0257] In certain embodiments, the CD8α signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 496, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 496, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 496, or is a codon-degenerate variant of SEQ ID NO: 496.

[0258] In certain embodiments, the CAR is linked to a mouse Ig VH region 3 signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the mouse Ig VH region 3 signal peptide has the amino acid sequence of SEQ ID NO: 497, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 497. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 497 and / or is a conservatively substituted variant of SEQ ID NO: 497.

[0259] In certain embodiments, the mouse Ig VH region 3 signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 498, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 498, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 498, or is a codon-degenerate variant of SEQ ID NO: 498.

[0260] In certain embodiments, the CAR is linked to a β2M signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the β2M signal peptide has the amino acid sequence of SEQ ID NO: 499, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 499. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 499 and / or is a conservatively substituted variant of SEQ ID NO: 499.

[0261] In certain embodiments, the β2M signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 500, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 500, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 500, or is a codon-degenerate variant of SEQ ID NO: 500.

[0262] In certain embodiments, the CAR is linked to an azurocidin signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the azurocidin signal peptide has the amino acid sequence of SEQ ID NO:501, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO:501. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:501 and / or is a conservatively substituted variant of SEQ ID NO:501.

[0263] In certain embodiments, the azurocidin signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 502, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 502, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 502, or is a codon-degenerate variant of SEQ ID NO: 502.

[0264] In certain embodiments, the CAR is linked to a human serum albumin signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the human serum albumin signal peptide has the amino acid sequence of SEQ ID NO: 503, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 503. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 503 and / or is a conservatively substituted variant of SEQ ID NO: 503.

[0265] In certain embodiments, the human serum albumin signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 504, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 504, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 504, or is a codon-degenerate variant of SEQ ID NO: 504.

[0266] In certain embodiments, the CAR is linked to an A2M receptor-associated protein signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the A2M receptor-associated protein signal peptide has the amino acid sequence of SEQ ID NO:505, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO:505. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:505 and / or is a conservatively substituted variant of SEQ ID NO:505.

[0267] In certain embodiments, the A2M receptor-associated protein signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 506, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 506, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 506, or is a codon-degenerate variant of SEQ ID NO: 506.

[0268] In certain embodiments, the CAR is linked to an IGHV3-23 signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the IGHV3-23 signal peptide has the amino acid sequence of SEQ ID NO:507, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO:507. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:507 and / or is a conservatively substituted variant of SEQ ID NO:507.

[0269] In certain embodiments, the IGHV3-23 signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 508, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 508, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 508, or is a codon-degenerate variant of SEQ ID NO: 508.

[0270] In certain embodiments, the CAR is linked to an IGKV1-D33 signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the IGKV1-D33 signal peptide has the amino acid sequence of SEQ ID NO:509, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO:509. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:509 and / or is a conservatively substituted variant of SEQ ID NO:509.

[0271] In certain embodiments, the IGKV1-D33 signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 510, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 510, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 510, or is a codon-degenerate variant of SEQ ID NO: 510.

[0272] In certain embodiments, the CAR is linked to an IGHV3-33(L14F) signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the IGHV3-33(L14F) signal peptide has the amino acid sequence of SEQ ID NO:511, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO:511. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:511 and / or is a conservatively substituted variant of SEQ ID NO:511.

[0273] In certain embodiments, the IGHV3-33(L14F) signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 512, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 512, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 512, or is a codon-degenerate variant of SEQ ID NO: 512.

[0274] In certain embodiments, the CAR is linked to a TVB2 (T21A) signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the TVB2 (T21A) signal peptide has the amino acid sequence of SEQ ID NO: 513, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 513. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 513 and / or is a conservatively substituted variant of SEQ ID NO: 513.

[0275] In certain embodiments, the TVB2 (T21A) signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 514, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 514, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 514, or is a codon-degenerate variant of SEQ ID NO: 514.

[0276] In certain embodiments, the CAR is linked to a CD52 signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the CD52 signal peptide has the amino acid sequence of SEQ ID NO: 515, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 515. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 515 and / or is a conservatively substituted variant of SEQ ID NO: 515.

[0277] In certain embodiments, the CD52 signal peptide, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 516, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 516, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 516, or is a codon-degenerate variant of SEQ ID NO: 516.

[0278] In certain embodiments, the CAR is linked to an LNGFR signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the LNGFR signal peptide has the amino acid sequence of SEQ ID NO:517, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO:517. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:517 and / or is a conservatively substituted variant of SEQ ID NO:517.

[0279] In certain embodiments, the LNGFR signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 518, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 518, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 518, or is a codon-degenerate variant of SEQ ID NO: 518.

[0280] F. Exemplary CAR Constructs By way of example, and not limitation, a CAR can comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, and 629, or a conservatively substituted variant thereof. As a further example, and without limitation, a polynucleotide encoding a CAR can comprise a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, and 630, a sequence that hybridizes under stringent hybridization conditions to the complement of any one of such sequences, or a codon-degenerate variant of any one of such sequences.

[0281] In certain embodiments, a CAR may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 623, or a conservatively substituted variant thereof. In certain such embodiments, a polynucleotide encoding a CAR may comprise a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 624, a sequence that hybridizes under stringent hybridization conditions to the complement of any one of such sequences, or a codon-degenerate variant of any one of such sequences.

[0282] CAR and CAR structures and compositions are also described, for example, below. ● Chimeric Antigen Receptor(CAR)T-Cell Therapies for Cancer:A Practical Guide,Edited by:Daniel W.Lee and Nirali N.Shah,2020(ISBN 978-0-323-66181-2;DOI https: / / doi.org / 10.1016 / C2017-0-04066-1); ● Second Generation Cell and Gene-based Therapies,Biological Advances,Clinical Outcomes and Strategies for Capitalisation,Editors-in-Chief:Alain A.Vertes,Devyn M.Smith,Nathan J.Dowden,2020(ISBN 978-0-12-812034-7;DOI https: / / doi.org / 10.1016 / C2016-0-02070-3); ● Basics of Chimeric Antigen Receptor(CAR)Immunotherapy,Author:Mumtaz Yaseen Balkhi,2020(ISBN 978-0-12-819573-4,DOI https: / / doi.org / 10.1016 / C2018-0-05356-6); ● Engineering and Design of Chimeric Antigen Receptors,Authors:Sonia Guedan,Hugo Calderon,Avery D.Posey,Jr.,and Marcela V.Maus,Molecular Therapy:Methods & Clinical Development,Vol.12,March(2019)(https: / / www.cell.com / molecular-therapy-family / methods / pdf / S2329-0501(18)30133-5.pdf); ● Chimeric Antigen Receptor T Cell Therapy Pipeline at a Glance:A Retrospective and Systematic Analysis from Clinicaltrials.Gov,Authors:Eider F Moreno Cortes,Caleb K Stein,Paula A Lengerke Diaz,Cesar A Ramirez-Segura,Januario E.Castro,MD,Blood(2019)134(Supplement_1):5629(https: / / doi.org / 10.1182 / blood-2019-132273); ● WO2020209934(PCT / US2020 / 017794)-Novel chimeric antigen receptors and libraries(MIT); ● WO2020037142(PCT / US2019 / 046691)-Compositions and methods for high-throughput activation screening to boost t cell effector function(Yale); ● WO2015123642(PCT / US2015 / 016057)-Chimeric antigen receptors and methods of making(Univ.TX); ● WO2020014366(PCT / US2019 / 041213)-Ror-1 specific chimeric antigen receptors and uses thereof(Precigen); ● WO2019236577(PCT / US2019 / 035384)-Muc16 specific chimeric antigen receptors and uses thereof(Precigen) ● WO2019079486(PCT / US2018 / 056334)-Polypeptide compositions comprising spacers(Precigen) ● WO2017214333(PCT / US2017 / 036440)-Cd33 specific chimeric antigen receptors(Precigen)

[0283] V. Cytokines In some embodiments, the modified immune effector cells of the present invention can include a cytokine. The cytokine can be encoded, for example, by a polynucleotide of the present disclosure. For example, the polynucleotide can encode a miRNA, a CAR and a cytokine, or a miRNA and a cytokine.

[0284] In some cases, the cytokine comprises at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or variants or combinations thereof. In certain embodiments, the cytokine is an interferon, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factor, hGH, and / or a ligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, IFN-alpha, IFN-beta, or IFN-gamma.

[0285] In certain embodiments, the cytokine is an interleukin. In some cases, the interleukin is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, or a functional variant or fragment thereof.

[0286] In certain embodiments, the cytokine may be IL-12, or a functional fragment or variant thereof. In some embodiments, the IL-12 is single chain IL-12 (scIL-12), protease sensitive IL-12, destabilized IL-12, membrane bound IL-12, intercalated IL-12. In some examples, the IL-12 variant is as described in WO2015 / 095249, WO2016 / 048903, WO2017 / 062953.

[0287] In certain embodiments, the cytokine may be IL-15, or a functional fragment or variant thereof. In certain embodiments, the IL-15, or a functional fragment or variant thereof, is membrane bound. This may occur when IL-15, or a functional fragment or variant thereof, is bound to membrane bound IL-15Rα, or a functional fragment or variant thereof. Thus, certain embodiments of the invention may include a fusion protein comprising IL-15 and IL-15Rα, or their respective functional fragments or variants.

[0288] In certain embodiments, the IL-15, or functional fragment or variant thereof, comprises the amino acid sequence of SEQ ID NO: 519, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 519. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 519 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 519.

[0289] In certain embodiments, the IL-15, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 520, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 520, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 520, or is a codon-degenerate variant of SEQ ID NO: 520.

[0290] In certain embodiments, the IL-15Rα, or functional fragment or variant thereof, comprises the amino acid sequence of SEQ ID NO: 521, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 521. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 521 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 521.

[0291] In certain embodiments, the IL-15Rα, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 522, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 522, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 522, or is a codon-degenerate variant of SEQ ID NO: 522.

[0292] In certain embodiments, the IL-15, or a functional fragment or variant thereof, is linked to the IL-15Rα, or a functional fragment thereof, via a linker.

[0293] In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 529, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than SEQ ID NO: 529. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 529 and / or is a conservatively substituted variant of SEQ ID NO: 529.

[0294] In certain embodiments, the linker is encoded by a nucleic acid comprising SEQ ID NO: 530, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 530, hybridizes under stringent hybridization conditions to SEQ ID NO: 530, or is a codon-degenerate variant of SEQ ID NO: 530.

[0295] In certain embodiments, a fusion protein comprising IL-15 and IL-15Rα comprises the amino acid sequence of SEQ ID NO: 523, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 523. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 523 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 523.

[0296] In certain embodiments, a fusion protein comprising IL-15 and IL-15Rα is encoded by a nucleic acid comprising SEQ ID NO: 524, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 524, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 524, or is a codon-degenerate variant of SEQ ID NO: 603.

[0297] In certain embodiments, the cytokine is linked to a signal peptide.Any signal for use in eukaryotic cells, including those described above for use in CAR, can be linked to the cytokine.In certain embodiments, the cytokine is linked to an IgE signal peptide.

[0298] In certain embodiments, a fusion protein comprising IL-15 and IL-15Rα comprises the amino acid sequence of SEQ ID NO: 525, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 525. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 525 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 525.

[0299] In certain embodiments, a fusion protein comprising IL-15 and IL-15Rα is encoded by a nucleic acid comprising SEQ ID NO: 526, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 526, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 526, or is a codon-degenerate variant of SEQ ID NO: 526.

