Improving immune cell function

Abstract

To provide a method utilizing the use of cytokine signaling as means for improving the efficacy of immune cell-based immunotherapies.SOLUTION: A vector comprising a nucleic acid encoding a membrane bound IL-15-IL-15Rα sushi domain chimeric receptor and a nucleic acid encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the function of the CAR and / or the TCR is improved by the membrane bound IL-15-IL-15Rα sushi domain chimeric receptor, is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 159,610, filed March 11, 2021, and U.S. Provisional Patent Application No. 63 / 210,300, filed June 14, 2021, which are incorporated herein in their entireties for all purposes.

[0002] The present disclosure relates to the fields of immunology and cell therapy, and more particularly to improving T cell and natural killer (NK) cell-based immunotherapies containing chimeric antigen receptors (CARs) and / or T cell receptors (TCRs) by modulating cytokine signaling. [Background technology]

[0003] The immune system provides a natural defense against cancer through its ability to search for, seek out, and destroy malignant cells throughout the body. However, a key caveat to this defense is that certain cancers can induce an immunosuppressive microenvironment that reduces the robustness of antitumor immune responses (Beatty et al., Clin Cancer Res, (21)(4):687-632 (2015)). These immune evasion mechanisms present challenges to the implementation and efficacy of cellular immunotherapies, including the use of engineered cell therapy technologies such as chimeric antigen receptor (CAR) T-cell therapy, T cell receptor (TCR) T-cell therapy, and / or natural killer cell-based immunotherapy.

[0004] Because overall T cell function and proliferation depend on cytokine signaling, it is theorized that the use of cytokines may improve the overall quality and efficacy of T cell-based therapies. Previous studies have demonstrated the success of using IL-2 as a means to expand T cell-based therapy, but drawbacks included both T cell exhaustion and reduced T cell persistence. (Gattinoni et al., J Clin Invest, (115): 1616-1626 (2005)). Other studies have shown improved efficacy of CAR-T cells by using IL-7 and IL-15 together (Xu et al., Blood, (123): 3750-3759 (2014)). CAR-T efficacy was also reported to be improved by the use of IL-21 (Singh et al., Cancer Res, (71) 3516-3527 (2011)). Similarly, IL-2 has been found to enhance NK cell cytotoxicity (Hu et al., Front. Immunol., (20) 1205 (2019)).

[0005] Therefore, there is a need to exploit the use of cytokine signaling as a means to improve the efficacy of immune cell-based immunotherapies. Summary of the Invention

[0006] Membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric receptors are disclosed. In embodiments, the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor comprises an IL-15 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6, a first linker connecting the IL-15 domain to the IL-15Rα sushi domain polypeptide set forth in SEQ ID NO:7 or SEQ ID NO:95, and a transmembrane domain comprising a FAS transmembrane domain or a dimerization domain, e.g., an IL-7 transmembrane domain.

[0007] In embodiments, a second nucleotide sequence linking the IL-15 polypeptide and the IL-15Rα sushi domain is The first linker comprises the amino acid sequence set forth in SEQ ID NO: 8. In an embodiment, the first linker comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0008] In embodiments, the IL-15Rα sushi domain polypeptide is linked to the transmembrane domain by a second linker. In embodiments, the second linker comprises the amino acid sequence set forth in SEQ ID NO: 24. In embodiments, the second linker comprises the amino acid sequence set forth in SEQ ID NO: 26.

[0009] In embodiments, the transmembrane domain is a FAS transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 22. In embodiments, the transmembrane domain is a FAS transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 42. In embodiments, the IL-7 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0010] In embodiments, the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, and 94. In embodiments, the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 30.

[0011] In embodiments, the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor further comprises a signaling sequence. In embodiments, the signaling sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 12 to 20. In embodiments, the signaling sequence comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0012] Disclosed herein are nucleic acids encoding the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptors described herein. In embodiments, the nucleic acid encoding the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor comprises a nucleic acid sequence set forth in a sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:100.

[0013] Disclosed are recombinant vectors comprising nucleic acids encoding the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptors described herein.

[0014] In embodiments, the recombinant vector or nucleic acid further comprises a nucleic acid encoding a chimeric antigen receptor (CAR) or a T-cell receptor (TCR). In embodiments, the CAR or TCR binds to a tumor antigen. In embodiments, the tumor antigen is selected from the group consisting of 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A-kinase anchoring protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), β2-integrin, BAFF, B lymphoma cells, B-cell maturation antigen (BCMA), bcr-abl (an oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia cell oncogene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), BST2, C242 antigen, 9-O-acetyl-CA19-9 marker, CA-125, CAEX, calreticulin, carbonic anhydrase 9 (CAIX), C-MET, CCR4, CCR5, CCR8, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD16, CD19, CD20, CD22, CD23 (IgE receptor), CD24 , CD25, CD27, CD28, CD30(TNFRSF8), CD33, CD34, CD38, CD40, CD40L, CD41, CD44, CD44V6, CD49f, CD 51, CD52, CD56, CD63, CD70, CD72, CD74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD12 5, CD133, CD137, CD138, CD150, CD152 (CTLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PD-L1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), claudin 6 (CLDN6), C-type Lectin-like molecule-1 (CLL-1 or CLECL1), C-type lectin domain family 12 member A (CLEC12A), cytomegalovirus (CMV)-infected cell antigen, CNT0888, CRTAM (CD355), CS-1 (also known as CD2 subset 1, CRACC, CD319, and 19A24), CTLA-4, cyclin B1, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B 1 (CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neoepitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 variant (ELF2M), endosialin, epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EphA2), ephrin B2, receptor tyrosine-protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), E RBB, ERBB2 (Her2 / neu), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS transgene mutant gene 6 (ETV6-AML) located on chromosome 12p, Fc fragment of IgA receptor (FCAR or CD89), fibroblast activation protein alpha (FAP), FBP, Fc receptor-like 5 (FcRL5), fetal acetylcholine receptor (AChR), fibronectin extra domain B, Fms-like tyrosine kinase 3 (FLT3), folate binding protein (FBP), folate receptor 1, folate receptor beta, folate receptor gamma, Fos-related antigen 1, fucosyl, fucosylGM1;GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF18), GM1, ganglioside GM3, the hexasaccharide moiety of GloboH glycoceramide (GloboH), glycoprotein 75, glypican-3 (GPC 3), glycoprotein 100 (gpl00), GPNMB, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), hepatitis A virus cellular receptor 1 (HAVCR1), human epidermal growth factor receptor 2 (HER-2), HER2 / neu, HER3, HER4, HGF, high molecular weight melanoma-associated antigen (HMWMAA), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), heat shock protein 70-2 mutant (mut hsp70-2), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgG1, immunoglobulin lambda-like polypeptide 1 (IGLL1), IL-6, interleukin-11 receptor alpha (IL-11Rα), IL-13, interleukin-13 receptor subunit alpha-2 (IL-13Rα2 or CD213A2), insulin-like growth factor I receptor (IGF1-R), integrin α5β1, integrin ανβ3, intestinal carboxylesterase, kappa light chain, KCS1, kinase insert domain receptor (KDR), K IR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KG2D ligand, KIT (CD117), KLRGI, LAGE-la, LAG3, lymphocyte-specific protein tyrosine kinase (LCK), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), legumain, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis (Y) antigen, LeY, LG, LI cell adhesion molecule (LI-CAM), LIGHT, LMP2, lymphocyte antigen 6 complex, LTBR, ​​locus K9 (LY6K), Ly-6, lymphocyte antigen 75 (LY75), melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2), MAGE, melanoma-associated antigen 1 (MAGE-A1), T cell; MAGE-A3 melanoma antigen 1 (MelanA or MARTI), MelanA / MARTl, mesothelin, MAGE A3, melanoma inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin 1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory factor (MIS) receptor type II, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), N-glycolylamino acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, mammary differentiation antigen (NY-BR-1), NY-ESO-1, carcinoembryonic antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, polySA, proacrosin-binding protein sp32 (OY-TES 1), p53, p53 mutant, pannexin 3 (PANX3), prostatic acid phosphatase (PAP), paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), prostate cancer tumor antigen-1 (PCTA-1 or galectin-8), PD-1H, platelet-derived growth factor receptor alpha (PDGFR-alpha), PDGFR-beta, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostase, prostate cancer cells, prostein, protease serine 21 (testisin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (Prosome,Macropain subunit, beta type, receptor for advanced glycation end products (RAGE-1), RANKL, Ras mutant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), sarcoma translocation breakpoint, squamous cell carcinoma antigen recognized by T cells 3 (SART3), SAS, SDC1, SLAMF 7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, sonic hedgehog (SHH), sperm protein 17 (SPA17), stage-specific embryonic antigen 4 (SSEA-4), STEAP, sTn antigen, synovial sarcoma X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), TCR5β, TCRα, TCRβ, TCRδ, and TCRγ. Alternative reading frame protein (TARP), telomerase, TIGIT, TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin-C, TGF-β1, TGF-β2, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TIM1, TIM2, TIM3, TnAg, TRAIL-R1, TRAIL-R2, tyrosinase-related protein 2 (TRP-2), thyroid-stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosinase, uroplakin 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2), and vimentin, TACI, Wilms' tumor protein (WT1), or X-antigen family member 1A (XAGE1).

[0015] Disclosed are immune cells comprising the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor, recombinant vector, or nucleic acid described herein. In embodiments, the immune cells are T cells or natural killer (NK) cells. Disclosed are pharmaceutical compositions comprising the immune cells described herein.

[0016] Disclosed are methods of treating cancer associated with expression of a tumor antigen in a subject, the methods comprising administering to the subject an effective amount of the immune cells or pharmaceutical compositions disclosed herein.

[0017] A method for inducing an immune response in a subject or immunizing a subject against cancer is disclosed, comprising administering to the subject an effective amount of an immune cell or pharmaceutical composition described herein. is shown.

[0018] A method for improving immune cell function is disclosed that involves engineering immune cells to express a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric receptor. DETAILED DESCRIPTION OF THE INVENTION

[0019] term In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions of the following terms, as well as other terms, are found throughout the specification.

[0020] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0021] As used herein, unless otherwise stated or clear from context, the term "or" is understood to be inclusive and encompasses both "or" and "and."

[0022] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include A and B, A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0023] As used herein, the term "for example" is used merely as an example, is not intended to be limiting, and should not be construed to refer only to the items explicitly listed herein.

[0024] Terms such as "greater than," "at least," and "greater than," e.g., "at least one," are used to mean, but are not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 5, 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, 1 This is understood to include 38, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more, as well as any larger numbers or fractions therebetween.

[0025] Conversely, the term "less than" includes every value less than the recited value. For example, "100 nucleotides or less" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 4 Included are 5, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or fractional number in between is also included.

[0026] Terms such as "plurality," "at least two," "two or more," and "at least a second" are intended to mean, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 5, 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, 1 This is understood to include 38, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more, as well as any larger numbers or fractions therebetween.

[0027] Throughout this specification, the words "comprising" or "comprises" or "comprising" and other variations thereof are understood to mean the inclusion of the stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also presented.

[0028] Unless specifically stated or clear from the context, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within one or more standard deviations as practiced in the art. "About" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term can mean a value that is up to an order of magnitude or up to 5 times greater. When a particular value or composition is presented in this disclosure, unless otherwise specified, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[0029] As described herein, any concentration range, percentage range, ratio Ranges, or integer ranges, unless otherwise stated, should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (such as integer tenths and hundredths).

[0030] Units, prefixes and symbols used herein are presented using the format accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range.

[0031] 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. See, e.g., Juo, "The Concise Dictionary of Biomedicine and Molecular Biology," 2 nd ed.,(2001),CRC Press, “The Dictionary of Cell&Molecular Biology”, 5 th ed., (2013), Academic Press, and "The Oxford Dictionary of Biochemistry "And Molecular Biology", Cammack et al. eds., 2 nd ed., (2006), Oxford University Press, provides one of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0032] "Administering" refers to the physical introduction of an agent, such as an engineered T cell or NK cell disclosed herein, into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, e.g., intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0033] The terms "activated" and "activation" refer to the state of T cells or NK cells that have been sufficiently stimulated to induce detectable cell proliferation. In one embodiment, activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to proliferating T cells. The term "activated NK cells" refers, inter alia, to proliferating NK cells. Signals generated through the TCR alone may be insufficient for full activation of T cells; one or more secondary or costimulatory signals may also be required. Thus, T cell activation includes a primary stimulatory signal via the TCR / CD3 complex and one or more secondary costimulatory signals. Costimulation can be evidenced by proliferation and / or cytokine production by T cells that have received a primary activation signal, such as stimulation through the TCR / CD3 complex.

[0034] The term "agent" can refer to any class of molecule or entity, or multiple molecules or entities, any of which can be, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, cell (such as a T cell or NK cell or precursor of such a cell), or organism (e.g., a fraction or extract thereof) or component thereof. In some embodiments, an agent can be utilized in isolated or pure form. In some embodiments, an agent can be utilized in crude or impure form. In some embodiments, an agent can be utilized as part of a population, collection, or other entity that can be screened, for example, to identify or characterize members present therein. Or it may be provided as a library.

[0035] The term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, for example, allogeneic T cell transplantation.

[0036] The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are embedded in more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Generally, human antibodies are tetrameric entities of approximately 150 kD, composed of two identical heavy (H) chain polypeptides (each approximately 50 kD) and two identical light (L) chain polypeptides (each approximately 25 kD) that associate together in what is commonly referred to as a "Y-shaped" structure. The heavy and light chains are linked or connected to each other by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions together, resulting in the dimers being connected to each other to form a tetramer. Naturally produced antibodies, e.g., C H It is glycosylated on two domains.

[0037] The term "human antibody" is intended to include antibodies having variable and constant domain sequences generated, assembled, or derived from human immunoglobulin sequences or sequences indistinguishable therefrom. In some embodiments, antibodies (or antibody components) can be considered "human" even if their amino acid sequences include residues or elements not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "humanized" is intended to include antibodies having variable domains having sequences derived from the variable domain of a non-human species (e.g., murine) that have been modified to more closely resemble human germline coding sequences. In some embodiments, a "humanized" antibody comprises one or more framework domains having substantially the amino acid sequence of a human framework domain and one or more complementarity-determining regions having substantially the amino acid sequence of a non-human antibody. In some embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant domain. In some embodiments, a humanized antibody comprises the C, C, D, E, E, F ... H 1. Hinge, C H 2. C H 3, and optionally, C H It may contain four regions.

[0038] Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFvs), camelized antibodies, affinities, and the like. Examples of antibodies include antibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In certain embodiments, antibodies as described herein refer to polyclonal antibody populations. Antibodies can also include, for example, Fab' fragments, Fd' fragments, Fd fragments, isolated CDRs, single chain Fvs, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, single chain or tandem diabodies (TandAb®), Anticalins®, Nanobodies® minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®.

[0039] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, IgE, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass encoded by the heavy chain constant region genes (e.g., IgM or IgG1). The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless explicitly stated and unless the context dictates otherwise, the term "antibody" includes antigen-binding fragments or portions of any of the foregoing immunoglobulins, including monovalent and bivalent fragments or portions, as well as single-chain antibodies.

[0040] An "antigen-binding molecule," "antigen-binding portion," "antigen-binding fragment," or "antibody fragment," or "antigen-binding domain" refers to any molecule that comprises an antigen-binding portion (e.g., CDR) of the antibody from which the molecule is derived. An antigen-binding molecule may comprise an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules comprising a peptide-binding domain) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or a viral or bacterial antigen. In certain embodiments, the antigen-binding molecule is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In certain embodiments, the antigen-binding molecule or domain is an antibody fragment that specifically binds to an antigen and comprises one or more of its complementarity-determining regions (CDRs). In further embodiments, the antigen-binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen-binding molecule or domain comprises or consists of an avimer.

[0041] In some examples, the CDRs are substantially identical to those found in a reference antibody (e.g., an antibody of the present disclosure) and / or the sequence of the CDRs provided in this disclosure. In some embodiments, the CDRs are substantially identical to the reference CDRs, either in that they are identical in sequence or in that they contain 1, 2, 3, 4, or 5 (e.g., 1-5) amino acid substitutions relative to the reference CDR. In some embodiments, the CDRs are substantially identical to the reference CDRs, either in that they are identical in sequence or contain 1, 2, 3, 4, or 5 (e.g., 1-5) amino acid substitutions relative to the reference CDR. A CDR is substantially identical to a reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some embodiments, a CDR is substantially identical to a reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, a CDR is substantially identical to a reference CDR in that one amino acid within the CDR has been deleted, added, or substituted compared to the reference CDR, while the CDR otherwise has an amino acid sequence that is identical to the amino acid sequence of the reference CDR. In some embodiments, the CDR is substantially identical to a reference CDR in that 2, 3, 4, or 5 (e.g., 2-5) amino acids within the CDR have been deleted, added, or substituted relative to the reference CDR, while the CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In various embodiments, the antigen-binding fragment binds to the same antigen as the reference antibody. In various embodiments, the antigen-binding fragment cross-competes with the reference antibody, e.g., binds to substantially the same or identical epitope as the reference antibody.

[0042] Antigen-binding fragments can be produced by any means. For example, in some embodiments, antigen-binding fragments can be enzymatically or chemically produced by fragmentation of an intact antibody. In some embodiments, antigen-binding fragments can be recombinantly produced (such as by expression of an engineered nucleic acid sequence). In some embodiments, antigen-binding fragments can be wholly or partially synthetically produced. In some embodiments, antigen-binding fragments can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, and in some embodiments, at least about 200 amino acids (e.g., 50-100, 50-150, 50-200, or 100-200 amino acids).

[0043] The terms "variable region" and "variable domain" are used interchangeably. A variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically the amino-terminal 110-120 amino acids of a mature heavy chain and approximately 90-115 amino acids of a mature light chain, which vary significantly in sequence among antibodies and are used to determine the binding and specificity of a particular antibody for its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions within the variable domain are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FR).

[0044] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or antigen-binding molecule thereof.

[0045] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or antigen-binding molecule thereof.

[0046] Many definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modeling software. The contact definition is based on an analysis of available complex crystal structures. [Table 1]

[0047] Terms such as "Kabat numbering" are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding molecule thereof. In certain embodiments, the CDRs of an antibody can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 31-35 (which can optionally include one or two additional amino acids following 35, designated 35A and 35B in the Kabat numbering scheme) (CDR1), 50-65 (CDR2), and 95-102 (CDR3). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0048] In certain embodiments, the CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226. Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is located at heavy chain amino acids 26-32, 33, or 34; the Chothia CDR-H2 loop is located at heavy chain amino acids 52-56; and the Chothia CDR-H3 loop is located at heavy chain amino acids 52-56. The CDR-H3 loop is located at amino acids 95-102 of the heavy chain, while the Chothia CDR-L1 loop is located at amino acids 24-34 of the light chain, the Chothia CDR-L2 loop is located at amino acids 50-56 of the light chain, and the Chothia CDR-L3 loop is located at amino acids 89-97 of the light chain. When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). In certain embodiments, the CDRs of the antibodies described herein are Determined according to the Chothia numbering scheme.

[0049] The terms "constant region" and "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0050] When used in reference to an antibody, the term "heavy chain" may refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which types give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0051] When used in reference to an antibody, the term "light chain" can refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0052] "Antigen" refers to a compound, composition, or substance that can stimulate antibody production or T-cell responses in humans or animals, including compositions (such as those containing tumor-specific proteins) injected or absorbed into humans or animals. Antigens react with specific humoral or cellular immune products, including those elicited by heterologous antigens such as the disclosed antigens. A "target antigen" or "target antigen of interest" is an antigen that is substantially not found on the surface of other normal (desired) cells and to which the binding domain of a TCR or CAR contemplated herein is designed to bind. Those skilled in the art will readily understand that virtually any macromolecule, including all proteins or peptides, can function as an antigen. Antigens can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. Antigens can be specific to a particular tissue, such as cancer cells, or can be broadly expressed. Furthermore, fragments of larger molecules can act as antigens. A "target" is any molecule that is bound by a binding motif, CAR, TCR, or antigen-binding agent, e.g., an antibody.

[0053] “Antigen-specific targeting region” (ASTR) is a "Targeting region" refers to the region of a CAR or TCR that targets a specific antigen. The targeting region on a CAR or TCR is extracellular. In some embodiments, the antigen-specific targeting region comprises an antibody or its functional equivalent, or a fragment or derivative thereof, with each targeting region targeting a different antigen. The targeting region may comprise a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a diabody, each of which is specific for a target antigen. However, numerous alternatives exist, such as linked cytokines (resulting in recognition of cells bearing cytokine receptors), affibodies, ligand-binding domains derived from naturally occurring receptors, soluble protein / peptide ligands for receptors (e.g., on tumor cells), peptides, and vaccines that stimulate an immune response, each of which may be used in various embodiments of the present disclosure. In fact, as will be appreciated by those skilled in the art, almost any molecule that binds with high affinity to a given antigen may be used as an antigen-specific targeting region.

