Improvement of immune cell function

Abstract

The present disclosure relates to the field of cell therapy, and more specifically, to improving CAR and / or TCR function through improved cytokine signaling.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 159,610, filed on March 11, 2021, and U.S. Provisional Patent Application No. 63 / 210,300, filed on June 14, 2021, which are hereby incorporated by reference in their entirety for all purposes.

[0002] The present disclosure relates to the fields of immunology and cell therapy, and more specifically, to improving immunotherapies based on T cells and natural killer (NK) cells that include chimeric antigen receptors (CARs) and / or T cell receptors (TCRs) by modulating cytokine signaling.

Background Art

[0003] The immune system provides natural defense against cancer through its ability to search for, detect, and destroy malignant cells throughout the body. However, a point to note in this defense mechanism is that certain cancers can induce an immunosuppressive microenvironment that reduces the robustness of the anti - tumor immune response. (Beatty et al., Clin Cancer Res, (21)(4):687 - 632(2015)). These immune - evasion mechanisms pose challenges to the implementation and effectiveness of cell - based immunotherapies, including the use of engineered cell - therapy techniques such as chimeric antigen receptor (CAR) T - cell therapy, T - cell receptor (TCR) T - cell therapy, and / or natural killer cell - based immunotherapies.

[0004] Since the overall function and proliferation of T cells depend on cytokine signaling, the use of cytokines has been theorized to improve the overall quality and efficacy of T cell-based therapies. Past studies have demonstrated the success of using IL-2 as a means to expand T cell-based therapies, but the 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). The efficacy of CAR-T has also been 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 the cytotoxicity of NK cells. (Hu et al., Front. Immunol., (20)1205(2019)).

[0005] Therefore, it is necessary to utilize the use of cytokine signaling as a means to improve the effectiveness of immune cell-based immunotherapies.

Summary of the Invention

[0006] A membrane-bound interleukin 15 (IL-15)-IL-15Rα succinimide domain chimeric receptor is disclosed. In an embodiment, the membrane-bound IL-15-IL-15Rα succinimide domain chimeric receptor comprises an IL-15 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6, a first linker that links the IL-15 domain to an IL-15Rα succinimide 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, for example, an IL-7 transmembrane domain.

[0007] In an embodiment, the first linker that links the IL-15 polypeptide and the IL-15Rα sushi domain contains the amino acid sequence set forth in SEQ ID NO: 8. In an embodiment, the first linker contains the amino acid sequence set forth in SEQ ID NO: 10.

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

[0009] In an embodiment, the transmembrane domain is the FAS transmembrane domain containing the amino acid sequence set forth in SEQ ID NO: 22. In an embodiment, the transmembrane domain is the FAS transmembrane domain containing the amino acid sequence set forth in SEQ ID NO: 42. In an embodiment, the IL-7 transmembrane domain contains the amino acid sequence set forth in SEQ ID NO: 23.

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

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

[0012] Disclosed is a nucleic acid encoding the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor described herein. In an embodiment, the nucleic acid encoding the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor contains 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] A recombinant vector is disclosed that contains a nucleic acid encoding the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor 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 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenergic receptor beta 3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), beta2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (a cancer gene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral 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 ImprintedSites), 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, 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 (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 called CD2 subset 1, CRACC, CD319, and 19A24), CTLA-4, cyclin B1, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 (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), Endocan, Epithelial Cell Adhesion Molecule (EPCAM), Ephrin A-type Receptor 2 (EphA2), Ephrin B2, Receptor Tyrosine-Protein Kinase erb-B2, 3,4 (erb-B2,3,4), ERBB, ERBB2 (Her2 / neu), ERG (Transmembrane Protease, Serine 2 (TMPRSS2) ETS Fusion Gene), ETA, ETS Variant 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), Folic Acid Binding Protein (FBP), Folic Acid Receptor 1, Folic Acid Receptor Beta, Folic Acid Receptor Gamma, 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 Portion of Globosylceramide (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 Variant (muthsp70-2), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgGl, 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, κ 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 recognized by T cells (MelanA or MARTI), MelanA / MART1, mesothelin, MAGE A3, melanoma inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin1 (MUC1), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Mullerian-inhibiting substance (MIS) type II receptor, v-myc avian myelocytomatosis virus oncogene neuroblastoma-derived homolog (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-PIA, NPC-1C, NTB-A, breast differentiation antigen (NY-BR-1), NY-ESO-1, cancer fetal antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, poly SA, proacrosin-binding protein sp32 (OY-TES 1), p53, p53 variant, pannexin 3 (PANX3), prostate 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, prostain, 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, advanced glycation end product receptor (RAGE-1), RANKL, Ras variant, 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 3 recognized by T cells (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), TCR5β, TCRα, TCRβ, TCRδ, TCRγ. It is selected from the group consisting of 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 (Tie2), TIM1, TIM2, TIM3, Tn Ag, TRAIL-R1, TRAIL-R2, tyrosine-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 a membrane-bound IL-15-IL-15Rα succinamide 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 is a method of treating cancer associated with the expression of a tumor antigen in a subject, the method comprising administering to the subject an effective amount of the immune cells or pharmaceutical composition disclosed herein.

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

[0018] Disclosed is a method for improving immune cell function, comprising engineering immune cells to express a membrane-bound interleukin 15 (IL-15)-IL-15Rα succinamide domain chimeric receptor.

DETAILED DESCRIPTION OF THE INVENTION

[0019] TERMS To more readily understand the present disclosure, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout this 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 indicates otherwise.

[0021] As used herein, unless otherwise specified or clear from the 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 construed 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 in phrases such as "A and / or B" herein 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 include 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 by way of example and is not intended to be limiting and should not be construed as referring only to the items expressly recited herein.

[0024] Terms such as "above", "at least", "more than", for example, "at least one", although not limiting, means 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, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more of the recited value, and is understood to include any larger number or fraction therebetween.

[0025] Conversely, the term "less than" includes each value that is less than the recited value. For example, "less than 100 nucleotides" 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, 45, 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 lesser number or fraction therebetween is also included.

[0026] Terms such as "a plurality", "at least two", "two or more", "at least a second", etc., are not limiting, but include 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, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. Any larger number or fraction therebetween is also included.

[0027] Throughout this specification, the term "comprising" or variations such as "comprises" or "comprising" means including the stated element, integer or step, or group of elements, integers or steps, but does not mean excluding any other element, integer or step, or group of elements, integers or steps. When an aspect is described in this specification in the language of "comprising", it is understood that other similar aspects described in the terms of "consisting of" and / or "consisting essentially of" are also presented.

[0028] Unless specifically stated or obvious from the context, the term "about" refers to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which depends to some extent on how the value or composition is measured or determined, i.e., the limitations of the measuring system. For example, "about" or "consisting essentially of" may mean within the range of one or more standard deviations of the practice in the art. "About" or "consisting essentially of" may mean within 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 from 4.5 mg to 5.5 mg. Further, especially with respect to biological systems or processes, this term can mean up to one order of magnitude or up to five times the value. When a particular value or composition is presented in this disclosure, unless otherwise specified, it is necessary to assume that the meaning of "about" or "consisting essentially of" is within the acceptable error range for that particular value or composition.

[0029] As described herein, any range of concentrations, percentage ranges, ratio ranges, or integer ranges should be understood to include any integer value within the recited range, and, as appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise specified.

[0030] The units, prefixes, and symbols used herein are presented in the form accepted in the Systeme International de Unites (SI). Numerical ranges include 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. For example, 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 provide a general dictionary for many of the terms used in this disclosure to those of ordinary skill in the art.

[0032] "Administering" refers to the physical introduction of an agent, such as a modified T cell or NK cell disclosed herein, to 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, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration by injection or infusion. The phrase "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, arterial, intrathecal, lymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered orally, for example, via a non-parenteral route. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, such as intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be carried out, 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 a T cell or NK cell that has 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 cell" refers, inter alia, to a proliferating T cell. The term "activated NK cell" refers, inter alia, to a proliferating NK cell. Signals generated only through the TCR may be insufficient for complete activation of T cells, and one or more secondary or co-stimulatory signals may also be required. Thus, T cell activation includes a primary stimulatory signal through the TCR / CD3 complex and one or more secondary co-stimulatory signals. Co-stimulation can be evidenced by the proliferation and / or cytokine production of 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 a plurality of 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 a precursor of such a cell), or an organism (such as a fraction or extract thereof) or a component thereof. In some embodiments, the agent can be utilized in isolated form or pure form. In some embodiments, the agent can be utilized in crude form or impure form. In some embodiments, the agent can be provided as a population, collection or library that can be screened, for example, to identify or characterize members present therein.

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

[0036] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Generally, an antibody can include at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding molecule. Each H chain includes a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region includes three constant domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region includes one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) incorporated into more conserved regions called framework regions (FRs). Each VH and VL includes three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus toward the carboxy terminus. The variable regions of the heavy and light chains include a binding domain that interacts with the antigen. The constant region of the Ab can 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, a human antibody is a tetrameric agent of about 150 kD composed of two identical heavy (H) chain polypeptides (each about 50 kD) and two identical light (L) chain polypeptides (each about 25 kD), which are generally associated with each other in a structure commonly referred to as a "Y shape". 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 to each other, resulting in the connection of the dimers to each other to form a tetramer. Naturally produced antibodies are also glycosylated, for example, on the C H domain 2.

[0037] The term "human antibody" is intended to include antibodies having variable and constant domain sequences that are generated, assembled, or derived from human immunoglobulin sequences, or sequences that are indistinguishable therefrom. In some embodiments, an antibody (or antibody component) may be considered "human" even if its amino acid sequence includes residues or elements not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by somatic mutations in vivo). The term "humanized" is intended to include antibodies having a variable domain having a sequence derived from the variable domain of a non-human species (e.g., mouse) that has been modified to be more similar to human germline-encoded sequences. In some embodiments, a "humanized" antibody includes 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 includes at least a portion of the immunoglobulin constant region (Fc), generally that of a human immunoglobulin constant domain. In some embodiments, a humanized antibody includes the C H 1, hinge, C H 2, C H 3, and optionally, C H 4 regions.

[0038] Examples of 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-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 Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab’) 2Examples include fragments, disulfide - linked Fv (sdFv), 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 foregoing. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations. Antibodies also include, for example, Fab' fragments, Fd' fragments, Fd fragments, isolated CDRs, single - chain Fv, polypeptide - Fc fusions, single - domain antibodies (such as shark single - domain antibodies like IgNAR or fragments thereof), camelid antibodies, single - chain or tandem diabodies (TandAb®), Anticalins®, Nanobodies®, minibodies, BiTE®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR - like antibodies, Adnectins®, Affilins®, Trans - bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®.