[0300] VI. Cell tagging In some embodiments, the modified immune effector cells of the present invention may include a cell tag. The cell tag may be encoded, for example, by a polynucleotide of the present disclosure. For example, the polynucleotide may encode the miRNA, CAR and / or cytokine described herein and the cell tag. In some aspects, the cell tag is used as a kill switch, a selection marker, a biomarker, or a combination thereof.

[0301] In certain embodiments, the cell tag can be bound by a predetermined binding partner. In certain such embodiments, the cell tag is non-immunogenic. In certain such embodiments, the cell tag comprises a polypeptide that is truncated such that it is non-immunogenic.

[0302] In certain embodiments, administration of a given binding partner allows depletion of infused CAR-T cells. For example, administration of cetuximab or any antibody that recognizes HER1 allows removal of cells expressing a cell tag that contains a truncated non-immunogenic HER1. Truncation of the HER1 sequence eliminates the possibility of EGF ligand binding, EGFR homo- and heterodimerization, and / or EGFR-mediated signaling, while still retaining cetuximab binding capacity (Ferguson, K., 2008. A structure-based view of Epidermal Growth Factor Receptor regulation. Annu Rev Biophys, Volume 37, pp. 353-373).

[0303] In certain embodiments, the cell tag comprises at least one of a truncated non-immunogenic HER1 polypeptide, a truncated non-immunogenic LNGFR polypeptide, a truncated non-immunogenic CD20 polypeptide, or a truncated non-immunogenic CD52 polypeptide, or a functional fragment or variant thereof.

[0304] In certain embodiments, the cell tag comprises a truncated non-immunogenic HER1 polypeptide comprising HER1 domain III and a truncated HER1 domain IV. Such domains and nucleic acid sequences encoding same include those described in WO2018 / 226897.

[0305] In certain embodiments, the HER1 domain III comprises the amino acid sequence of SEQ ID NO: 604, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 565. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 565 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 565.

[0306] In certain embodiments, the HER1 domain III, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 566, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 566, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 566, or is a codon-degenerate variant of SEQ ID NO: 566.

[0307] In certain embodiments, the truncated HER1 domain IV comprises the amino acid sequence of SEQ ID NO: 567, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 567. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 567 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 567.

[0308] In certain embodiments, the truncated HER1 domain IV, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 568, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 568, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 568, or is a codon-degenerate variant of SEQ ID NO: 568.

[0309] In certain such embodiments, the truncated non-immunogenic HER1 comprises the amino acid sequence of SEQ ID NO: 569, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 569. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 569, and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 569.

[0310] In certain embodiments, the truncated, non-immunogenic HER1, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 570, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 570, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 570, or is a codon-degenerate variant of SEQ ID NO: 570.

[0311] In certain embodiments, the cell tag comprises a truncated non-immunogenic CD20, or CD20t-1, or a functional fragment or variant thereof. In certain such embodiments, the cell tag comprises the amino acid sequence of SEQ ID NO:573, 575, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO:573 or SEQ ID NO:575. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:573 or SEQ ID NO:575, and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO:573 or SEQ ID NO:575.

[0312] In certain embodiments, the truncated non-immunogenic CD20, or CD20t-1, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 574 or SEQ ID NO: 576, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 574 or SEQ ID NO: 576, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 574 or SEQ ID NO: 576, or is a codon-degenerate variant of SEQ ID NO: 574 or SEQ ID NO: 576.

[0313] In certain embodiments, the cell tag further comprises a transmembrane domain. The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from, for example, any membrane-bound or transmembrane protein. Suitable transmembrane domains may include the transmembrane domains of the alpha, beta or zeta chains of the T cell receptor, or the transmembrane domains from CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154, or functional fragments or variants thereof. Alternatively, the transmembrane domain may be synthetic and may include hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan and valine are found at one or both ends of the synthetic transmembrane domain.

[0314] In some embodiments, the transmembrane domain comprises the CD28 transmembrane domain, or a functional fragment or variant thereof. In certain such embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 477, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 477. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 477 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 477.

[0315] In certain embodiments, the CD28 transmembrane domain, or a functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 478, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 478, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 478, or is a codon-degenerate variant of SEQ ID NO: 478.

[0316] In certain embodiments, the cell tag comprises a truncated HER1, or a functional fragment or variant thereof, and a transmembrane domain, or a functional fragment or variant thereof. In some embodiments, the cell comprises the amino acid sequence of SEQ ID NO:571, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO:571. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:571 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO:571.

[0317] In certain embodiments, the cell tag is encoded by a nucleic acid comprising SEQ ID NO: 572, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 572, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 572, or is a codon-degenerate variant of SEQ ID NO: 572.

[0318] In certain embodiments, the cell tag is linked to a signal peptide.The signal peptide can be any signal peptide suitable for use in eukaryotic cells, including those described herein for CAR.In certain embodiments, the signal peptide is an Igκ signal peptide comprising the amino acid sequence of SEQ ID NO: 491, or a functional fragment or variant thereof.

[0319] VII. Genetic Constructs Exemplary polynucleotides are provided that can be used as templates for expression of various genes and other regulatory elements in cells, as shown in Figure 16. It is understood that various elements can be included or omitted in the polynucleotide, and different options are shown for various exemplary sites within the polynucleotide.

[0320] As shown, the polynucleotide may include an integration signal for attP / attB phage integration of the polynucleotide into the bacterial genome. The polynucleotide may further include a 5' homology arm or 5' terminal repeat, and a 3' homology arm or 3' terminal repeat. The polynucleotide may further include an insulator, a border element, and an S / MAR located 3' adjacent to the 5' homology arm or 5' terminal repeat, and 5' adjacent to the 3' homology arm or 3' terminal repeat. Between the insulators, boundary elements, or S / MARs, the polynucleotide may include, from 5' to 3', a promoter, which may include silencers, enhancers, TF binding modules, and a core promoter; a 5' untranslated region, which may include stability modules, translation control elements, and intron integration elements such as miRNA coding sequences; one or more genes, which may include signal peptides, extracellular domains, transmembrane domains, signaling domains, antibody domains, peptide linkers, inteins, and epitope tags; and a 3' untranslated region, which may include stability modules, translation control, 3' end processing signals, and transcription terminators. It is understood that the expressed genes may be separated by an IRES, a cleavage peptide, or a ribosome skipping peptide.

[0321] The CAR may be encoded in the same genetic construct as the miRNA, cytokine, and / or cell tag. The advantage of having two or more of such components expressed using one genetic construct is the stoichiometric expression of such components.

[0322] A. Linker In certain embodiments, the polypeptides of the invention (e.g., CAR, cytokine, and cell tag) are linked by a linker polypeptide. Linkers can also be used to link domains of a polypeptide (e.g., the VH and VL domains of a CAR, the truncated HER1 and transmembrane domains of a cell tag, and the IL-15 and IL-15Rα domains).

[0323] Linkers suitable for the present invention include flexible linkers, rigid linkers, and in vivo cleavable linkers. In some cases, the linker acts to link functional domains together (as in flexible and rigid linkers) or to release free functional domains in vivo (as in in vivo cleavable linkers).

[0324] As mentioned above, in some cases, the linker sequence may include a flexible linker. When the binding domains require some degree of movement or interaction, a flexible linker can be applied. The flexible linker may be composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The flexible linker may have a sequence consisting mainly of a stretch of Gly and Ser residues ("GS" linker). An example of a flexible linker may have the sequence (Gly-Gly-Gly-Gly-Ser)n. By adjusting the copy number "n", the length of this exemplary GS linker can be optimized to achieve appropriate separation of functional domains or to maintain the necessary inter-domain interactions. For example, (Gly-Gly-Gly-Gly-Ser)n (where n is 4) is the (G4S)4 linker shown in SEQ ID NO: 608 or its conservatively substituted amino acid sequence. In addition to the GS linker, other flexible linkers can be utilized in the recombinant fusion protein. In some cases, the flexible linker may contain additional amino acids such as Thr and Ala to maintain flexibility, in other cases polar amino acids such as Lys and Glu may be used to improve solubility.

[0325] Flexible linkers may be a suitable choice when specific movement or interaction is desired for the fusion protein domains. In addition, flexible linkers do not have a rigid structure, but in some cases, they can function as passive linkers to keep the distance between functional domains. The length of the flexible linker can be adjusted to allow proper folding or to achieve optimal biological activity of the fusion protein.

[0326] Rigid linkers can be used to maintain a fixed distance between domains of a polypeptide. Examples of rigid linkers include alpha-helix-forming linkers, Pro-rich sequences, (XP)n, X-Pro scaffolds, A(EAAAK)nA (n=2-5) (SEQ ID NO: 563), and functional fragments and variants thereof, to name a few. Rigid linkers can exhibit a relatively rigid structure in some cases by adopting an α-helical structure or by containing multiple Pro residues.

[0327] Linkers useful in the present invention may be cleavable in some cases. In other cases, the linker is non-cleavable. Non-cleavable linkers can covalently link functional domains to function as one molecule through in vivo or ex vivo processes. Linkers may also be cleavable in vivo. Cleavable linkers can be introduced to release free functional domains in vivo.

[0328] Cleavable linkers can be cleaved by the presence of reducing agents, proteases, to name a few. For example, reduction of disulfide bonds can be utilized to generate cleavable linkers. In the case of disulfide linkers, a cleavage event by disulfide exchange with a thiol, such as glutathione, can result in cleavage. In some cases, cleavable linkers can allow targeted cleavage. For example, in vivo cleavage of the linker in a recombinant fusion protein can also be performed by proteases that can be expressed in vivo under pathological conditions (e.g., cancer or inflammation), in specific cells or tissues, or constrained in specific cellular compartments. Cleavable linkers can include hydrazones, peptides, disulfides, or thioesters. For example, hydrazones can confer serum stability. In other cases, hydrazones can allow cleavage in acidic compartments. The acidic compartments can have a pH of up to 7. Linkers can also include thioethers. Thioethers can be non-reducible. Thioethers can be designed for intracellular proteolysis. Examples of cleavable linkers include Furinlink, fmdv, and 2A linkers (e.g., P2A, GSG-P2A, FP2A, T2A, and Furin-T2A), or functional fragments or variants thereof.

[0329] The fmdv linker may comprise the amino acid sequence of SEQ ID NO: 539, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 539. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 539 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 539.

[0330] In certain embodiments, the fmdv linker is encoded by a nucleic acid comprising SEQ ID NO: 540, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 540, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 540, or is a codon-degenerate variant of SEQ ID NO: 540.

[0331] The P2A linker may comprise the amino acid sequence of SEQ ID NO: 547, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 547. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 547 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 547.

[0332] In certain embodiments, the P2A linker is encoded by a nucleic acid comprising SEQ ID NO: 548, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 548, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 548, or is a codon-degenerate variant of SEQ ID NO: 548.

[0333] The GSG-P2A linker may comprise the amino acid sequence of SEQ ID NO: 549, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 549. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 549 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 549.

[0334] In certain embodiments, the GSG-P2A linker is encoded by a nucleic acid comprising SEQ ID NO: 550, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 550, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 550, or is a codon-degenerate variant of SEQ ID NO: 550.

[0335] The FP2A linker may comprise the amino acid sequence of SEQ ID NO: 555, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 555. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 555 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 555.

[0336] In certain embodiments, the FP2A linker is encoded by a nucleic acid comprising SEQ ID NO: 556, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 556, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 556, or is a codon-degenerate variant of SEQ ID NO: 556.

[0337] The T2A linker may comprise the amino acid sequence of SEQ ID NO: 541, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 541. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 541 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 541.

[0338] In certain embodiments, the T2A linker is encoded by a nucleic acid comprising SEQ ID NO: 475428, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 542, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 54, or is a codon-degenerate variant of SEQ ID NO: 542.