[0054] "Antigen-presenting cells" or "APCs" refer to cells that process and present antigens to T cells. Exemplary APCs include dendritic cells, macrophages, B cells, certain activated epithelial cells, and other cell types capable of TCR stimulation and appropriate T cell costimulation.

[0055] "Anti-tumor effect" refers to a biological effect that can be manifested as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with tumors. Anti-tumor effect can also refer to the prevention of tumor development.

[0056] Two events or entities are "associated" with one another if the presence, level, and / or form of one correlates with the presence, level, and / or form of the other. For example, an entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). For example, two or more entities are physically "associated" with one another if they directly or indirectly interact with one another, such that they come into and / or remain in physical proximity (e.g., bound to) one another. In a further example, two or more entities that are physically associated with one another may be covalently linked or connected to one another or non-covalently associated, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0057] The term "autologous" refers to any material derived from the same individual that is later reintroduced. For example, the engineered autologous cell therapy (eACT™) method described herein involves the collection of lymphocytes from a patient, which are then engineered to express, for example, a CAR construct, and then administered to the same patient.

[0058] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A K D is k off / k onis calculated from the quotient of A is k on / k off It is calculated from the quotient of k on refers to the association rate constant of, for example, an antibody to an antigen, and k off refers to, for example, the dissociation of an antibody against an antigen. on and k off can be determined by techniques known to those skilled in the art, such as BIACORE® or KinExA.

[0059] The term "KD" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. D There is an inverse relationship between the binding affinity and the K D The smaller the value, the higher or stronger the affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, and therefore a smaller K D Conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction and therefore a larger K D In some situations, a higher binding affinity (or K ) of a particular molecule (e.g., an antibody) to its interaction partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., an antibody) to another interaction partner molecule (e.g., antigen Y) may be observed. D ) is larger than K D (lower or weaker affinity) with a smaller K D The binding affinity may be expressed as a binding ratio determined by dividing by the binding affinity (higher or stronger affinity), for example, as a 5-fold or 10-fold greater binding affinity in some cases.

[0060] "k d The term "(sec-1 or 1 / s)" refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. 0i r Also called the value.

[0061] "k a The term "(M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody binding fragment.

[0062] "K A The term "(M-1 or 1 / M)" refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is k a k d It is obtained by dividing by

[0063] The term "binding" generally refers to a non-covalent association between two or more entities. Direct binding involves physical contact between the entities or moieties. "Indirect" binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can be assessed in any of a variety of contexts, such as when the interacting entities or moieties are studied alone or in a more complex system (e.g., while covalently or otherwise associated with a carrier entity and / or within a biological system such as a cell).

[0064] The terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex) as such binding is understood by those of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined, for example, by immunoassays, BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen has a K A At least 2 log, 2.5 log, 3 log, 4 log, or more than K Aand binds to its antigen with a binding affinity of about 10. Binding can include preferential association of a binding motif, antibody, or antigen-binding system with the target of the binding motif, antibody, or antigen-binding system compared to association of the binding motif, antibody, or antigen-binding system with an entity that is not the target (i.e., a non-target). In some embodiments, a binding motif, antibody, or antigen-binding system selectively binds to a target if the binding of the binding motif, antibody, or antigen-binding system with the target is more than 2-fold, more than 5-fold, more than 10-fold, more than 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more than 100-fold greater than binding of the binding motif, antibody, or antigen-binding system with the non-target. In some embodiments, a binding motif, antibody, or antigen-binding system has a binding affinity of about 10 -5 Less than M, about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M or about 10 -9 If it is less than M, it will selectively bind to the target.

[0065] In another embodiment, the molecules that specifically bind to an antigen are about 1 x 10 -7 Dissociation constant of M (K d In some embodiments, the antigen-binding molecule binds at K d is about 1×10 -9 M ~ approx. 5×10 -9 In some embodiments, an antigen-binding molecule specifically binds an antigen with a "high affinity" when the K d is 1×10 -10 M ~ approx. 5×10 -10 In one embodiment, the antigen-binding molecule specifically binds to an antigen with "very high affinity" when the -9 K of M d In one embodiment, the dissociation rate is about 1×10 -5 is less than.

[0066] In certain embodiments, provided herein are antibodies or antigen-binding molecules thereof that bind to a target human antigen, e.g., in certain embodiments, the antigen-binding molecule binds to the target antigen with 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more affinity than to the target antigen of another species, as measured, for example, by radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In specific embodiments, the antibodies or antigen-binding molecules thereof described herein that bind to a target human antigen can be used in a variety of applications, including, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assays. The antibody or antigen-binding molecule thereof binds to a target antigen of another species with less than 10%, 15%, or 20% of the binding to the human antigen as measured by surface plasmon resonance or kinetic exclusion assays.

[0067] The term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and may invade nearby tissues. Examples of cancers that may be treated by the methods of the present disclosure include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, myeloma, and other white blood cell malignancies. In some embodiments, the methods of the present disclosure may be used to treat cancers of the immune system, including, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma, and ovarian cancer. Diffuse large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, central nervous system cancer The present invention may be used to reduce tumor size in tumors resulting from neoplasms of the central nervous system (CNS), primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of the above cancers. In one particular embodiment, the cancer is multiple myeloma. Certain cancers may be responsive to chemotherapy or radiation therapy, or certain cancers may be refractory. Refractory cancers refer to cancers that are not amenable to surgical intervention; refractory cancers either do not respond to chemotherapy or radiation therapy initially, or the cancer becomes non-responsive over time. Cancers may also include diffuse large B-cell lymphoma (DLBCL), not otherwise specified, primary mediastinal large B-cell lymphoma after two or more lines of systemic therapy, and primary mediastinal large B-cell lymphoma after two or more lines of systemic therapy. This includes relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including lymphoma, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma.

[0068] The terms "cancerous cell," "cancer cell," "tumor cell," or variants thereof, refer to individual cells of a carcinoma or tissue. A tumor generally refers to a swelling or lesion formed by the abnormal proliferation of cells, and can be benign, pre-malignant, or malignant. Most cancers form tumors, but some cancers, such as leukemia, do not necessarily form tumors. In the case of cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors.

[0069] A "chemokine" is a type of cytokine that mediates chemotaxis or directional movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1α, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0070] A "chimeric antigen receptor" or "CAR" is a molecule that binds to an immune cell (e.g., naive T cells, central memory T cells, effector memory T cells, NK cells, or CAR refers to a molecule engineered to contain a means for activating T cells (e.g., T cells ...

[0071] "Decrease" or "lower" or "alleviate" or "reduce" or "attenuate" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a lower physiological response (i.e., downstream effect) compared to the response caused by either the vehicle alone (i.e., active moiety) or a control molecule / composition. A "decrease" or "reduced" amount is typically a "statistically significant" amount and can include a 1.1, 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (e.g., 1.5, 1.6, 1.7, 1.8, etc., above 1, including all integers and decimals in between) decrease in the response produced by the vehicle, the control composition (reference response).

[0072] "Extracellular domain" (or "ECD") refers to a portion of a polypeptide that, when present in a cell membrane, is understood to reside outside the cell membrane, in the extracellular space.

[0073] The term "extracellular ligand-binding domain," as used herein, refers to an oligo- or polypeptide capable of binding to a ligand, e.g., a cell surface molecule. For example, the extracellular ligand-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state (e.g., cancer). Examples of cell surface markers that can act as ligands include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0074] The binding domain of a CAR may be followed by a "spacer" or "hinge," which refers to a region that moves the antigen-binding domain away from the effector cell surface to allow proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). The hinge region in a CAR is generally located between the transmembrane (TM) domain and the binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region, which can be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary hinge regions for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8 alpha, CD4, CD28, and CD7, which can be the wild-type hinge regions of these molecules or can be modified.

[0075] The "transmembrane" region or domain is the portion of the CAR that anchors the extracellular binding moiety to the plasma membrane of an immune effector cell and facilitates binding of the binding domain to a target antigen. The transmembrane domain can be a CD3 zeta transmembrane domain; however, other transmembrane domains that can be used include those obtained from CD8 alpha, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain is that of CD137. In certain embodiments, the transmembrane domain is synthetic, in which case it contains primarily hydrophobic residues such as leucine and valine.

[0076] The term "intracellular signaling domain" or "signaling domain" refers to a portion of a chimeric antigen receptor protein that is involved in transmitting the message of effective CAR binding to a target antigen to the interior of immune effector cells and inducing effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells or other cellular responses elicited by antigen binding to the extracellular CAR domain. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, aid or activity, including cytolytic activity or cytokine secretion. Thus, the terms "intracellular signaling domain" or "signaling domain," used interchangeably herein, refer to the portion of a protein that transmits an effector function signal and directs the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, as long as it transduces the effector function signal. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal. The intracellular signaling domain, also known as the "signaling domain," is typically derived from a portion of the human CD3 or FcRy chain.

[0077] It is known that signals generated through the T cell receptor alone are insufficient for full activation of T cells; secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the T cell receptor (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Cytoplasmic signaling sequences that act in a costimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0078] Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present disclosure include those derived from DAP10, DAP12, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0079] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or an Fc receptor, that provides a second signal required for efficient activation and function of T lymphocytes upon antigen binding. Examples of such costimulatory molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and CD83. Thus, although the present disclosure provides an exemplary costimulatory domain derived from 4-1BB, other costimulatory domains are contemplated. The inclusion of one or more costimulatory signaling domains can enhance the efficacy and proliferation of T cells expressing the CAR receptor. The intracellular signaling and costimulatory signaling domains can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order.

[0080] While scFv-based CARs engineered to contain signaling domains from CD3 or FcR gamma have been shown to deliver potent signals for T cell activation and effector function, they are not sufficient to induce signals that promote T cell survival and proliferation in the absence of concomitant costimulatory signals. The presence of one or more costimulatory signaling domains (e.g., 4-1BB, CD28, CD137, CD134, and CD278) is not sufficient to induce signals that promote T cell survival and proliferation in the absence of concomitant costimulatory signals. Other CARs containing binding domains, hinges, transmembrane and signaling domains derived from CD3 zeta or FcR gamma, along with intracellular costimulatory domains derived from CD3 zeta or FcR gamma, can more effectively direct anti-tumor activity and increased cytokine secretion, lytic activity, survival and proliferation in CAR-expressing T cells in vitro, as well as in animal models and cancer patients (Milone et al., Molecular Therapy, 2009;17:1453-1464; Zhong et al., Molecular Therapy, 2010;18:413-420; Carpenito et al., PNAS, 2009;106:3360-3365).

[0081] A "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in a T cell response, including, but not limited to, proliferation and / or up-regulation or down-regulation of key molecules.

[0082] "Costimulatory ligands" include molecules on antigen-presenting cells that specifically bind to cognate costimulatory molecules on T cells. Binding of a costimulatory ligand provides a signal that mediates T cell responses, including, but not limited to, proliferation, activation, and differentiation. Costimulatory ligands induce signals, for example, by binding of the T cell receptor (TCR) / CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules, in addition to the primary signal provided by the stimulatory molecule. Costimulatory ligands include, but are not limited to, 3 / TR6, 4-1BB ligand, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT) 3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), B7-H3, and others. Ligands that bind differently include lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), and MHC class I chain-related protein A (MICA). Protein B (MHC class I chain-related protein B, MICB), OX40 ligand, P Examples of costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds to CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0083] A "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, "costimulatory molecules" that are cognate binding partners on a T cell that specifically bind to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, CD45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, and CD 84, CD86, CD8 alpha, CD8 beta, CD9, CD96 (Tactile), CDl-la, CDl-lb, CDl-lc, CDl-ld, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14, TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1(CD1) la / CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1, CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0084] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within a CDR or framework region of the antibody or antigen-binding molecule thereof can be replaced with an amino acid residue having a similar side chain. In general, two sequences are generally considered "substantially similar" if they contain conservative amino acid substitutions at corresponding positions. For example, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "non-polar" side chains. Substitution of another amino acid of the same type may be considered a conservative substitution. Exemplary amino acid classifications are summarized in Tables 2 and 3 below. [Table 2] [Table 3]

[0085] "Combination therapy" refers to those situations in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic moieties) simultaneously. In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered before any dose of a second regimen); and in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may include administration of one or more agents or modalities to a subject receiving other agents or modalities in combination. For clarity, combination therapy does not require that individual agents be administered together (or necessarily simultaneously) in a single composition, although in some embodiments, two or more agents or active portions thereof may be administered together in a combination composition or even as a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0086] "Corresponding to" may be used to indicate the location / identity of a structural element in a molecule or composition through comparison to an appropriate reference molecule or composition. For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, for purposes of simplicity, residues in a polypeptide may be designated using a standard numbering system based on the reference related polypeptide, so that, for example, an amino acid "corresponding to" a residue at position 100 need not actually be the 100th amino acid in the amino acid chain, as long as it corresponds to the residue found at position 100 in the reference polypeptide. For example, to identify "corresponding" residues in polypeptides and / or nucleic acids according to the present disclosure, A variety of sequence alignment strategies are available, including software programs such as BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE, which can be used to align sequences.

[0087] An antigen-binding molecule, such as an antibody, its antigen-binding fragment, CAR, or TCR, "cross-competes" with a reference binding molecule, such as an antibody or its antigen-binding fragment, if the interaction between the antigen and a first antigen-binding molecule blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule to interact with the antigen. Cross-competition can be complete, e.g., binding of the antigen-binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or it can be partial, e.g., binding of the antigen-binding molecule to the antigen reduces the ability of the reference antigen-binding molecule to bind to the antigen. In certain embodiments, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to the same or overlapping epitope as the reference antigen-binding molecule. In other embodiments, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to a different epitope than the reference antigen-binding molecule. Many types of competitive binding assays can be used to determine whether one antigen-binding molecule competes with another antigen-binding molecule: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (ELISA), or other methods. immunoassay, EIA), sandwich competition assay (Stahli et al., 1983, Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct label assay, solid-phase direct label sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct label RIA using 1-125 label (Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552), and direct label RIA (Moldenhauer et al. al., 1990, Scand. J. Immunol. 32:77-82).

[0088] "Cytokine" refers to a non-antibody protein released by one cell in response to contact with a specific antigen, where the cytokine interacts with a second cell and mediates a response in the second cell. Cytokines can be endogenously expressed by a cell or administered to a subject. Cytokines can be released by immune cells, such as macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, proinflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines such as interleukin (IL) 7 and IL-15 Cytokines can promote the survival and proliferation of immune cells, and proinflammatory cytokines can promote the inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of proinflammatory cytokines include IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), and soluble intercellular adhesion molecules. 1 (soluble intercellular adhesion molecule 1, sICAM-1), soluble intercellular adhesion molecule 1, Soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor These include, but are not limited to, vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include C-reactive protein (C-reactive These include CRP (chemically reactive protein) and serum amyloid A (SAA). , but not limited to these.

[0089] "Decrease" or "lower" or "alleviate" or "reduce" or "attenuate" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a lower physiological response (i.e., downstream effect) compared to the response caused by either the vehicle alone (i.e., active moiety) or a control molecule / composition. A "decrease" or "reduced" amount is typically a "statistically significant" amount and can include a 1.1, 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (e.g., 1.5, 1.6, 1.7, 1.8, etc., above 1, including all integers and decimals in between) decrease in the response produced by the vehicle, the control composition (reference response).

[0090] The term "domain" refers to a portion of an entity. In some embodiments, a "domain" is associated with a structural and / or functional characteristic of an entity, e.g., such that when the domain is physically separated from the rest of its parent entity, the domain substantially or completely retains the structural and / or functional characteristic. In some embodiments, a domain can comprise a portion of an entity that, when separated from its (parent) entity and linked or connected to a different (recipient) entity, substantially retains and / or confers on the recipient entity one or more structural and / or functional characteristics characterized in the parent entity, for example. In some embodiments, a domain is a portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a portion of a polypeptide. In some such embodiments, a domain is characterized by a structural element (e.g., an amino acid sequence or sequence motif, an α-helical character, a β-sheet character, a coiled-coil character, a random-coil character, etc.) and / or by a functional characteristic (e.g., a binding activity, an enzymatic activity, a folding activity, a signaling activity, etc.).

[0091] The term "dosage form" can be used to refer to a physically discrete unit of active agent (e.g., an antigen-binding system or antibody) for administration to a subject. Generally, each such unit contains a predetermined amount of active agent. In some embodiments, such amount is a unit dose (whole fraction thereof) appropriate for administration according to a dosing regimen determined to correlate with a desired or beneficial outcome when administered to a relevant population. The total amount of a therapeutic composition or agent to be administered to a subject is determined by one or more physicians and may involve administration of more than one dosage form.

[0092] The term "dosing regimen" may be used to refer to a set of one or more unit doses administered individually to a subject. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated in time from the other doses. In some embodiments, a dosing regimen includes multiple doses, where consecutive doses are separated from each other by periods of equal length; in some embodiments, a dosing regimen includes multiple doses, where consecutive doses are separated from each other by periods of at least two different lengths. In some embodiments, all doses within a dosing regimen are the same unit dose amount. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first dose at a first dose amount, followed by one or more additional doses at a second dose amount that is different from the first dose amount. In some embodiments, the dosage regimen is adjusted periodically to achieve desired or beneficial results.

[0093] "Effector cell" refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. In some embodiments, effector cells may include, but are not limited to, one or more of monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, T lymphocytes, and B lymphocytes. Effector cells may be from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys.

[0094] "Effector function" refers to the biological outcome of the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). Effector functions can be antigen binding-dependent, antigen binding-independent, or both. ADCC refers to the lysis of antibody-bound target cells by immune effector cells. Without wishing to be bound by any theory, ADCC is generally thought to be a cytotoxic effect of Fc receptors (Fc receptors, Fc It is understood that effector cells, including antigen-specific receptors (e.g., receptors R), are responsible for recognizing and subsequently killing antibody-coated target cells (e.g., cells expressing on their surface the antigen to which the antibody binds). Effector cells that mediate ADCC can include immune cells, including, but not limited to, one or more of natural killer (NK) cells, macrophages, neutrophils, and eosinophils.

[0095] The term "engineered autologous cell therapy," also known as adoptive cell transfer and abbreviated as "eACT™," refers to the process of harvesting a patient's own T cells and subsequently genetically modifying them to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered, for example, to express a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen linked to an intracellular signaling moiety comprising at least one costimulatory domain and at least one activation domain. The costimulatory domain can be derived from a naturally occurring costimulatory domain or a variant thereof, for example, a variant with a truncated hinge domain ("truncated hinge domain, THD"), and the activation domain can be derived from, for example, CD3-zeta. In certain embodiments, CARs are engineered to have two, three, four, or more costimulatory domains. CAR scFvs can be engineered for targeting.

[0096] In some embodiments, the CAR is engineered such that the costimulatory domain is expressed as a separate polypeptide chain. Exemplary CAR T cell therapies and constructs are described in U.S. Patent Application Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated by reference in their entireties. "Adoptive cell therapy" or "Adoptive cell therapy, AC" ACT involves the transplantation of immune cells with anti-tumor activity into a subject, e.g., a cancer patient. In some embodiments, ACT is a therapeutic approach that involves the use of lymphocytes (e.g., engineered lymphocytes) with anti-tumor activity.

[0097] The terms "enhance" or "promote" or "increase" or "expand" generally refer to an increase in a response relative to either a vehicle or a control molecule / composition. "Increased" refers to the ability of the compositions contemplated herein to generate, induce, or cause a greater physiological response (e.g., downstream effects). A measurable physiological response can include increased T cell expansion, activation, persistence, and / or increased killing capacity of cancer cells, as understood in the art and from the description herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1, 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (e.g., 1.5, 1.6, 1.7, 1.8, etc., above 1, including all integers and decimal points in between) over the response generated by a vehicle or control composition.

[0098] "Epitope" refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (a linear or continuous epitope), or an epitope can be, for example, from a polypeptide or two or more non-contiguous regions of a polypeptide (a conformational, non-linear, discontinuous, or discontinuous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt4):339-350, McPherson A (1990) Eur J Biochem 189:1-23, Chayen NE (1997) Structure 5:1269-1274, McPherson A (1976) J Biol Chem 251:6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al., U.S. Patent Application Publication No. 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed. Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For example, see Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0099] "Endogenous" with respect to a gene, protein, and / or nucleic acid refers to the natural occurrence of that gene, protein, and / or nucleic acid in a cell, such as an immune cell.