[0039] The immunoglobulin 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 an Ab class or subclass encoded by a heavy chain constant region gene (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 that reduce their immunogenicity in humans. Unless explicitly stated otherwise or the context otherwise indicates, the term "antibody" includes any antigen-binding fragment or antigen-binding portion of any of the aforementioned immunoglobulins, including monovalent and divalent fragments or portions, and single-chain antibodies.

[0040] "Antigen-binding molecule", "antigen-binding portion", "antigen-binding fragment", "antibody fragment", or "antigen-binding domain" refers to any molecule that includes the antigen-binding portion (e.g., CDR) of an antibody from which the molecule is derived. An antigen-binding molecule may include antigen complementarity-determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules that include a peptide-binding domain) are another example of suitable antigen-binding molecules. 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 proliferative disorder or a viral antigen or a 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 includes 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 includes an avimer or consists of an avimer.

[0041] In some examples, the CDR is substantially identical to that found in a reference antibody (e.g., an antibody of the present disclosure) and / or the sequence of the CDR provided in the present disclosure. In some embodiments, the CDR is substantially identical to the reference CDR in that the sequences are identical or contain 1, 2, 3, 4, or 5 (e.g., 1-5) amino acid substitutions. In some embodiments, the CDR is substantially identical to the 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%) to the reference CDR. In some embodiments, the CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, the CDR is substantially identical to the reference CDR in that one amino acid within the CDR has been deleted, added, or substituted, 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 the reference CDR in that 2, 3, 4, or 5 (e.g., 2-5) amino acids within the CDR have been deleted, added, or substituted, while the CDR otherwise has an amino acid sequence that is identical to the amino acid sequence of the reference CDR. In various embodiments, the antigen-binding fragment binds the same antigen as the reference antibody. In various embodiments, the antigen-binding fragment cross-competes with the reference antibody and binds, for example, 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 produced enzymatically or chemically by fragmentation of intact antibodies. In some embodiments, antigen-binding fragments can be produced recombinantly (such as by expression of engineered nucleic acid sequences). In some embodiments, antigen-binding fragments can be produced wholly or in part synthetically. In some embodiments, antigen-binding fragments may 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, may have a length of at least about 200 amino acids (e.g., 50-100, 50-150, 50-200, or 100-200 amino acids).

[0043] The terms "variable region" or "variable domain" are used interchangeably. The variable region typically refers to a part of an antibody, generally part of the light or heavy chain, typically the amino-terminal 110-120 amino acids of the mature heavy chain and about 90-115 amino acids of the mature light chain, which vary greatly in sequence between antibodies and are used for the binding and specificity of a particular antibody to its specific antigen. The variability of the sequence is concentrated in regions called complementarity-determining regions (CDRs), and the more highly conserved regions within the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, the CDRs of the light and heavy chains are thought to be mainly responsible for the antibody's interaction and specificity with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes rodent or mouse 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 includes rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

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

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

[0046] Many definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between two used by Oxford Molecular's AbM antibody modeling software. The contact definition is based on the 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 the amino acid residues of the heavy and light chain variable regions of an antibody or its antigen-binding molecule. 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, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within the antibody heavy chain molecule are typically at amino acid positions 31-35 (optionally including one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3). Using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 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 the antibodies can be determined according to the Chothia numbering scheme that refers to the positions of the 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 rules, the Chothia CDR-H1 loop is present at heavy chain amino acids 26-32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52-56, the Chothia CDR-H3 loop is present at heavy chain amino acids 95-102, while the Chothia CDR-L1 loop is present at light chain amino acids 24-34, the Chothia CDR-L2 loop is present at light chain amino acids 50-56, and the Chothia CDR-L3 loop is present at light chain amino acids 89-97. When numbered using the Kabat numbering rules, the end of the Chothia CDR-HI loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B, and if neither 35A nor 35B is 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 usual 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, and these types are related to subclasses of IgG, e.g., IgG 1 , IgG 2 , IgG 3 and IgG 4 The IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, include:

[0051] When used in reference to an antibody, the term "light chain" may 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 the production of antibodies or T cell responses in humans or animals, including compositions (such as those containing tumor-specific proteins) that are injected or absorbed by humans or animals. Antigens react with the products of specific humoral or cellular immunity, including those induced by heterologous antigens such as the disclosed antigens. A "target antigen" or "target antigen of interest" is an antigen that is not substantially found on the surface of other normal (desired) cells and is designed to be bound by the binding domain of a TCR or CAR contemplated herein. One of ordinary skill in the art will readily understand that virtually any macromolecule, including all proteins or peptides, can function as an antigen. Antigens may be expressed endogenously, i.e., by genomic DNA, or recombinantly. Antigens can be specific for certain tissues such as cancer cells or can be widely expressed. Additionally, 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, such as an antibody.

[0053] The "antigen-specific targeting region" (ASTR) refers to the region of a CAR or TCR that targets a specific antigen. The targeting region on the CAR or TCR is extracellular. In some embodiments, the antigen-specific targeting region comprises an antibody or a functional equivalent or a fragment or a derivative thereof, and each of the targeting regions targets a different antigen. The targeting region can include 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 the target antigen. However, there are numerous alternatives such as linked cytokines (which result in the recognition of cells having 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 promote an immune response, each of which can be used in various embodiments of the present disclosure. Indeed, as will be understood by those skilled in the art, almost any molecule that binds to a given antigen with high affinity can be used as an antigen-specific targeting region.

[0054] "Antigen-presenting cell" or "APC" refers to a cell that processes and presents an antigen to a T cell. Exemplary APCs include dendritic cells, macrophages, B cells, certain activated epithelial cells, and other cell types capable of TCR stimulation and appropriate T cell co-stimulation.

[0055] "Anti-tumor effect" refers to a biological effect that can be presented as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall survival or progression-free survival, an extension of mean lifespan, or an improvement in various physiological symptoms associated with the tumor. The anti-tumor effect can also refer to the prevention of tumor development.

[0056] Two events or entities are "associated" with each other 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 (e.g., across a relevant population) with the incidence and / or susceptibility of the disease, disorder, or condition. For example, two or more entities are physically "associated" with each other if they interact directly or indirectly such that they are physically proximate to each other (e.g., by binding, etc.) and / or remain physically proximate. In a further example, two or more entities that are physically associated with each other are covalently linked or connected to each other or non-covalently associated with each other, for example, by hydrogen bonds, 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 re-introduced later. For example, the methods of engineered autologous cell therapy (eACT™) described herein include 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 total strength 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 that 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 can generally be represented by the dissociation constant (K D ). Affinity can be measured and / or expressed in many ways known in the art, including, but not limited to, the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ). K D is k off / k onis calculated from the quotient, while K A is k on / k off is calculated from the quotient of.k on refers to, for example, the association rate constant of an antibody to an antigen, and k off refers to, for example, the dissociation of an antibody from an antigen.k 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 specific antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment that binds to an antigen.K D has an inverse relationship with the binding affinity, and thus, the smaller the K D value, the higher, i.e., stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, and thus a smaller K D value, and conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, and thus a larger K D value. In some situations, compared to the binding affinity of a molecule (e.g., an antibody) to another interacting partner molecule (e.g., antigen Y), a higher binding affinity (or K D ) of a specific molecule (e.g., an antibody) to an interacting partner molecule (e.g., antigen X) can be expressed as a binding ratio determined by dividing a larger K D (lower, or weaker affinity) by a smaller K D (higher, or stronger affinity), and can be expressed, for example, in some cases, as 5-fold or 10-fold higher binding affinity.

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

[0061] "k" a The term "(M-1×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 obtained by dividing k a by k d .

[0063] The term "binding" generally refers to non-covalent association between two or more entities. Direct binding involves physical contact between the entities or moieties. "Indirect" binding involves physical interactions through physical contact with one or more intermediate entities. Binding between two or more entities can be evaluated in any of a variety of situations, such as when the interacting entities or moieties are studied alone or in more complex systems (such as when associated with a carrier entity by covalent or other means and / or within a biological system such as a cell).

[0064] The terms "immunologically specifically bind", "immunologically specifically recognize", "specifically bind", and "specifically recognize" are similar terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or immune complex) as understood by one of ordinary skill in the art. For example, a molecule that specifically binds to an antigen generally binds with a lower affinity to other peptides or polypeptides, as determined by, for example, an immunoassay, 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 that is at least 2 log, 2.5 log, 3 log, 4 log, or more greater than the K Aand binds to the antigen. The binding may include a preferential association of the binding motif, antibody, or antigen-binding system with the target of the binding motif, antibody, or antigen-binding system as compared to the association of the binding motif, antibody, or antigen-binding system with an entity that is not the target (i.e., non-target). In some embodiments, the binding of the binding motif, antibody, or antigen-binding system to the target is more than 2-fold, more than 5-fold, more than 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more than 100-fold as compared to the binding of the binding motif, antibody, or antigen-binding system to the non-target, and the binding motif, antibody, or antigen-binding system binds selectively to the target. In some embodiments, the binding motif, antibody, or antigen-binding system binds selectively to the target when the binding affinity is less than about 10 -5 M, less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M.

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

[0066] In certain embodiments, provided herein is an antibody or antigen-binding molecule thereof that binds to a target human antigen. For example, in certain embodiments, the antigen-binding molecule binds to the target antigen with a greater affinity by, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more, than it binds to another species of target antigen, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In specific embodiments, the antibody or antigen-binding molecule thereof described herein that binds to the target human antigen binds to another species of target antigen at less than 10%, 15%, or 20% of the binding of the antibody or antigen-binding molecule to the human antigen, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.

[0067] The term "cancer" generally relates to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Examples of cancers that can be treated by the methods of the present disclosure include, but are not limited to, cancers of the immune system, such as lymphoma, leukemia, myeloma, and other white blood cell malignancies. In some embodiments, the methods of the present disclosure can be used, for example, to reduce the tumor size of bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric 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 (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small intestine cancer, cancers of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers including those induced by asbestos, other B cell malignancies, and tumors derived from combinations of the above cancers. In one particular embodiment, the cancer is multiple myeloma. Certain cancers can be responsive to chemotherapy or radiation therapy, or certain cancers can be refractory.Refractory cancer refers to cancer that is not suitable for surgical intervention. Refractory cancer either does not respond to chemotherapy or radiotherapy from the beginning, or the cancer becomes non-responsive over time. Cancer further includes recurrent or refractory diffuse large B-cell lymphoma (DLBCL) after two or more systemic therapies, including DLBCL arising from diffuse large B-cell lymphoma, mediastinal primary large B-cell lymphoma after two or more systemic therapies, high-grade B-cell lymphoma, and follicular lymphoma.

[0068] The terms "cancerous cell", "cancer cell", "tumor cell", or variants thereof refer to individual cells of a cancer tumor or tissue. A tumor generally refers to a swelling or lesion formed by abnormal cell growth 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 the tumors.