[0339] In some embodiments, the linker comprises a furin polypeptide and a 2A polypeptide, and the furin polypeptide and the 2A polypeptide are connected by a polypeptide linker comprising at least three hydrophobic amino acids. Such a linker is referred to as a "Furin-T2A" linker. In some cases, the at least three hydrophobic amino acids are selected from the list consisting of glycine (Gly) (G), alanine (Ala) (A), valine (Val) (V), leucine (Leu) (L), isoleucine (Ile) (I), proline (Pro) (P), phenylalanine (Phe) (F), methionine (Met) (M), tryptophan (Trp) (W). In some cases, the polypeptide linker can comprise one or more GS linker sequences, such as (GS)n, (SG)n, and (GSG)n, where n can be any number from 0 to 30.

[0340] The Furin-T2A linker may comprise the amino acid sequence of SEQ ID NO: 543 or 545, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is at most 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues shorter at the N-terminus and / or C-terminus than the amino acid sequence of SEQ ID NO: 543 or 545. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 543 or 545 and / or is a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 543 or 545.

[0341] In certain embodiments, the Furin-T2A linker is encoded by a nucleic acid comprising SEQ ID NO: 544 or 546, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 544 or 546, hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 544 or 546, or is a codon-degenerate variant of SEQ ID NO: 544 or 546.

[0342] The linker may be an engineered linker. For example, the linker may be designed to include chemical features such as hydrophobicity. The method of designing the linker may be computational. In some cases, the computational method may include graphic techniques. The computational method may be used to search for suitable peptides from a library of three-dimensional peptide structures derived from a database. For example, the Brookhaven Protein Data Bank (PDB) may be used to measure the distance in space between selected amino acids of the linker.

[0343] Further exemplary linkers are provided in the Sequence Listing.

[0344] In some cases, at least two linker sequences can be included in the same protein. For example, the polypeptides of interest in a fusion protein can be separated by at least two linkers. In some cases, the polypeptides can be separated by 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 linkers.

[0345] The CAR, cell tag, and / or cytokine of the present invention can be expressed as a fusion protein. In such embodiments, such components can be linked together using a self-cleaving peptide, such as the 2A peptide.

[0346] In certain embodiments, the self-cleaving peptide is a T2A peptide, or a functional fragment or variant thereof.

[0347] In certain embodiments, the self-cleaving peptide is a Furin-T2A peptide, or a functional fragment or variant thereof. In certain such embodiments, the CAR, cytokine, and cell tag are expressed as fusion proteins with the CAR and cytokine linked by a self-cleaving linker, e.g., one that comprises Furin-T2A, and the cytokine and cell tag linked by a self-cleaving linker, e.g., one that comprises T2A.

[0348] B. Promoter The polynucleotide of the present invention may be present in the construct in operable linkage with a promoter. An appropriate promoter may be selected based on the host cell and the effect desired. Suitable promoters include constitutive and inducible promoters. The promoter may be tissue-specific, and such promoters are well known in the art.

[0349] Examples of constitutive promoters for use in the present invention include early cytomegalovirus (CMV) promoter, human elongation growth factor 1 alpha 1 (hEF1A1), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter, and functional fragments and variants thereof.

[0350] In certain embodiments, the promoter is a hEF1A1 promoter. The hEF1A1 promoter may comprise the sequence of SEQ ID NO:577, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:577, or hybridizes under stringent hybridization conditions with the complement of SEQ ID NO:577. In certain embodiments, the promoter is a CMV promoter. The CMV promoter may comprise the sequence of SEQ ID NO:578, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:578, or hybridizes under stringent hybridization conditions with the complement of SEQ ID NO:578.

[0351] In contrast to constitutive promoters, the use of inducible promoters provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In one aspect, the inducible promoter can be a gene switch ligand-inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible two polypeptide ecdysone receptor-based gene switch, such as a RHEOSWITCH® gene switch.

[0352] VIII. Vectors and Delivery Systems In certain embodiments, the polynucleotides of the present invention may be delivered to target cells by any suitable delivery system, including non-viral and viral delivery systems. In some embodiments, the vector may comprise a polynucleotide of the present disclosure encoding an miRNA, a CAR, a cytokine, a cell tag, or any combination thereof.

[0353] In certain cases, the miRNA, CAR, cytokine, and / or cell tag are expressed in separate vectors. In other aspects, the miRNA, CAR, cytokine, and / or cell tag are expressed from one single vector. In certain cases, the CAR and the miRNA are expressed in separate vectors. In other aspects, the miRNA, CAR and cytokine are expressed from one single vector. In certain cases, the vector can be a lentiviral vector, a retroviral vector, a Sleeping Beauty transposon, or a vector containing a sequence for serine recombinase-mediated integration. In some aspects, the vector is a plasmid, a minicircle DNA, or a nanoplasmid.

[0354] In certain embodiments, where the vector is a plasmid, minicircle DNA or nanoplasmid, the plasmid, minicircle DNA or nanoplasmid may further comprise a bacterial origin of replication. In certain embodiments, the bacterial origin of replication may be from a ColE1 plasmid. In certain embodiments, the bacterial origin of replication comprises the sequence of SEQ ID NO: 579, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 579 or hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 579.

[0355] The expression vector introduced into the cells to assess the expression of one or more miRNAs and CARs or portions thereof described herein can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a population of cells to be transfected or infected via a viral or non-viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA or used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as the neomycin resistance gene (neo) and the ampicillin resistance gene. In some embodiments, a truncated epidermal growth factor receptor (HER1t or HER1t-1) tag can be used as a selectable marker gene.

[0356] Reporter genes can be used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by the recipient organism or tissue and that code for a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., FEBS Letters 479:79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or can be obtained commercially. In general, the construct with the minimal 5' flanking region that shows the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0357] In some embodiments, the viral vectors described herein may contain a hEF1A1 promoter to drive expression of the transgene, a bovine growth hormone polyA sequence to enhance transcription, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and an LTR sequence derived from the pFUGW plasmid.

[0358] A. Methods for Introducing Nucleic Acids into Cells Methods for introducing and expressing genes into cells are well known. In the context of an expression vector, the vector can be easily introduced into a host cell, such as a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0359] 1.Physical method Physical methods for introducing polynucleotides into host cells, such as immune effector cells, include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)). In some embodiments, the method for introducing polynucleotides into host cells is calcium phosphate transfection or polyethyleneimine (PEI) transfection. In some embodiments, the method for introducing polynucleotides into host cells is electroporation.

[0360] a. Electroporation (EP) buffer A variety of buffers can be used for electroporation. The buffers disclosed herein have been found to have improved properties, including enhanced transfection capacity, even though these buffers contain fewer components compared to other known electroporation buffers.

[0361] Table 4 provides different amounts of monobasic phosphate and dibasic phosphate used as buffering agents. Table 5 provides buffers 1-20 containing buffering agents and glucose. Table Z provides buffers 21-37 containing buffering agents and mannitol. Table 7 provides the pH, conductivity, and osmolality of buffers 1, 2, and 3 compared to a control buffer (Mirus Bio™ Ingenio™ Electroporation Solution, catalog number MIR-50117; Mirus Bio LLC, Madison, WI, USA) ("Control 1"). [Table 4-2] [Table 5] [Table 6] [Table 7]

[0362] In some embodiments, the buffer comprises a solvent such as water. In some embodiments, the water may be purified and / or sterilized. For example, the water may undergo deionization (e.g., capacitive deionization or electrodeionization), reverse osmosis, carbon filtration, microfiltration, ultrafiltration, and / or ultraviolet sterilization. In some embodiments, the water is deionized. In some embodiments, the water is of a quality designated as "water for injection," also known as "sterile water for injection." Water for injection is generally made by distillation or reverse osmosis. Water for injection is a preparation of sterile, non-pyrogenic, solute-free water and is chemically designated as "sterile water for injection." 2 0" and has a pH of about 5.0 to about 7.0, preferably about 5.5.

[0363] In some embodiments, the solvent comprises 0.1% to 99.9% by volume of the total buffer volume. For example, the solvent may comprise at least about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.1% by volume of the total buffer volume.

[0364] In some embodiments, the buffer comprises a solute, e.g., a sugar or an organic compound derived from a sugar, e.g., a sugar alcohol. In embodiments in which the buffer comprises a sugar, the sugar may comprise a monosaccharide, a disaccharide, and / or a polysaccharide. In some embodiments, the sugar comprises a monosaccharide, e.g., glucose, fructose, and / or galactose. In some embodiments, the sugar comprises a disaccharide, e.g., sucrose, lactose, and maltose. In some embodiments, the sugar comprises a polysaccharide, e.g., cellulose or starch. In embodiments in which the buffer comprises a sugar alcohol, the sugar alcohol may comprise mannitol, sorbitol, xylitol, lactitol, isomalt, maltitol, and / or hydrogenated starch hydrolysate (HSH).

[0365] In some embodiments, the sugar is present in an amount less than about 50 millimolar (mM). For example, the sugar may be present in an amount less than about 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM. In some embodiments, the sugar is present in an amount ranging from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 10 mM to about 20 mM, or from about 25 mM to about 35 mM. In some embodiments, the sugar is present in an amount of about 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, or 35 mM.

[0366] In some embodiments, the sugar is glucose. In these embodiments, glucose may be present in an amount less than about 50 millimolar (mM). For example, glucose may be present in an amount less than about 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM. In some embodiments, glucose is present in an amount ranging from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 10 mM to about 20 mM, or from about 25 mM to about 35 mM. In some embodiments, glucose is present in an amount of about 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, or 35 mM. In certain embodiments, glucose is present in an amount of about 30 mM or 31 mM.

[0367] In some embodiments, the sugar is mannitol. In these embodiments, mannitol may be present in an amount less than about 50 millimolar (mM). For example, mannitol may be present in an amount less than about 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM. In some embodiments, mannitol is present in an amount ranging from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 10 mM to about 20 mM, or from about 25 mM to about 35 mM. In some embodiments, mannitol is present in an amount of about 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, or 35 mM.

[0368] In some embodiments, the EP buffer contains one or more chloride salts, such as potassium chloride (KCl) and / or magnesium chloride (MgCl 2 In some embodiments, the buffer comprises one or more buffering agents, such as Na 2 HPO 4 , NaH 2 PO 4 , or Na 2 HPO 4 / NaH 2 PO 4 In some embodiments, the buffer further comprises one or more of HEPES and / or DMSO. In other embodiments, the buffer specifically excludes one or more buffering agents commonly found in commercially available electroporation (EP) buffers. For example, in some embodiments, the buffer excludes one or both of DMSO and / or HEPES.

[0369] In some embodiments, the buffer is water (H 2 O), glucose, KCl, MgCl 2 , and Na 2 HPO 4 / NaH 2 PO 4 In some embodiments, the buffer comprises water (H 2 O), glucose, KCl, MgCl2 , Na 2 HPO 4 / NaH 2 PO 4 In another embodiment, the buffer comprises water (H 2 O), glucose, KCl, MgCl 2 , Na 2 HPO 4 / NaH 2 PO 4 , HEPES, and DMSO.

[0370] In some embodiments, the buffer is water (H 2 O), glucose, KCl, MgCl 2 , and Na 2 HPO 4 / NaH 2 PO 4 In some embodiments, the buffer consists essentially of water (H 2 O), glucose, KCl, MgCl 2 , Na 2 HPO 4 / NaH 2 PO 4 In another embodiment, the buffer consists essentially of water (H 2 O), glucose, KCl, MgCl 2 , Na 2 HPO 4 / NaH 2 PO 4 , HEPES, and DMSO.