[0100] "Exogenous" refers to an agent, such as a nucleic acid, gene, or protein, being introduced into a cell, e.g., from an external source. A nucleic acid introduced into a cell is exogenous even if it encodes a protein that is naturally found in the cell. Such exogenous introduction of a nucleic acid encoding a protein can be used to increase expression of the protein above levels that would naturally be found in the cell under similar conditions, e.g., without the introduction of the exogenous nucleic acid.

[0101] The term "excipient" refers to an agent that may be included in a composition to, for example, provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc.

[0102] As used herein, "expansion" refers to expanding a population of transduced immune cells for a predetermined period of time to produce a population of engineered immune cells. The predetermined period of expansion can be any suitable period that allows for the production of (i) a sufficient number of cells in the population of engineered immune cells for at least one dose to be administered to a patient, (ii) a population of engineered immune cells with a favorable proportion of immature cells compared to typical longer processes, or (iii) both (i) and (ii). This period will depend on the cell surface receptors expressed by the immune cells, the vector used, the dose required to have a therapeutic effect, and other variables. Thus, in some embodiments, the predetermined period of expansion can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days.

[0103] A "fragment" or "portion" of a material or entity described herein has a structure that comprises a distinct portion of a whole, e.g., a physical or abstract entity. In some embodiments, a fragment lacks one or more portions found in the whole. In some embodiments, a fragment consists of or comprises a characteristic structural element, domain, or portion found in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) found throughout the polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polymer (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). The whole material or entity may, in some embodiments, be referred to as the "parent" of the fragment.

[0104] The term "fusion polypeptide" or "fusion protein" generally refers to a polypeptide comprising at least two segments. Generally, a polypeptide containing at least two such segments is considered to be a fusion polypeptide if the two segments are (1) not inherently contained in the same peptide, and / or (2) not previously linked or connected to each other in a single polypeptide, and / or (3) linked or connected to each other through the action of the human hand. In embodiments, a CAR is a fusion protein. In one embodiment, a membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide is a fusion protein.

[0105] The term "gene product" or "expression product" generally refers to the RNA (before and / or after processing) transcribed from a gene or the polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene.

[0106] The terms "genetically engineered" or "engineered" refer to methods of modifying a cell's genome, including, but not limited to, deleting a coding or non-coding region or portion thereof, or inserting a coding region or portion thereof. In some embodiments, the modified cell is a lymphocyte, e.g., a T cell or NK cell, and can be obtained from either a patient or a donor. The cell can be engineered to express an exogenous construct, e.g., a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR), that is integrated into the cell's genome. Engineering generally includes manipulation by the hand of man. For example, a polynucleotide is considered "engineered" if it has been manipulated by the hand of man so that two or more sequences that are not naturally linked or connected in that order are directly linked or connected to each other in the engineered polynucleotide. In the context of manipulating cells by molecular biology techniques, a cell or organism is considered "engineered" if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, e.g., by transformation, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution or deletion mutation, or by other protocols). Engineered cells can be modified to express exogenous constructs, e.g., membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric receptors, chimeric antigen receptors (CARs), or T-cell receptors (TCRs), that are integrated into the genome of the cell. Progeny of engineered polynucleotides or binding agents are generally referred to as "engineered," even if the actual manipulation was performed on the previous entity. In some embodiments, "engineered" refers to a designed and manufactured entity. The term "designed" refers to an agent that (i) has a structure selected by or through the human hand, (ii) is produced by a process requiring the human hand, and / or (iii) is distinct from natural substances and other known agents.

[0107] "T cell receptor" or "TCR" refers to the antigen-recognition molecule present on the surface of T cells. During normal T cell development, each of the four TCR genes, α, β, γ, and δ, can rearrange to give rise to a wide variety of TCR proteins.

[0108] The term "heterologous" refers to a sequence derived from any source other than a naturally occurring sequence. For example, a heterologous sequence included as part of a costimulatory protein is an amino acid that does not naturally occur as a wild-type human costimulatory protein, i.e., does not align with the wild-type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than the nucleotide sequence of the wild-type human costimulatory protein coding sequence.

[0109] The term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for calculating the percent identity between two provided polypeptide sequences are known. For example, calculating the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. The comparison or alignment of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, such as BLAST (Basic Local Alignment Search Tool). In this context, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

[0110] To calculate percent identity, the sequences to be compared are typically aligned in a way that maximizes the correspondence between the sequences. An example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387, Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose percent sequence identity is to be determined. The sequences are aligned for optimal matching of each amino acid or nucleotide (the "matched span" determined by the algorithm). In certain embodiments, standard comparison matrices (for the PAM250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919) are also used in the algorithm. Other algorithms are available for comparing amino acid or nucleic acid sequences, including those available in commercially available computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences.Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying similar sequences, the above programs generally provide an indication of the degree of similarity. In some embodiments, two sequences are considered to be substantially similar if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more of the corresponding residues are similar and / or identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) over a relevant stretch of residues. In some embodiments, the relevant stretch is the entire sequence.In some embodiments, the related runs are at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 1. At least 25, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 or more residues. Sequences having substantial sequence similarity may be homologs of each other.

[0111] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is at least about 95%, e.g., about 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0112] The terms "substantial similarity" or "substantially similar," as applied to polypeptides, mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, and even at least 98% or 99% sequence identity. Typically, residue positions that are not identical differ by conservative amino acid substitutions.

[0113] The terms "improve," "increase," "inhibit," and "reduce" refer to values ​​relative to a baseline or other reference standard value. In some embodiments, suitable baseline measurements may include measurements in a particular system (e.g., in a single individual) under otherwise equivalent conditions in the absence (e.g., before and / or after) of an agent or treatment, or in the presence of an appropriate equivalent reference agent. In some embodiments, suitable baseline measurements may include measurements in an equivalent system known or expected to respond in an equivalent manner in the presence of the relevant agent or treatment.

[0114] "Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including Abs, cytokines, and complement) produced either by these cells or the liver, resulting in the selective targeting, binding, damaging, destroying, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0115] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or recurring with a disease by methods that involve inducing, enhancing, suppressing, or otherwise modifying the immune response. Examples of immunotherapies include, but are not limited to, NK cell and T cell therapy. T cell therapy includes adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered T cell therapy, and other immunotherapy. These conditioning regimens may include enhanced autologous cell therapy (eACT™), and allogeneic T cell transplantation. However, one skilled in the art will appreciate that the conditioning methods disclosed herein will enhance the efficacy of any transplanted T cell therapy. Examples of T cell therapies are described in U.S. Patent Application Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388, and WO 2008 / 081035.

[0116] Immunotherapeutic T or NK cells can be derived from any source known in the art. For example, T and NK cells can be differentiated in vitro from hematopoietic stem cell populations. Alternatively, T cells and NK cells can be obtained from a subject. T cells and NK cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood drawn from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0117] The term "in vitro" refers to events that take place in an artificial environment, such as a test tube, reaction vessel, cell culture, etc., rather than within a multicellular organism. The term "in vitro cell" refers to any cell that is cultured ex vivo. An in vitro cell can include a T cell or an NK cell. The term "in vivo" refers to events that take place within a multicellular organism, such as a human or non-human animal.

[0118] The term "isolated" refers to (1) a substance that is separated from at least some components with which it was previously associated or with which it would otherwise be associated, and / or (2) a substance that is present in a composition that includes a limited or defined amount or concentration of one or more known or unknown contaminants. An isolated substance may, in some embodiments, be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of other components that are not the substance with which the substance was previously associated, such as other components or contaminants with which the substance would previously or otherwise be associated. In certain instances, a substance is isolated when it is present in a composition containing limited or reduced amounts or concentrations of the same or similar types of molecules. For example, in certain instances, a nucleic acid, DNA, or RNA substance is isolated when it is present in a composition containing a limited or reduced amount or concentration of the nucleic acid, DNA, or RNA molecule that is not the substance. For example, in certain instances, a polypeptide substance is isolated when it is present in a composition containing a limited or reduced amount or concentration of the polypeptide molecule that is not the substance. In certain embodiments, the amount can be, for example, an amount measured relative to the amount of the desired substance present in the composition. In certain embodiments, a limited amount can be an amount that is 100% or less of the amount of the substance in the composition, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or less (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of the amount of the substance in the composition. In certain instances, the composition is pure or substantially pure with respect to the selected substance.In some embodiments, an isolated material is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) pure. A material is "pure" if it is substantially free of other components or contaminants. In some embodiments, a material may still be considered "isolated" or "pure" even after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percent isolation or purity of the material is calculated without including such carriers or excipients.

[0119] "Linker" (L) or "linker domain" or "linker region" refers to an oligo- or polypeptide region, about 1 to 100 amino acids in length, that links together, for example, any of the domains / regions of a membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide, a chimeric antigen receptor and / or an scFv, or one or more of these polypeptides. Linkers can be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to one another. Longer linkers may be used if it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof, and combinations thereof. In some embodiments, the linker comprises a picornavirus 2A-like linker, a porcine teschovirus (P2A), a viral (T2A) CHYSEL sequence (SEQ ID NO: 1), or combinations, variants, and functional equivalents thereof. In other embodiments, the linker sequence is Asp-Val / Ile-Glu-X-Asn-Pro-Gly, which results in cleavage between the 2A glycine and the 2B proline. (2A) -Pro (2B)The linker may contain the motif (SEQ ID NO: 2). Other linkers include non-cleavable linkers. In order to realize the present disclosure, a number of linkers are used, including "flexible linkers." The latter are glycine-rich. Klein et al., Protein Engineering, Design & Selection Vol. 27, No. 10, pp. 325-330, 2014, Priyanka et al., Protein Sci., 2013 Feb;22(2):153-167. In some embodiments, the linker is a synthetic linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In one embodiment, the linker has the amino acid sequence AGS (SEQ ID NO: 3). In one embodiment, the linker has the amino acid sequence GGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 4). In one embodiment, the linker has the amino acid sequence GGGGSGGGGS (SEQ ID NO: 5).

[0120] A linker can be part of a multi-component agent that connects different elements to one another. For example, a polypeptide comprising two or more functional or structural domains can include a stretch of amino acids between such domains that connects them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, where S1 and S2, which can be the same or different, represent two domains associated with one another by the linker. The linker can connect or link any of the domains / regions of a membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide, a chimeric antigen receptor, and / or an scFv. In some embodiments, the polypeptide linker comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 11 The linker may be 0, 95, 100, or more amino acids in length (e.g., 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, or 10-100 amino acids in length). In one example, a linker is used to connect or join the C-terminus of an IL-15 polypeptide to the N-terminus of an IL-15Rα sushi domain polypeptide. In another example, a linker is used to join the C-terminus of an IL-15Rα sushi domain polypeptide to a transmembrane domain. In some embodiments, the linker is characterized by its tendency not to adopt a rigid three-dimensional structure, but instead providing flexibility to the polypeptide. In another example, they can be used to link one or more expressed polypeptides, such as the CAR, TCR, and / or membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptides disclosed herein. In some examples, the CAR or TCR and membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptides can be used to link one or more expressed polypeptides, such as the CAR, TCR, and / or membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptides disclosed herein, for example, when they are used alone. The proteins are expressed as a single peptide and then linked by a cleavable linker so that they can be cleaved intracellularly.

[0121] The term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte that represent a component of the genetic immune system. NK cells reject tumor- and virus-infected cells. They act through the process of apoptosis, or programmed cell death. They were called "natural killers" because they do not require activation to kill cells. T cells play a role in cell-mediated immunity (without antibody involvement). Their T cell receptors (TCRs) differentiate from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells (i.e., stem memory T cells), and cytotoxic T cells (TCs). SCM (ii) central memory T cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, similar to naive cells, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting multiple functional attributes unique to memory cells; CM (iii) effector memory T cells express L-selectin and CCR7 and secrete IL-2, but they do not secrete IFNγ or IL-4. EM There are various types of T cells: CD4+CD25+ regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. B cells, on the other hand, play a role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, hence the name.

[0122] The term "neutralizing" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the biological action of that ligand. In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or otherwise alters the ability of the ligand to bind through indirect means (such as a structural or energetic change in the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it is bound from performing its biological function.

[0123] "Nucleic acid" refers to any polymeric chain of nucleotides. Nucleic acids can be DNA, RNA, or a combination thereof. In some embodiments, nucleic acids comprise one or more naturally occurring nucleic acid residues. In some embodiments, nucleic acids are comprised of one or more nucleic acid analogs. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis (in vivo or in vitro) by polymerization based on a complementary template, reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, nucleic acids comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, In some embodiments, the nucleic acid is partially or entirely single-stranded, and in some embodiments, the nucleic acid is partially or entirely double-stranded. In a nucleic acid, a nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide.

[0124] "Operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. For example, a control element "operably linked" to a functional element is associated such that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In embodiments, a promoter is operably linked to a nucleic acid.

[0125] A "patient" includes any human suffering from cancer. The terms "subject" and "patient" are used interchangeably herein.

[0126] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, with no limit on the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, e.g., commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, generally referred to in the art as proteins, of which there are many varieties. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0127] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not harmful to the recipient or the benefits to the recipient outweigh any harmful effects. With respect to carriers, diluents, or excipients used to formulate the compositions disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not harmful to the recipient, or any harmful effects must be outweighed by the benefits to the recipient. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting a drug from one part of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient, or the benefits to the recipient must outweigh any harmful effects. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution, ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0128] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is administered to a relevant subject or population. It is present in a unit dosage amount suitable for administration in a treatment regimen that, when administered, exhibits a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition may be formulated for administration in solid or liquid form, including, but not limited to, forms adapted for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, such as sterile solutions or suspensions, or sustained-release formulations, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, such as creams, ointments, or sustained-release patches or sprays applied to the skin, lungs, or oral cavity; vaginal or rectal administration, such as pessaries, creams, or foams; sublingual; ophthalmic; transdermal; or nasal, pulmonary, and other mucosal surfaces.

[0129] The term "proliferation" refers to an increase in cell division, either symmetric or asymmetric division of cells. In some embodiments, "proliferation" refers to the symmetric or asymmetric division of T cells. "Increased proliferation" occurs when there is an increase in the number of cells in a treated sample compared to cells in an untreated sample.

[0130] The term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control that is an agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or determined substantially contemporaneously with the test, measurement, or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Generally, a reference or control is determined or characterized under conditions or circumstances comparable to those under evaluation, where similarity is sufficient to justify reliance on and / or comparison to the selected reference or control.

[0131] "Regulatory T cells" ("Tregs," "Treg cells," or "Tregs") refer to a lineage of CD4+ T lymphocytes involved in controlling certain immune activities, such as responses to autoimmunity, allergies, and infections. Regulatory T cells can modulate the activity of T cell populations and can also influence specific innate immune system cell types. Tregs can be identified by expression of the biomarkers CD4, CD25, and Foxp3, as well as low expression of CD127. Naturally occurring Treg cells typically comprise approximately 5-10% of peripheral CD4+ T lymphocytes. However, Treg cells within the tumor microenvironment (i.e., tumor-infiltrating Treg cells) can comprise as much as 20-30% of the total CD4+ T lymphocyte population.

[0132] The term "sample" generally refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological source or an environmental source. In some embodiments, the source of interest may include a cell or organism, such as a cell population, a tissue, or an animal (e.g., a human). In some embodiments, the source of interest comprises a biological tissue or fluid. In some embodiments, the biological tissue or fluid may include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chyme, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural effusion, pus, catarrhal secretions, saliva, sebum, semen, semen, semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or components thereof. In some embodiments, biological fluids may include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymphatic fluid, and / or cellular fluid. In some embodiments, biological fluids may include phytoexudates. In some embodiments, biological tissues or samples may be obtained by, for example, aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral swab, nasal swab, skin swab, or vaginal swab), scraping, surgery, lavage (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other lavage). In some embodiments, In the present context, a biological sample includes cells obtained from an individual. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. In some embodiments, as the context will dictate, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). Such a "processed sample" can include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to one or more techniques, such as nucleic acid amplification or reverse transcription, isolation and / or purification of specific components, etc.

[0133] "Single-chain variable fragment," "single-chain antibody variable fragment," or "scFv" antibody refers to a form of antibody that contains only the variable regions of the heavy and light chains connected by a linker peptide.

[0134] The term "cancer stage" refers to a qualitative or quantitative assessment of the level of progression of cancer. In some embodiments, criteria used to determine the stage of cancer may include, but are not limited to, one or more of the following: where the cancer is located in the body, tumor size, whether the cancer has spread to lymph nodes, whether the cancer has spread to one or more different parts of the body, etc. In some embodiments, cancer may be staged using the so-called TNM system, where T refers to the size and extent of the main tumor, usually called the primary tumor, N refers to the number of nearby lymph nodes that have cancer, and M refers to whether the cancer has metastasized. In some embodiments, cancer may be referred to as Stage 0 (abnormal cells are present without spreading to nearby tissues, also known as carcinoma in situ or CIS; CIS is not cancer but may become cancer), Stage I-III (cancer is present, and the larger the number, the larger the tumor and the more it has spread to nearby tissues), or Stage IV (cancer has spread to distant parts of the body). In some embodiments, the cancer may be assigned a stage selected from the group consisting of: in situ, localized (cancer is limited to where it began and there is no sign it has spread), regional (cancer has spread to nearby lymph nodes, tissues, or organs), distal (cancer has spread to distant parts of the body), and unknown (there is not enough information to determine the stage).

[0135] "Stimulation" refers to a primary response induced by binding of a stimulatory molecule to its cognate ligand, which mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, e.g., a T cell receptor (TCR) / CD3 complex, that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.), specifically binds to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, such as, but not limited to, activation, initiation of an immune response, or proliferation. Stimulatory ligands include, but are not limited to, anti-CD3 antibodies (e.g., OKT3), peptide-loaded MHC class I molecules, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies.

[0136] The phrase "therapeutic agent" may refer to any agent that induces a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a model organism or a population of human subjects. In some embodiments, the appropriate population may be defined by various criteria, such as a particular age group, sex, genetic background, pre-existing clinical conditions, depending on the presence or absence of biomarkers, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, reduce, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent is an agent that has been, or needs to be, approved by a government agency before it can be commercially available for administration to humans. In this embodiment, the therapeutic agent is a drug that requires a medical prescription for administration to a human.

[0137] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective administration amount" of a therapeutic agent, e.g., engineered CAR T cells or NK cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from developing disease or promotes regression of disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those of skill in the art, such as by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0138] The terms "transduction" and "transduced" refer to the process by which foreign DNA is introduced into a cell via a viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0139] "Transformation" refers to any process by which exogenous DNA is introduced into a host cell. Transformation can occur under natural or artificial conditions using a variety of methods. Transformation can be achieved using any known method for the insertion of foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. In some embodiments, some transformation methodologies are selected based on the host cell to be transformed and / or the nucleic acid to be inserted. Methods of transformation may include, but are not limited to, viral infection, electroporation, and lipofection. In some embodiments, a "transformed" cell is stably transformed in that the inserted DNA is capable of replicating as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, the transformed cell can express the introduced nucleic acid.

[0140] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the goal of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or biochemical manifestations associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission. In some embodiments, treatment can be treatment of a subject who does not exhibit symptoms of the associated disease, disorder, and / or condition and / or who exhibits only early signs of the disease, disorder, and / or condition. In some embodiments, such treatment can be treatment of a subject who exhibits one or more definitive signs of the associated disease, disorder, and / or condition. In some embodiments, treatment can be treatment of a subject who has been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment can be treatment of a subject who is known to have one or more susceptibility factors statistically correlated with an increased risk of developing the associated disease, disorder, and / or condition.

[0141] The term "vector" refers to a recipient nucleic acid molecule that contains or has been modified to incorporate a provided nucleic acid sequence. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA can be ligated. Another type of vector is a viral vector, in which additional DNA segments are ligated into the viral genome. Certain vectors can be inserted into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors contain sequences that direct the expression of inserted genes to which they are operably linked. Such vectors may be referred to herein as "expression vectors." Standard techniques can be used to manipulate vectors, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference.

[0142] A "binding protein" is a protein that can non-covalently bind to another molecule. A binding protein can bind, for example, to a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein), and / or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it can bind to itself (forming a homodimer, homotrimer, etc.) and / or to one or more molecules of different proteins. A binding protein can have more than one type of binding activity.