[0069] "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 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0070] "Chimeric antigen receptor" or "CAR" refers to a molecule engineered to include means for activating a binding motif and immune cells (e.g., T cells such as naive T cells, central memory T cells, effector memory T cells, NK cells, or combinations thereof) upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. In some embodiments, a CAR includes a binding motif, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. T cells genetically engineered to express a chimeric antigen receptor may be referred to as CAR T cells. Similarly, NK cells genetically engineered to express a chimeric antigen receptor may be referred to as CAR NK cells.

[0071] "Decrease" or "reduce" or "alleviate" or "diminish" 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 a response caused by either vehicle alone (i.e., the active moiety) or a control molecule / composition. A "decrease" or "reduced" amount is typically a "statistically significant" amount, being a decrease 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-fold, or more (e.g., 500-fold, 1000-fold) (e.g., including all integers and fractions in between greater than 1 such as 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by the vehicle, which is the control composition (reference response).

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

[0073] As used herein, the term "extracellular ligand-binding domain" refers to an oligo or polypeptide that can bind 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 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] Following the binding domain of the CAR, there may be a "spacer" or "hinge", which refers to a region that moves the antigen-binding domain away from the effector cell surface to allow for 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 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 used in the CARs described herein include hinge regions derived from the extracellular regions of type I 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 part of the CAR that anchors the extracellular binding portion to the plasma membrane of the immune effector cell and promotes binding of the binding domain to the target antigen. The transmembrane domain can be the CD3 zeta transmembrane domain, however, other transmembrane domains that can be used include those derived from CD8 alpha, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain is the transmembrane domain of CD137. In certain embodiments, the transmembrane domain is synthetic, in which case it mainly contains hydrophobic residues such as leucine and valine.

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

[0077] Signals generated only through the T cell receptor are known to be insufficient for complete activation of T cells, and secondary or co-stimulatory signals are also required. Thus, T cell activation can be said to be mediated by two different 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 co-stimulatory signals (secondary cytoplasmic signaling sequences). Cytoplasmic signaling sequences that act in a co-stimulatory 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 terms "costimulatory signaling domain" or "costimulatory domain" refer to the 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 Fc receptor that provides a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen. 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. Accordingly, the present disclosure provides an exemplary costimulatory domain derived from 4-1BB, although other costimulatory domains are contemplated. 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 tandemly linked in any order at the carboxyl terminus of the transmembrane domain.

[0080] ScFv-based CARs engineered to contain signaling domains derived from CD3 or FcR gamma have been shown to deliver potent signals for T cell activation and effector function, but they are not sufficient to induce signals that promote T cell survival and proliferation in the absence of accompanying costimulatory signals. Other CARs containing a binding domain, hinge, transmembrane, and signaling domain derived from CD3 zeta or FcR gamma, together with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from 4-1BB, CD28, CD137, CD134, and CD278), can more effectively direct antitumor activity as well as 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] "Costimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, but not limited to, results in a T cell response such as proliferation and / or upregulation or downregulation of important molecules.

[0082] "Costimulatory ligand" includes molecules on antigen-presenting cells that specifically bind to cognate costimulatory molecules on T cells. Binding of costimulatory ligands provides signals, including but not limited to, mediating T cell responses such as proliferation, activation, and differentiation. Costimulatory ligands, in addition to the primary signal provided by stimulatory molecules, can induce signals, for example, by the binding of the T cell receptor (TCR) / CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules. Examples of costimulatory ligands include, but are not limited to, 3 / TR6, 4-1BB ligand, agonists or antibodies that bind to Toll-like 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), ligands that specifically bind to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed death (PD) L1.Examples of co-stimulatory ligands include, but are not limited to, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, 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 and thereby mediates a costimulatory response by the T cell, such as, but not limited to, proliferation. The costimulatory molecule includes, but is not limited to, the fact that the "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the T cell, such as, but not limited to, proliferation.Co-stimulatory molecules include, but are not limited to, 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, CD84, CD86, CD8 alpha, CD8 beta, CD9, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CD5, CEACAM1, CRTAM, 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 (CD11a / CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signal transduction lymphocyte activation molecule, SLAM (SLAMF1, CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavage products, or combinations thereof.

[0084] "Conservative amino acid substitutions" are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains are defined in the art. These families include amino acids having 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 within the framework region of its antibody or antigen-binding molecule can be replaced with amino acid residues having similar side chains. Generally, two sequences are generally considered "substantially the same" if they contain conservative amino acid substitutions at corresponding positions. For example, a particular amino acid is generally classified as a "hydrophobic" or "hydrophilic" amino acid and / or as having a "polar" or "nonpolar" side chain. Substitution of another amino acid of the same type can 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 where a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more treatment components). In some embodiments, two or more regimens may be administered simultaneously, and in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered prior to the administration of any dose of the second regimen), and in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administration" of combination therapy may include administration of one or more agents or modalities to a subject that is receiving administration of other agents or modalities in combination. For clarity, combination therapy does not require that the individual agents be administered together (or necessarily simultaneously) in a single composition, although in some embodiments, two or more agents or their active moieties 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" can be used to indicate the position / identity of a structural element in a molecule or composition through comparison with an appropriate reference molecule or composition. For example, in some embodiments, monomer residues in a polymer (e.g., amino acid residues in a polypeptide or nucleic acid residues in a polynucleotide) can be identified as those that "correspond" to residues in an appropriate reference polymer. For example, for purposes of simplification, residues in a polypeptide may be designated using a standard numbering system based on a reference related polypeptide, such that, for example, the amino acid that "corresponds" to the residue at position 100 is not necessarily actually the 100th amino acid in the amino acid chain, so long as it corresponds to the residue found at position 100 in the reference polypeptide. For example, various sequence alignment strategies can be utilized to identify "corresponding" residues in polypeptides and / or nucleic acids according to the present disclosure, including, for example, 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.

[0087] When the interaction between an antigen and a first antigen-binding molecule blocks, limits, inhibits, or otherwise reduces the ability of a reference binding molecule to interact with the antigen, an antigen-binding molecule such as an antibody, an antigen-binding fragment thereof, a CAR or a TCR "cross-competes" with a reference binding molecule such as an antibody or an antigen-binding fragment thereof. Cross-competition can be complete; for example, the 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; for example, the 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 epitopes as the reference antigen-binding molecule. In other embodiments, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to epitopes that are different from those of the reference antigen-binding molecule. A number of different types of competitive binding assays can be used to determine whether one antigen-binding molecule competes with another: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme 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 labeled assay, solid-phase direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeled 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 labeled RIA (Moldenhauer et 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, and the cytokine interacts with a second cell to mediate a response in the second cell. Cytokines can be endogenously expressed by cells 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, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines such as interleukin (IL)-7 and IL-15 promote the survival and proliferation of immune cells, and pro-inflammatory cytokines can promote an 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 pro-inflammatory cytokines include, but are not limited to, 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), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), 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, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0089] "Decrease" or "reduce" or "mitigate" or "lower" or "weaken" 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 a response caused by either vehicle alone (i.e., the active moiety) or a control molecule / composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount, being a decrease 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-fold, or more (e.g., 500-fold, 1000-fold) (e.g., including all integers and fractions in between greater than 1 such as 1.5, 1.6, 1.7, 1.8, etc.) of a response produced by a vehicle, which is a control composition (reference response).

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

[0091] The term "dosage form" can be used to refer to a physically distinct unit of an active agent (e.g., an antigen-binding system or antibody) for administration to a subject. Generally, each such unit contains a predetermined amount of the active agent. In some embodiments, such amount is the unit dosage amount (in whole or in part) appropriate for administration according to an administration regimen determined to correlate with a desired or beneficial result when administered to the 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 can include the administration of two or more dosage forms.

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

[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 can 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 can be from any organism including, but not limited to, humans, mice, rats, rabbits, and monkeys.

[0094] "Effector function" refers to the biological consequences of the interaction between the Fc region of an antibody and 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 function 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 understood to involve effector cells, including Fc receptors (FcR), recognizing and then killing antibody-coated target cells (e.g., cells expressing an antigen to which the antibody is bound) on their surface. Effector cells mediating 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", which is also known as adoptive cell transfer and may be abbreviated as "eACT™", is a process in which a patient's own T cells are harvested and subsequently genetically modified to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. The 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 that includes at least one co-stimulatory domain and at least one activation domain. The co-stimulatory domain can be derived from a naturally occurring co-stimulatory domain or a variant thereof, such as a variant having a truncated hinge domain ("THD"), and the activation domain can be derived from, for example, CD3-zeta. In certain embodiments, the CAR is designed to have two, three, four, or more co-stimulatory domains. The CAR scFv can be designed to target.

[0096] In some embodiments, the CAR is engineered such that the co-stimulatory 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 herein by reference in their entirety. "Adoptive cell therapy" or "ACT" involves transplanting immune cells having anti-tumor activity into a subject, such as a cancer patient. In some embodiments, ACT is a therapeutic approach that includes the use of lymphocytes having anti-tumor activity (e.g., engineered lymphocytes).

[0097] The terms "enhance", "promote", "increase", or "augment" generally refer to the ability of the compositions contemplated herein to produce, induce, or cause a greater physiological response (e.g., downstream effects) as compared to the response caused by either the vehicle or the control molecule / composition. Measurable physiological responses can include, as will be apparent from an understanding of the art and the description herein, an increase in T cell expansion, activation, persistence, and / or an increase in the killing ability of cancer cell death. An "increased" or "enhanced" amount is typically a "statistically significant" amount, being 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-fold, or more (e.g., 500-fold, 1000-fold) (e.g., all integers and decimal points in between greater than 1 such as 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by the vehicle or control composition.

[0098] An "epitope" refers to the localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or continuous epitope), or an epitope can be, for example, a combination from two or more non - contiguous regions of a polypeptide or polypeptide (conformation, non - linear, discontinuous, or discontinuous epitope). In certain embodiments, the epitope to which an antibody binds can be determined, for example, by NMR spectroscopy, X - ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange combined 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 and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations Inc.; see, for example, 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 achieved 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 descriptions of mutagenesis techniques including alanine scanning mutagenesis technology.

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

[0100] "Exogenous" refers to a drug such as a nucleic acid, gene, or protein being introduced into a cell, for example, 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 the expression of the protein beyond the level naturally found in the cell under similar conditions, for example, without the introduction of the exogenous nucleic acid.

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

[0102] As used herein, "expansion" refers to expanding a population of transduced immune cells over a predetermined period to produce a population of engineered immune cells. The predetermined period for expansion can be (i) a sufficient number of cells in a population of engineered immune cells for at least one dose to be administered to a patient, (ii) a population of engineered immune cells having a preferred proportion of naive cells compared to a typical longer process, or (iii) any suitable period that enables the production of both (i) and (ii). This period depends on 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 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.