[0371] In some embodiments, the buffer is water (H 2 O), glucose, KCl, MgCl 2 , and Na 2 HPO 4 / NaH 2 PO 4 In some embodiments, the buffer comprises water (H 2 O), glucose, KCl, MgCl 2 , Na 2 HPO 4 / NaH 2 PO 4In another embodiment, the buffer consists of water (H 2 O), glucose, KCl, MgCl 2 , Na 2 HPO 4 / NaH 2 PO 4 , HEPES, and DMSO.

[0372] In some embodiments, the buffer has a pH in the range of about 6.0-8.0, 6.5-8.0, 7.0-8.0, 7.5-8.0, 6.0-7.5, 6.0-7.0, 6.0-6.5, 6.5-7.5, or 6.5-7.0. In some embodiments, the buffer has a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0373] In some embodiments, the buffer solution comprising one or more buffering agents has a pH in the range of about 6.0-8.0, 6.5-8.0, 7.0-8.0, 7.5-8.0, 6.0-7.5, 6.0-7.0, 6.0-6.5, 6.5-7.5, or 6.5-7.0. In some embodiments, the buffer solution has a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0374] In some embodiments, the buffer is Na 2 HPO 4 and / or NaH 2 PO 4 In embodiments where the buffer contains both buffering agents, the ratio of the two (i.e., Na 2 HPO 4 / NaH 2 PO 4 ) can be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 9:1, 8:1, 7:1, 6:1 5:1, 4:1, 3:1, 2:1, or 2:3. 2 HPO 4 / NaH 2PO 4 have a pH of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9.

[0375] In some embodiments, Na 2 HPO 4 and NaH 2 PO 4 A mixture of (Na 2 HPO 4 / NaH 2 PO 4 " or "sodium phosphate") may be present in the buffer in an amount ranging from about 50 mM to 160 mM, 60 mM to 150 mM, 70 mM to 140 mM, 75 mM to 130 mM, 80 mM to 125 mM, 90 mM to 125 mM, 90 mM to 120 mM, 90 mM to 115 mM, or 90 mM to 105 mM. In some embodiments, Na 2 HPO 4 / NaH 2 PO 4 is present in an amount of at least 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. 2 HPO 4 / NaH 2 PO 4 is present in an amount of 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 109 mM, 110 mM, 111 mM, 112 mM, 113 mM, 114 mM, 115 mM, 116 mM, 117 mM, 118 mM, 119 mM, or 120 mM. 2 HPO 4 / NaH 2 PO 4 is present in an amount of 90 mM or 105 mM.

[0376] In embodiments where the buffer comprises KCl, KCl may be present in an amount less than about 30 mM. For example, KCl may be present in an amount less than about 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM. In some embodiments, KCl is present in an amount ranging from about 1 mM to about 30 mM, from about 2 mM to about 25 mM, from about 3 mM to about 20 mM, from about 4 mM to about 15 mM, from about 5 mM to about 10 mM, or from about 5 mM to about 15 mM. In some embodiments, KCl is present in an amount of about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM. In certain embodiments, KCl is present in an amount of about 5 mM or 10 mM. In some embodiments, the KCl has a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9.

[0377] Buffer is MgCl 2 In embodiments comprising MgCl 2 may be present in an amount less than about 50 mM. For example, MgCl 2 may be present in an amount less than about 45 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM. 2 In some embodiments, MgCl is present in an amount ranging from about 5 mM to about 50 mM, from about 6 mM to about 45 mM, from about 7 mM to about 40 mM, from about 8 mM to about 35 mM, from about 9 mM to about 30 mM, from about 10 mM to about 25 mM, or from about 15 mM to about 25 mM. 2 is present in an amount of about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM. 2 is present in an amount of about 15 mM or 20 mM. 2has a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9.

[0378] In embodiments where the buffer comprises HEPES, HEPES may be present in an amount less than about 30 mM. For example, HEPES may be present in an amount less than about 25 mM, 20 mM, 15 mM, 10 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 0.5 mM, or 0.1 mM. In some embodiments, HEPES is present in an amount ranging from about 1 mM to about 30 mM, from about 2 mM to about 25 mM, from about 3 mM to about 20 mM, from about 4 mM to about 15 mM, or from about 5 mM to about 10 mM. In some embodiments, HEPES is present in an amount of about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM. In certain embodiments, HEPES is present in an amount of 0 mM, 5 mM, or 10 mM. In some embodiments, HEPES has a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9.

[0379] In embodiments where the buffer comprises DMSO, DMSO may be present in an amount of 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% or less by volume of the total buffer volume. In some embodiments, DMSO is present in about 0% to about 2.5% by volume of the total buffer volume. In some embodiments, DMSO is present in an amount ranging from about 0.1% to 5%, 1% to 5%, 2% to 5%, 3% to 5%, or 4% to 5% by volume of the total buffer volume. In some embodiments, the DMSO has a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9. In other embodiments, no DMSO is present in the buffer at all.

[0380] In certain embodiments, the buffer contains sugar in an amount of 50 mM or less, HEPES in an amount of 25 mM or less, Na in an amount of 160 mM or less. 2 HPO 4 / NaH 2 PO 4 , 10 mM or less of KCl, 20 mM or less of MgCl 2 and DMSO in an amount of 5% or less by volume of the total buffer volume. In some of these embodiments, the sugar may include a monosaccharide and / or a sugar alcohol. In some of these embodiments, the sugar is mannitol and / or glucose. In some of these embodiments, the sugar is glucose. In some embodiments, the buffer does not include DMSO.

[0381] In certain embodiments, the buffer comprises a sugar in an amount of at least about 15 mM, HEPES in an amount of no more than 25 mM, Na in an amount of at least about 90 mM, 2 HPO 4 / NaH 2 PO 4 , KCl in an amount of at least about 2 mM, MgCl in an amount of at least 15 mM 2 and DMSO in an amount of 5% or less by volume of the total buffer volume. In some of these embodiments, the sugar may include a monosaccharide and / or a sugar alcohol. In some of these embodiments, the sugar is mannitol and / or glucose. In some of these embodiments, the sugar is glucose. In some embodiments, the buffer does not include DMSO.

[0382] In certain embodiments, the buffer comprises sugar in an amount ranging from about 15 mM to about 35 mM, KCl in an amount ranging from about 5 mM to about 10 mM, MgCl in an amount ranging from about 10.5 mM to about 20 mM. 2 , in an amount ranging from about 90 mM to about 105 mM Na 2 HPO 4 / NaH 2 PO 4, HEPES in an amount of 25 mM or less, and DMSO in an amount of 5% or less by volume of the total buffer volume. In some of these embodiments, the sugar may include a monosaccharide and / or a sugar alcohol. In some of these embodiments, the sugar is mannitol and / or glucose. In some of these embodiments, the sugar is glucose. In some embodiments, the buffer does not include DMSO.

[0383] In certain embodiments, the buffer contains glucose in an amount of about 15 mM, KCl in an amount of about 6 mM, MgCl in an amount of about 10.5 mM. 2 Na in an amount of about 105 mM 2 HPO 4 / NaH 2 PO 4 , HEPES in an amount ranging from about 15 mM, and DMSO in an amount of about 2.5% by volume of the total buffer volume.

[0384] In certain embodiments, the buffer contains glucose in an amount of about 30 mM, KCl in an amount of about 10 mM, MgCl in an amount of about 20 mM. 2 , Na in an amount of about 105 mM 2 HPO 4 / NaH 2 PO 4 and HEPES in an amount of about 5 mM. In some embodiments, DMSO is specifically excluded from the buffer.

[0385] In a particular embodiment, the buffer contains glucose in an amount of about 31 mM, KCl in an amount of about 5 mM, and MgCl in an amount of about 15 mM. 2 , and Na in an amount of about 90 mM 2 HPO 4 / NaH 2 PO 4 In some embodiments, one or more of HEPES and DMSO are specifically excluded from the buffer.

[0386] In a particular embodiment, the buffer contains glucose in an amount of about 30 mM, KCl in an amount of about 5 mM, and MgCl in an amount of about 15 mM. 2 , Na in an amount of about 90 mM 2 HPO4 / NaH 2 PO 4 and HEPES in an amount of about 10 mM. In some embodiments, DMSO is specifically excluded from the buffer.

[0387] In certain embodiments, the buffer contains glucose in an amount of about 25 mM, KCl in an amount of about 15 mM, and MgCl in an amount of about 25 mM. 2 , Na in an amount of about 120 mM 2 HPO 4 / NaH 2 PO 4 and HEPES in an amount of about 10 mM. In some embodiments, DMSO is specifically excluded from the buffer.

[0388] In certain embodiments, the pH of the buffer may be adjusted. In some embodiments, the buffer is adjusted to a pH of 6.5-8. In some embodiments, the buffer is adjusted to a pH of about 7.0-7.6. In some embodiments, the buffer is adjusted to a pH of about 6.9-7.2, or about 7.0-7.1. In some embodiments, the buffer is adjusted to a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In certain embodiments, the buffer is adjusted to a pH of about 7.0 or 7.1.

[0389] In certain embodiments, the conductivity of the buffer solution is about 7.0 ms / cm to about 16.0 ms / cm, about 9.0 ms / cm to about 16.0 ms / cm, about 11.0 ms / cm to about 16.0 ms / cm, or about 13.0 ms / cm to about 16.0 ms / cm. In some embodiments, the conductivity of the buffer solution is about 7.0 ms / cm to about 15.0 ms / cm, about 9.0 ms / cm to about 15.0 ms / cm, about 11.0 ms / cm to about 15.0 ms / cm, or about 13.0 ms / cm to about 15.0 ms / cm.In some embodiments, the conductivity of the buffer solution is about 7.0ms / cm, about 7.1ms / cm, about 7.2ms / cm, about 7.3ms / cm, about 7.4ms / cm, about 7.5ms / cm, about 7.6ms / cm, about 7.7ms / cm, about 7.8ms / cm, about 7.9ms / cm, about 8.0ms / cm, about 8.1ms / cm, about 8.2ms / cm, about 8.3ms / cm, about 8.4ms / cm, about 8.5ms / cm, about 8.6ms / cm, about 8.7ms / cm, about 8.8ms / cm, about 8.9ms / cm, about 9.0ms / cm, about 9.1ms / cm, about 9.2ms / cm m, approximately 9.3ms / cm, approximately 9.4ms / cm, approximately 9.5ms / cm, approximately 9.6ms / cm, approximately 9.7ms / cm, approximately 9.8ms / cm, approximately 9.9ms / cm, approximately 10.0ms / cm, approximately 10.1ms / cm, approximately 10.2ms / cm, approximately 10.3ms / cm, approximately 10.4ms / cm, approximately 10.5ms / cm, approximately 10.6ms / cm, approximately 10.7ms / cm, approximately 10.8ms / cm, approximately 10.9ms / cm, approximately 11.0ms / cm, approximately 11.1ms / cm, approximately 11.2ms / cm, approximately 11.3ms / cm, approximately 11.4ms / cm, approximately 11.5ms / cm, approximately 11.6ms / cm, approximately 11.7ms / cm, approximately 11.8ms / cm, approximately 11.9ms / cm, approximately 12.0ms / cm, approximately 12.1ms / cm, approximately 12.2ms / cm, approximately 12.3ms / cm, approximately 12.4ms / cm, approximately 12.5ms / cm, approximately 12.6ms / cm, approximately 12. 7ms / cm, approximately 12.8ms / cm, approximately 12.9ms / cm, approximately 13.0ms / cm, approximately 13.1ms / cm, approximately 13.2ms / cm, approximately 13.3ms / cm, approximately 13.4ms / cm, approximately 13.5ms / cm, approximately 13.6ms / cm, approximately 13.7ms / cm, approximately 13.8ms / cm, about 13.9 ms / cm, about 14.0 ms / cm, about 14.1 ms / cm, about 14.2 ms / cm, about 14.3 ms / cm, about 14.4 ms / cm, about 14.5 ms / cm, about 14.6 ms / cm, about 14.7 ms / cm, about 14.8 ms / cm, about 14.9 ms / cm, about 15.0 ms / cm, about 15.1 ms / cm, about 15.2 ms / cm, about 15.3 ms / cm, about 15.4 ms / cm, about 15.5 ms / cm, about 15.6 ms / cm, about 15.7 ms / cm, about 15.8 ms / cm, about 15.9 ms / cm, or about 16.0 ms / cm.In certain embodiments, the conductivity of the buffer is about 11.6, 12.8, or 14.3.