[0143] A "transmembrane domain" is a domain of a polypeptide that comprises at least one contiguous amino acid sequence that spans a lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell. For example, a transmembrane domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid sequences that each span a lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell. A transmembrane domain may comprise, for example, at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) contiguous amino acid sequence that has an α-helical secondary structure in a lipid bilayer (which spans the lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell). In some embodiments, a transmembrane domain may comprise two or more contiguous amino acid sequences that form a β-barrel secondary structure in a lipid bilayer (which each spans the lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell). Non-limiting examples of transmembrane domains are described herein. Further examples of transmembrane domains are known in the art.

[0144] The phrase "extracellular side of the plasma membrane," when used to describe the location of a polypeptide, means that the polypeptide contains at least one transmembrane domain that spans the plasma membrane and at least one domain (e.g., at least one antigen-binding domain) that is located in the extracellular space. In one example, the IL-15 and IL-15Rα portions of a membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide are presented on the extracellular side of the plasma membrane.

[0145] By "signal sequence" is meant a peptide sequence generally present at the N-terminus of a newly synthesized protein that directs entry into the secretory pathway.

[0146] A "membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide" means that an IL-15 polypeptide is linked to an IL-15Rα sushi domain. The membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide is further tethered to the cell membrane (and is not secreted) by linking to a transmembrane domain as described herein.

[0147] The term "persistent" refers to, for example, the ability of one or more transplanted immune cells, or their progeny (e.g., NK cells or differentiated or mature T cells) administered to a subject to persist for a period of time. "Increasing the persistence of one or more transplanted immune cells, or their progeny (e.g., NK cells or differentiated or mature T cells), refers to extending the period during which the transplanted immune cells are detectable in a subject following administration. For example, the in vivo persistence of one or more transplanted immune cells can be extended by at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months. Additionally, the persistence of the one or more transplanted immune cells in vivo may be increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to one or more transplanted immune cells not prepared by the methods disclosed herein.

[0148] The present disclosure may employ, unless specifically indicated to the contrary, methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the skill of the art, many of which are described below by way of example. Such techniques are explained fully in the literature, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience, Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985), Anand, Techniques for the Analysis of Complex See journal monographs such as Genomes, (Academic Press, New York, 1992), Transcription and Translation (B. Hames & S. Higgins, Eds., 1984), Perbal, A Practical Guide to Molecular Cloning (1984), Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Current Protocols in Immunology QE Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology.

[0149] Other features, objects, and advantages of the present disclosure will be apparent from the following detailed description, but it should be understood that the detailed description, while indicating embodiments of the present disclosure, is given by way of illustration only, and not by way of limitation.

[0150] Membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polymerase Disclosed herein are membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptides. In embodiments, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptides contain a signal peptide and, when expressed from their corresponding nucleic acids, are thought to be targeted to a cell membrane by the signal sequence. In embodiments, the signal sequence is cleaved so that the displayed membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide does not contain the signal sequence. In embodiments, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptides contain a transmembrane domain that anchors the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide to a cell membrane.

[0151] Interleukin-15 (IL-15) is a cytokine with structural similarity to interleukin-2 (IL-2). Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (γ-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) after infection with a virus or viruses. This cytokine induces the proliferation of natural killer cells, cells of the innate immune system whose primary role is to kill virus-infected cells. The protein encoded by this gene is a cytokine that regulates the activation and proliferation of T cells and natural killer cells. This cytokine and interleukin-2 share many biological activities. They have been found to bind to a common hematopoietin receptor subunit, compete for the same receptor, and may therefore negatively regulate each other's activity. The number of CD8+ memory cells has been shown to be controlled by the balance between this cytokine and IL-2. This cytokine induces activation of JAK kinases and phosphorylation and activation of the transcriptional activators STAT3, STAT5, and STAT6. As used herein, unless otherwise specified, the term "IL-15" refers to the mature form of IL-15 (i.e., without the signal peptide) or an active fragment thereof. The protein product of IL-15 can have any amino acid sequence known in the art, for example, that available at NCBI Gene ID: 3600 (updated February 6, 2021), which is specifically incorporated herein by reference.

[0152] In certain embodiments, an IL-15 polypeptide refers to a polypeptide having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the mature form of IL-15 or a fragment thereof that has similar activity to the full-length mature form. In embodiments, an IL-15 polypeptide has an amino acid sequence that has at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:6. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 6).

[0153] The interleukin-15 receptor subunit alpha (also known as CD125 or IL-15Rα) is a cytokine receptor that specifically binds interleukin-15 (IL-15) with high affinity. IL-15 and IL-2 receptors share two subunits, IL2R beta and IL2R gamma. IL-15Rα is structurally related to an additional IL2-specific α subunit, IL2Rα, for high-affinity IL2 binding. Unlike IL2RA, IL-15Rα can bind IL-15 with high affinity independently of other subunits, suggesting different roles between IL-15 and IL2. This receptor has been reported to enhance cell proliferation and the expression of the apoptosis inhibitors BCL2L1 / BCL2-XL and BCL2. As used herein, In this context, unless otherwise specified, the term "IL-15Rα" refers to the mature form of IL-15Rα (i.e., without the signal peptide). The protein product of IL-15Rα can have any amino acid sequence known in the art, e.g., that available at NCBI Gene ID: 3601 (updated February 3, 2021), which is specifically incorporated herein by reference. Furthermore, unless otherwise stated, the IL-15Rα sushi domain refers to the sushi domain of IL-15Rα, e.g., comprising or consisting of amino acid residues 49-162 of the full-length IL-15Rα polypeptide.

[0154] In certain embodiments, an IL-15Rα polypeptide refers to a polypeptide having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the mature form of IL-15Rα, or a fragment thereof that has similar activity as the full-length mature form. In embodiments, the IL-15Rα polypeptide comprises the active form of IL-15Rα polypeptide from amino acids 49 to 162. In embodiments, the IL-15Rα sushi domain subunit has an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 7. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRD (SEQ ID NO: 7). In a further embodiment, the IL-15Rα sushi domain subunit has an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 95. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPG (SEQ ID NO: 95).

[0155] In certain embodiments, IL-15 and IL-15Rα sushi domain subunits can be linked as described herein. In certain embodiments, the linker sequence comprises a set of glycine and serine repeats, such as Ser(Gly4Ser)n (SEQ ID NO: 8), where n is a positive integer greater than or equal to 1 and less than 10. In one embodiment, the linker is Ser(Gly4Ser)3 (SEQ ID NO: 9) or Ser(Gly3Ser)1 (Gly4Ser) n (Gly3Ser)1 (SEQ ID NO: 10). In an embodiment, the linker sequence comprises or consists of SGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 11). Additional sequences can be used as linker sequences.

[0156] In embodiments, the polypeptides disclosed herein include a signal sequence, e.g., a heterologous signal sequence, e.g., an IgE signal sequence, a kappa signal sequence, a CD8 signal sequence, or any peptide with essentially equivalent activity.

[0157] Exemplary signal sequences are provided in Table 4 below. [Table 4]

[0158] In embodiments, the signal sequence is linked to the IL-15 subunit using a linker, such as one of the linkers described herein. In one embodiment, the linker is an AGS (SEQ ID NO: 4) linker. In embodiments, a Myc sequence is used alone or in combination with any of the above linkers. In some embodiments, the amino acid sequence of the Myc sequence is EQKLISEEDL (SEQ ID NO: 21).

[0159] In embodiments, the polypeptides disclosed herein comprise a transmembrane domain sequence, e.g., a heterologous transmembrane domain, e.g., a FAS transmembrane domain sequence, or an IL-7 transmembrane domain sequence, or a peptide with essentially equivalent activity.

[0160] In embodiments, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide comprises a FAS transmembrane domain sequence, which is believed to confer surface expression of the monomeric membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide. In embodiments, the FAS transmembrane domain sequence comprises an amino acid sequence having at least 75% sequence identity (at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:22:LGWLCLLLLPIPLIVWV (SEQ ID NO:22).

[0161] In embodiments, the FAS transmembrane domain sequence comprises an amino acid sequence having at least 75% sequence identity (at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 42: RSNLGWLCLLLLPIPLIVWVKRKEVQKT (SEQ ID NO: 42).

[0162] In embodiments, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide comprises a heterodimerization domain such that the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide forms a homodimer when expressed. In embodiments, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide comprises an IL-7 transmembrane domain sequence. In embodiments, the IL-7 transmembrane domain sequence comprises a CPT sequence motif. This sequence is believed to result in surface expression of the homodimeric membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide, which forms a stable homodimer through disulfide formation. In embodiments, the IL-7 transmembrane domain sequence comprises an amino acid sequence having at least 75% sequence identity (at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 23. PILLTCPTISILSFFSVALLVILACVLW (SEQ ID NO: 23).

[0163] In certain embodiments, the IL-15Rα sushi domain subunit can be linked to a transmembrane anchor domain as described herein. In certain embodiments, the linker sequence is (Gly4Ser) n (SEQ ID NO: 24), where n is a positive integer greater than or equal to 1 and less than 10. In one embodiment, the linker can be (Gly4Ser)1 (SEQ ID NO: 25) or (Gly4Ser)2 (SEQ ID NO: 26) or (Gly4Ser)3 (SEQ ID NO: 27). In one embodiment, the linker sequence comprises or consists of GGGGSGGGGS (SEQ ID NO: 26).

[0164] In one embodiment, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide has an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95% or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:27. MDWTWILFLVAAATRVHSEQKLISEEDLAGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDGGGGSGGGGSRSNLGWLCLLLLPIPLIVWVKRKEVQKT (SEQ ID NO: 27).

[0165] In one embodiment, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide has an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95% or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 43. MDWTWILFLVAAATRVHSAGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDGGGGSGGGGSRSNLGWLCLLLLPIPLIVWVKRKEVQKT (SEQ ID NO: 43).

[0166] In one embodiment, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide has an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:28. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDGGGGSGGGGSRSNLGWLCLLLLPIPLIVWVKRKEVQKT (SEQ ID NO: 28).

[0167] In one embodiment, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide has at least 75% sequence identity (at least 75%, at least MDWTWILFLVAAATRVHSAGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDGGGGSGGGGSPILLTCPTISILSFFSVALLVILACVLW (SEQ ID NO: 44).

[0168] In one embodiment, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide has an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:29. MDWTWILFLVAAATRVHSEQKLISEEDLAGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDGGGGSGGGGSPILLTCPTISILSFFSVALLVILACVLW (SEQ ID NO: 29).

[0169] In one embodiment, the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide has an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 30. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDGGGGSGGGGSPILLTCPTISILSFFSVALLVILACVLW (SEQ ID NO: 30).

[0170] In one embodiment, the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide has an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 94. MDWTWILFLVAAATRVHSEQKLISEEDLAGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGGSGGGGSGGG GSGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTA SASHQPPGGGGGSGGGGSRSNLGWLCLLLLPIPLIVWVKRKEVQKT (SEQ ID NO: 94).

[0171] The present disclosure provides nucleic acids encoding the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptides described herein. In embodiments, the nucleic acids encoding the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptides include nucleic acids encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:6. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:7. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:8. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:9.In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 10. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 11. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 12. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 13. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%) to SEQ ID NO: 14. In embodiments, the nucleic acid encoding the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide includes a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 15. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 16. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 17. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 18.In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 19. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 20. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 21. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 22.In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 23. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 24. The nucleic acid encoding the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide includes a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 25. In embodiments, the nucleic acid encoding the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide includes a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 25. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:26. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:27. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:28.In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:29. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 30. In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid encoding an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 94.

[0172] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 31. GCTGGAAGCAATTGGGTGAACGTGATCTCCGACCTCAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGATGCCACACTCTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTTACAGCCATGAAATGCTTTCTGCTGGAGCTGCAAGTGATC TCTCTGGAGTCCGGAGATGCTTCCATCCACGACACAGTGGAGAATCTGATCATTCTGGCTAACAACTCCCTCTCCAGCAACGGCAATGTCACAGAGTCCGGCTGCAAAGAGTGTGAAGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTCCAGATGTTCATCAACACCTCCTCC (SEQ ID NO: 31).

[0173] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 32. GCCGGCAGCAACTGGGTCAACGTGATCTCCGATCTGAAGAAGATCGAAGATCTGATCCAGTCCATGCACATCGATGCCACACTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAAGTTACAGCCATGAAGTGCTTTCTGCTCGAACTGCAAGTGATTTCTCTGGAGAGCGGAGATGCCA GCATCCACGACACCGTGGAGAATCTGATCATTCTGGCCAACAACTCTCTGAGCAGCAACGGCAATGTGACAGAGTCCGGCTGTAAGGAGTGCGAGGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATTGTCCAAATGTTCATCAACACCAGCAGC (SEQ ID NO: 32).

[0174] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 33. ATTACATGCCCTCCCCCCATGTCCGTGGAACATGCCGACATCTGGGTGAAGTCCTACTCTCTGTACTCGCGTGAACGTTATATCTGCAACAGCGGCTTTAAGAGGAAGGCCGGAACCTCCTCTGACCGAATGTGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTAGCCTCAAGTGTATTAGGGAC (SEQ ID NO: 33).

[0175] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 34: AGAAGCAATCTGGGCTGGCTGTGTCTGCTGCTGCTCCCCATCCCTCTGATTGTGTGGGTCAAGAGGAAGGAGGTCCAGAAAACC (SEQ ID NO: 34).

[0176] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 35. CCTATTCTGCTGACATGCCCCACCATCTCCATCCTGTCTTTT TTTTCTGTTGCTCTGCTGGTGATTCTGGCTTGCGTGCTGTGG (SEQ ID NO: 35).

[0177] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 36: GGCGGCGGCAGCGGCGGCGGCGGATCCGGCGGAGGAGGCAGCGGAGGAGGAGGAAGCGGAGGAGGCTCC (SEQ ID NO: 36).

[0178] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 37. GGCGGCGGCTCCGGCGGCGGAGGCTCCGGCGGAGGCGGATCCGGCGGCGGCGGATCCGGCGGAGGATCC (SEQ ID NO: 37).

[0179] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 38. GGCGGCGGAGGATCCGGAGGAGGCGGATCT (SEQ ID NO: 38).

[0180] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 39. GGCGGCGGAGGAAGCGGAGGAGGAGGAAGC (SEQ ID NO: 39). Monomer

[0181] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 40. GCTGGAAGCAATTGGGTGAACGTGATCTCCGACCTCAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGATGCCACACTCTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTTACAGCCATGAAATGCTTTCTGCTGGAGCTGCAAGTGATCTCTGGAGTCCGGAGATGCTTCCATCCACGACACAGTGGAGAATCTGATCATTCTGGCT AACAACTCCCTCTCCAGCAACGGCAATGTCACAGAGTCCGGCTGCAAAGAGTGTGAAGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTCCAGATGTTCATCAACACCTCCTCCGGCGGCGGCAGCGGCGGCGGCGGATCCGGCGGAGGAGGCAGCGGAGGAGGAGGAAGCGGAGGAGGCTCCATTACATGCCCTCCCC CCATGTCCGTGGAACATGCCGACATCTGGGTGAAGTCCTACTCTCTGTACTCGCGTGAACGTTATATCTGCAACAGCGGCTTTAAGAGGAAGGCCGGAACCTCCTCTGACCGAATGTGTGCTGAACAAGGCCACCAATGTGGCTCACTGG ACCACACCTAGCCTCAAGTGTATTAGGGACGGCGGCGGAGGATCCGGAGGAGGCGGATCTAGAAGCAATCTGGGCTGGCTGTGTCTGCTGCTGCTCCCCATCCCTCTGATTGTGTGGGTCAAGAGGAAGGAGGTCCAGAAAACC (SEQ ID NO: 40).

[0182] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 42. (SEQ ID NO: 42)

[0183] In embodiments, a nucleic acid encoding a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 41. GCCGGCAGCAACTGGGTCAACGTGATCTCCGATCTGAAGAAGATCGAAGATCTGATCCAGTCCATGCACATCGATGCCCACACTGTACACCGAGAGCGACGTGCACCCAGCTGCAAAGTTACAGCCATGAAGTGCTTTCTGCTCGAACTGCAAGTGATTTCTCTGGAGAGCGGAGATGCCAGCATCCACGACACCGTGGAGAATCTGATCATTCTGGCC AACAACTCTCTGAGCAGCAACGGCAATGTGACAGAGTCCGGCTGTAAGGAGTGCGAGGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATTGTCCAAATGTTCATCAACACCAGCAGCGGCGGCGGCTCCGGCGGCGGAGGCTCGGCGGAGGCGGATCCGGCGGCGGCGATCGGCGGAGGATCCATTACATGCCCCCCTC CCATGTCCGTGGAACACGCCGACATCTGGGTGAAGAGCTACTCTCTGTACAGCAGAGAGCGTTACATCTGCAACAGCGGCTTTAAGAGGAAAGCCGGCACCAGCAGCCTCACAGAGTGCGTGCTCAACAAGGCCACCAACGTCGCCCATTGGACCACCCCCTCTCTGAAGTGTATTAGGGACGGCGGCGGAGGAAGCGGAGGAGGAAGCCCTATTCTGCTGACATGCCCACCATCTCCATCCTGTCTTTTTTTCTGTTGCTCTGCTGGTGATTCTGGCTTGCGTGTGGTGA (SEQ ID NO: 41).

[0184] In embodiments, the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide (comprising a FAS transmembrane domain and no myc tag) comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 96. ATGGACTGGACATGGATTCTGTTTCTGGTGGCCGCCGCCACAAGAGTGCACAGCAATTGGGTGAACGTGATCTCCGACCTCAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGATGCCACACTCTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTTACAGCCATGAAATGCTTTCTGCTGGAGCTGCAAGTGATCTCTCTGGAGTCCGGAGATGCTTCCATCCACGACACAGTGGAGAATCTGATCATTCTGGCTAACAACTCCCTCTCCAGCAACGGCAATGTCACAGAGTCCGGCTGCAAAGAGTGTGAAGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTCCAGATGTTCATCAACACCTCCTCCGGCGGCGGCAGCGGCGGCGGCGGATCCGGCGGAGGAGGCAGCGGAGGAGGAGGAAGCGGAGGAGGCTCCATTACATGCCCTCCCCCCATGTCCGTGGAACATGCCGACATCTGGGTGAAGTCCTACTCTCTGTACTCGCGTGAACGTTATATCTGCAACAGCGGCTTTAAGAGGAAGGCCGGAACCTCCTCTCTGACCGAATGTGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTAGCCTCAAGTGTATTAGGGACGGCGGCGGAGGATCCGGAGGAGGCGGATCTAGAAGCAATCTGGGCTGGCTGTGTCTGCTGCTGCTCCCCATCCCTCTGATTGTGTGGGTCAAGAGGAAGGAGGTCCAGAAAACCTAA(SEQ ID NO: 96).

[0185] In embodiments, the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide (comprising an IL-7 transmembrane domain and no myc tag) comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 97. ATGGACTGGACATGGATTCTGTTTCTGGTGGCCGCCGCCACAAGAGTGCACTCCAACTGGGTCAACGTGATCTCCGATCTGAAGAAGATCGAAGATCTGATCCAGTCCATGCACATCGATGCCACACTGTACACCGAGAGCGACGTGCACCCCAGCTGC AAAGTTACAGCCATGAAGTGCTTTCTGCTCGAACTGCAAGTGATTTCTCTGGAGAGCGGAGATGCCAGCATCCACGACACCGTGGAGAATCTGATCATTCTGGCCAACAACTCTCTGAGCAGCAACGGCAATGTGACAGAGTCCGGCTGTAAGGAGTGCG AGGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATTGTCCAAATGTTCATCAACACCAGCAGCGGCGGCGGCTCCGGCGGCGGAGGCTCCGGCGGAGGCGGATCCGGCGGCGGCGGATCCGGCGGAGGATCCATTACATGCCCCCCTCCCATGTCCGTGGAACACGCCGACATCTGGGTGAAGAGCTACTCTCTGTACAGCAGAGAGCGTTACATCTGC AACAGCGGCTTTAAGAGGAAAGCCGGCACCAGCAGCCTCACAGAGTGCGTGCTCAACAAGGCCACCAACGTCGCCCATTGGACCACCCCCTCTCTGAAGTGTATTAGGGACGGCGGCG GAGGAAGCGGAGGAGGAGGAAGCCCTATTCTGCTGACATGCCCCACCATCTCCATCCTGTCTTTTTTTTCTGTTGCTCTGCTGGTGATTCTGGCTTGCGTGCTGTGGTGA (SEQ ID NO: 97).

[0186] In embodiments, a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide (in the context of an anti-CD19 CAR, comprising a FAS transmembrane domain and no myc tag) comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95% or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 98.