[0103] A "fragment" or "portion" of a material or entity described herein has a structure that includes individual parts of the whole, for example, of a physical or abstract entity. In some embodiments, the fragment lacks one or more parts seen in the whole. In some embodiments, the fragment consists of or includes characteristic structural elements, domains, or portions seen 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 monomer units (e.g., residues) seen in the whole 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 monomer units (e.g., residues) seen in the whole polymer (e.g., 85 - 90%, 85 - 95%, 85 - 100%, 90 - 95%, 90 - 100%, or 95 - 100%). The whole material or entity can, 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 a fusion polypeptide if the two segments are (1) parts not essentially contained in the same peptide, and / or (2) parts not pre-linked or connected to each other in a single polypeptide, and / or (3) parts linked or connected to each other through the action of human hands. In embodiments, the CAR is a fusion protein. In one embodiment, the 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 RNA (before and / or after processing) transcribed from a gene, or a polypeptide (before and / or after modification) encoded by RNA transcribed from a gene.

[0106] The terms "genetically engineered" or "engineered", without limitation, refer to methods of modifying the genome of a cell, such as deleting a coding region or non-coding region or a part thereof, or inserting a coding region or a part thereof. In some embodiments, the modified cell is a lymphocyte, such as a T cell or an NK cell, and can be obtained from either a patient or a donor. The cell can be modified to express an exogenous construct, such as a membrane-bound interleukin-15 (IL-15)-IL-15Rα succin domain chimeric receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR) that is integrated into the genome of the cell. Manipulation generally includes manipulation by human hands. For example, a polynucleotide is considered "engineered" if two or more sequences that are not linked or connected in that order in nature are manipulated by human hands so as to be directly linked or connected to each other in the engineered polynucleotide. In the context of manipulating cells by techniques of molecular biology, a cell or organism is considered "engineered" if its genetic information is altered (e.g., a new genetic material that did not previously exist is introduced by, for example, transformation, somatic cell hybridization, transfection, transduction, or other mechanisms, or a previously existing genetic material is changed or removed by, for example, substitution or deletion mutations, or other protocols). The engineered cell can be modified to express an exogenous construct, such as a membrane-bound interleukin-15 (IL-15)-IL-15Rα succin domain chimeric receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR) that is integrated into the genome of the cell. The progeny of an engineered polynucleotide or agent are generally referred to as "engineered", even if the actual manipulation was performed on a previous entity. In some embodiments, "engineered" refers to an entity that is designed and manufactured. The term "designed" refers to an agent that (i) has its structure selected by or through human hands, (ii) is produced by a process that requires human hands, and / or (iii) is different 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 the development of normal T cells, each of the four TCR genes, α, β, γ, and δ, can rearrange to yield a very diverse set of TCR proteins.

[0108] The term "heterologous" means 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 occur naturally as a wild-type human costimulatory protein, i.e., an amino acid that 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, such as 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, the calculation of 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). Next, the nucleotides or amino acids at corresponding positions are compared. If the 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 optionally a function of the number of identical positions shared by the sequences, 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 the determination of the percent identity between two sequences can be achieved using a mathematical algorithm such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered to be "homologous" to each other 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 the percent identity, the sequences to be compared are typically aligned in a way that gives the maximum match between the sequences. An example of a computer program that can be used to determine the 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 for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acids or nucleotides (the "matched span" determined by the algorithm). In certain embodiments, standard comparison matrices (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM62 comparison matrix) are also used in the algorithm. Other algorithms are available for comparing amino acid sequences 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 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 over a relevant stretch of residues (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some embodiments, the relevant stretch is the complete sequence.In some embodiments, a contiguous stretch is 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 125, 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 can be homologs of each other.

[0111] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as discussed below, are measured by any well-known algorithm for sequence identity such as FASTA, BLAST, or Gap, and indicate the presence of at least about 95%, e.g., about 95%, about 96%, 97%, 98%, or 99% nucleotide sequence identity. 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] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 95% sequence identity, and even at least 98% or 99% sequence identity, when optimally aligned by a program such as GAP or BESTFIT using default gap weights. Typically, non-identical residue positions differ by conservative amino acid substitutions.

[0113] The terms "improve", "increase", "inhibit", and "reduce" refer to values relative to a baseline or other reference value. In some embodiments, an appropriate reference measurement may include measurements in a particular system (e.g., in a single individual) in the absence of the agent or treatment (e.g., before and / or after), or in the presence of an appropriate equivalent reference agent, otherwise under equivalent conditions. In some embodiments, an appropriate reference measurement 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 by any of these cells or the liver, which results in the selective targeting, binding, damage, destruction, 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 having or at risk of having or recurring with a disease by a method that includes inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, NK cell and T cell therapies. T cell therapies can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), and allogeneic T cell transplantation. However, one of ordinary skill in the art will understand that the pretreatment methods disclosed herein enhance the effectiveness of any adoptive 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 International Publication No. 2008 / 081035.

[0116] The T cells or NK cells for immunotherapy can be derived from any source known in the art. For example, T cells and NK cells can be differentiated in vitro from a hematopoietic stem cell population or 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, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. Further, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a blood unit collected from a subject using various techniques known to those of skill in the art, such as FICOLL™ separation and / or apheresis. Further 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 occur 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. In vitro cells can include T cells or NK cells. The term "in vivo" refers to events that occur within a multicellular organism, such as a human or non-human animal.

[0118] The term "isolated" refers to a substance that is separated from at least some of the components with which it was previously associated or would otherwise be associated, and / or a substance that is present in a composition containing one or more known or unknown contaminants in a limited or defined amount or concentration. In some embodiments, an isolated substance is separated from other components that are not the substance with which it was previously associated, for example, other components or contaminants with which the substance was previously or would otherwise be associated, by 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%). In certain instances, a substance is isolated when it is present in a composition containing the same or similar types of molecules in a limited or reduced amount or concentration. For example, in certain instances, a nucleic acid, DNA, or RNA substance is isolated when it is present in a composition containing nucleic acid, DNA, or RNA molecules that are not the substance in a limited or reduced amount or concentration. For example, in certain instances, a polypeptide substance is isolated when it is present in a composition containing polypeptide molecules that are not the substance in a limited or reduced amount or concentration. 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 is an amount that is 100% or less of the amount of the substance in the composition, for example, 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, the isolated substance has a purity of 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%). A substance is "pure" if it is substantially free of other components or contaminants. In some embodiments, a substance 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.), and in such embodiments, the isolation or percent purity of the substance is calculated without including such carriers or excipients.

[0119] A "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 succimeric polypeptide, a chimeric antigen receptor, and / or an scFv, or one or more of these polypeptides. The linker can be composed of flexible residues such as glycine and serine such that adjacent protein domains can move freely relative to each other. A longer linker may be used if it is desired to ensure that two adjacent domains do not sterically interfere with each other. The linker may or may not be 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, porcine teschovirus (P2A), the CHYSEL sequence of equine rhinitis A virus (T2A) (SEQ ID NO: 1), or combinations, variants, and functional equivalents thereof. In other embodiments, the linker sequence Asp-Val / Ile-Glu-X-Asn-Pro-Gly (2A) -Pro (2B)It may contain the motif (SEQ ID NO: 2). Other linkers include non-cleavable linkers. To implement the present disclosure, a number of linkers including "flexible linkers" are used. The latter is rich in glycine. 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 - element agent that connects different elements to each other. For example, a polypeptide containing two or more functional or structural domains can include a stretch of amino acids between such domains that link them to each other. In some embodiments, a polypeptide containing a linker element has an overall structure of the general form S1 - L - S2, where S1 and S2 can be the same or different and represent two domains associated with each other 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 is 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, 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 link 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 connect the C - terminus of an IL - 15Rα sushi - domain polypeptide to a transmembrane domain. In some embodiments, the linker tends not to adopt a rigid three - dimensional structure and instead is characterized by providing flexibility to the polypeptide. In another example, it can be used to link to one or more polypeptides expressed such as CARs, TCRs, and / or membrane - bound IL - 15 - IL - 15Rα sushi - domain chimeric polypeptides disclosed herein. In some examples, CARs or TCRs and membrane - bound IL - 15 - IL - 15Rα sushi - domain chimeric polypeptides are linked by a cleavable linker such that, for example, they are expressed as a single peptide and then cleaved intracellularly.

[0121] The term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cytotoxic) lymphocyte that represents a component of the innate immune system. NK cells reject tumor and virus-infected cells. It acts through the process of apoptosis or programmed cell death. Since they do not require activation to kill cells, they are called "natural killer". T cells play a role in cell-mediated immunity (without antibody involvement). Its T cell receptor (TCR) differentiates from other lymphocyte types. The thymus, a differentiated organ of the immune system, is mainly responsible for the maturation of T cells. Six types of T cells, namely, helper T cells (e.g., CD4+ cells), cytotoxic T cells (TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells, also known as), memory T cells ((i) stem memory T SCM cells are, like naive cells, CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3 and LFA-1 and exhibit a number of functional attributes unique to memory cells), (ii) central memory T CM cells express L-selectin and CCR7 and secrete IL-2, but they do not secrete IFNγ or IL-4, (iii) effector memory T EM cells do not express L-selectin or CCR7 and produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (Natural Killer T cells, NKT) and gamma delta T cells exist. On the other hand, B cells play a role in humoral immunity (with antibody involvement). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and turn into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, and their name is derived from here.

[0122] The term "neutralize" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the biological activity 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, either directly or through indirect means (such as a conformational 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. The nucleic acid can be DNA, RNA, or a combination thereof. In some embodiments, the nucleic acid comprises one or more natural nucleic acid residues. In some embodiments, the nucleic acid is composed of one or more nucleic acid analogs. In some embodiments, the nucleic acid is 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, regeneration in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid has a length of 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, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues (e.g., 20-100, 20-500, 20-1000, 20-2000, or 20-5000, or more residues). In some embodiments, the nucleic acid is partially or wholly single-stranded, and in some embodiments, the nucleic acid is partially or wholly double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is a complement of a sequence encoding a polypeptide.

[0124] "Operably linked" refers to an arrangement where the recited components are in a relationship that enables them to function in their intended manner. For example, a control element "operably linked" to a functional element is associated such that the expression and / or activity of the functional element is achieved under conditions compatible with the control element. In an embodiment, the promoter is operably linked to nucleus a.