[0390] In some embodiments, the osmolality of the buffer is lower than the osmolality of the cells to be transfected (i.e., also known as "intracellular osmolality"). In some embodiments, the osmolality of the buffer is about 250 mOsm / kg H 2 O ~ approx. 1255mOsm / kg H 2 O, about 250mOsm / kg H 2 O~about 1100mOsm / kg H 2 O, about 250mOsm / kg H 2 O~about 900mOsm / kg H 2 O, about 250mOsm / kg H 2 O~about 700mOsm / kg H 2 O, about 250mOsm / kg H 2 O~about 500mOsm / kg H 2 O, about 250mOsm / kg H 2 O~about 400mOsm / kg H 2 O, or approximately 250 mOsm / kg H 2 O~about 360mOsm / kg H 2 In some embodiments, the osmolality is in the range of about 360 mOsm / kg H 2 O ~ approx. 1255mOsm / kg H 2 O, about 360mOsm / kg H 2 O~about 1100mOsm / kg H 2 O, about 360mOsm / kg H 2 O~about 900mOsm / kg H 2 O, about 360mOsm / kg H 2 O~about 700mOsm / kg H 2 O, about 360mOsm / kg H 2 O~about 500mOsm / kg H 2 O, about 360mOsm / kg H 2 O~about 400mOsm / kg H 2 In some embodiments, the osmolality is about 250 mOsm / kg H 2 O2, 255 mOsm / kg H2 O2, 260 mOsm / kg H 2 O2, 270 mOsm / kg H 2 O2, 275 mOsm / kg H 2 O, about 280mOsm / kg H 2 O2, approximately 285 mOsm / kg H 2 O, about 290mOsm / kg H 2 O, about 300mOsm / kg H 2 O, about 305mOsm / kg H 2 O, about 310mOsm / kg H 2 O, about 315mOsm / kg H 2 O, about 320mOsm / kg H 2 O, about 325mOsm / kg H 2 O, about 330mOsm / kg H 2 O, about 335mOsm / kg H 2 O, about 340mOsm / kg H 2 O, about 345mOsm / kg H 2 O, about 350mOsm / kg H 2 O, about 355mOsm / kg H 2 O, about 360mOsm / kg H 2 O, about 365mOsm / kg H 2 O, about 370mOsm / kg H 2 O, about 375mOsm / kg H 2 O, about 380mOsm / kg H 2 O, about 385mOsm / kg H 2 O, about 390mOsm / kg H 2 O, about 395mOsm / kg H 2 O, or approximately 400 mOsm / kg H 2 In a particular embodiment, the osmolality is about 280 mOsm / kg H 2 O, about 292mOsm / kg H 2 O, about 340mOsm / kg H 2 O, or approximately 362 mOsm / kg H 2 It is O.

[0391] In some embodiments, the buffer is selected from one or more of the exemplary buffers shown in Tables 5 and 6. In certain embodiments, the buffer is selected from Buffer 1, Buffer 2, or Buffer 3.

[0392] In some embodiments, the buffers of the present invention are used in conjunction with UltraPorator™ electroporation devices and cartridges (or cassettes). See PCT / US20 / 59984 (filed November 11, 2020) and U.S. Patent Application No. 17 / 095,028 (filed November 11, 2020). The device is designed to enable rapid manufacturing for a variety of gene and cell therapies. The UltraPorator™ is a high-throughput, semi-closed electroporation system for electroporating large numbers of cells in a single operation. The UltraPorator™ system is an advancement over current electroporation devices by significantly reducing processing time and contamination risk. For example, the UltraPorator can be utilized as a scale-up and commercialization solution for distributed chimeric antigen receptor (CAR) T cell manufacturing, such as UltraCAR-T™ manufacturing of T cells reprogrammed to target cancer antigens in vivo.

[0393] The buffers of the present invention are surprisingly effective at producing high cell transfection efficiencies when electroporation is performed using the buffers in an UltraPorator™ electroporation device and / or cartridge (or cassette). See PCT / US20 / 59984 (filed November 11, 2020) and U.S. Patent Application No. 17 / 095,028.

[0394] b. Methods utilizing EP buffers and recombinant cells produced using those methods In another aspect of the invention, there is provided a method of introducing biologically active material (e.g., DNA or RNA) into cells via electrical current (i.e., electroporation) utilizing a buffer according to the invention. The method comprises forming a suspension by combining cells obtained from a human with an exogenous biological material in a buffer according to the invention, and then applying an electrical current in the form of a voltage pulse to the suspension, thereby facilitating the introduction of the biological material into the cells.

[0395] In certain embodiments, the voltage pulse may have a field strength of up to 1-10 kV*cm-1, and a duration of 5-250 μs, and a current density of at least 2 A*cm-2. In certain embodiments, the voltage pulse allows for the direct transfection of biologically active substances (e.g., DNA) into the cell nuclei of animal and human cells. In certain embodiments, a current having a current density of 2-14 A*cm-2, preferably up to 5 A*cm-2, and a duration of 1-100 ms may also be applied following the voltage pulse without interruption.

[0396] The methods according to the invention may be used to optimize transfection of biologically active substances into cells, including animal cell nuclei, where biologically active substances (e.g., nucleic acids, polypeptides, etc.) can be introduced with high efficiency into quiescent or dividing animal cells.

[0397] In some embodiments, the cells are exposed to the buffer for less than 10 minutes. For example, the cells can be exposed to the buffer for less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute.

[0398] In some embodiments, the method is used to introduce biologically active substances into primary human blood cells, human blood multipotent progenitor cells, and primary human fibroblasts and endothelial cells. In some embodiments, the cells are human blood cells, e.g., immune cells. In certain embodiments, the immune cells are neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells), or some combination thereof. In some embodiments, the lymphocytes are T cells. In some embodiments, the cells are isolated from a patient.

[0399] In some embodiments, the biological material comprises a nucleic acid, a peptide, a polypeptide, a protein, an enzyme, an RNP, or some combination thereof. In some embodiments, the biological material is heterologous to the cell. In some embodiments, the biological material is partially or completely synthetic.

[0400] In some embodiments, the nucleic acid is selected from DNA or RNA. In some embodiments, the DNA may comprise cDNA. In some embodiments, the RNA may comprise mRNA, tRNA, rtRNA, lncRNA, sRNA, or combinations thereof. In some embodiments, the nucleic acid is a recombinant nucleic acid. In some embodiments, the peptide comprises a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, or combinations thereof. In some embodiments, the peptide is recombinant.

[0401] Methods utilizing the buffers of the present invention provide desirable high transfection yields, particularly when compared to methods utilizing other electroporation buffers. In some embodiments, the transfection yield with the buffers of the present invention is at least about 1.1 times higher than the transfection yield with a control (prior art) buffer. For example, the transfection yield with the buffers of the present invention can be about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 2.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 times higher than the transfection yield with a control (prior art) buffer. In some embodiments, the transfection yield with the buffer of the invention can be more than 5 times, e.g., 6, 7, 8, 9, or 10 times, higher than the transfection yield of a control (prior art) buffer. In certain embodiments, the transfection yield with the buffer of the invention is 1.35, 1.41, 1.46, 1.97, 1.98, 2.05, 2.12, 2.40, or 2.44 times higher than the transfection yield of a control (prior art) buffer.

[0402] Methods utilizing the buffers of the present invention provide desirable high transfected cell recovery rates, particularly when compared to methods utilizing other electroporation buffers, and in some embodiments, transfected cell recovery rates with the buffers of the present invention are at least about 1.1-fold higher than transfected cell recovery rates with control (prior art) buffers. For example, the transfected cell recovery with the buffer of the invention may be about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 2.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 times higher than the transfected cell recovery with the buffer of the invention. In some embodiments, the transfected cell recovery with the buffer of the invention may be more than 5 times higher than the transfected cell recovery with the buffer of the control (prior art). In certain embodiments, transfected cell recovery with the buffer of the invention is 1.53, 1.66, 1.72, 1.80, 2.06, 2.17, 2.23, 2.34, or 2.61 times higher than the transfected cell recovery with a control (prior art) buffer.

[0403] In another aspect of the present invention, recombinant cells are provided. In some embodiments, recombinant immune cells are produced using the methods of the present invention. In certain embodiments, the recombinant immune cells are modified T cells. In some embodiments, the modified T cells are chimeric antigen receptor (CAR) T cells. In some embodiments, the CAR-T cells are administered to patients for therapeutic purposes.

[0404] c. Electroporation devices and methods for their use An exemplary electroporation apparatus includes one or more chambers configured to store buffer and cells during the electroporation process, one or more electrode pairs configured to generate an electric field in the one or more chambers during the electroporation process, each electric field corresponding to one chamber, and a flow channel configured to transport cells during a cell collection process after the electroporation process.

[0405] In some embodiments, the device comprises one chamber, two chambers, three chambers, four chambers, five chambers, six chambers, seven chambers, eight chambers, nine chambers, ten chambers, or more than ten chambers, hi certain embodiments, the device utilizes a continuous flow or microfluidic system.

[0406] In some embodiments, the electroporation apparatus further comprises a pump for pumping liquid medium from the flow channel to at least one of the chambers during the harvesting process, the liquid medium being obtained at the inlet port. In some embodiments, the pump comprises a valve or valves connecting one or more chambers to the flow channel. In some embodiments, the valve or valves are opened one at a time. In some embodiments, the valve or valves allow only one-way flow of fluid. In some embodiments, each valve corresponds to one chamber. In some embodiments, each valve corresponding to a chamber valve is a pinch valve or pinch type valve. In some embodiments, each of the valves operates using a spring, lever, or piston motion.

[0407] In some embodiments, the one or more chambers comprise a given chamber, each electrode of the electrode pair is located on an opposite side of the given chamber, and each electrode of the electrode pair comprises both an interior portion within the given chamber and an exterior portion outside the given chamber.

[0408] In some embodiments, the electroporation apparatus further comprises an inlet port, an outlet port, and one or more adjacent flow channels connecting the inlet port and the outlet port to the flow channel.

[0409] In some embodiments, the electroporation apparatus further comprises a pump for pumping liquid medium from the flow channel to at least one of the chambers during the collection process, the liquid medium being obtained at the inlet port.

[0410] In some embodiments, the electroporation apparatus further comprises a surface including one or more openings leading to the one or more chambers, and air flow channels below the openings and connecting the air flow between the chambers.

[0411] In some embodiments, the electroporation apparatus further comprises a vent or air filter connecting the air flow channel to the exterior of the electroporation apparatus.

[0412] In some embodiments, the electroporation apparatus further comprises a seal configured to cover one or more openings. In some embodiments, each chamber in the electroporation apparatus comprises a narrowing shape toward a respective valve. In some embodiments, the electroporation apparatus further comprises a pair of electrodes, each electrode of the electrode pair being disposed on an opposite side of each chamber. The distance between the two electrodes in the electrode pair is referred to as the "gap distance" or "separation distance." This distance spans the width of the chamber.