[0187] In embodiments, a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide (in the context of an anti-CD19 CAR, comprising an IL-7 transmembrane domain and no myc tag) comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95% or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 99. ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGA CATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCT GGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAA ACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGG

[0188] In embodiments, the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide (comprising a FAS transmembrane domain and not a myc tag) has at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity) to SEQ ID NO: 100, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95- 100%).

[0189] In embodiments, a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide (in the context of an anti-CD19 CAR, comprising a FAS transmembrane domain and no myc tag) comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95% or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 101. ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGG CAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTAT TCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCGGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGATCCGGCAAGCCCGGATC TGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGC CTTCTTTTGCTGCCCATACCGTTGATCGTCTGGGTTAAGCGCAAAGAAGTCCAGAAAACTTAA (SEQ ID NO: 101).

[0190] The present disclosure provides methods and compositions for improving the efficacy of antigen-binding systems, such as CARs and TCRs, that contain binding motifs that bind to antigens of interest, e.g., tumor antigens. In certain embodiments, the antigen-binding system is a chimeric antigen receptor (CAR). In certain embodiments, the antigen-binding system is a T cell receptor (TCR). The antigen-binding system can bind to tumor antigens or pathogen antigens.

[0191] Chimeric antigen receptors (CARs) are engineered receptors that can direct or redirect T cells (e.g., patient or donor T cells) to target a selected antigen. CARs can be engineered to recognize an antigen and, upon binding to that antigen, activate immune cells to attack and destroy cells bearing that antigen. If these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. CARs generally contain an extracellular binding motif that mediates antigen binding, a transmembrane domain, which is understood to span or translocate the cell membrane if the antigen-binding system is present on the cell surface or membrane, and an intracellular (or cytoplasmic) signaling domain.

[0192] According to at least one non-limiting aspect, there are at least three "generations" of CAR compositions. In first-generation CARs, a binding motif (e.g., a single-chain fragment variable binding motif) is linked or connected to a signaling domain (e.g., CD3ζ) via a transmembrane domain, optionally including a hinge domain and one or more spacers. In second-generation CARs, a costimulatory domain (such as CD28, 4-1BB, or OX-40) is introduced along with the signaling domain (e.g., CD3ζ). Third-generation CARs include a second costimulatory domain.

[0193] TCR is a heterodimer composed of an α chain and a β chain. TCR signaling requires the recruitment of signaling proteins to generate an immune synapse. In addition, localization of the TCR at the plasma membrane depends on the CD3 complex expressed in T cells. Engineered single-chain TCRs can be generated using, for example, the transmembrane and signaling domains of CAR constructs, known methods and constructs (e.g., sTCR and TCR-CAR molecules, e.g., fusions of the TCR β chain with CD28 TM and CD28 and CD3ζ signaling modules).

[0194] The antigen binding system can comprise a VH and a VL. In some embodiments, the VH and VL are connected by a linker (L).

[0195] In some embodiments, the antigen binding system further comprises a costimulatory domain, and / or an extracellular domain (e.g., a "hinge" or "spacer" region), and / or a transmembrane domain, and / or an intracellular (signaling) domain, and / or a CD3 zeta or CD3 epsilon activation domain.

[0196] One or more antigen binding motifs determine the target of the antigen binding system. The binding motif of the antigen binding system can include any binding motif. The binding motif can be engineered to be expressed as part of a single chain with other CAR components, and is therefore used at least in part in chimeric antigen receptors. For example, see U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136; Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998 , 161:2791-2797, each of which is incorporated herein by reference with respect to binding motif domains in CARs. A binding motif or scFv is a single-chain antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain and the light chain variable domain are linked or connected to each other. See, e.g., U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al. See, e.g., et al., Cancer Immunol Immunotherapy (1997) 45:131-136, each of which is incorporated herein by reference with respect to the binding motif domain. When derived from a parent antibody, the binding motif may retain some, all, or essentially retain the parent antibody's binding of the target antigen.

[0197] In various embodiments, the binding motif binds to a tumor antigen. In certain embodiments, the tumor antigen is 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A-kinase anchoring protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), β2-integrin, BAFF, B lymphoma cells, B-cell maturation antigen (BCMA), bcr-abl (an oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia cell oncogene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), BST2, C242 antigen, 9-O-acetyl-CA19-9 marker, CA-125,CAEX, calreticulin, carbonic anhydrase 9 (CAIX), C-MET, CCR4, CCR5, CCR8, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD16, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD25, CD27, CD28, CD30 (TNFRSF8), CD33, CD34, CD38, CD40, CD 40L, CD41, CD44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, CD72, CD74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD125, CD133, CD137, CD138, CD150, CD152(CTLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), claudin 6 (CLDN6), TACI, C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-type lectin domain family 12 member A (CLEC12A), cytomegalovirus (CMV)-infected cell antigen, CNT0888, CRTAM (CD355), CS-1 (also known as CD2 subset 1, CRACC, CD319, and 19A24), CTLA-4, cyclin B1, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B 1 ​​(CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neoepitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 variant (ELF2M), endosialin, epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EphA2), ephrin B2, receptor tyrosine-protein kinase erb-B2, 3, 4 (erb-B2, 3, 4,4), ERBB, ERBB2 (Her2 / neu), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS transgene mutant gene 6 (ETV6-AML) located on chromosome 12p, Fc fragment of IgA receptor (FCAR or CD89), fibroblast activation protein alpha (FAP), FBP, Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor (AChR), fibronectin extra domain B, Fms-like tyrosine kinase, 3 (FLT3), folate binding protein (FBP), folate receptor 1, folate receptor α, folate receptor β, Fos-related antigen 1, fucosyl, fucosyl GM1; GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), GP 100, hexasaccharide moiety of GloboH glycoceramide (GloboH), glycoprotein 75, glypican-3 (GPC3), glycoprotein 100 (gpl00), GPNMB, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), hepatitis A virus cellular receptor 1 (HAVCR1), human epidermal growth factor receptor 2 (HER-2), HER2 / neu, HER3, HER4, HGF, high molecular weight melanoma-associated antigen (HMWMAA), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), heat shock protein 70-2 mutant (mut hsp70-2), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgG1, immunoglobulin lambda-like polypeptide 1 (IGLL1), IL-6, interleukin-11 receptor alpha (IL-1 lRa), IL-13, interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2), insulin-like growth factor I receptor (IGF1-R), integrin α5β1, integrin ανβ3, intestinal carboxylesterase, kappa light chain, KCS1, kinase insert domain receptor (KDR), KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KG2D ligand, KIT (CD117), KLRGI, LAGE- la, LAG3, lymphocyte-specific protein tyrosine kinase (LCK), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), legumain, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis (Y) antigen, LeY, LG, LI cell adhesion molecule (LI-CAM), LIGHT, LMP2, lymphocyte antigen 6 complex, LTBR, ​​locus K9 (LY6K), Ly-6, lymphocyte antigen 75 (LY75), melanoma cancer testis antigen-1 (MAD-CT-1);Melanoma cancer testis antigen-2 (MAD-CT-2), MAGE, melanoma-associated antigen 1 (MAGE-A1), MAGE-A3 melanoma antigen 1 (MelanA or MARTI) recognized by T cells, MelanA / MARTl, mesothelin, MAGE A3, melanoma inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory factor (MIS) receptor type II, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), N-glycolylamine acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, mammary differentiation antigen (NY-BR-1), NY-ESO-1, carcinoembryonic antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, polySA, proacrosin-binding protein sp32 (OY-TES 1), p53, p53 mutant, pannexin 3 (PANX3), prostatic acid phosphatase (PAP), paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), prostate cancer tumor antigen-1 (PCTA-1 or galectin-8), PD-1H, platelet-derived growth factor receptor alpha (PDGFR-alpha), PDGFR-beta, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostase, prostate cancer cell, prostein, protease serine 21 (testisin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (prosome, macropain) subunit, beta type, receptor for advanced glycation end products (RAGE-1), RA; NKL, Ras mutant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), kidney ubiquitous 1 (RU1), kidney ubiquitous 2 (RU2), sarcoma translocation breakpoint, squamous cell carcinoma antigen recognized by T cells 3 (SART3), SAS, SDC1, SLAMF7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, sonic hedgehog (SHH), sperm protein 17 (SPA17), stage-specific embryonic Antigen 4 (SSEA-4), STEAP, sTn antigen, synovial sarcoma X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), TCRa, TCRb, TCR5γ, TCR gamma alternative reading frame protein (TARP), telomerase, TIGIT, TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin-C, TGF-β2, TGF-β, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TIM1, TIM2, TIM3, Tn Ag, TRAIL-R1, TRAIL-R2, tyrosinase-related protein 2 (TRP-2), thyroid-stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosinase, ROR1, TAG-72, uroplakin 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2), and vimentin, Wilms' tumor protein (WT1), or X-antigen family member 1A (XAGE1). See also International Patent Application Publication No. WO 2015 / 142675.

[0198] A CAR may comprise one or more antigen-binding domains that bind to a target antigen. In certain embodiments, the antigen-binding domain binds to CD19. In certain embodiments, the antigen-binding domain binds to CD20. In some embodiments, the CAR further comprises a costimulatory domain, and / or an extracellular domain (e.g., a "hinge" or "spacer" region), and / or a transmembrane domain, and / or an intracellular (signaling) domain, and / or a CD3 activation domain. In some embodiments, the CAR comprises at least a binding domain that binds to a target antigen, a costimulatory domain, an extracellular domain, a transmembrane domain, and a CD3-zeta or CD3-epsilon activation domain.

[0199] In certain embodiments, CARs contemplated herein may include linker residues added between various domains, for example, between the VH and VL domains, for proper spacing conformation of the molecule. CARs contemplated herein may include one, two, three, four, or five or more linkers. In some embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids in length.

[0200] Illustrative examples of linkers include glycine polymers (G), glycine-serine polymers (G 1~5 S 1~5)n (where n is an integer of at least 1, 2, 3, 4, or 5), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can function as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine also has better access to phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Other linkers contemplated herein include the Whitlow linker (see Whitlow, Protein Eng. 6(8):989-95 (1993)). Those skilled in the art will appreciate that the design of CARs in some embodiments may include a fully or partially flexible linker, thereby allowing for a more flexible linker. It will be appreciated that the linker can include flexible linkers as well as one or more moieties that confer less flexible structure to provide a desired CAR structure. In one embodiment, any of the constructs described herein can include a "GS" linker. In another embodiment, any of the constructs described herein includes a "GSG" linker. In one example, the glycine-serine linker comprises or consists of the amino acid sequence GS (SEQ ID NO: 43). In one example, the glycine-serine linker comprises or consists of the amino acid sequence GGGSGGGS (SEQ ID NO: 44). In another embodiment, the CAR described herein comprises an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 45 (GSTSGSGKPGSGEGSTKG (SEQ ID NO: 45)). In one embodiment, the linker is encoded by a nucleic acid sequence having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid sequence set forth below. GGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGC (SEQ ID NO: 46) GGGAGCACTAGCGGCTCTGGCAAACCTGGATCTGGCGAGGGATCTACCAAGGGC (SEQ ID NO: 47), GGCTCCACCAGCGGAAGCGGCAAGCCAGGCTCAGGCGAAGGATCTACAAAAGGC (SEQ ID NO: 48), or GGGAGCACAAGCGGCTCTGGCAAACCTGGATCCGGCGAGGGATCTACCAAGGGC (SEQ ID NO: 49).

[0201] In embodiments, the CAR comprises an scFv that further comprises a variable region linking sequence. A "variable region linking sequence" is an amino acid sequence that connects the heavy chain variable region to the light chain variable region and provides a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same light and heavy chain variable regions. In one embodiment, the variable region linking sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids in length.

[0202] In embodiments, the binding domain of the CAR is followed by one or more "spacer domains," which refer to regions that move the antigen binding domain away from the effector cell surface to allow for proper cell / cell contact, antigen binding, and activation (Patel et al. al., Gene Therapy, 1999;6:412-419). Spacer domains can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0203] The binding domain of a CAR can generally be followed by one or more "hinge domains," which serve to position the antigen-binding domain away from the effector cell surface to allow for proper cell-cell contact, antigen binding, and activation. The hinge can be the extracellular domain of the antigen-binding system located between the binding motif and the transmembrane domain. The hinge can also be referred to as an extracellular domain or a "spacer." The hinge can contribute to the expression, activity, and / or stability of the receptor. In some embodiments, the hinge domain is located between the binding motif and the transmembrane domain. The hinge also provides flexibility for accessing the target antigen. The hinge comprises an immunoglobulin-like hinge domain. CARs generally comprise one or more hinge domains between the binding domain and the transmembrane domain. The hinge domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. In some embodiments, the antigen binding system may comprise a hinge that is, is from, or is derived from an immunoglobulin-like hinge domain (e.g., comprising all or a fragment of an immunoglobulin-like hinge domain). In some embodiments, the hinge domain is from or is derived from an immunoglobulin. In some embodiments, the hinge domain is selected from an IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM hinge, or a fragment thereof.

[0204] The hinge may be derived from natural or synthetic sources. In some embodiments, the antigen binding system is selected from the group consisting of CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8 alpha, CD8 beta, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49 d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB) , CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LI GHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK -p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD11a / CD18), NKG2C,The hinge may be, be from, or be derived from (e.g., including all or a fragment thereof) DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, or Toll ligand receptor, or a fragment or combination thereof.

[0205] In some embodiments, a CAR may comprise a hinge that is, is from, or is derived from (e.g., including all or a fragment thereof) a hinge of CD8alpha. In some embodiments, a CAR may comprise a hinge that is, is from, or is derived from (e.g., including all or a fragment thereof) a hinge of CD28. In some embodiments, a hinge is a fragment or a fragment of a hinge of CD8alpha. is, is from, or is derived from, a fragment of the hinge of CD28, wherein the fragment is less than the entire hinge. In some embodiments, the fragment of CD8 alpha hinge or fragment of CD28 hinge comprises an amino acid sequence that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids of the CD8 alpha hinge, or at the N-terminus or C-terminus, or both, of the CD28 hinge.

[0206] In embodiments, the hinge domain comprises a CD28 hinge region. In embodiments, the CD28 hinge domain has an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 230. (IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 50)). In embodiments, the CD28 hinge domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having a sequence according to the following: attgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 51).

[0207] In embodiments, the hinge domain comprises a truncated CD28 hinge region (CD28T) hinge region, as disclosed in International Patent Application No. PCT / US2017 / 025351, filed March 31, 2017, which is incorporated by reference in its entirety. In embodiments, the CAR comprises a CD28T hinge domain having an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 52. (LDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 52)). In embodiments, the CD28T hinge domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having a sequence according to the following: ctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacaccttttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 53).

[0208] In embodiments, the hinge domain comprises a CD8α hinge region. In embodiments, a CAR described herein comprises a hinge domain derived from CD8α having an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 54 (FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 54)). In embodiments, The hinge domain derived from CD8α is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having the sequence set forth below. TTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGAC (SEQ ID NO: 55).

[0209] The polynucleotide and polypeptide sequences of these hinge domains are known. In some embodiments, a polynucleotide encoding a hinge domain comprises a nucleotide sequence that is 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 about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known nucleotide sequence. In some embodiments, the polypeptide sequence of the hinge domain comprises a polypeptide sequence that is 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 about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known polypeptide sequence.

[0210] Generally, a "transmembrane domain" (e.g., of an antigen-binding system) refers to a domain that, when present in a molecule on the cell surface or in the cell membrane, has the property of being intramembrane (e.g., spanning part or all of the cell membrane). The costimulatory domain of the antigen-binding system of the present disclosure may further comprise a transmembrane domain and / or an intracellular signaling domain. Not all amino acids in a transmembrane domain need be present in the membrane. For example, in some embodiments, a transmembrane domain is characterized by a specified stretch or portion of a protein being located substantially within the membrane. Amino acid or nucleic acid sequences can be analyzed using various algorithms to predict the subcellular localization (e.g., transmembrane localization) of proteins. The programs psort (PSORT.org) and Prosite (prosite.expasy.org) are examples of such programs.

[0211] The type of transmembrane domain included in the antigen-binding system described herein is not limited to any particular type. In some embodiments, a transmembrane domain that is naturally associated with a binding motif and / or an intracellular domain is selected. In some examples, the transmembrane domain contains one or more amino acid modifications (e.g., deletions, insertions, and / or substitutions) to, for example, prevent binding of such domains to transmembrane domains of the same or different surface membrane proteins and minimize interaction with other members of the receptor complex.

[0212] The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains include the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8 beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / C, D137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD1 9a, CD2, CD247, CD276(B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96(Tactile), CDS, CEACAM1, CRTAM, Cytocar In receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KI RDS2, LAT, LFA-1, LFA-1, ligands that bind to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules The transmembrane domain may be derived from (e.g., may comprise at least the transmembrane domain of) a molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof. In some embodiments, the transmembrane domain may be synthetic (and may comprise primarily hydrophobic residues, such as, for example, leucine and valine). In some embodiments, a phenylalanine, tryptophan, and valine triplet is included at both ends of the synthetic transmembrane domain.In some embodiments, the transmembrane domain is directly linked or connected to the cytoplasmic domain. In some embodiments, a short oligo- or polypeptide linker (e.g., 2-10 amino acids in length) may form the link between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine doublet.

[0213] In embodiments, a CAR described herein comprises a TM domain derived from CD28 having an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 56 (FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 56)). In embodiments, the TM domain derived from CD28 is encoded by a nucleic acid with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having a sequence according to the following: ttttgggtgctggtggtggttgggggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg (SEQ ID NO: 57).

[0214] In embodiments, a CAR described herein comprises a TM and intracellular domain derived from CD8α having an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 58. IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 58)). In embodiments, the TM domain from CD8α is at least 5'-seq to a nucleic acid having a sequence according to the following: The nucleic acid sequence is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). ATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAAC (SEQ ID NO: 59).

[0215] The polynucleotide and polypeptide sequences of the transmembrane domains provided herein are known. In some embodiments, a polynucleotide encoding a transmembrane domain comprises a nucleotide sequence that is 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 about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known nucleotide sequence. In some embodiments, the polypeptide sequence of the transmembrane domain comprises a polypeptide sequence that is 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 about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known polypeptide sequence. Optionally, a short spacer can form a link between any or some of the extracellular domain, transmembrane domain, and intracellular domain of the CAR.

[0216] The intracellular domain (or cytoplasmic domain) comprises one or more signaling domains that, upon binding of a target antigen to the binding motif, trigger and / or mediate an intracellular signal that, for example, activates one or more immune cell effector functions (e.g., natural immune cell effector functions). In some embodiments, the signaling domain of the intracellular domain mediates activation of at least one of the normal effector functions of an immune cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity including secretion of cytokines. In some embodiments, the signaling domain of the intracellular domain mediates T cell activation, proliferation, survival, and / or other T cell function. The intracellular domain can comprise a signaling domain that is an activation domain. The intracellular domain can comprise a signaling domain that is a costimulatory signaling domain.

[0217] Intracellular signaling domains that can transmit signals upon antigen binding to immune cells are known, any of which can be included in the antigen-binding system of the present disclosure. For example, the cytoplasmic sequence of the T cell receptor (TCR) is known to initiate signal transduction after TCR binding to an antigen (see, e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

[0218] In some embodiments, a CAR contemplated herein comprises an intracellular signaling domain. "Intracellular signaling domain" refers to the portion of a CAR that is responsible for transmitting the message of effective CAR binding to a target antigen to the interior of an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors into the CAR-bound target cell or other cellular responses elicited by antigen binding to the extracellular CAR domain. In some embodiments, the signaling domain and / or activation domain comprises an immunoreceptor tyrosine-based activation domain (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 ip These include those derived from Chiron, CD5, CD22, CD79a, CD79b, and CD66d (see, e.g., Love et al., Cold Spring Harb. Perspect. Biol. 2:a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol. 27:591-619 (2009)). In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA- 1, CD1-1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0219] The term "effector function" refers to a specialized function of a cell. Effector functions of T cells can be, for example, aid or activities including cytolytic activity or cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, so long as it transduces the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0220] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals may also be required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains (e.g., the TCR / CD3 complex), which initiate primary activation in an antigen-dependent manner via the TCR, and costimulatory signaling domains, which act antigen-independently to provide secondary or costimulatory signals. In some embodiments, CARs contemplated herein comprise an intracellular signaling domain comprising one or more "costimulatory signaling domains" and "primary signaling domains."