[0125] "Patient" includes any human being 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 a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of the protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, which are generally also referred to as peptides, oligopeptides, and oligomers in the art, and long chains, which are generally referred to as proteins in the art, and there are many types of them. "Polypeptide" includes, for example, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include 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 that recipient or the benefit to the recipient outweighs 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 components of the composition and not be harmful to the recipient or the benefit to the recipient must outweigh any harmful effects. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material involved in the transport or delivery of 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 benefit to the recipient must outweigh any harmful effects. Some examples of materials that can function 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed 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; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffering 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 present in a unit dosage amount suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, the pharmaceutical composition may be formulated for administration in solid or liquid form, including, but not limited to, forms adapted for the following: oral administration, such as aqueous solutions (aqueous or non-aqueous solutions or suspensions), tablets, such as buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection as a sterile solution or suspension, or as a sustained release formulation; topical administration, such as in the form of creams, ointments, or sustained release patches or sprays applied to the skin, lung, or oral cavity; intravaginal or rectal, such as in the form of pessaries, creams, or foam; sublingual; for the eye; transdermal; or for the nasal, lung, and other mucosal surfaces.

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

[0130] The term "reference" describes the standard or control against which a comparison is made. For example, in some embodiments, a target agent, animal, individual, population, sample, sequence, or value 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 simultaneously with the target test, measurement, or determination. In some embodiments, the reference or control is optionally a reference or control from the past embodied in a tangible medium. Generally, the reference or control is determined or characterized under conditions or circumstances equivalent to those under evaluation. Where the similarity is sufficient to justify the dependence on and / or comparison to the selected reference or control.

[0131] "Regulatory T cells" ("Tregs", "Treg cells", or "Tregs") refers to a lineage of CD4+ T lymphocytes that are involved in the control of specific immune activities, such as responses to autoimmunity, allergy, and infection. Regulatory T cells can regulate the activity of T cell populations and can also affect certain innate immune cell types. Tregs can be identified by the expression of the biomarkers CD4, CD25, and Foxp3, as well as low expression of CD127. Naturally occurring Treg cells typically constitute about 5-10% of peripheral CD4+ T lymphocytes. However, Treg cells within the tumor microenvironment (i.e., tumor-infiltrating Treg cells) can constitute up to 20-30% of the total CD4+ T lymphocyte population.

[0132] The term "sample" generally refers to an aliquot of a material obtained from 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 can include cells or organisms such as a cell population, a tissue, or an animal (e.g., a human). In some embodiments, the source of interest includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid can include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chyme, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, ascites, pleural fluid, pus, catarrhal secretions, saliva, sebum, semen, serous fluid, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or components thereof. In some embodiments, the biological fluid can include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, the biological fluid can include plant exudates. In some embodiments, the biological tissue or sample can be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral swab, nasal swab, skin swab, or vaginal swab), scraping, surgery, washing (e.g., bronchoalveolar, duct, nasal, eye, oral, uterine, vaginal, or other wash). In some embodiments, the biological sample includes cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as apparent from the context, the term "sample" refers to a preparation obtained by processing a 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 obtained by extracting from the sample or 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" antibodies, "single-chain antibody variable fragments", or "scFv" antibodies refer to forms of antibodies that contain only the variable regions of the heavy and light chains linked by a linker peptide.

[0134] The term "cancer stage" refers to a qualitative or quantitative assessment of the level of cancer progression. In some embodiments, the criteria used to determine cancer stage can include, but are not limited to, one or more of where in the body the cancer is located, 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, so-called TNM systems can be used for staging, according to which 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 with cancer, and M refers to whether the cancer has metastasized. In some embodiments, cancer can be referred to as stage 0 (abnormal cells are present without spreading to nearby tissue, also called carcinoma in situ or CIS, which is not cancer but can become cancer), stages I - III (cancer is present, and the larger the number, the larger the tumor and the more it has spread to nearby tissue), or stage IV (cancer has spread to distant parts of the body). In some embodiments, cancer can be assigned to a stage selected from the group consisting of: in situ, localized (cancer is limited to where it started and shows no signs of spreading), regional (cancer has spread to nearby lymph nodes, tissue, or organs), distant (cancer has spread to distant parts of the body), and unknown (not enough information to determine the stage).

[0135] "Stimulation" refers to the primary response induced by the binding of a stimulatory molecule to its cognate ligand, and the binding mediates signal transduction events. A "stimulatory molecule" is a molecule on a T cell, such as the 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., APC, dendritic cell, B cell, etc.), specifically binds to a stimulatory molecule on a T cell and thereby can mediate, but is not limited to, primary responses by the T cell such as activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, anti-CD3 antibodies (such as OKT3), MHC class I molecules loaded with peptides, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies.

[0136] The term "therapeutic agent" can refer to any agent that induces a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be a population of model organisms or human subjects. In some embodiments, the appropriate population can be defined by various criteria such as specific age groups, genders, genetic backgrounds, existing clinical conditions, etc., depending on the presence or absence of biomarkers, etc. In some embodiments, a therapeutic agent is a substance that can be used for the alleviation, improvement, reduction, inhibition, prevention, delay in onset, reduction in severity, and / or reduction in 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 approved or needs to be approved by a government agency before it can be commercially available for administration to humans. In some embodiments, a therapeutic agent is an agent that requires a medical prescription for administration to humans.

[0137] The "therapeutically effective amount", "effective dose", "effective amount", or "therapeutically effective dosage" 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 the development of a disease or promotes regression of a disease as demonstrated by a decrease in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or prevention of a disorder or disability resulting from the disease. The ability of a therapeutic agent to promote regression of a disease can be evaluated using a variety of methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by assaying the activity of the agent 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, DNA vector, RNA vector, adenoviral vector, baculoviral vector, Epstein-Barr viral vector, papovaviral vector, vaccinia viral vector, herpes simplex viral vector, adeno-associated vector, 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 being transformed and / or the nucleic acid being inserted. Methods of transformation can 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 can replicate as part of an autonomously replicating plasmid or the host chromosome. In some embodiments, transformed cells can express the introduced nucleic acid.

[0140] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on, or administration of an active agent to, a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or a biochemical hallmark 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 of a subject who does not exhibit signs of a related disease, disorder, and / or condition, and / or of a subject who exhibits only early signs of a disease, disorder, and / or condition. In some embodiments, such treatment can be of a subject who exhibits one or more definitive signs of a related disease, disorder, and / or condition. In some embodiments, treatment can be of a subject diagnosed as having a related disease, disorder, and / or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing a related disease, disorder, and / or condition.

[0141] The term "vector" refers to a recipient nucleic acid molecule that contains or has been modified to incorporate the 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, into which an additional DNA segment can be ligated to the viral genome. Certain vectors are capable of autonomous replication in the host cells 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) may integrate into the genome of the host cell upon introduction into the host cell and are thereby replicated with the host genome. Further, certain vectors contain sequences that direct the expression of an inserted gene to which they are operably linked. Such vectors may be referred to herein as "expression vectors". Standard techniques can be used to manipulate vectors, as can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference.

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

[0143] A "transmembrane domain" is a domain of a polypeptide that, when present in the corresponding endogenous polypeptide when expressed in mammalian cells, comprises at least one continuous amino acid sequence that traverses the lipid bilayer. For example, a transmembrane domain, when present in the corresponding endogenous polypeptide when expressed in mammalian cells, can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 continuous amino acid sequences, each of which traverses the lipid bilayer. A transmembrane domain can comprise, for example, at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) continuous amino acid sequence having an α-helix secondary structure in the lipid bilayer (when present in the corresponding endogenous polypeptide when expressed in mammalian cells, traversing the lipid bilayer). In some embodiments, a transmembrane domain can comprise two or more continuous amino acid sequences (when present in the corresponding endogenous polypeptide when expressed in mammalian cells, each traversing the lipid bilayer) that form a β-barrel secondary structure in the lipid bilayer. 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 comprises at least one transmembrane domain that traverses 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] A "signal sequence" means a peptide sequence that is generally present at the N-terminus of a newly synthesized protein that induces entry into the secretory pathway.

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

[0147] The term "persistence" refers to the ability to maintain, for a period of time, one or more transplanted immune cells administered to a subject, or their progeny (e.g., NK cells or differentiated or mature T cells), at a detectable level within the subject. As used herein, 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 detectable period of the transplanted immune cells in the subject after 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. In addition, the in vivo persistence of one or more transplanted immune cells can be extended 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 can use methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology, which are within the scope of the art unless otherwise indicated, and many of which are described below for purposes of illustration. Such techniques are fully described in the literature.For example, see the monographs of journals such as 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 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, N.Y., 1998) Current Protocols in Immunology Q.E. 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. However, it should be understood that the detailed description is given by way of illustration only and not limitation, although it shows embodiments of the present disclosure.

[0150] A membrane-bound interleukin-15 (IL-15)-IL-15Rα succinimide domain chimeric polypeptide is disclosed. In embodiments, the membrane-bound IL-15-IL-15Rα succinimide domain chimeric polypeptide comprises a signal peptide and is thought to be directed to the cell membrane by that signal sequence when expressed from its corresponding nucleic acid. In embodiments, the signal sequence is cleaved so that the presented membrane-bound IL-15-IL-15Rα succinimide domain chimeric polypeptide does not include a signal sequence. In embodiments, the membrane-bound IL-15-IL-15Rα succinimide domain chimeric polypeptide comprises a transmembrane domain for anchoring the membrane-bound IL-15-IL-15Rα succinimide domain chimeric polypeptide to the cell membrane.

[0151] Interleukin 15 (IL-15) is a cytokine that has structural similarity to interleukin-2 (IL-2). Similar to IL-2, IL-15 binds to a complex composed of the IL-2 / IL-15 receptor β chain (CD122) and the common γ chain (γ-C, CD132) and signals through it. IL-15 is secreted by monocytes (and some other cells) after infection with a virus (or viruses). This cytokine induces the proliferation of natural killer cells, i.e., cells of the innate immune system whose main 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 thus may negatively regulate each other's activities. The number of CD8+ memory cells has been shown to be controlled by the balance between this cytokine and IL2. This cytokine induces the activation of JAK kinases and the phosphorylation and activation of the transcription 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 may have any amino acid sequence known in the art, for example, those available at NCBI Gene ID:3600 (updated February 6, 2021), which is specifically incorporated herein by reference.

[0152] In certain embodiments, the 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the mature form of IL-15 or a fragment thereof having an activity similar to that of the full-length mature form. In an embodiment, the IL-15 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 6. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 6).

[0153] 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. The receptors for IL-15 and IL-2 share two subunits, IL2R beta and IL2R gamma. IL-15Rα is structurally related to IL2Rα, an additional IL2-specific α subunit 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 apoptosis inhibitors BCL2L1 / BCL2-XL and BCL2. As used herein, 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, for example, those available at NCBI Gene ID:3601 (updated February 3, 2021), which is specifically incorporated herein by reference. Further, unless otherwise stated, the IL-15Rα sushi domain refers to the sushi domain of IL-15Rα that includes, or consists of, amino acid residues 49-162 of the full-length IL-15Rα polypeptide.