[0413] In some embodiments, each of the one or more chambers comprises a gap distance of about 0.1 mm to about 20 mm, about 0.5 mm to about 10 mm, about 1 mm to about 7 mm, or about 1 mm to about 4 mm. In some embodiments, the gap distance is about 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, 5.0 mm, 5.5 mm, 6.0 mm, 6.5 mm, 7.0 mm, 7.5 mm, or 8.0 mm. In some embodiments, the gap distance is about 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, or 4.0 mm. In some embodiments, the gap distance is less than about 4 mm, less than about 3.5 mm, less than about 3.0 mm, less than about 2.5 mm, less than about 2.0 mm, less than about 1.5 mm, or less than about 1.0 mm. In some embodiments, a gap distance of less than about 4.0 mm improves the electroporation performance of the buffers provided herein.

[0414] In some embodiments, each electrode of a pair of electrodes of an electroporation apparatus comprises an interior portion within a given chamber and an exterior portion outside the given chamber, and each pair of electrodes is configured to connect to an electrical circuit, hi some embodiments, the interior portion within the given chamber has an elliptical surface and includes a gold coating.

[0415] In some embodiments, each chamber of the electroporation apparatus is configured to store a volume of at least about 50 μL, at least about 100 μL, at least about 150 μL, at least about 200 μL, at least about 250 μL, at least about 300 μL, at least about 350 μL, at least about 400 μL, at least about 450 μL, at least about 150 μL, at least about 500 μL, at least about 550 μL, at least about 600 μL, at least about 650 μL, at least about 700 μL, at least about 750 μL, at least about 800 μL, at least about 850 μL, at least about 900 μL, at least about 950 μL, or at least about 1000 μL (1.0 mL).

[0416] In some embodiments, the chambers of the electroporation apparatus, in combination, can accommodate at least about 500 μL, at least about 1.0 mL, at least about 1.2 mL, at least about 1.4 mL, at least about 1.6 mL, at least about 1.8 mL, at least about 2.0 mL, at least about 2.2 mL, at least about 2.4 mL, at least about 2.6 mL, at least about 2.8 mL, at least about 3.0 mL, at least about 3.2 mL, at least about 3.4 mL of cells in liquid suspension for electroporation. , at least about 3.6 mL, at least about 3.8 mL, at least about 4.0 mL, at least about 4.2 mL, at least about 4.4 mL, at least about 4.6 mL, at least about 4.8 mL, at least about 5.0 mL, at least about 5.2 mL, at least about 5.4 mL, at least about 5.6 mL, at least about 5.8 mL, at least about 6.0 mL, at least about 6.2 mL, at least about 6.4 mL, at least about 6.6 mL, at least about 6.8 mL, or at least about 7.0 mL.

[0417] In some embodiments, the cells involved in the electroporation process are at least 1×10 8 cells, at least 2 x 10 8 cells, at least 3 x 10 8 cells, at least 4 x 10 8cells, at least 5 x 10 8 cells, at least 6 x 10 8 cells, at least 7 x 10 8 cells, at least 8 x 10 8 cells, at least 9 x 10 8 cells, at least 1 x 10 9 cells, at least 2 x 10 9 cells, at least 3 x 10 9 cells, at least 4 x 10 9 cells, at least 5 x 10 9 cells, at least 6 x 10 9 cells, at least 7 x 10 9 cells, at least 8 x 10 9 cells, at least 9 x 10 9 cells, at least 1 x 10 10 cells, at least 2 x 10 10 cells, at least 3 x 10 10 cells, at least 4 x 10 10 cells, at least 5 x 10 10 cells, at least 6 x 10 10 cells, at least 7 x 10 10 cells, at least 8 x 10 10 cells, at least 9 x 10 10 cells, at least 1 x 10 11 cells, at least 2 x 10 11 cells, at least 3 x 10 11 cells, at least 4 x 10 11 cells, at least 5 x 10 11 cells, at least 6 x 10 11 cells, at least 7 x 10 11 cells, at least 8 x 10 11 cells, at least 9 x 10 11 cells, at least 1 x 10 12 cells, at least 2 x 10 12 cells, at least 3 x 10 12 cells, at least 4 x 10 12 cells, at least 5 x 10 12cells, at least 6 x 10 12 cells, at least 7 x 10 12 cells, at least 8 x 10 12 cells, and at least 9 × 10 12 The population comprises a population selected from the group consisting of cells.

[0418] In some embodiments, the device of the invention comprises an UltraPorator™ electroporation device and cartridge (see PCT / US20 / 59984 and U.S. Patent Application No. 17 / 095,028). As described above, the UltraPorator™ electroporation device is designed to enable rapid manufacturing for a variety of gene and cell therapies. This device can be utilized as a scale-up and commercialization solution for decentralized CAR T cell manufacturing, such as UltraCAR-T™ manufacturing of T cells reprogrammed to target cancer antigens in vivo.

[0419] In some embodiments, the device of the present invention is used in a method of electroporation, the method including performing an electroporation process by generating an electric field in a chamber using a pair of electrodes, the chamber being configured to store a buffer and cells during the electroporation process, and performing a cell collection process by opening a valve connected to the chamber and transporting the buffer and cells to an outlet port using a flow channel connected to the valve, each of the chamber, electrode pair, valve, outlet port, and flow channel being disposed within an electroporation device.

[0420] In some embodiments, the step of performing a cell collection process further comprises pumping the liquid medium from the flow channel into the chamber using a pump, the liquid medium being obtained at an inlet port, and the inlet port and outlet port being connected to the flow channel by adjacent flow channels in the electroporation device.

[0421] In some embodiments, the cell collection process further includes venting the chamber into a flow channel, and pressure within the chamber is maintained via a vent or air filter connected to an air flow channel flowing between the chamber and another chamber.

[0422] In some embodiments, the method of electroporation further includes depositing cells into an opening leading to a chamber holding a buffer solution, applying a seal to the opening, and connecting the electrode pair to at least one circuit, for example, by inserting the electroporation apparatus into a docking station.

[0423] In some embodiments, the method utilizes one or more of the exemplary buffers shown in Tables 5 and 6. In certain embodiments, the method utilizes Buffer 1, Buffer 2, or Buffer 3.

[0424] In some embodiments, the method is performed in an UltraPorator™ electroporation apparatus (see PCT / US20 / 59984 and U.S. Patent No. 17 / 095,028). In certain embodiments, the method is performed in an UltraPorator™ electroporation apparatus and utilizes one or more of the exemplary buffers shown in Tables 5 and 6. In certain embodiments, the method is performed in an UltraPorator™ electroporation apparatus and utilizes Buffer 1, Buffer 2, or Buffer 3 (shown in Table 5).

[0425] d. Electroporation system In another aspect of the present invention, a system for electroporation is provided. In some embodiments, the system for electroporation includes an electroporation device as described herein and an electroporation buffer as described herein. In some embodiments, the electroporation system includes an UltraPorator™ electroporation device and cartridge (see PCT / US20 / 59984 and US 17 / 095,028). As described above, the UltraPorator™ electroporation device is designed to enable rapid manufacturing for various gene and cell therapies. This device can be utilized as a scale-up and commercialization solution for distributed CAR T cell manufacturing, such as UltraCAR-T™ manufacturing of T cells reprogrammed to target cancer antigens in vivo.

[0426] In some embodiments, the system for electroporation further comprises a buffer, which is from one or more of the exemplary buffers shown in Tables 5 and 6. In certain embodiments, the system for electroporation comprises a buffer selected from Buffer 1, Buffer 2, or Buffer 3. It has been found that a system comprising an UltraPorator™ device and one of Buffers 1, 2, or 3 results in surprisingly high cell transfection efficiency compared to a system comprising an UltraPorator™ device and a control buffer.

[0427] e. Kit for electroporation In another aspect of the present invention, a kit for electroporation is provided. The kit may include any of the buffers described herein. In some embodiments, the kit includes one or more of the exemplary buffers shown in Tables 5 and 6. In certain embodiments, the kit includes a buffer selected from Buffer 1, Buffer 2, or Buffer 3.

[0428] In some embodiments, the kit comprises one or more containers filled with the buffer solution according to the present invention and other suitable reagents and / or devices. For example, the kit may further comprise a vector containing the nucleic acid of interest. In some embodiments, the kit may comprise a dropper, a pipette, and / or a cuvette. In some embodiments, the buffer solution may be packaged in aliquoted containers or as a stock solution. In some embodiments, the kit further comprises packaging for safe shipping of the buffer and any additional reagents and / or devices. In some embodiments, the kit comprises information regarding the contents of the buffer and any additional reagents. Additionally, the kit may comprise written materials, such as a user manual or answers to frequently asked questions.

[0429] 2.Chemical method Chemical methods for introducing polynucleotides into host cells 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 exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0430] 3. Viral Vectors and Delivery Methods When a viral delivery system is utilized, an exemplary delivery vehicle is a liposome. Lipid formulations can be used for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid can be associated with a lipid. The nucleic acid associated with a lipid can be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped within the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained as a suspension in lipid, contained in or complexed with micelles, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist as a bilayer structure, a micelle, or in a "collapsed" structure. They may also simply be dispersed in the solution, or in some cases form aggregates that are not uniform in size or shape.Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids.For example, lipids include the naturally occurring lipid droplets in the cytoplasm, as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0431] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo., dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY), cholesterol ("Chol") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can 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 is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of single and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an 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, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5:505-10 (1991)). However, compositions that have structures in solution that differ from the usual vesicular structure are also encompassed. For example, lipids may assume a micellar structure or may exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0432] Also provided herein is a viral-based delivery system into which the nucleic acid of the present invention is inserted. Representative viral expression vectors include, but are not limited to, adenovirus-based vectors (e.g., the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), adeno-associated virus-based vectors, lentivirus-based vectors (e.g., the lentivirus-based pLPI from Life Technologies (Carlsbad, Calif.)), retrovirus vectors (e.g., pFB-ERV+pCFB-EGSH), and herpes virus-based vectors. In one embodiment, the viral vector is a lentivirus vector. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentivirus vectors have an additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. Generally, and in embodiments, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0433] In some embodiments, a lentiviral vector is provided that includes a backbone and a nucleic acid sequence encoding one or more miRNAs and a CAR. Optionally, the vector further includes a nucleic acid encoding a cytokine and a cell tag, such as a truncated HER1, CD20t-1, or full-length CD20.

[0434] In some embodiments, the nucleic acid encoding one or more miRNAs and a CAR is cloned into a vector comprising a lentiviral backbone component. Exemplary backbone components include, but are not limited to, pFUGW and pSMPUW. The pFUGW lentiviral vector backbone is a self-inactivating (SIN) lentiviral vector backbone, which removes unnecessary HIV-1 viral sequences, thereby reducing the possibility of tumor formation, harmful mutations, and the development of infectious particle reproduction. In some embodiments, the vector encoding one or more miRNAs and a CAR also encodes a cytokine in a single construct. In some embodiments, one or more miRNAs and a CAR and a cytokine are encoded in two separate lentiviral vectors. In some embodiments, the cytokine is expressed in a cell tag. In some embodiments, one or more miRNAs and a CAR can be co-expressed with a cytokine and a cell tag from a single lentiviral vector. In further embodiments, one or more miRNAs and a CAR can be under the control of an inducible promoter. In another embodiment, the cytokine can be under the control of an inducible promoter. In one aspect, the inducible promoter can be a gene switch ligand inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible, two polypeptide ecdysone receptor-based gene switch, such as the RHEOSWITCH® gene switch.