[0221] In some embodiments, the signaling domain and / or activation domain is an immunoreceptor. The CAR comprises a receptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d (see, e.g., Love et al., Cold Spring Harb. Perspect. Biol. 2:a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol. 27:591-619 (2009)). In some embodiments, the CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order. In one embodiment, the CAR has a CD3ζ domain having an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 60. VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 60). In embodiments, the CD3 zeta domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having the sequence set forth below. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGGCGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCC TGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 61). In embodiments, the CD3 zeta domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having a sequence set forth below. AGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCC TGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGA (SEQ ID NO: 61).

[0222] CARs contemplated herein comprise one or more costimulatory signaling domains to enhance the efficacy and proliferation of T cells expressing the CAR receptor. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule.

[0223] In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DAP-12, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 ( LFA-1, CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof.

[0224] CARs can include, for example, costimulatory signaling domains to enhance signaling efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012), Kalos et al., Sci Transl. Med. 3:95 (2011), Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross See, e.g., et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016). Signals generated through the TCR alone may be insufficient to fully activate T cells, and secondary or costimulatory signals may increase activation. Thus, in some embodiments, the signaling domain further comprises one or more additional signaling domains (e.g., costimulatory signaling domains) that activate one or more immune cell effector functions (e.g., natural immune cell effector functions described herein). In some embodiments, a portion of such a costimulatory signaling domain may be used, so long as that portion transduces an effector function signal. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences of a T cell co-receptor (or fragment thereof). Non-limiting examples of costimulatory domains include 4-1BB (also known as TNFRSF9, CD137, CDw137, ILA, and tumor necrosis factor receptor superfamily member 9), 4-1BBL / CD137, BAFFR, BLAME (SLAMF8), activating NK receptor, BTLA (also known as CD272 and BTLA1), CARD11, CD2 (also known as LFA-2, SRBC, T11, and CD2 molecules). CD3 gamma, CD3 delta, CD3 epsilon, CD4, CD7 (also known as GP40, LEU-9, TP41, Tp40, and CD7 molecules), CD8 alpha, CD8 beta, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD19a, CD27 (also known as S152, S152, LPFS2, T14, TNFRSF7, and Tp55), CD28 (also known as Tp44) CD40 (also known as Bp50, CDW40, TNFRSF5, p50, CD40 (protein), and CD40 molecule), CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD80 (B7, B7-1, B7.1, BB1, CD28LG, CD CD83 (and ligands that specifically bind to CD83), CD84, CD86, CD96 (antennary), CD100 (SEMA4D), CD103, CD160 (also known as BY55, NK1, NK28, and CD160 molecules), CD244 (also known as 2B4, NAIL, NKR2B4, Nmrk, SLAMF4, and CD244 molecules), CD247, CD276 (also known as B7-H3, 4Ig-B7-H3, B7H3, B7RP-2), CD366, CDS, CEACAM1, CRT AM, cytokine receptor, DAP10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR (TNFRSF18, RP5-902P8.2, also known as AITR, CD357, and GITR-D), GITRL, HVEM (TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2), ICAM-1, ICOS (inducible T cell costimulatory, also known as AILIM, CD278, and CVID1), Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, immunoglobulin-like proteins, integrins, ITGA4, IA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB 1, ITGB2, ITGB7, KIRDS2, LAG3 (also known as CD223 and lymphocyte activation 3), LAT, LFA-1 (also known as lymphocyte function-associated antigen 1), and CD1 la / CD18), LIGHT (also known as TNFSF14, CD258, HVEML, LTg, TR2, TNLG1D, and tumor necrosis factor superfamily member 14), LTBR, ​​Ly9 (CD229), MHC class I molecules, NKG2C (also known as CD314, D12S2489E, KLR, NKG2-D, NKG2D, and killer cell lectin-like receptor K1), NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40 (TNFRSF4, ACT35, RP5-902P8).3, IMD16, CD134, TXGP1L, and tumor necrosis factor receptor superfamily member 4), PAG / Cbp, PD-1 (also known as PDCD1, CD279, PD-1, SLEB2, hPD-1, hPD-1, hSLE1, and programmed cell death 1), PD-L1 (also known as CD274, B7-H, B7H1, PD-L1, PDCD1L1, PDCD1LG1, PDL1, CD274 molecule, and programmed cell death 1 ligand 1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM) proteins, e.g., SLAM (SLAMF1, CD15), Exemplary costimulatory proteins include, but are not limited to, SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), and SLAMF7), SLP76, TIM3 (also known as HAVCR2, HAVcr-2, KIM-3, TIM3, TIMD-3, TIMD3, Tim-3, and Hepatitis A Virus Cellular Receptor 2), TNF receptor protein, TNFR2, Toll Ligand Receptor, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TNFR2, trans- / RANKL, TRIM, VLA1, VLA-6, and ZAP70. Exemplary costimulatory proteins have the amino acid sequence of a costimulatory protein naturally found on T cells, the complete native amino acid sequence of which is set forth in NCBI Reference Sequence: NP_006130.1. In certain instances, the CAR comprises a 4-1BB costimulatory domain.

[0225] In embodiments, the CAR has at least 75% sequence identity (at least 75%, at least 80%, at least 90%, at least 95%, or 100%) to SEQ ID NO: 62. The CD28 costimulatory domain includes a CD28 costimulatory domain having an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having a sequence according to the following: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 62). In embodiments, the CD28 costimulatory domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having a sequence according to the following: aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc (SEQ ID NO: 63).

[0226] In embodiments, the CAR comprises a 4-1BB costimulatory domain having an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 64: RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 64). In embodiments, the 4-IBB costimulatory domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having a sequence set forth below. AGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTG (SEQ ID NO: 65).

[0227] The engineered CARs described herein may also include an N-terminal signal peptide or tag at the N-terminus of the scFv or antigen-binding domain. In one embodiment, a heterologous signal peptide can be used. The antigen-binding domain or scFV can be fused to a leader or signal peptide that directs the nascent protein to the endoplasmic reticulum and subsequently to the cell surface. It is understood that when a polypeptide containing a signal peptide is expressed on the cell surface, the signal peptide is generally proteolytically removed during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, polypeptides such as the CAR constructs described herein are generally expressed on the cell surface as mature proteins lacking a signal peptide, although precursor forms of the polypeptides contain a signal peptide. Any suitable signal sequence known in the art can be used. Similarly, any known tag sequence known in the art can also be used. In one embodiment, the signal sequence is the CSF2RA signal sequence. In embodiments, the invention includes a CSF2RA having an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 66), MEWTWVFLFLLSVTAGVHS (SEQ ID NO: 67), or MALPVTALLLPLALLLHAARP (SEQ ID NO: 68).

[0228] The polynucleotide and polypeptide sequences of the signaling domains provided herein are known. In some embodiments, the polynucleotides encoding the signaling domains are at least about 60%, at least about 65%, or at least about 69% identical to the known nucleotide sequences. The nucleotide sequences of the sequences are 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 about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical. In some embodiments, the polypeptide sequence of the signaling domain comprises a polypeptide sequence that is 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 about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known polypeptide sequence.

[0229] Components of a CAR can be exchanged or "swapped" for equivalent components using routine techniques of biotechnology. In some non-limiting examples, a CAR of the present disclosure can include a binding motif provided herein in combination with a hinge provided herein and a costimulatory domain provided herein. In certain examples, a CAR of the present disclosure can include a leader sequence, along with a binding motif provided herein, in combination with a hinge provided herein and a costimulatory domain provided herein.

[0230] A variety of CAR sequences, components, and / or frameworks are known, including, but not limited to, sequences of hinges, spacers, transmembrane domains, costimulatory domains, stimulatory domains, binding motifs, and variants of each, and, for example, when heavy chain variable domain or CDR sequences and light chain variable domain or CDR sequences are provided, a CAR with the desired binding and components or structure can be readily constructed.

[0231] "Polypeptide," "polypeptide fragment," "peptide," and "protein," unless specified to the contrary, are used in accordance with their conventional meaning, i.e., as a sequence of amino acids. A polypeptide is not limited to a particular length and can include, for example, a full-length protein sequence or a fragment of a full-length protein, and can include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications, both naturally occurring and non-naturally occurring, known in the art. In various embodiments, a polypeptide contemplated herein includes a signal (or leader) sequence at the N-terminal end of the protein, which directs translocation of the protein co- or post-translationally.

[0232] Polypeptides include "polypeptide variants." Polypeptide variants can differ from naturally occurring polypeptides in one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or synthetically produced, for example, by modifying one or more of the above polypeptide sequences. For example, in some embodiments, it may be desirable to improve the binding affinity and / or other biological properties of engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptors and CARs and TCRs by introducing one or more substitutions, deletions, additions, and / or insertions. Preferably, polypeptides of the present disclosure include polypeptides having at least about 50%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acid identity thereto. Polypeptides of the present disclosure include variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences set forth herein (see, e.g., Sequence Listing), and typically, variants retain at least one biological activity of the reference sequence. Polypeptides include "polypeptide fragments." Polypeptide fragments can be derived from amino-terminal deletions, carboxyl-terminal deletions, and / or fragments of naturally occurring or recombinantly produced polypeptides. "A polypeptide fragment" refers to a polypeptide that may be monomeric or multimeric, having internal deletions or substitutions of the polypeptide. In certain embodiments, a polypeptide fragment may comprise an amino acid chain of at least 5 to about 500 amino acids in length. It will be appreciated that in certain embodiments, the fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length.

[0233] The polypeptides can also be fused or conjugated in-frame to linkers or other sequences to facilitate synthesis, purification, or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. As noted above, the polypeptides of the present disclosure can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence changes are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492); Kunkel et al., (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, J.D. et al., (Molecular Biology of See, for example, "The Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987" and the references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest is provided in Dayhoff et al., (1978) Atlas of A model of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0234] In certain embodiments, variants comprise conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid with similar properties such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic properties of the polypeptide to remain substantially unchanged. Modifications can be made to the polynucleotide and polypeptide structures of the present disclosure and still obtain functional molecules that encode variant or derivative polypeptides with desirable properties.

[0235] Polypeptide variants also include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules). Covalent variants can be prepared by linking functional groups found on the amino acid chain or the N- or C-terminal residues, as is known in the art. Variants also include allelic variants, species variants, and muteins. Truncation or deletion of regions that do not affect the functional activity of the protein are also variants.

[0236] If expression of two or more polypeptides is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence. In another embodiment, the two or more polypeptides may be expressed as a fusion protein containing one or more self-cleaving polypeptide sequences, such as a T2A polypeptide. In other embodiments, they may be expressed from different promoters and present in two or more vectors. In some embodiments, the anti-CD19 CAR is encoded in the same vector as the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor, and the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor is encoded in the same vector as the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor. and the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor is operably linked to the same promoter as the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor, where the sequences are separated by an IRES sequence. In some embodiments, the anti-CD19 CAR or TCR is encoded in the same vector as the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor, where the sequences are separated by an IRES sequence or a cleavable linker. In certain embodiments, the anti-CD19 and / or anti-CD20 CAR or TCR is expressed on a cell that has also been engineered to express the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor. In some embodiments, the anti-CD19 and / or anti-CD20 CAR or TCR is encoded in the same vector as the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor, where the sequences are separated by an IRES sequence or a cleavable linker. In some aspects, the anti-CD19 and / or anti-CD20 CAR CAR or TCR is encoded in the same vector as the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor, which is operably linked to a promoter that is different from the promoter of the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor. In some embodiments, the anti-CD19 CAR-linked CAR and / or the anti-CD20 CAR-linked CAR is encoded in a different vector as the engineered membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor.

[0237] Anti-CD19 CARs can comprise antigen-binding sequences found in the antibodies described herein (see, e.g., Table 5). In some embodiments, anti-CD19 CARs of the present disclosure comprise antigen-binding fragments provided herein.

[0238] In various embodiments, the anti-CD19 CAR comprises at least one HCDR disclosed in Table 5. In various embodiments, the anti-CD19 CAR comprises at least two HCDRs disclosed in Table 5. In various embodiments, the anti-CD19 CAR comprises three HCDRs disclosed in Table 5.

[0239] In various embodiments, the anti-CD19 binding motif comprises at least one LCDR disclosed in Table 5. In various embodiments, the anti-CD19 CAR comprises at least two LCDRs disclosed in Table 5. In various embodiments, the anti-CD19 CAR comprises three LCDRs disclosed in Table 5.

[0240] In various embodiments, the anti-CD19 CAR comprises at least one HCDR disclosed in Table 5 and at least one LCDR disclosed in Table 5. In various embodiments, the anti-CD19 binding motif comprises at least two HCDRs disclosed in Table 5 and at least two LCDRs disclosed in Table 5. In various embodiments, the anti-CD19 binding motif comprises three HCDRs disclosed in Table 5 and three LCDRs disclosed in Table 5.

[0241] In various embodiments, the anti-CD19 binding motif comprises at least one heavy chain framework region (heavy chain FR) of a heavy chain variable domain disclosed in Table 5. In various embodiments, the anti-CD19 binding motif comprises at least two heavy chain FRs of a heavy chain variable domain disclosed in Table 5. In various embodiments, the anti-CD19 binding motif comprises three heavy chain FRs of a heavy chain variable domain disclosed in Table 5.

[0242] In various embodiments, the anti-CD19 binding motif comprises at least one light chain FR of a light chain variable domain disclosed in Table 5. In various embodiments, the anti-CD19 binding motif comprises at least two light chain FRs of a light chain variable domain disclosed in Table 5. In various embodiments, the anti-CD19 binding motif comprises three light chain FRs of a light chain variable domain disclosed in Table 5.

[0243] In various embodiments, an anti-CD19 CAR comprises at least one heavy chain FR from a heavy chain variable domain disclosed in Table 5 and at least one light chain FR from a light chain variable domain disclosed in Table 5. In various embodiments, an anti-CD19 CAR comprises at least two heavy chain FRs from a heavy chain variable domain disclosed in Table 5 and at least two light chain FRs from a light chain variable domain disclosed in Table 5. In various embodiments, an anti-CD19 CAR comprises three heavy chain FRs from a heavy chain variable domain disclosed in Table 5 and three light chain FRs from a light chain variable domain disclosed in Table 5.

[0244] In various embodiments, the anti-CD19 CAR comprises one, two, or three FRs that together or each individually have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the corresponding FR(s) of the heavy chain variable domain of a heavy chain variable domain disclosed in Table 5. In various embodiments, the anti-CD19 CAR comprises one, two, or three FRs that together or each individually have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the corresponding FR(s) of the light chain variable domain of a light chain variable domain disclosed in Table 5.

[0245] In various embodiments, the anti-CD19 CAR comprises at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in Table 5. In various embodiments, the anti-CD19 CAR comprises at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in Table 5.

[0246] In various embodiments, the anti-CD19 CAR comprises at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in Table 5, and at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in Table 5.

[0247] In certain embodiments, an anti-CD19 CAR comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a linker having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 45. In certain embodiments, an anti-CD19 CAR comprises a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a linker having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or and a leader sequence having 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). [Table 5]

[0248] In one embodiment, the anti-CD19 CAR has an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95% or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 90. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSG EGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 90). In embodiments, the anti-CD19 CAR is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having a sequence according to the following: ACGACGTTCTGGATAAGAGAAGGGCAGAGATCCCGAAATGGGGCAAGCCCAGACGCAAGAACCCTCAGGAGGGGCTTTACAACGAACTGCAGAAGGATAAGATGGCTGAGGCTTACTCGGAGATTGGATGAAGGGGAGAGAAGGCGGGGCAAGGGACACACGATGGCTTATACCAGGGCTGAGCACCGCCACCAAGGACACATACGCAGCCTTCATATGCAGGCTCTGCCCCAAGA (SEQ ID NO: 91).

[0249] In one embodiment, the anti-CD19 CAR linked to a membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor has an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 92 (SEQ ID NO: 92).In embodiments, the anti-CD19 CAR linked to a membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95% or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having a sequence according to: GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCA AGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCGGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAG

[0250] Exemplary anti-CD20 antibodies and fragments thereof suitable for use in the CARs, vectors, cells, and methods disclosed herein can be found in International Patent Publication No. WO / 2020 / 123691, published June 18, 2020, which is incorporated herein by reference in its entirety. The entities are specifically incorporated herein by reference. In embodiments, the anti-CD20 CAR has an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 90. QVQLVQSGAEVKKPGASVKVSCKASGYTFKEYGISWVRQAPGQGLEWMGWISAYSGHTYYAQKLQGRVTMTTDTSSTTAYMELRSLRSDDTAVYYCARGPHYDDWSGFIIWFDPWGQGTLVTVSSG STSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRFPPTFGQGTKVEIKAA AFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 90). In embodiments, the anti-CD20 CAR is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having a sequence according to the following: CCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGA ACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGA (SEQ ID NO: 91).

[0251] The present disclosure also provides nucleic acids encoding any of the various membrane-bound IL-15-IL-15Rα chimeric polypeptides described herein, or any of the CARs or TCRs. In one embodiment, a recombinant nucleic acid construct comprises a nucleic acid molecule encoding a membrane-bound IL-15-IL-15Rα chimeric polypeptide, optionally together with one or more CARs or TCRs.

[0252] The present disclosure includes vectors comprising a nucleic acid of the present disclosure and / or encoding a membrane-bound IL-15-IL-15Rα chimeric polypeptide of the present disclosure, optionally in combination with a nucleic acid encoding either a CAR or a TCR as described herein. Any vector may be suitable for the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeT cDNA, pBABE-hygro-hTERT, pMKO.1, and the like. These include GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-DsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0253] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include vectors designed for propagation and amplification, or for expression, or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, MD), pBluescript series (Stratagene, La Jolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors useful in the context of the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, a bicistronic IRES vector (e.g., from Clontech) is used to express the nucleic acid encoding the antigen binding system and the nucleic acid encoding the antigen binding system described herein. and an inducible expression construct for both.

[0254] Recombinant expression vectors can be prepared using standard recombinant DNA techniques, as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, 1994. Circular or linear expression vector constructs can be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0255] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for recombinant expression vectors include, for example, the neomycin / G418 resistance gene, the puromycin resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0256] Vectors useful in the context of the present disclosure can be "naked" nucleic acid vectors (i.e., vectors with little or no proteins, sugars, and / or lipids encapsulating them) or vectors complexed with other molecules. Other molecules that may be suitably combined with vectors include, but are not limited to, viral coats, cationic lipids, liposomes, polyamines, gold particles, and targeting moieties such as ligands, receptors, or antibodies that target cellular molecules.

[0257] In certain embodiments, the membrane-bound IL-15-IL-15Rα chimeric polypeptide and the CAR or TCR can be assembled in a single multicistronic expression cassette, in multiple expression cassettes in a single vector, or in multiple vectors. In one embodiment, the present disclosure provides a set of vectors comprising a first vector comprising a sequence encoding any of the membrane-bound IL-15-IL-15Rα chimeric polypeptides described herein and a second vector comprising a sequence encoding a CAR or TCR. In some embodiments, one or both of the first and second vectors are lentiviral, retroviral, or adenoviral vectors. In some embodiments, the second vector further comprises a promoter and / or enhancer sequence operably linked to the sequence encoding the CAR or TCR. In some embodiments, the second vector further comprises a poly(A) sequence operably linked to the sequence encoding the CAR or TCR. In one embodiment, the present disclosure provides a polycistronic expression cassette. Examples of elements that create polycistronic expression cassettes include various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, and the like). Examples of suitable IRES include, but are not limited to, VEGF IRES, IGF-II IRES, NF-KB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). Combinations of retroviral vectors with appropriate packaging lines are also suitable, and the capsid proteins can function to infect human cells. A variety of amphotropic virus-producing cell lines are known, including PA12 (Miller et al., 1985, Mol. Cell. Biol. 5:431-437); PA317 ( and CRIP (Danos et al., 1988, Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114, or GALV envelopes and any others known in the art, are also suitable.

[0258] Vector DNA can be introduced into cells, e.g., immune cells, via conventional transformation, transfection, or transduction techniques. The terms "transformation" and "transfection" encompass a variety of art-recognized techniques for introducing foreign nucleic acids (e.g., DNA) into cells, such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, gene guns, nanoparticle-mediated delivery, or electroporation. Transduction includes viral delivery of vectors into cells, e.g., by vectors disclosed herein, including, but not limited to, retroviruses, lentiviruses, and AAVs.

[0259] The present disclosure includes cells that contain, express, or have been engineered (e.g., transformed or transduced) to contain or express at least one vector or nucleic acid of the present disclosure. In one embodiment, the present disclosure provides a cell (1) that contains (a) a CAR or TCR and (b) a membrane-bound IL-15-IL-15Rα chimeric polypeptide. An immune cell can be transduced with the CAR or TCR and the membrane-bound IL-15-IL-15Rα chimeric polypeptide such that the cell expresses the CAR or TCR and the membrane-bound IL-15-IL-15Rα chimeric polypeptide.