[0154] In certain embodiments, the 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the mature form of IL-15Rα or a fragment thereof having activity similar to the full-length mature form. In embodiments, the IL-15Rα polypeptide comprises the active form of the IL-15Rα polypeptide of amino acids 49-162. 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 7. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRD (SEQ ID NO: 7). In further 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, for example, 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 the IL-15Rα sushi domain subunit can be linked as described herein. In certain embodiments, the linker sequence is Ser(Gly 4It contains a set of glycine and serine repeats such as Ser)n(SEQ ID NO: 8), where n is a positive integer of 1 or more and less than 10. In one embodiment, the linker is Ser(Gly 4 Ser) 3 (SEQ ID NO: 9) or Ser(Gly 3 Ser) 1 (Gly 4 Ser) n (Gly 3 Ser) 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 the linker sequence.

[0156] In embodiments, the polypeptides disclosed herein include a signal sequence, such as a heterologous signal sequence, such as an IgE signal sequence, a kappa signal sequence, a CD8 signal sequence, or any peptide having 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 the AGS (SEQ ID NO: 4) linker. In embodiments, the 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 include a transmembrane domain sequence, such as a heterologous transmembrane domain, such as a FAS transmembrane domain sequence, or an IL-7 transmembrane domain sequence, or any peptide having essentially equivalent activity.

[0160] In an embodiment, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide comprises a FAS transmembrane domain sequence. This sequence is thought to result in surface expression of the monomeric membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide. In an embodiment, the FAS transmembrane domain sequence has 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) and comprises an amino acid sequence.

[0161] In an embodiment, the FAS transmembrane domain sequence has 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) and comprises an amino acid sequence.

[0162] In embodiments, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide comprises a heterodimerization domain such that when expressed, the membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide forms a homodimer. 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 thought to result in surface expression of a homodimeric membrane-bound IL-15-IL-15Rα sushi domain chimeric polypeptide that forms a stable homodimer by 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 comprises a set of glycine and serine repeats such as (Gly 4 Ser) n (SEQ ID NO: 24), where n is a positive integer from 1 or more to less than 10. In one embodiment, the linker is (Gly 4 Ser) 1 (SEQ ID NO: 25) or (Gly 4 Ser) 2 (SEQ ID NO: 26) or (Gly 4 Ser) 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, for example, 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, for example, 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, for example, 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 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 44. 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, for example, 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, for example, 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 94. MDWTWILFLVAAATRVHSEQKLISEEDLAGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGGGGGSGGGGSRSNLGWLCLLLLPIPLIVWVKRKEVQKT (SEQ ID NO: 94).

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

[0172] In embodiments, the nucleic acid encoding the 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 31. GCTGGAAGCAATTGGGTGAACGTGATCTCCGACCTCAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGATGCCACACTCTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTTACAGCCATGAAATGCTTTCTGCTGGAGCTGCAAGTGATCTCTCTGGAGTCCGGAGATGCTTCCATCCACGACACAGTGGAGAATCTGATCATTCTGGCTAACAACTCCCTCTCCAGCAACGGCAATGTCACAGAGTCCGGCTGCAAAGAGTGTGAAGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTCCAGATGTTCATCAACACCTCCTCC (SEQ ID NO: 31).

[0173] In an embodiment, the nucleic acid encoding the 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 32. GCCGGCAGCAACTGGGTCAACGTGATCTCCGATCTGAAGAAGATCGAAGATCTGATCCAGTCCATGCACATCGATGCCACACTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAAGTTACAGCCATGAAGTGCTTTCTGCTCGAACTGCAAGTGATTTCTCTGGAGAGCGGAGATGCCAGCATCCACGACACCGTGGAGAATCTGATCATTCTGGCCAACAACTCTCTGAGCAGCAACGGCAATGTGACAGAGTCCGGCTGTAAGGAGTGCGAGGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATTGTCCAAATGTTCATCAACACCAGCAGC (SEQ ID NO: 32).

[0174] In an embodiment, the nucleic acid encoding the 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 33. ATTACATGCCCTCCCCCCATGTCCGTGGAACATGCCGACATCTGGGTGAAGTCCTACTCTCTGTACTCGCGTGAACGTTATATCTGCAACAGCGGCTTTAAGAGGAAGGCCGGAACCTCCTCTCTGACCGAATGTGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTAGCCTCAAGTGTATTAGGGAC (SEQ ID NO: 33).

[0175] In an embodiment, the nucleic acid encoding the 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, for example, 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 an embodiment, the nucleic acid encoding the 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 35. CCTATTCTGCTGACATGCCCCACCATCTCCATCCTGTCTTTTTTTTCTGTTGCTCTGCTGGTGATTCTGGCTTGCGTGCTGTGG (SEQ ID NO: 35).

[0177] In an embodiment, the nucleic acid encoding the 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, for example, 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 an embodiment, the nucleic acid encoding the 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, for example, 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 an embodiment, the nucleic acid encoding the 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, for example, 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 an embodiment, the nucleic acid encoding the 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, for example, 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 an embodiment, the nucleic acid encoding the 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 40. GCTGGAAGCAATTGGGTGAACGTGATCTCCGACCTCAAGAAGATCGAGGATCTGATCCAGTCCATGCACATCGATGCCACACTCTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTTACAGCCATGAAATGCTTTCTGCTGGAGCTGCAAGTGATCTCTCTGGAGTCCGGAGATGCTTCCATCCACGACACAGTGGAGAATCTGATCATTCTGGCTAACAACTCCCTCTCCAGCAACGGCAATGTCACAGAGTCCGGCTGCAAAGAGTGTGAAGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTCCAGATGTTCATCAACACCTCCTCCGGCGGCGGCAGCGGCGGCGGCGGATCCGGCGGAGGAGGCAGCGGAGGAGGAGGAAGCGGAGGAGGCTCCATTACATGCCCTCCCCCCATGTCCGTGGAACATGCCGACATCTGGGTGAAGTCCTACTCTCTGTACTCGCGTGAACGTTATATCTGCAACAGCGGCTTTAAGAGGAAGGCCGGAACCTCCTCTCTGACCGAATGTGTGCTGAACAAGGCCACCAATGTGGCTCACTGGACCACACCTAGCCTCAAGTGTATTAGGGACGGCGGCGGAGGATCCGGAGGAGGCGGATCTAGAAGCAATCTGGGCTGGCTGTGTCTGCTGCTGCTCCCCATCCCTCTGATTGTGTGGGTCAAGAGGAAGGAGGTCCAGAAAACC(SEQ ID NO: 40).

[0182] In an embodiment, the nucleic acid encoding the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide comprises a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO: 42 (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%). GCTGGAAGCAATTGGGTGAACGTGATCTCCGACCTCAAAAAGATCGAGGATCTGATCCAGTCCATGCACATCGATGCCACACTCTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTTACAGCAATGAAATGCTTTCTGCTGGAGTTGCAAGTAATCTCCCTGGAGTCCGGAGATGCTTCCATCCACGACACAGTGGAGAATTTAATCATTCTGGCTAACAATTCCCTCTCGTCTAATGGCAATGTCACTGAGAGCGGCTGTAAAGAGTGTGAAGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTCCAAATGTTCATCAACACCTCGTCCGGGGGCGGCTCCGGGGGAGGAGGATCGGGGGGAGGAGGAAGCGGAGGTGGAGGAAGCGGTGGAGGGTCCATTACATGCCCTCCCCCCATGTCCGTGGAACATGCCGACATATGGGTAAAGTCCTACTCTCTGTACTCGCGGGAACGTTATATCTGCAACAGCGGCTTTAAGAGAAAGGCCGGAACATCtTCTCTGACCGAATGTGTGCTGAACAAGGCCACAAATGTGGCTCACTGGACCACGCCTAGCCTCAAGTGTATTAGGGACGGCGGCGGAGGTTCCGGTGGCGGGGGCTCTAGATCGAATCTGGGCTGGCTGTGTCTGCTGCTGCTCCCCATCCCTCTGATTGTGTGGGTTAAGCGAAAAGAGGTCCAGAAAACCTAA(SEQ ID NO: 42)

[0183] In an embodiment, the nucleic acid encoding the 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 41. GCCGGCAGCAACTGGGTCAACGTGATCTCCGATCTGAAGAAGATCGAAGATCTGATCCAGTCCATGCACATCGATGCCACACTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAAGTTACAGCCATGAAGTGCTTTCTGCTCGAACTGCAAGTGATTTCTCTGGAGAGCGGAGATGCCAGCATCCACGACACCGTGGAGAATCTGATCATTCTGGCCAACAACTCTCTGAGCAGCAACGGCAATGTGACAGAGTCCGGCTGTAAGGAGTGCGAGGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATTGTCCAAATGTTCATCAACACCAGCAGCGGCGGCGGCTCCGGCGGCGGAGGCTCCGGCGGAGGCGGATCCGGCGGCGGCGGATCCGGCGGAGGATCCATTACATGCCCCCCTCCCATGTCCGTGGAACACGCCGACATCTGGGTGAAGAGCTACTCTCTGTACAGCAGAGAGCGTTACATCTGCAACAGCGGCTTTAAGAGGAAAGCCGGCACCAGCAGCCTCACAGAGTGCGTGCTCAACAAGGCCACCAACGTCGCCCATTGGACCACCCCCTCTCTGAAGTGTATTAGGGACGGCGGCGGAGGAAGCGGAGGAGGAGGAAGCCCTATTCTGCTGACATGCCCCACCATCTCCATCCTGTCTTTTTTTTCTGTTGCTCTGCTGGTGATTCTGGCTTGCGTGCTGTGGTGA (SEQ ID NO: 41).

[0184] In an embodiment, the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide (including the FAS transmembrane domain and not including the 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, for example, 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 an embodiment, the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide (including the IL-7 transmembrane domain and not including the 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 97. ATGGACTGGACATGGATTCTGTTTCTGGTGGCCGCCGCCACAAGAGTGCACTCCAACTGGGTCAACGTGATCTCCGATCTGAAGAAGATCGAAGATCTGATCCAGTCCATGCACATCGATGCCACACTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAAGTTACAGCCATGAAGTGCTTTCTGCTCGAACTGCAAGTGATTTCTCTGGAGAGCGGAGATGCCAGCATCCACGACACCGTGGAGAATCTGATCATTCTGGCCAACAACTCTCTGAGCAGCAACGGCAATGTGACAGAGTCCGGCTGTAAGGAGTGCGAGGAGCTGGAGGAGAAAAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATTGTCCAAATGTTCATCAACACCAGCAGCGGCGGCGGCTCCGGCGGCGGAGGCTCCGGCGGAGGCGGATCCGGCGGCGGCGGATCCGGCGGAGGATCCATTACATGCCCCCCTCCCATGTCCGTGGAACACGCCGACATCTGGGTGAAGAGCTACTCTCTGTACAGCAGAGAGCGTTACATCTGCAACAGCGGCTTTAAGAGGAAAGCCGGCACCAGCAGCCTCACAGAGTGCGTGCTCAACAAGGCCACCAACGTCGCCCATTGGACCACCCCCTCTCTGAAGTGTATTAGGGACGGCGGCGGAGGAAGCGGAGGAGGAGGAAGCCCTATTCTGCTGACATGCCCCACCATCTCCATCCTGTCTTTTTTTTCTGTTGCTCTGCTGGTGATTCTGGCTTGCGTGCTGTGGTGA (SEQ ID NO: 97).