[0435] Other suitable vectors include integrating expression vectors that can randomly integrate into the DNA of a host cell, or can include recombination sites that allow specific recombination between the expression vector and the host cell chromosome. Such integrating expression vectors can utilize endogenous expression control sequences of the host cell chromosome to effect expression of the desired protein. Examples of vectors that integrate in a site-specific manner include, for example, components of the flp-in system (e.g., pcDNA™5 / FRT) from Invitrogen (Carlsbad, Calif.), or the cre-lox system, such as that found in the pExchange-6 core vector from Stratagene (La Jolla, Calif.). Examples of vectors that randomly integrate into a host cell chromosome include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Invitrogen (Carlsbad, Calif.), and pCI or pFN10A(ACT)FLEXI™ from Promega (Madison, Wis.). Additional promoter elements, e.g. enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although many promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often 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 appear to be able to function either cooperatively or independently to activate transcription.

[0436] In some embodiments, the cell tag gene is cloned into the lentiviral plasmid backbone in frame with the CAR gene. In other embodiments, the cell tag is cloned into a separate lentiviral vector.

[0437] 4. Non-viral Vectors and Delivery Systems a. Sleeping Beauty transposon system Polynucleotides encoding one or more miRNAs alone or one or more miRNAs and chimeric receptors, cytokines, and / or cell tags described herein can be introduced into immune effector cells using a non-viral based delivery system such as the "Sleeping Beauty (SB) transposon system", which refers to a synthetic DNA transposon system for introducing DNA sequences into vertebrate chromosomes. Exemplary Sleeping Beauty transposon systems are described, for example, in U.S. Patent Nos. 6,489,458 and 8,227,432. As used herein, Sleeping Beauty transposon systems can include Sleeping Beauty transposase polypeptides and derivatives, functional fragments, and variants thereof, as well as Sleeping Beauty transposon polynucleotides, derivatives, and functional variants, and fragments thereof. In certain embodiments, the Sleeping Beauty transposase is encoded by an mRNA. In some embodiments, the mRNA encodes SB10, SB11, SB100x, or SB110 transposase. In some embodiments, the mRNA includes a cap and a polyA tail.

[0438] DNA transposons translocate from one DNA site to another in a simple cut-and-paste fashion. Transposition is the precise process by which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or a different DNA molecule or genome. Like other Tc1 / mariner-type transposases, SB transposase inserts the transposon at a TA dinucleotide base pair in the recipient DNA sequence. The insertion site can be elsewhere in the same DNA molecule or in another DNA molecule (or chromosome). There are approximately 200 million TA sites in the mammalian genome, including human. The TA insertion site is duplicated in the process of transposon integration. This duplication of the TA sequence is a hallmark of transposition and is used to confirm its mechanism in some experiments. The transposase may be encoded within the transposon or it may be supplied by another source, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons are most useful as genetic tools because they cannot continue to excise and reinsert independently after insertion. The Sleeping Beauty transposon is envisioned for use as a non-viral vector for gene transfer into the genome of vertebrates and for gene therapy.

[0439] Briefly, the Sleeping Beauty system (Hackett et al., Mol Ther 18:674-83, (2010)) was adapted to genetically modify immune effector cells (Cooper et al., Blood 105:1622-31, (2005)). In one embodiment, this involves two steps. (i) electrotransfer of DNA plasmids expressing the Sleeping Beauty transposon (Jin et al., Gene Ther 18:849-56, (2011); Kebriai et al., Hum Gene Ther 23:444-50, (2012)) and the Sleeping Beauty transposase, and (ii) growth and expansion of T cells stably expressing the integrants on designer artificial antigen presenting cells (AaPCs) derived from the K562 cell line (also known as AaPCs (activated and proliferating cells). In another embodiment, the second step (ii) is eliminated and the genetically modified T cells are cryopreserved or immediately infused into the patient.

[0440] In one embodiment, the Sleeping Beauty transposon system is described, for example, in Hudecek et al., Critical Reviews in Biochemistry and Molecular Biology, 52:4, 355-380 (2017), Singh et al., Cancer Res(8):68 (2008). April 15, 2008, and Maiti et al., J Immunother. 36(2):112-123 (2013).

[0441] In some embodiments, one or more miRNAs, CARs, cytokines, and cell tags can be encoded by a single transposon DNA plasmid vector. In some embodiments, one or more miRNAs, CARs, cytokines, and cell tags can be encoded by different transposon DNA plasmid vectors. In further embodiments, one or more miRNAs and CARs can be under the control of an inducible promoter. In another embodiment, the cytokine can be under the control of an inducible promoter. In one aspect, the inducible promoter can be a gene switch ligand inducible promoter. In some cases, the inducible promoter can be a small molecule ligand inducible two polypeptide ecdysone receptor-based gene switch, such as a RHEOSWITCH® gene switch, further described below. In certain embodiments, the CARs, cytokines, and cell tags can be configured in one, two, or more transposons.

[0442] In some embodiments, MUC16, CD33, or ROR1 specific CARs and other genetic elements are expressed using a SB11 transposon system, a SB100X transposon system, a SB110 transposon system, a piggyBac transposon system (see, e.g., U.S. Pat. Nos. 6,489,458, 8,227,432, 9,228,180; Wilson et al, “PiggyBac Transposon-mediated Gene Transfer in Human Cells,” Molecular Therapy 15:139-145 (2007) and WO2016 / 145146), and / or a piggyBat transposon system (see, e.g., Mitra et al., “Functional characterization of piggyBat from the bat Myotis lucifugus unveils an active mammalian DNA transposon,” Proc. Natl. Acad. Sci USA 110:234-239 (2013). Additional transposase and transposon systems are provided in U.S. Pat. Nos. 7,148,203, 8,227,432, U.S. Patent Publication No. 2011 / 0117072, Mates et al., Nat Genet, 41(6):753-61 (2009). doi:10.1038 / ng.343. Epub 2009 May 3, Gene Ther., 18(9):849-56 (2011). doi:10.1038 / gt.2011.40. Epub 2011 Mar 31, and Ivics et al., Cell. 91(4):501-10, (1997).

[0443] In certain embodiments, the vector is a Sleeping Beauty plasmid comprising a left transposon repeat region and a right transposon repeat region. In certain embodiments, the left transposon repeat region comprises a nucleic acid comprising SEQ ID NO: 580, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 580, or hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 580. In certain embodiments, the right transposon repeat region comprises a nucleic acid comprising SEQ ID NO: 581, or a functional fragment or variant thereof. In certain embodiments, a functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:581 or hybridizes under stringent hybridization conditions to the complement of SEQ ID NO:581.

[0444] b. Recombinase-Based Delivery Systems In other embodiments, nucleic acids encoding one or more miRNAs, CARs, cytokines, and / or cell tags can be integrated into the DNA of immune effector cells via a recombinase and an integrating expression vector. Such vectors can be integrated randomly into the DNA of the host cell or can include recombination sites to allow specific recombination between the expression vector and the host cell chromosome. Such integrating expression vectors can utilize endogenous expression control sequences of the host cell chromosome to effect expression of the desired protein. In some embodiments, targeted integration is promoted by the presence of sequences on the donor polynucleotide that are homologous to sequences flanking the integration site. For example, targeted integration using the donor polynucleotides described herein can be achieved according to conventional transfection techniques, such as those used to create gene knockouts or knockins by homologous recombination. In other embodiments, targeted integration is promoted both by the presence of sequences on the donor polynucleotide that are homologous to sequences flanking the integration site and by contacting the cell with the donor polynucleotide in the presence of a site-specific recombinase. Site-specific recombinase, or simply recombinase, refers to a polypeptide that catalyzes conservative site-specific recombination between its compatible recombination sites. As used herein, site-specific recombinase includes naturally occurring polypeptides that retain activity, as well as derivatives, variants, and / or fragments, and naturally occurring polynucleotides, derivatives, variants, and / or fragments that encode the recombinase that retain activity.

[0445] The recombinase can be introduced into the target cell before, simultaneously with, or after the introduction of the targeting vector. The recombinase can be directly introduced into the cell as a protein, for example, using liposomes, coated particles, or microinjection. Alternately, a polynucleotide, either DNA or messenger RNA, encoding the recombinase can be introduced into the cell using a suitable expression vector. The above targeting vector components are useful for constructing an expression cassette containing a sequence encoding the recombinase of interest. However, the expression of the recombinase can be regulated in other ways, for example, by placing the expression of the recombinase under the control of a regulatable promoter (i.e., a promoter whose expression can be selectively induced or repressed).

[0446] Recombinases can be from the integrase or resolvase family. The integrase family of recombinases has more than 100 members, including, for example, FLP, Cre, and lambda integrase. The integrase family, also called the tyrosine family or lambda integrase family, uses the hydroxyl group of catalytic tyrosine for nucleophilic attack on DNA phosphodiester bond. Typically, members of the tyrosine family first cleave DNA and then form a double-strand break. Examples of tyrosine family integrases include Cre, FLP, SSV1, and lambda (λ) integrase. In the resolvase family, also known as the serine recombinase family, a conserved serine residue forms a covalent bond to the DNA target site (Grindley, et al., (2006) Ann Rev Biochem 16:16).

[0447] In one embodiment, the recombinase is an isolated polynucleotide sequence comprising a nucleic acid sequence encoding a recombinase selected from the group consisting of SPβc2 recombinase, SF370.1 recombinase, Bxb1 recombinase, A118 recombinase, and φRv1 recombinase. Examples of serine recombinases are described in detail in U.S. Patent No. 9,034,652.

[0448] Recombinases for use in the practice of the invention can be recombinantly produced or purified as described above. Polypeptides having the desired recombinase activity can be purified to the desired purity by methods known in the art of purification, including, but not limited to, protein ammonium sulfate precipitation, size fractionation, affinity chromatography, HPLC, ion exchange chromatography, heparin agarose affinity chromatography (e.g., Thorpe & Smith, Proc. Nat. Acad. Sci. 95:5505-5510, 1998).

[0449] In one embodiment, the recombinase can be introduced into a eukaryotic cell containing a recombination attachment site where recombination is desired by any suitable method. For example, methods of introducing functional proteins into cells by microinjection or other methods are well known in the art. The introduction of purified recombinase protein ensures the transient presence of the protein and its function, which is often a preferred embodiment. Alternatively, the gene encoding the recombinase can be included in an expression vector used to transform the cell, where the polynucleotide encoding the recombinase is operably linked to a promoter that mediates expression of the polynucleotide in the eukaryotic cell. The recombinase polypeptide can also be introduced into the eukaryotic cell by a messenger RNA encoding the recombinase polypeptide. In general, it is preferred that the recombinase is present only for such time as is necessary for the insertion of the nucleic acid fragment into the genome being modified. Thus, the lack of persistence associated with most expression vectors is not expected to be detrimental. The recombinase gene can be introduced into the cell before, after, or simultaneously with the introduction of the exogenous polynucleotide of interest. In one embodiment, the recombinase gene is present in a vector carrying the polynucleotide to be inserted, and the recombinase gene may also be contained within the polynucleotide.

[0450] In one embodiment, a method for site-specific recombination comprises providing a first recombination site and a second recombination site; and contacting the first recombination site and the second recombination site with a prokaryotic recombinase polypeptide to effect recombination between the recombination sites, wherein the recombinase polypeptide is capable of mediating recombination between the first recombination site and the second recombination site, wherein the first recombination site is attP or attB and the second recombination site is attB or attP, and wherein the recombinase is selected from the group consisting of Listeria monocytogenes phage recombinase, Streptococcus pyogenes phage recombinase, Bacillus subtilis phage recombinase, Mycobacterium tuberculosis phage recombinase, and Mycobacterium smegmatis phage recombinase, with the proviso that if the first recombination attachment site is attB, then the second recombination attachment site is attP, and if the first recombination attachment site is attP, then the second recombination attachment site is attB.