[0260] Chimeric antigen receptors (CARs or CAR-Ts) and engineered T cell receptors (TCRs) can be readily inserted into and expressed by immune cells, such as T cells. In certain embodiments, cells (e.g., immune cells such as T cells, NK cells, or induced pluripotent stem cells (iPSCs)) are obtained from a donor subject. In some embodiments, the donor subject is a human patient suffering from cancer or a tumor. In other embodiments, the donor subject is a human patient not suffering from cancer or a tumor. In some embodiments, the engineered cells are autologous to the subject. In some embodiments, the engineered cells are allogeneic to the subject.

[0261] In certain embodiments, the immune cells of the present disclosure have increased secretion of anti-tumor cytokines (e.g., including but not limited to, IL-18, IL-2, IFN-γ, and TNF-α). In certain embodiments, the immune cells have decreased secretion of cytokines associated with cytokine release syndrome (CRS), e.g., IL-6.

[0262] Any cell can be used as a host cell for the polynucleotides, vectors, or polypeptides of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae, such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans and Shigella; Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cells are T cells, B cells, tumor infiltrating lymphocytes (TILs), TCR-expressing cells, natural killer (NK) cells, dendritic cells, The immune cells are selected from the group consisting of cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes, macrophages, platelets, thymocytes, and myeloid cells. In one embodiment, the immune cells are T cells. In another embodiment, the immune cells are NK cells. In certain embodiments, the T cells are tumor-infiltrating lymphocytes (TILs), autologous T cells, engineered autologous T cells (eACT™), allogeneic T cells, xenogeneic T cells, iPSC cells, or any combination thereof.

[0263] In one embodiment, the membrane-bound IL-15-IL-15Rα chimeric polypeptide and / or CAR or TCR provided herein are introduced into T cells. T cells can be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. In some embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. In some embodiments, cells are washed with PBS. As will be appreciated, the washing step may be performed, for example, using a semi-automated flow-through centrifuge, such as a Cobe™ 2991 cell processor, a Baxter CytoMate™, or the like. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers or other saline solutions, with or without buffers. In some embodiments, undesirable components of the apheresis sample are removed. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication Nos. 2013 / 0287748, 2015 / 120096, and 2017 / 070395, all of which are incorporated by reference in their entirety for purposes of describing these methods.

[0264] In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, using centrifugation through a PERCOLL™ gradient. In some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA+, and CD45RO+ T cells, are further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. In some embodiments, cell sorting and / or selection may be performed by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are performed to isolate a cell population of interest for use in the present disclosure.

[0265] In some embodiments, PBMCs are used directly for genetic modification of immune cells using the methods described herein. In some embodiments, after isolating PBMCs, T lymphocytes are further isolated and both cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subpopulations before or after genetic modification and / or expansion. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, expression of phenotypic markers of central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and are negative for granzyme B. In some embodiments, central memory T cells are CD8+, CD45RO+, and CD62L+ T cells. In some embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. In some embodiments, CD4+ T cells are further classified into subpopulations. For example, CD4+ T helper cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens.

[0266] In some embodiments, immune cells, e.g., NK cells or T cells, are genetically modified after isolation using known methods, or the immune cells are activated and expanded (or differentiated, in the case of progenitor cells) in vitro before being genetically modified. In another embodiment, immune cells, e.g., NK cells or T cells, are genetically modified with a CAR or TCR described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding the CAR or TCR), optionally genetically modified with a membrane-bound IL-15-IL-15Rα chimeric polypeptide (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a membrane-bound IL-15-IL-15Rα chimeric polypeptide), and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent Nos. 6,905,874, 6,867,041, and 6,797,514, as well as International Patent Application Publication No. 2012 / 079000, the entire contents of which are incorporated herein by reference. Generally, such methods involve contacting PBMCs or isolated T cells with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, bound to beads or other surfaces in a culture medium containing appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies bound to the same beads function as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In other embodiments, T cells are activated and stimulated to expand with feeder cells and appropriate antibodies and cytokines, for example, using methods described in U.S. Pat. Nos. 6,040,177 and 5,827,642 and International Patent Application Publication No. WO 2012 / 129514, the contents of which are incorporated herein by reference in their entireties.

[0267] The methods described herein can further include enriching a population of lymphocytes obtained from a donor. Enrichment of a population of lymphocytes, e.g., one or more T cells, can be achieved using a separation medium (e.g., FICOLL-PAQUE™, ROSETTESEP™ HLA Total Lymphocyte Enrichment cocktail, Lymphocyte This can be achieved by cell size, shape or density separation by filtration or elution, immunomagnetic separation (e.g., magnetic activated cell sorting system, MACS), fluorescence separation (e.g., fluorescence activated cell sorting system, FACS), or bead-based column separation.

[0268] The methods described herein can further include stimulating the population of lymphocytes with one or more T cell stimulatory agents to generate a population of activated T cells under appropriate conditions. The population of activated T cells can be generated using any combination of one or more suitable T cell stimulatory agents, including, but not limited to, antibodies or functional fragments thereof that target T cell stimulatory molecules or costimulatory molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, or functional fragments thereof), or any other suitable mitogens (e.g., tetradecanoylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), or natural ligands for T cell stimulatory molecules or costimulatory molecules.

[0269] Suitable conditions for stimulating lymphocyte populations described herein can include temperature, duration, and / or the presence of a certain level of CO2. In certain embodiments, the temperature for stimulation is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In certain embodiments, the temperature for stimulation is about 34-38°C. In certain embodiments, the temperature for stimulation is about 35-37°C. In certain embodiments, the temperature for stimulation is about 36-38°C. In certain embodiments, the temperature for stimulation is about 36-37°C or about 37°C.

[0270] Another condition for stimulating a lymphocyte population described herein can include the duration of stimulation. In some embodiments, the duration of stimulation is about 24 to 72 hours. In some embodiments, the duration of stimulation is about 24 to 36 hours, about 30 to 42 hours, about 36 to 48 hours, about 40 to 52 hours, about 42 to 54 hours, about 44 to 56 hours, about 46 to 58 hours, about 48 to 60 hours, about 54 to 66 hours, or about 60 to 72 hours. In one specific embodiment, the duration of stimulation is about 48 hours or at least about 48 hours. In other embodiments, the duration of stimulation is about 44 to 52 hours. In certain embodiments, the duration of stimulation is about 40 to 44 hours, about 40 to 48 hours, about 40 to 52 hours, or about 40 to 56 hours.

[0271] Another condition for stimulating lymphocyte populations described herein can include CO2 levels. In some embodiments, the CO2 level for stimulation is about 1.0-10% CO2. In some embodiments, the CO2 level for stimulation is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the CO2 level for stimulation is about 3-7% CO2. In other embodiments, the CO2 level for stimulation is about 4-6% CO2. In one embodiment, the CO2 level for stimulation is about 4.5-5.5% CO2. In one specific embodiment, the CO2 level for stimulation is about 5% CO2.

[0272] The conditions for stimulating the population of lymphocytes can further include any combination of temperature, duration of stimulation, and / or in the presence of a level of CO. For example, stimulating the population of lymphocytes can include stimulating the population of lymphocytes with one or more T cell stimulatory agents at a temperature of about 36-38° C. for about 44-52 hours in the presence of CO at a level of about 4.5-5.5% CO.

[0273] The concentration of lymphocytes useful in the methods herein is about 1.0 to 10.0 x 10 6 In certain embodiments, the lymphocyte concentration is about 1.0-2.0 x 10 cells / mL. 6 cells / mL, approximately 1.0~3.0×10 6 cells / mL, approximately 1.0~4.0×10 6 cells / mL, approximately 1.0~5.0×10 6 cells / mL, approximately 1.0~6.0×10 6 cells / mL, approximately 1.0~7.0×10 6 cells / mL, approximately 1.0~8.0×10 6 cells / mL, 1.0~9.0×10 6 cells / mL, or approximately 1.0–10.0 × 10 6 In certain embodiments, the lymphocyte concentration is about 1.0-2.0 x 10 cells / mL. 6 In certain embodiments, the lymphocyte concentration is about 1.0-1.2 x 10 cells / mL. 6 cells / mL, approximately 1.0~1.4×10 6 cells / mL, approximately 1.0~1.6×10 6 cells / mL, approximately 1.0~1.8×10 6 cells / mL, or approximately 1.0-2.0 x 10 6 In certain embodiments, the lymphocyte concentration is at least about 1.0 x 10 cells / mL. 6 cells / mL, at least approximately 1.1 x 10 6 cells / mL, at least approximately 1.2 x 10 6 cells / mL, at least approximately 1.3 x 10 6 cells / mL, at least approximately 1.4 x 10 6 cells / mL, at least approximately 1.5 x 106 cells / mL, at least approximately 1.6 x 10 6 cells / mL, at least approximately 1.7 x 10 6 cells / mL, at least approximately 1.8 x 10 6 cells / mL, at least approximately 1.9 x 10 6 cells / mL, At least about 2.0 x 10 6 cells / mL, at least approximately 4.0 x 10 6 cells / mL, at least approximately 6.0 × 10 6 cells / mL, at least approximately 8.0 × 10 6 cells / mL, or at least about 10.0 x 10 6 cells / mL.

[0274] Anti-CD3 antibodies (or functional fragments thereof), anti-CD28 antibodies (or functional fragments thereof), or a combination of anti-CD3 and anti-CD28 antibodies can be used in accordance with the step of stimulating a lymphocyte population. Any soluble or immobilized anti-CD2, anti-CD3, and / or anti-CD28 antibodies or functional fragments thereof (e.g., clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), clone 15E8 (anti-CD28)) can be used. In some embodiments, antibodies are available from Miltenyi Biotec, BD Biosciences, and others. Anti-CD3 antibodies and / or anti-CD28 antibodies can be commercially purchased from vendors known in the art, including, but not limited to, Biosciences (e.g., MACS GMP CD3 pure 1 mg / mL, Part No. 170-076-116) and eBioscience, Inc. Additionally, one of skill in the art would understand how to manufacture anti-CD3 antibodies and / or anti-CD28 antibodies by standard methods. In some embodiments, the one or more T cell stimulatory agents used in accordance with the step of stimulating a lymphocyte population comprise an antibody or functional fragment thereof that targets a T cell stimulatory or costimulatory molecule in the presence of a T cell cytokine. In one aspect, the one or more T cell stimulatory agents comprise an anti-CD3 antibody and IL-2. In certain embodiments, the T cell stimulatory agent comprises an anti-CD3 antibody at a concentration of about 20 ng / mL to 100 ng / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In one particular embodiment, the concentration of the anti-CD3 antibody is about 50 ng / mL. In alternative embodiments, T cell activation is not required. In such embodiments, the step of stimulating the population of lymphocytes to produce a population of activated T cells is omitted from the method, and the population of lymphocytes that can be enriched for T lymphocytes is transduced according to the following steps:

[0275] The methods described herein can include transducing a population of activated T cells with a viral vector comprising a membrane-bound IL-15-IL-15Rα chimeric polypeptide and / or a nucleic acid molecule encoding a CAR or TCR using single-cycle transduction to produce a population of transduced T cells. In embodiments utilizing a viral vector with a membrane-bound IL-15-IL-15Rα chimeric polypeptide, the viral vector can be separate from the viral vector encoding the CAR or TCR, or the viral vector can encode both the membrane-bound IL-15-IL-15Rα chimeric polypeptide and the CAR or TCR. Transduction of a population of activated immune cells described herein can be performed at any combination of time period, specific temperature, and / or in the presence of specific levels of CO2: at a temperature of about 36-38°C for about 16-24 hours, in the presence of CO2 at a level of about 4.5-5.5% CO2. Immune cells can be prepared by a combination of any one of the methods of the present application with any manufacturing method for preparing T cells for immunotherapy, including, but not limited to, International Patent Application Publication Nos. WO 2015 / 120096 and WO 2017 / 070395, which are incorporated by reference in their entireties for purposes of describing these methods; any and all methods used in the preparation of Axicabtagene ciloleucel, Brexucabtagene autoleucel, Liso-cel; any and all methods used in the preparation of Tisagenlecleucel; any method used in the preparation of "off-the-shelf" T cells for immunotherapy; and any other method for preparing lymphocytes for administration to humans. The manufacturing process can be adapted to remove circulating tumor cells from cells obtained from the patient.

[0276] Several recombinant viruses are used as viral vectors to deliver genetic material into cells. The viral vector used in accordance with the transduction step can be any ecotropic or amphotropic viral vector, including, but not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated viral (AAV) vectors. In some embodiments, the method further comprises retrovirally transducing one or more NK cells or T cells. In one embodiment, the viral vector used to transduce the population of NK cells or activated T cells is an MSGV1γ retroviral vector. In certain embodiments, the viral vector used to transduce the population of NK cells or activated T cells is the PG13-CD19-H3 vector described by Kochenderfer, J. Immunother. 32(7):689-702 (2009). According to one aspect of this embodiment, the viral vector is propagated in suspension culture in a medium specific for viral vector production, referred to herein as a "viral vector inoculum." Any suitable growth medium and / or supplements for propagating viral vectors can be used in the viral vector inoculum according to the methods described herein. According to some embodiments, the viral vector inoculum is then added to the serum-free culture medium described below during the transduction step.

[0277] Conditions for transducing a population of NK cells or activated T cells described herein can include a specific time period, a specific temperature, and / or the presence of a specific level of CO2. In certain embodiments, the temperature for transduction is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In one embodiment, the temperature for transduction is about 34-38°C. In another embodiment, the temperature for transduction is about 35-37°C. In another embodiment, the temperature for transduction is about 36-38°C. In yet another embodiment, the temperature for transduction is about 36-37°C. In one embodiment, the temperature for transduction is about 37°C.

[0278] In certain embodiments, the time period for transduction is about 12 to 36 hours. In some embodiments, the time period for transduction is about 12 to 16 hours, about 12 to 20 hours, about 12 to 24 hours, about 12 to 28 hours, or about 12 to 32 hours. In other embodiments, the time period for transduction is about 20 hours or at least about 20 hours. In one embodiment, the time period for transduction is about 16 to 24 hours. In other embodiments, the time period for transduction is at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, or at least about 26 hours.

[0279] In certain embodiments, the CO2 level for transduction is about 1.0-10% CO2. In other embodiments, the CO2 level for transduction is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the CO2 level for transduction is about 3-7% CO2. In another embodiment, the CO2 level for transduction can be about 4-6% CO2. In another embodiment, the CO2 level for transduction is about 4.5-5.5% CO2. In one embodiment, the CO2 level for transduction is about 5% CO2.

[0280] In some embodiments, transduction of a population of activated T cells described herein can be performed for any combination of a specific time period, a specific temperature, and / or in the presence of a specific level of CO2: at a temperature of about 36-38°C for about 16-24 hours in the presence of CO2 at a level of about 4.5-5.5% CO2.

[0281] The methods described herein identify one or more populations of transduced NK cells or T cells. The method can include expanding the NK cells or T cells for a period of time to produce a population of engineered NK cells or T cells. The predetermined period for expansion can be any suitable period that allows for the production of (i) a sufficient number of cells in at least one dose of engineered NK cells or T cells for administration to a patient, (ii) a population of engineered T cells with a favorable proportion of immature cells compared to typical longer processes, or (iii) both (i) and (ii). This period will depend on the cell surface receptors expressed by the NK cells or T cells, the vector used, the dose required to have a therapeutic effect, and other variables. Thus, in some embodiments, the predetermined period for expansion can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. In some aspects, the period for expansion is shorter than expansion methods known in the art. For example, the predetermined time period for expansion may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or more than 75% shorter. In one embodiment, the time period for expansion is about 3 days and the time period from enrichment of the lymphocyte population to production of engineered NK cells or T cells is about 6 days.

[0282] Conditions for expanding a population of transduced NK cells or T cells can include temperature and / or the presence of a certain level of CO2. In certain embodiments, the temperature is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In one embodiment, the temperature is about 34-38°C. In another embodiment, the temperature is about 35-37°C. In another embodiment, the temperature is about 36-38°C. In yet another embodiment, the temperature is about 36-37°C. In one embodiment, the temperature is about 37°C. In certain embodiments, the CO2 level is 1.0-10% CO2. In other embodiments, the CO2 level is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the level of CO2 is about 4.5-5.5% CO2. In another embodiment, the level of CO2 is about 5% CO2. In other embodiments, the level of CO2 is about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO2. In some embodiments, conditions for expanding a population of transduced NK cells or T cells can include any combination of temperature and / or the presence of a level of CO2. For example, conditions for expanding a population of transduced T cells include a temperature of about 36-38°C and the presence of CO2 at a level of about 4.5-5.5% CO2.

[0283] Each step of the manufacturing process described herein can be performed in a closed system. In certain embodiments, the closed system is a closed-bag culture system using any suitable cell culture bag (e.g., Miltenyi Biotec MACS® GMP Cell Differentiation Bags, Origen Biomedical PermaLife Cell Culture bags). In some embodiments, the cell culture bag used in the closed-bag culture system is coated with a recombinant human fibronectin fragment during the transduction process. The recombinant human fibronectin fragment may contain three functional domains: a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. The recombinant human fibronectin fragment can be used to increase the gene efficiency of retroviral transduction of immune cells by assisting in colocalization of the target cell with the viral vector. In certain embodiments, the recombinant human fibronectin fragment is RETRONECTIN® (Takara Bio, Japan). In certain embodiments, the cell culture bag is coated with the recombinant human fibronectin fragment at a concentration of about 1 to 60 μg / mL or about 1 to 40 μg / mL. In another embodiment, the cell culture bag contains about 1-20 μg / m In some embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 11 μg / mL, about 12 μg / mL, about 13 μg / mL, about 14 μg / mL, about 15 μg / mL, about 16 μg / mL, about 17 μg / mL, about 18 μg / mL, about 19 μg / mL, or about 20 μg / mL. In other embodiments, the cell culture bag is coated with about 2-5 μg / mL, about 2-10 μg / mL, about 2-20 μg / mL, about 2-25 μg / mL, about 2-30 μg / mL, about 2-35 μg / mL, about 2-40 μg / mL, about 2-50 μg / mL, or about 2-60 μg / mL of the recombinant human fibronectin fragment. In certain embodiments, the cell culture bag is coated with at least about 2 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, at least about 15 μg / mL, at least about 20 μg / mL, at least about 25 μg / mL, at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL, or at least about 60 μg / mL of the recombinant human fibronectin fragment. In one specific embodiment, the cell culture bag is coated with at least about 10 μg / mL of recombinant human fibronectin fragment. The cell culture bag used in the closed-bag culture system can be optionally blocked with human serum albumin (HSA) during the transduction process. In another embodiment, the cell culture bag is not blocked with HSA during the transduction process.

[0284] The population of engineered immune cells produced by the above methods can optionally be cryopreserved to allow for later use of the cells. Methods for cryopreserving a population of engineered immune cells are also provided herein. Such methods can include washing and concentrating the population of engineered immune cells with a diluent. For example, the diluent can be saline, 0.9% saline, PlasmaLyte A (PL), 5% dextrose / 0.45% NaCl saline solution (D5), human serum albumin (HSA), or a combination thereof. HSA can also be added to the washed and concentrated cells to improve cell viability and cell recovery after thawing. In another embodiment, the washing solution is saline, and the washed and concentrated cells are supplemented with HSA (5%). The method can also include generating a cryopreservation mixture, wherein the cryopreservation mixture comprises the diluted cell population in the diluent and a suitable cryopreservation solution. The cryopreservation solution can be CryoStor10 (BioLife). The cryopreservation solution can be any suitable solution, including, but not limited to, HSA (Hyperphosphate Degradation Solutions), mixed with the dilution of engineered immune cells at a 1:1 or 2:1 ratio. HSA can be added to provide a final concentration of about 1.0-10%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 1-3% HSA, about 1-4% HSA, about 1-5% HSA, about 1-7% HSA, about 2-4% HSA, about 2-5% HSA, about 2-6% HSA, about 2-7% HSA, or about 2.5% HSA in the cryopreserved mixture. Cryopreservation of the engineered immune cell population can include washing the cells with 0.9% saline, adding HSA to the washed cells at a final concentration of 5%, and diluting the cells 1:1 with CryoStor™ CS10 (final concentration of 2.5% HSA in the final cryopreservation mixture). In some embodiments, the method also includes freezing the cryopreservation mixture. The cryopreservation mixture can also include freezing the engineered immune cell population at a final concentration of about 1 x 10 HSA in the cryopreservation mixture. 6 ~Approx. 1.5×10 7The cells are frozen in a controlled-rate freezer using a defined freezing cycle at a cell concentration of 1000 cells / mL. The method may also include storing the cryopreservation mixture in vapor-phase liquid nitrogen.