[0186] In an embodiment, the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide (in the context of an anti-CD19 CAR, including the FAS transmembrane domain and not including the myc tag) comprises a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO: 98 (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%).

[0187] In an embodiment, the membrane-bound interleukin-15 (IL-15)-IL-15Rα succinimide domain chimeric polypeptide (in the context of the anti-CD19 CAR, including the IL-7 transmembrane domain and not including the myc tag) comprises a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO: 99 (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%).

[0188] In an embodiment, the membrane-bound interleukin-15 (IL-15)-IL-15Rα succinimide domain chimeric polypeptide (including the FAS transmembrane domain and not including the myc tag) comprises a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO: 100 (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%).

[0189] In an embodiment, the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric polypeptide (in the context of an anti-CD19 CAR, including the FAS transmembrane domain and not including the myc tag) comprises a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO: 101 (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%).

[0190] The present disclosure provides methods and compositions for improving the effectiveness of antigen-binding systems such as CARs and TCRs, including binding motifs that bind to a target antigen, such as a tumor antigen. 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 a tumor antigen or a pathogen antigen.

[0191] A chimeric antigen receptor (CAR) is an engineered receptor that can direct or redirect T cells (e.g., patient or donor T cells) to target a selected antigen. A CAR can be engineered to recognize an antigen and, upon binding to that antigen, activate immune cells to attack and destroy cells bearing that antigen. When these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. A CAR generally includes an extracellular binding motif that mediates antigen binding, a transmembrane domain that is understood to span or traverse the cell membrane when the antigen-binding system is present on the cell surface or cell 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, the 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 that optionally includes a hinge domain and one or more spacers. In second-generation CARs, a co-stimulatory domain (such as CD28, 4-1BB, or OX-40) is introduced together with the signaling domain (e.g., CD3ζ). Third-generation CARs include a second co-stimulatory domain.

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

[0194] The antigen-binding system may include VH and VL. In some embodiments, VH and VL are linked by a linker (L).

[0195] In some embodiments, the antigen-binding system further includes a co-stimulatory 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. Since the binding motif can be engineered to be expressed as part of a single chain together with other CAR components, it is used at least in part in a chimeric antigen receptor. See, for example, U.S. Patent Nos. 7,741,465 and 6,319,494, and 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 the binding motif domain in a CAR. The binding motif or scFv is a single-chain antigen-binding fragment that includes a heavy-chain variable domain and a light-chain variable domain, and the heavy-chain variable domain and the light-chain variable domain are linked or connected to each other. See, for example, U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar 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 parental antibody, the binding motif may retain some, all, or essentially all of the binding of the parental antibody to 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, adrenergic receptor beta 3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), beta2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (a cancer gene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral 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 ImprintedSites), 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, 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 called CD2 subset 1, CRACC, CD319, and 19A24), CTLA-4, cyclin B1, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 (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), Endocan, Epithelial Cell Adhesion Molecule (EPCAM), Ephrin A-type Receptor 2 (EphA2), Ephrin B2, Receptor Tyrosine-Protein Kinase erb-B2, 3,4 (erb-B2,3,4), ERBB, ERBB2 (Her2 / neu), ERG (Transmembrane Protease, Serine 2 (TMPRSS2) ETS Fusion Gene), ETA, ETS Variant 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), Folic Acid Binding Protein (FBP), Folic Acid Receptor 1, Folic Acid Receptor Alpha, Folic Acid 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 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), GP 100, Hexasaccharide Portion of GloboH Glycosphingolipid (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 (HPVE7), heat shock protein 70-2 variant (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, IgGl, immunoglobulin lambda-like polypeptide 1 (IGLL1), IL-6, interleukin 11 receptor alpha (IL-11Ra), IL-13, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), insulin-like growth factor I receptor (IGF1-R), integrin alpha5beta1, integrin alphavbeta3, 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 recognized by T cells (MelanA or MARTI), MelanA / MARTl, mesothelin, MAGEA3, 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 inhibiting factor (MIS) receptor type II, v-myc avian myelocytomatosis virus oncogene neuroblastoma-derived homolog (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, breast differentiation antigen (NY-BR-1), NY-ESO-1, cancer fetal antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, polySA, proacrosin-binding protein sp32 (OY-TES1), p53, p53 variants, pannexin 3 (PANX3), prostate 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, prostain, 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 variants, 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 (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-alpha precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGFβ2, TGF-beta, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TIM1, TIM2, TIM3, TnIt is selected from the group consisting of Ag, TRAIL-R1, TRAIL-R2, tyrosine-related protein 2 (TRP-2), thyroid-stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosine kinase, 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. 2015 / 142675.

[0198] The CAR may include one or more antigen-binding domains that bind to the 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 includes a co-stimulatory 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 includes at least a binding domain that binds to the target antigen, a co-stimulatory domain, an extracellular domain, a transmembrane domain, and a CD3-zeta or CD3-epsilon activation domain.

[0199] In certain embodiments, the CAR contemplated herein may include linker residues added between various domains, for example, between the VH domain and the VL domain, for proper spatial conformation of the molecule. The CAR contemplated herein may include one, two, three, four, or five or more linkers. In some embodiments, the length of the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or amino acids of 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] Exemplary examples of linkers include glycine polymers (G)n, 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 can thus function as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine further accesses the phi-psi space more 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)). One of ordinary skill in the art will recognize that the design of the CAR in some embodiments may include all or in part a flexible linker, whereby the linker may include a flexible linker and one or more moieties that impart a lower flexible structure to provide the desired CAR structure. In one embodiment, any of the constructs described herein may include a "GS" linker. In another embodiment, any of the constructs described herein may include 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 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 the amino acid sequence of 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 described below. GGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGC (SEQ ID NO: 46) GGGAGCACTAGCGGCTCTGGCAAACCTGGATCTGGCGAGGGATCTACCAAGGGC (SEQ ID NO: 47), GGCTCCACCAGCGGAAGCGGCAAGCCAGGCTCAGGCGAAGGATCTACAAAAGGC (SEQ ID NO: 48), or GGGAGCACAAGCGGCTCTGGCAAACCTGGATCCGGCGAGGGATCTACCAAGGGC (SEQ ID NO: 49).

[0201] In an embodiment, the CAR comprises an scFv further comprising a variable region linker sequence. The "variable region linker sequence" 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 the specific binding affinity to the same target molecule as an antibody comprising the same light and heavy chain variable regions. In one embodiment, the variable region linker 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, one or more "spacer domains" follow the binding domain of the CAR, which refers to a region that moves the antigen-binding domain away from the effector cell surface to allow for proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). The spacer domain can be derived from any of natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is part of an immunoglobulin and includes, but is not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0203] The binding domain of the 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 an 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 "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 can also provide flexibility to access the target antigen. The hinge includes an immunoglobulin-like hinge domain. The CAR generally includes one or more hinge domains between the binding domain and the transmembrane domain. The hinge domain can be derived from any of natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. In some embodiments, the antigen-binding system is an immunoglobulin-like hinge domain, from an immunoglobulin-like hinge domain, or includes a hinge derived from an immunoglobulin-like hinge domain (e.g., including all or a fragment of the immunoglobulin-like hinge domain). In some embodiments, the hinge domain is from an immunoglobulin or derived from an immunoglobulin. In some embodiments, the hinge domain is selected from the hinge of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, or a fragment thereof.

[0204] The hinge can be derived from a natural source or a synthetic source. In some embodiments, the antigen-binding system is 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), CD49d (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 (LIGHT), 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 co-stimulatory factor (ICOS), LFA-1 (CD11a / CD18), NKG2C,It can include a hinge that is 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, or is from these, or is derived from these (e.g., including all or a fragment thereof).

[0205] In some embodiments, the CAR can include a hinge that is the hinge of CD8 alpha, or is from the hinge, or is derived from the hinge (e.g., including all or a fragment thereof). In some embodiments, the CAR can include a hinge that is the hinge of CD28, or is from the hinge, or is derived from the hinge (e.g., including all or a fragment thereof). In some embodiments, the hinge is, or is from, or is derived from a fragment of the hinge of CD8 alpha or the hinge of CD28, and the fragment is smaller than the whole. In some embodiments, the fragment of the CD8 alpha hinge or the CD28 hinge includes 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 at the N-terminus and / or C-terminus of the CD8 alpha hinge or the CD28 hinge.

[0206] In an embodiment, the hinge domain includes a CD28 hinge region. In an embodiment, 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 230. (IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 50)). In an embodiment, 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having the sequence as follows. attgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 51).

[0207] In an embodiment, the hinge domain includes a truncated CD28 hinge region (CD28T) hinge region as disclosed in International Patent Application No. PCT / US2017 / 025351, filed on March 31, 2017, which is hereby incorporated by reference in its entirety. In an embodiment, the CAR includes a CD28T hinge 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 52. (LDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 52)). In an embodiment, the CD28 hinge domain has 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 as follows and is encoded by a nucleic acid having at least 75% sequence identity to the nucleic acid having the sequence as follows. ctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 53).

[0208] In an embodiment, the hinge domain includes a CD8α hinge region. In an embodiment, the CAR described herein includes 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, for example, 85 - 90%, 85 - 95%, 85 - 100%, 90 - 95%, 90 - 100%, or 95 - 100%) to SEQ ID NO: 54 (FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 54)). In an embodiment, the hinge domain derived from CD8α has 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 described below and is encoded by a nucleic acid having at least 75% sequence identity to the nucleic acid having the sequence described below. TTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGAC (SEQ ID NO: 55).