[0451] Further embodiments provide for the introduction of a site-specific recombinase into a cell whose genome is to be modified. One embodiment relates to a method for obtaining site-specific recombination in a eukaryotic cell, the method comprising providing a eukaryotic cell comprising a first recombination attachment site and a second recombination attachment site, and contacting the first recombination attachment site and the second recombination attachment site with a prokaryotic recombinase polypeptide to effect recombination between the recombination attachment sites, the recombinase polypeptide being capable of mediating recombination between the first recombination attachment site and the second recombination attachment site, the first recombination attachment site being a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB), the second recombination attachment site being attB or attP, and the recombinase being selected from Listeria monocytogenes phage recombinase, Streptococcus pyogenes phage recombinase, Bacillus subtilis phage recombinase, Mycobacterium tuberculosis phage recombinase, and Mycobacterium smegmatis phage recombinase, with the proviso that if the first recombination attachment site is attB, then the second recombination attachment site is attP, and if the first recombination attachment site is attP, then the second recombination attachment site is attB. In one embodiment, the recombinase is selected from the group consisting of A118 recombinase, SF370.1 recombinase, SPβc2 recombinase, φRv1 recombinase, and Bxb1 recombinase. In one embodiment, the recombination results in integration.

[0452] c. Other non-viral delivery systems In other embodiments, nucleic acids encoding one or more miRNAs, CARs, cytokines, and / or cell tags can be incorporated into the DNA of immune effector cells via gene editing systems utilizing CRISPR, TALEN, or zinc finger nucleases.

[0453] Regardless of the method used to introduce exogenous nucleic acids into a host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of a particular peptide by immunological means (ELISA and Western blot) or by the assays described herein to identify peptides or proteins or nucleic acids included within the scope of the present invention.

[0454] IX. Engineered T Cell Receptors (TCRs) In some embodiments, the chimeric receptor comprises an engineered T cell receptor. T cell receptors (TCRs) are composed of two chains (αβ or γδ) that pair to form a heterodimeric receptor on the surface of T cells. In some examples, αβTCRs are expressed on most T cells in the body and are known to be involved in the recognition of specific MHC-restricted antigens. Each α and β chain is composed of two domains: a constant domain (C) that anchors the protein to the cell membrane and associates with the invariant subunit of the CD3 signaling apparatus, and a variable domain (V) that confers antigen recognition through six loops called complementarity determining regions (CDRs). In some examples, each of the V domains comprises three CDRs, e.g., CDR1, CDR2, and CDR3, with CDR3 as the hypervariable region. These CDRs interact with a complex formed between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (pepMHC) (e.g., HLA-A, HLA-B, HLA-C, HLA-DPAl, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, or HLA-DRB1 complex). In some instances, the constant domain further comprises a joining region connecting the constant domain to the variable domain. In some instances, the beta chain further comprises a short diversity region that constitutes part of the joining region.

[0455] The T cell receptor α chain contains a constant (conserved region), i.e., the Va24-Ja18 junction (amino acid sequence GSTLGR or a conservatively substituted amino acid sequence thereof), but both the α and β chains are highly variable.

[0456] In some cases, such TCRs are reactive to specific tumor antigens, such as NY-ESO, Mage A3, Titin, MART-1, HPV, HBV, MAGE-A4, MAGE-A10, MAGE A3 / A6, gp100, MAGE-A1, or PRAME. In other cases, such TCRs are reactive to specific neoantigens expressed within the patient's tumor (i.e., patient-specific, somatic, non-synonymous mutations expressed by the tumor). In some cases, engineered TCRs may be affinity enhanced.

[0457] In some embodiments, TCRs are described using the International Immunogenetics and Genetics (IMGT) TCR nomenclature and a link to the IMGT public database of TCR sequences. For example, there may be several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions that are distinguished by their framework, CDR1, CDR2, and CDR3 sequences. Thus, the Vα types may be referenced in the IMGT nomenclature by a unique TRAV number. For example, "TRAV21" defines a TCR Vα region with a unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence that is partially defined by an amino acid sequence that is conserved from TCR to TCR, but also includes amino acid sequences that vary from TCR to TCR. Similarly, "TRBV5-1" defines a TCR Vβ region with a unique framework and CDR1 and CDR2 sequences, but only a partially defined CDR3 sequence.

[0458] In some cases, the beta chain diversity region is referred to in the IMGT nomenclature by the abbreviation TRBD.

[0459] In some instances, the unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field, e.g., they can be found in the IMGT public database and in "T cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8.

[0460] In some embodiments, the αβ heterodimeric TCR is transfected as a full-length chain, e.g., having both the cytoplasmic and transmembrane domains. In some cases, the TCR includes a disulfide bond introduced between residues of each constant domain, e.g., as described in WO2006 / 000830.

[0461] In some examples, the TCRs described herein are in a single chain format, see for example WO2004 / 033685. Single chain formats include αβTCR polypeptides of the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, Vα-Cα-L-Vβ-Cβ, where Vα and Vβ are the TCR α and β variable regions, respectively, Cα and Cβ are the TCR α and β constant regions, respectively, and L is a linker sequence. In certain embodiments, the single chain TCRs of the invention may have disulfide bonds introduced between residues of the respective constant domains, as described in WO2004 / 033685.

[0462] In some embodiments, the TCR can be an αβTCR. In some embodiments, the TCR can be a γδTCR. It is understood that the TCRs of the present disclosure can bind to any of the antigen targets described herein.

[0463] In some embodiments, the TCR may be any one of V51, V52, and V51negV52neg TCR subsets. In some embodiments, the engineered cells may express any combination of V51, V52, V53, V55, V57, or V58 TCR chains with Vy2, Vy3, Vy7, Vy8, Vy9, Vy10, or Vy11 TCR chains. In some embodiments, the engineered cells may have essentially identical genetic material. In one aspect, the engineered cells may not contain chimeric antigen receptors.

[0464] The TCRs described herein may be associated with a detectable label, a therapeutic agent, or a PK-modifying moiety. Exemplary detectable labels for diagnostic purposes include, but are not limited to, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and imaging reagents.

[0465] In some cases, each chain of a TCR disclosed herein, e.g., αβ or γδ, comprises a modified spacer region that connects the constant region of the TCR chain to the transmembrane region. In some cases, the spacer region of each chain of a TCR disclosed herein comprises 1) a stalk region and one or more stalk extension regions adjacent to the stalk region. The stalk and stalk extension regions may be, for example, as described above for use in chimeric antigen receptors. In some embodiments, each chain of a TCR disclosed herein incorporates a spacer that comprises a stalk region and up to 20 stalk extension regions.

[0466] In some examples, the stalk region comprises an extracellular hinge region from a TCR alpha or TCR beta chain, or the stalk region comprises a sequence having at least 80% homology to an extracellular hinge region from a TCR alpha or TCR beta chain. In alternative examples, the stalk region comprises any portion of the extracellular region of a TCR alpha or TCR beta constant region that has at least 80% homology to the extracellular region of the TCR alpha or TCR beta constant region, respectively. For example, the stalk region may comprise a sequence having at least 80%, 85%, 90%, 95%, or greater than 95% homology to any portion of the extracellular region of the TCR alpha or TCR beta constant region.

[0467] TCR chain heterodimers are formed by interchain disulfide bonds in the extracellular hinge regions of the α and β chains. In some embodiments, the stalk region comprises a dimerization site. The dimerization site may comprise a disulfide bond formation site. The dimerization site may comprise a cysteine ​​residue. The stalk region may allow for disulfide bond formation. Such disulfide bonds may be formed at the disulfide bond formation site or at the dimerization site. In some examples, dimerization occurs between the α and β chains of the TCR.

[0468] In some embodiments, the stalk extension region is used to link the stalk region to the transmembrane domains of the TCR alpha and beta chains. In certain embodiments, the stalk extension region is used to link the stalk region to the constant domains of the TCR alpha and beta chains. In certain embodiments, the stalk region and the stalk extension region may be connected via a linker.

[0469] In some instances, the stalk extension domain comprises a sequence that is partially homologous to the stalk region. In some instances, each of the stalk extension regions comprises a sequence that is homologous to the stalk region, except that the stalk extension region lacks the dimerization site of the stalk region. In some instances, each of the stalk extension regions comprises a sequence that is identical to the stalk region. In other instances, each of the stalk extension regions comprises a sequence that is identical to the stalk region with at least one amino acid residue substitution relative to the stalk region. In some instances, each of the stalk extension regions is unable to form disulfide bonds or dimerize with a homologous stalk extension region.

[0470] In other embodiments, one stalk extension region may be connected to another stalk extension region via a linker, such as a glycine-serine rich linker.

[0471] In some embodiments, the addition of the stalk extension region prevents mispairing of the transgenic TCR α and β chains with the native TCR α and β chains expressed by the genetically modified T cell.

[0472] In some embodiments, the modified immune effector cells of the present disclosure may comprise a TCR of the present disclosure and a cytokine of the present disclosure. In certain embodiments, the modified immune effector cells may comprise a fusion protein comprising a TCR and IL-15 and IL-15Rα, or a functional fragment or variant of such domains.

[0473] X. Immune Checkpoint Inhibitors In some embodiments, the engineered cells of the present disclosure may comprise an immune checkpoint inhibitor. In certain embodiments, the polynucleotides of the presen...

Claims

1. A non-naturally occurring polynucleotide encoding (a) an miRNA that inhibits expression of an immune checkpoint protein, and (b) a chimeric receptor.

2. The polynucleotide of claim 1, wherein the miRNA inhibits expression of PD-1.

3. 3. The polynucleotide of claim 2, comprising a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:267 or capable of hybridizing to the complement of SEQ ID NO:267 under stringent hybridization conditions.

4. The polynucleotide of claim 1 , wherein the chimeric receptor is a T cell receptor.

5. The polynucleotide of claim 1 , wherein the chimeric receptor is a chimeric antigen receptor.

6. The polynucleotide of claim 5, wherein the chimeric antigen receptor comprises an antigen-binding domain that binds to an epitope on ROR1.

7. 7. The polynucleotide of claim 6, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 465, or a functional fragment or variant thereof.

8. 6. The polynucleotide of claim 5, wherein the chimeric antigen receptor comprises a spacer comprising a stalk region comprising the amino acid sequence of SEQ ID NO: 467, or a functional fragment or variant thereof, and a stalk extension region comprising the amino acid sequence of SEQ ID NO: 473, or a functional fragment or variant thereof.

9. The polynucleotide of claim 1 further encoding a cytokine.

10. The polynucleotide of claim 9, wherein the cytokine is IL-15, or a functional fragment or variant thereof, and is membrane-bound.

11. The polynucleotide of claim 10, which encodes a fusion protein comprising IL-15, or a functional fragment or variant thereof, and IL-15Rα, or a functional fragment or variant thereof.

12. The polynucleotide of claim 11, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:523, or a functional fragment or variant thereof.

13. The polynucleotide of claim 1 further encoding a cell tag.

14. 14. The polynucleotide of claim 13, wherein the truncated HER1 comprises HER1 domain III, or a functional fragment or variant thereof, and a truncated HER1 domain IV, or a functional fragment or variant thereof.

15. The polynucleotide of claim 14, wherein the cell tag further comprises a CD28 transmembrane domain, or a functional fragment or variant thereof.

16. A vector comprising the polynucleotide according to any one of claims 1 to 15.

17. The vector of claim 16 , comprising a Sleeping Beauty transposon.

18. A modified immune effector cell comprising a polynucleotide according to any one of claims 1 to 15.

19. A composition comprising a polynucleotide according to any one of claims 1 to 15, or a cell according to claim 18.

20. A kit comprising the polynucleotide according to any one of claims 1 to 15 or the cell according to claim 18.

21. 20. Use of the modified immune effector cell of claim 18 in the manufacture of a medicament for the treatment of a disease or disorder.

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