[0285] The population of engineered immune cells produced by the methods described herein can be cryopreserved in a predetermined dose. The predetermined dose can be a therapeutically effective dose, which can be any of the therapeutically effective doses provided below. The predetermined dose of engineered immune cells can be a therapeutically effective dose of the engineered immune cells produced by the immune cells. The binding motif expressed by the engineered immune cells may depend on the binding motif expressed (e.g., the affinity and density of the binding motif expressed on the cell), the type of target cell, the nature of the disease or condition being treated, or a combination of both. The binding motif expressed by the engineered immune cells may be any antigen or molecule targeted by the CAR or TCR. In certain aspects, the predetermined dose of engineered immune cells expressing a CAR or TCR may be greater than about 1 million to less than about 3 million transduced engineered NK cells or T cells / kg. In one embodiment, the predetermined dose of engineered NK cells or T cells expressing a CAR or TCR may be about 1 million to about 2 million transduced engineered NK cells or T cells per kilogram of body weight (cells / kg). The predetermined dose of engineered NK cells or T cells expressing a CAR or TCR may be between about 1 million and about 2 million, at least about 2 million to less than about 3 million transduced engineered NK cells or T cells per kilogram of body weight (cells / kg). In one embodiment, a predetermined dose of engineered NK cells or T cells expressing a CAR or TCR can be about 2 million transduced engineered T cells / kg. In another embodiment, a predetermined dose of engineered NK cells or T cells expressing a CAR or TCR can be at least about 2 million transduced engineered NK cells or T cells / kg. Examples of predetermined doses of engineered NK cells or T cells expressing a CAR or TCR can be about 2.0 million, about 2.1 million, about 2.2 million, about 2.3 million, about 2.4 million, about 2.5 million, about 2.6 million, about 2.7 million, about 2.8 million, or about 2.9 million transduced engineered NK cells or T cells / kg. In one embodiment, a population of engineered T cells can be cryopreserved at a predetermined dose of about 1 million engineered NK cells or T cells per kilogram of body weight (cells / kg). In certain embodiments, the population of engineered NK cells or T cells may be cryopreserved at a predetermined dose of about 500,000 to about 1 million engineered NK cells or T cells / kg.In certain embodiments, populations of engineered NK cells or T cells may be cryopreserved at predetermined doses of at least about 1 million, at least about 2 million, at least about 3 million, at least about 4 million, at least about 5 million, at least about 6 million, at least about 7 million, at least about 8 million, at least about 9 million, at least about 10 million engineered NK cells or T cells / kg. In other aspects, the population of engineered NK cells or T cells may be cryopreserved at a predetermined dose of less than 1 million cells / kg, 1 million cells / kg, 2 million cells / kg, 3 million cells / kg, 4 million cells / kg, 5 million cells / kg, 6 million cells / kg, 7 million cells / kg, 8 million cells / kg, 9 million cells / kg, 10 million cells / kg, more than 10 million cells / kg, more than 20 million cells / kg, more than 30 million cells / kg, more than 40 million cells / kg, more than 50 million cells / kg, more than 60 million cells / kg, more than 70 million cells / kg, more than 80 million cells / kg, more than 90 million cells / kg, or more than 100 million cells / kg. In certain embodiments, the population of engineered NK cells or T cells may be cryopreserved at a predetermined dose of about 1 million to about 2 million engineered NK cells or T cells / kg. The population of engineered NK cells or T cells may be cryopreserved at a predetermined volume of about 1 million to about 2 million cells / kg, about 1 million to about 3 million cells / kg, about 1 million to about 4 million cells / kg, about 1 million to about 5 million cells / kg, about 1 million to about 6 million cells / kg, about 1 million to about 7 million cells / kg, about 1 million to about 8 million cells / kg, about 1 million to about 9 million cells / kg, or about 1 million to about 10 million cells / kg. The predetermined dose of the population of engineered NK cells or T cells may be calculated based on the subject's body weight. In one example, the population of engineered NK cells or T cells can be cryopreserved in about 0.5 to 200 mL of cryopreservation medium.Furthermore, the population of engineered T cells may be cryopreserved in about 0.5 mL, about 1.0 mL, about 5.0 mL, about 10.0 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, or about 100 mL, about 10-30 mL, about 10-50 mL, about 10-70 mL, about 10-90 mL, about 50-70 mL, about 50-90 mL, about 50-110 mL, about 50-150 mL, or about 100-200 mL of cryopreservation medium. In certain embodiments, the population of engineered NK cells or T cells may be cryopreserved in about 50-70 mL of cryopreservation medium.

[0286] The present disclosure also provides compositions (e.g., pharmaceutical compositions) comprising any of the nucleic acids, vectors, sets of nucleic acids, sets of vectors, or cells described herein. For example, provided herein are compositions comprising any of the nucleic acids or sets of nucleic acids described herein, or any of the vectors or sets of vectors provided herein, and a pharmaceutically acceptable solvent or carrier. Also provided herein are pharmaceutical compositions comprising any of the various sets of vectors provided herein (e.g., a set of vectors comprising a first vector comprising any of the nucleic acids encoding a membrane-bound IL-15-IL-15Rα chimeric polypeptide and a second vector comprising a nucleic acid sequence encoding a CAR or TCR) and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some embodiments, the composition comprises an excipient. In another embodiment, the composition comprises a CAR or TCR and NK cells or T cells comprising a membrane-bound IL-15-IL-15Rα chimeric polypeptide.

[0287] In other embodiments, the compositions are selected for delivery via the digestive tract, such as parenteral delivery, inhalation, or oral delivery. Preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8. In certain embodiments, when parenteral administration is intended, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising a composition described herein, with or without an additional therapeutic agent, in a pharmaceutically acceptable vehicle. In certain embodiments, the parenteral injection vehicle is sterile distilled water in which the composition described herein, with or without at least one additional therapeutic agent, is formulated as a properly preserved sterile, isotonic solution. In certain embodiments, preparation involves formulating the desired molecule using beads or liposomes, which are polymeric compounds (such as polylactic acid or polyglycolic acid) that allow for controlled or sustained release of the product, which are then delivered by depot injection. In certain embodiments, an implantable drug delivery device is used to introduce the desired molecule.

[0288] In some embodiments, the composition can be any of the cells described herein (e.g., any of the cells described herein that have been previously obtained from a subject, e.g., a subject identified or diagnosed with cancer). In one embodiment, the cells comprise a membrane-bound IL-15-IL-15Rα chimeric polypeptide and / or a nucleic acid encoding any of the CARs or TCRs described herein. In compositions comprising any of the cells described herein, the composition can further comprise cell culture medium or a pharmaceutically acceptable buffer (e.g., phosphate-buffered saline).

[0289] Pharmaceutical compositions can include CAR-expressing cells or TCR-expressing cells described herein, e.g., a plurality of TCR-expressing cells or CAR-expressing cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can further include buffers, such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.

[0290] The pharmaceutical compositions of the present disclosure may be formulated for administration according to any of the embodiments described herein, at least one non-limiting example of which is intravenous administration. The compositions may be formulated for intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, and / or subcutaneous administration routes. In embodiments, the compositions are formulated for parenteral administration routes. Compositions suitable for parenteral administration may be aqueous or non-aqueous isotonic sterile injection solutions, which may contain, for example, antioxidants, buffers, bacteriostats, and solutes that render the composition isotonic with the blood of the intended recipient. Aqueous or non-aqueous sterile suspensions may contain one or more suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. The pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented).

[0291] In various embodiments, the engineered NK cells or T cells described herein can be incorporated into pharmaceutical compositions. Pharmaceutical compositions comprising engineered T cells as disclosed herein can be in any form. Such forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.

[0292] Pharmaceutical compositions comprising the binding agents of the present disclosure can be formulated by known methods (e.g., Remington's Pharmaceutical Sciences, 17th Edition, Alfonso R. Gennaro, ed., Mack Publishing Company, Easton, PA (1985)). In various examples, pharmaceutical compositions comprising the binding agents of the present disclosure can be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, but are not limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Compositions comprising engineered T cells can include pharmaceutically acceptable salts, e.g., acid addition salts or base addition salts.

[0293] Sterile compositions for injection can be formulated using distilled water for injection as a vehicle according to conventional pharmaceutical practice. For example, aqueous solutions for injection can be prepared using isotonic solutions containing physiological saline or other adjuvants such as glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc., optionally in combination with suitable solubilizers, for example, alcohols such as ethanol, polyhydric alcohols such as propylene glycol, polyethylene glycol, and nonionic surfactants such as Polysorbate 80 (trademark), HCO-50, etc.

[0294] Non-limiting examples of oily liquids include sesame oil and soybean oil, which may be combined with benzyl benzoate or benzyl alcohol as a solubilizer. Other ingredients that may be included in the composition include buffers such as phosphate buffer or sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injections can be packaged in suitable ampoules.

[0295] In one embodiment, the pharmaceutical composition is substantially free of detectable levels of contaminants, such as endotoxin, mycoplasma, replication-competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, bacteria, and fungi. In one embodiment, the bacterium is at least one species selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and / or Streptococcus pyogenes group A.

[0296] In some embodiments, the engineered cells are treated ex vivo with interleukin-2 (IL-2) before infusion into the cancer patient, and the cancer patient is treated with IL-2 after infusion. Additionally, in some embodiments, the cancer patient may undergo preliminary lymphodepletion (temporary removal of the immune system) before administration of the binding agent. Combining IL-2 treatment with preliminary lymphodepletion Combining the two may enhance the persistence of the binding agent. In some embodiments, the engineered cells are transduced or transfected with a nucleic acid encoding a cytokine (e.g., a membrane-bound IL-15-IL-15Rα chimeric polypeptide), which may be engineered to provide constitutive, regulatable, or temporally controlled expression of the cytokine. Suitable cytokines include, for example, cytokines that act to enhance the survival of T lymphocytes during the contraction phase, which may facilitate the formation and survival of memory T lymphocytes.

[0297] In some embodiments, the dosage administered to a subject may vary depending on the embodiment, the composition used, the method of administration, and the site and subject being treated. However, the dosage must be sufficient to provide a therapeutic response. A clinician can determine the therapeutically effective amount of the composition to be administered to a human or other subject to treat or prevent a medical condition. The exact amount of the composition required to be therapeutically effective may depend on many factors, such as the activity of the binding agent and the route of administration.

[0298] Any suitable number of engineered cells containing a CAR or TCR can be administered to a subject. A single engineered cell described herein can expand and provide a therapeutic benefit, although in some embodiments, 10 2 More than one, e.g., 10 3 More than 10 pieces 4 More than 10 pieces 5 More than 10 8 In some embodiments, 10 or more engineered cells are administered. 12 10 or less, for example 11 Less than or equal to 10 9 Less than or equal to 10 7 No more than, or no more than 10 engineered cells described herein are administered to a subject. In some embodiments, no more than 10 engineered cells described herein are administered to a subject. 2 ~10 5 pieces, 10 4 ~10 7 pieces, 10 3 ~10 9 Pieces or 10 5 ~10 10 The pharmaceutical composition comprising the CAR or TCR-containing cells is administered in an amount of, for example, 10 4 ~10 9 cells / kg body weight (e.g., 10 5 ~10 6 In another embodiment, the therapeutically effective amount of T cells is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or approximately 10 8The pharmaceutical composition may contain, for example, about 2 x 10 cells. 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 10 7 It may be administered at a dose of cells / kg.

[0299] The dose of engineered T cells or NK cells described herein can be administered to a mammal once or in a series of subdoses administered over a suitable period of time, e.g., daily, semi-weekly, weekly, biweekly, semi-monthly, bimonthly, semi-annually, or yearly, as needed. A dosage unit containing an effective amount of the binding agent can be administered in a single daily dose, or the total daily dose can be administered in divided doses, two, three, four or more times daily, as needed.

[0300] The appropriate administration means can be selected by a physician. The administration route can be parenteral administration, for example, administration by injection, nasal administration, pulmonary administration, or transdermal administration. Administration can be systemic administration or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, the composition is selected for delivery via the digestive tract, such as parenteral delivery, inhalation, or oral administration. The dosage and administration method may vary depending on the subject's weight, age, condition, etc., and can be selected appropriately.

[0301] The selection or use of any form may depend in part on the intended mode of administration and therapeutic application. For example, compositions comprising engineered cells of the present disclosure intended for systemic or local delivery may be in the form of an injectable or infusible solution. Thus, engineered compositions of the present disclosure may be formulated for administration by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). Parenteral administration includes enteral and topical administration, usually by injection. refers to modes of administration other than intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0302] In various embodiments, pharmaceutical compositions containing engineered cells of the present disclosure can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of a composition containing engineered cells of the present disclosure into an appropriate solvent, optionally containing one or a combination of the ingredients listed above, followed by filtered sterilization. Generally, dispersions are prepared by incorporating a composition containing engineered cells of the present disclosure into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. The proper fluidity of the solution can be maintained, for examp...

Claims

1. 1. A membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric receptor comprising an IL-15 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6, a first linker connecting the IL-15 domain to an IL-15Rα sushi domain polypeptide set forth in SEQ ID NO:7 or SEQ ID NO:95, and a transmembrane domain comprising an IL-7 transmembrane domain or a FAS transmembrane domain.

2. 2. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 1, wherein the first linker comprises the amino acid sequence set forth in SEQ ID NO:

8.

3. 3. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 2, wherein the first linker comprises the amino acid sequence set forth in SEQ ID NO:

11.

4. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of any one of claims 1 to 3, wherein the IL-15Rα sushi domain polypeptide is linked to the transmembrane domain as a second linker.

5. 5. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 4, wherein the second linker comprises the amino acid sequence set forth in SEQ ID NO:

24.

6. 6. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 5, wherein the second linker comprises the amino acid sequence set forth in SEQ ID NO:

26.

7. 7. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 1, wherein the transmembrane domain is a FAS transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:

22.

8. 8. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 7, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28 and 94.

9. 7. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 1, wherein the transmembrane domain is an IL-7 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:

23.

10. 10. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 9, comprising the amino acid sequence set forth in SEQ ID NO:

30.

11. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of any one of claims 1 to 8, further comprising a signaling sequence.

12. 12. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 11, wherein the signaling sequence comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12-20.

13. 13. The membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 12, wherein said signaling sequence comprises the amino acid sequence set forth in SEQ ID NO:

12.

14. A nucleic acid encoding the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of any one of claims 1 to 13.

15. 15. The nucleic acid of claim 14, wherein the nucleic acid comprises a nucleic acid sequence set forth in a sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:

100.

16. A recombinant vector comprising the nucleic acid of claim 14.

17. 16. The recombinant vector or nucleic acid of claim 14 or 15, further comprising a nucleic acid encoding a chimeric antigen receptor or a T cell receptor.

18. 16. The recombinant vector or nucleic acid of claim 14 or 15, wherein the chimeric antigen receptor or T cell receptor binds to a tumor antigen.

19. The tumor antigen is selected from the group consisting of 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A-kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), β2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (an oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia cell oncogene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), BST2, C242 antigen, 9-O-acetyl-CA19-9 marker, CA-125,CAEX, calreticulin, carbonic anhydrase 9 (CAIX), C-MET, CCR4, CCR5, CCR8, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD16, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD25, CD27, CD28, CD30 (TNFRSF8), CD33, CD34, CD38, CD40, C D40L, CD41, CD44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, CD72, CD74, CD79a, CD79b, CD80, CD8 4, CD96, CD97, CD100, CD123, CD125, CD133, CD137, CD138, CD150, CD152 (CTLA-4), CD160, CD171, CD17 9a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PD-L1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), claudin 6 (CLDN6), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-type lectin domain family 12 member A (CLEC12A), cytomegalovirus (CMV)-infected cell antigen, CNT0888, CRTAM (CD355), CS-1 (CD2 subset 1, also known as CRACC, CD319, and 19A24), CTLA-4, cyclin B1, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B 1 ​​(CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neoepitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 variant (ELF2M), endosialin, epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EphA2), ephrinB2, receptor tyrosine-protein kinase erb-B2, 3, 4 (erb-B2, 3, 4,4), ERBB, ERBB2 (Her2 / neu), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS transduced mutant gene 6 located on chromosome 12p (ETV6-AML), Fc fragment of IgA receptor (FCAR or CD89), fibroblast activation protein, alpha (FAP), FBP, Fc receptor-like 5 (FcRL5), fetal acetylcholine receptor (AChR), fibronectin extra domain B, Fms-like tyrosine kinase 3 (FLT3), folate binding protein (FBP), folate receptor 1, folate receptor alpha, folate receptor beta, Fos-related antigen 1, fucosyl, fucosyl GM1;GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF18), GM1, ganglioside GM3, hexasaccharide moiety of GloboH glycoceramide (GloboH), glycoprotein 75, glypican-3 (GPC 3), glycoprotein 100 (gpl00), GPNMB, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), hepatitis A virus cellular receptor 1 (HAVCR1), human epidermal growth factor receptor 2 (HER-2), HER2 / neu, HER3, HER4, HGF, high molecular weight melanoma-associated antigen (HMWMAA), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), heat shock protein 70-2 mutant (mutated) hsp70-2), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgG1, immunoglobulin lambda-like polypeptide 1 (IGLL1), IL-6, interleukin-11 receptor alpha (IL-11Rα), IL-13, interleukin-13 receptor subunit alpha-2 (IL-13Rα2 or CD213A2), insulin-like growth factor I receptor (IGF1-R), integrin α5β1, integrin ανβ3, intestinal carboxylesterase, kappa light chain, KCS1, kinase insert domain receptor (KDR), K IR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KG2D ligand, KIT (CD117), KLRGI, LAGE-la, LAG3, lymphocyte-specific protein tyrosine kinase (LCK), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), legumain, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis (Y) antigen, LeY, LG, LI-cell adhesion molecule (LI-CAM), LIGHT, LMP2, lymphocyte antigen 6 complex, LTBR, ​​locus K9 (LY6K), Ly-6, lymphocyte antigen 75 (LY75), melanoma cancer testis antigen-1 (MAD-CT-1);Melanoma cancer testis antigen-2 (MAD-CT-2), MAGE, melanoma-associated antigen 1 (MAGE-A1), MAGE-A3 melanoma antigen 1 (MelanA or MART1), MelanA / MART1, mesothelin, MAGE A3, melanoma inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin 1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory factor (MIS) receptor type II, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), N-glycolylamine acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, mammary differentiation antigen (NY-BR-1), NY-ESO-1, carcinoembryonic antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, polySA, proacrosin-binding protein sp32 (OY-TES 1), p53, p53 mutant, pannexin 3 (PANX3), prostatic acid phosphatase (PAP), paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), PD-1H, platelet-derived growth factor receptor alpha (PDGFR-alpha), PDGFR-beta, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostase, prostate cancer cells, prostein, protease serine 21 (testisin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (prosome, macropain) subunit; Beta type, receptor for advanced glycation end products (RAGE-1), RANKL, Ras mutant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), sarcoma translocation breakpoint, squamous cell carcinoma antigen recognized by T cells 3 (SART3), SAS, SDC1, SLAMF7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, sonic hedgehog (SHH), sperm protein 17 (SPP) A17), stage-specific embryonic antigen 4 (SSEA-4), STEAP, sTn antigen, synovial sarcoma X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), TCR5γ, TCRα, TCRβ, TCRγ alternative reading frame protein (TARP), telomerase, TIGIT, TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin-C, TGF-β1, TGF-β2, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TACI, TIM1, TIM2, TIM3, Tn Ag, TRAIL-R1, TRAIL-R2, tyrosinase-related protein 2 (TRP-2), thyroid-stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosinase, uroplakin 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2), and vimentin, Wilms' tumor protein (WT1), or X-antigen family member 1A (XAGE1).

20. An immune cell comprising the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor, recombinant vector or nucleic acid of any one of claims 1 to 19.

21. The immune cell of claim 20, which is a T cell or a natural killer (NK) cell.

22. A pharmaceutical composition comprising the immune cells of claim 20 or 21.

23. 23. A method for treating cancer associated with expression of a tumor antigen in a subject, comprising administering to the subject an effective amount of an immune cell of claim 20 or 21 or a pharmaceutical composition of claim 22.

24. 23. A method of inducing an immune response in a subject or immunizing a subject against cancer, comprising administering to the subject an effective amount of an immune cell of claim 20 or 21 or a pharmaceutical composition of claim 22.

25. 14. A method of improving immune cell function, comprising engineering said immune cells to express the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric receptor of any one of claims 1 to 13.

26. 14. A method for increasing phospho-STAT5 levels in immune cells, comprising engineering the immune cells to express the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric receptor of any one of claims 1 to 13.

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