[0209] The polynucleotide sequences and polypeptide sequences of these hinge domains are known. In some embodiments, the polynucleotide encoding the 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 has the attribute of being present within the membrane when present in a molecule on the cell surface or cell membrane (e.g., spanning part or all of the cell membrane). The co-stimulatory domain of the antigen-binding system of the present disclosure may further comprise a transmembrane domain and / or an intracellular signaling domain. It is not necessary for all amino acids in the transmembrane domain to be present within the membrane. For example, in some embodiments, the transmembrane domain is characterized in that a specified stretch or portion of the protein is substantially located within the membrane. Amino acid sequences or nucleic acid sequences can be analyzed using various algorithms for predicting intracellular localization of proteins (e.g., transmembrane localization). Programs such as 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 type. In some embodiments, a transmembrane domain that is naturally associated with the binding motif and / or intracellular domain is selected. In some examples, the transmembrane domain includes one or more amino acid modifications (e.g., deletions, insertions, and / or substitutions) to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, for example, and to minimize interactions with other members of the receptor complex.

[0212] The transmembrane domain can be derived from either a natural or synthetic source. When 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 chains 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 / CD137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRTAM, 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 co-stimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand that binds to CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), 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, Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavages, or combinations thereof (e.g., may include at least a transmembrane domain). In some embodiments, the transmembrane domain can be synthetic (and can include mainly hydrophobic residues such as leucine and valine, for example). In some embodiments, a triplet of phenylalanine, tryptophan, and valine 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) can form a linkage between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine doublet.;

[0213] In embodiments, the CARs described herein include a TM domain derived from CD28 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 SEQ ID NO: 56 (FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 56)). In embodiments, the TM domain derived from CD28 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 the sequence: ttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg (SEQ ID NO: 57). ttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg(SEQ ID NO: 57).

[0214] In an embodiment, the CAR described herein comprises a TM and an intracellular domain derived from CD8α 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 58. IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 58)). In an embodiment, the TM 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 according to: ATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAAC (SEQ ID NO: 59).

[0215] The polynucleotide sequences and polypeptide sequences of the transmembrane domains provided in this specification are known. In some embodiments, the polynucleotide encoding the 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 linkage between any one or several of the extracellular domain, transmembrane domain, and intracellular domain of the CAR.

[0216] The intracellular domain (or cytoplasmic domain) includes one or more signaling domains that, upon binding of a target antigen to a binding motif, cause and / or mediate intracellular signals that activate, for example, one or more immune cell effector functions (such as innate immune cell effector functions). In some embodiments, the signaling domain of the intracellular domain mediates the activation of at least one of the normal effector functions of immune cells. The effector functions of T cells can be, for example, cytolytic activity or helper activity including the secretion of cytokines. In some embodiments, the signaling domain of the intracellular domain mediates the activation, proliferation, survival, and / or other T cell functions. The intracellular domain can include a signaling domain that is an activation domain. The intracellular domain can include a signaling domain that is a co-stimulatory signaling domain.

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

[0218] In some embodiments, the CARs contemplated herein include an intracellular signaling domain. An "intracellular signaling domain" refers to a part of the CAR that is involved in transmitting an effective CAR message that binds to a target antigen into the interior of an immune effector cell, thereby 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 induced by antigen binding to the extracellular CAR domain. In some embodiments, the signaling domain and / or activation domain includes an immunoreceptor tyrosine-based activation domain (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 certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptors, 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 receptors, DAP-10, DNAM1 (CD226), Fc gamma receptors, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell co-stimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, LIGHT, 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 proteins, 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 special function of a cell. The effector function of a T cell can be, for example, assistance or activity including cytolytic activity or secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the part of a protein that transmits an effector function signal and instructs the cell to perform a special function. Usually, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. As long as a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used instead of the entire domain as long as it converts the effector function signal. The term "intracellular signaling domain" means any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0220] It is known that signals generated only through the TCR are insufficient for complete activation of T cells and that secondary or co-stimulatory signals may also be required. Thus, T cell activation can be said to be mediated by two different classes of intracellular signaling domains: a primary signaling domain that antigen-dependently initiates primary activation via the TCR (e.g., the TCR / CD3 complex) and a co-stimulatory signaling domain that acts antigen-independently to provide a secondary or co-stimulatory signal. In some embodiments, the CAR contemplated herein includes an intracellular signaling domain that includes one or more "co-stimulatory signaling domains" and "primary signaling domains".

[0221] In some embodiments, the signaling domain and / or activation domain comprises an immunoreceptor 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, for example, 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 co-stimulatory signaling domains. The intracellular primary and co-stimulatory signaling domains can be tandemly linked in any order at the carboxyl terminus of the transmembrane domain. In one embodiment, the CAR has a CD3ζ domain having an amino acid sequence with at least 75% sequence identity to SEQ ID NO: 60 (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%). VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 60). In embodiments, the CD3ζ domain is encoded by a nucleic acid having at least 75% sequence identity to the nucleic acid having the sequence set forth below (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%). AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGGCGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 61). In an embodiment, the CD3ζ 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 described below. AGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGA (SEQ ID NO: 61).

[0222] The CARs contemplated herein include one or more co-stimulatory signaling domains for enhancing the efficacy and proliferation of T cells expressing the CAR receptor. As used herein, the terms "co-stimulatory signaling domain" or "co-stimulatory domain" refer to the intracellular signaling domain of a co-stimulatory molecule.

[0223] In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, 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 co-stimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, LIGHT, 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 fragments, cleavage, or combinations thereof.

[0224] The CAR may include a co-stimulatory signaling domain, for example, to enhance signal transduction efficacy. U.S. Patent Nos. 7,741,465 and 6,319,494, and 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 et al., Annu. Rev. Pharmacol. Toxicol.See 56:59-83(2016). Signals generated only through the TCR may be insufficient for complete activation of T cells, and secondary or co-stimulatory signals can increase activation. Thus, in some embodiments, the signaling domain further includes one or more additional signaling domains (e.g., co-stimulatory signaling domains) that activate one or more immune cell effector functions (e.g., the innate immune cell effector functions described herein). In some embodiments, a portion of such co-stimulatory signaling domains can be used so long as that portion transmits an effector function signal. In some embodiments, the cytoplasmic domains described herein include one or more cytoplasmic sequences of a T cell co-receptor (or a fragment thereof). Non-limiting examples of co-stimulatory 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), CARD1l, CD2 (also known as LFA-2, SRBC, T11, and CD2 molecule), CD3 gamma, CD3 delta, CD3 epsilon, CD4, CD7 (also known as GP40, LEU-9, TP41, Tp40, and CD7 molecule), 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), CD29, CD30 (also known as TNFRSF8, D1S166E, and Ki-1), CD40L (also known as CD40LG, CD154, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39, hCD40L, and CD40 ligand), 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, also known as CD28LG1, LAB7, and CD80 molecule; CD83 (and its ligand that specifically binds to CD83); CD84; CD86; CD96 (tactile); CD100 (SEMA4D); CD103; CD160 (also known as BY55, NK1, NK28, and CD160 molecule); CD244 (also known as 2B4, NAIL, NKR2B4, Nmrk, SLAMF4, and CD244 molecule); 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 (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D); GITRL; HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2); ICAM-1; ICOS (inducible T cell co-stimulator, also known as AILIM, CD278, and CVID1); Ig alpha (CD79a); IL2R beta; IL2R gamma; IL7R alpha; immunoglobulin-like protein; integrin; ITGA4, also known as IA4; ITGA6; ITGAD; ITGAE; ITGAL; ITGAM; ITGAX; ITGB1; ITGB2; ITGB7; KIRDS2; LAG3 (also known as CD223 and lymphocyte activation 3); LAT; LFA-1 (also known as lymphocyte function-associated antigen 1 and CD11a / CD18); LIGHT (also known as TNFSF14, CD258, HVEML, LTg, TR2, TNLG1D, and tumor necrosis factor superfamily member 14); LTBR; Ly9 (CD229); MHC class I molecule; 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 also known as tumor necrosis factor receptor superfamily member 4, PAG / Cbp, PD-1 (PDCD1, CD279, PD-1, SLEB2, hPD-1, hPD-1, hSLE1, and also known as programmed cell death 1), PD-L1 (CD274, B7-H, B7H1, PD-L1, PDCD1L1, PDCD1LG1, PDL1, CD274 molecule, and also known as programmed cell death 1 ligand 1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activation molecule (SLAM protein, for example, SLAM (SLAMF1, CD150, IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Lyl08), and SLAMF7), SLP76, TIM3 (HAVCR2, HAVcr-2, KIM-3, TIM3, TIMD-3, TIMD3, Tim-3, and also known as 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 state / RANKL, TRIM, VLA1, VLA-6, and ZAP70 are included, but not limited to these. Exemplary co-stimulatory proteins have the amino acid sequence of co-stimulatory proteins naturally found on T cells, and the complete native amino acid sequence of the co-stimulatory protein is described in NCBI reference sequence: NP_006130.1. In a particular example, the CAR includes a 4-1BB co-stimulatory domain.

[0225] In an embodiment, the CAR comprises a CD28 co-stimulatory 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 62. RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 62). In an embodiment, the CD28 co-stimulatory 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 as follows. aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc (SEQ ID NO: 63).

[0226] In an embodiment, the CAR comprises a 4-1BB co-stimulatory 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 64. RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 64). In an embodiment, the 4-IBB co-stimulatory 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 described 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 the signal peptide, although the precursor form of the polypeptide contains the 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, it includes CSF2RA 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 MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 66), MEWTWVFLFLLSVTAGVHS (SEQ ID NO: 67), or MALPVTALLLPLALLLHAARP (SEQ ID NO: 68).

[0228] The polynucleotide sequences and polypeptide sequences of the signaling domains provided herein are known. In some embodiments, the polynucleotide encoding the signaling 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 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] The components of the CAR can be exchanged or "swapped" for equivalent components using routine techniques of biotechnology. In some non-limiting and partial examples, the CARs of the disclosure can comprise the binding motifs provided herein in combination with the hinge provided herein and the co-stimulatory domain provided herein. In certain examples, the CARs of the disclosure can comprise a leader sequence, together with the binding motifs provided herein, in combination with the hinge provided herein and the co-stimulatory domain provided herein.

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

[0231] "Polypeptide", "polypeptide fragment", "peptide", and "protein" are, unless specified to the contrary, used in their conventional sense, 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 known in the art, both naturally occurring and non-naturally occurring. In various embodiments, the polypeptides contemplated herein include a signal (or leader) sequence at the N-terminal portion of the protein, which directs the movement of the protein during co-translation or post-translation.

[0232] Polypeptides include "polypeptide variants". A polypeptide variant may differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions, and / or insertions. Such variants may occur naturally or may be produced synthetically, for example, by modifying one or more of the polypeptide sequences described above. 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 as well as CARs and TCRs by introducing one or more substitutions, deletions, additions and / or insertions. Preferably, the 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. The 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 described herein (see, e.g., the Sequence Listing), and typically, the variants maintain at least one biological activity of the reference sequence. Polypeptides include "polypeptide fragments". A polypeptide fragment refers to a polypeptide that can be a monomer or multimer having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions or substitutions of a naturally occurring or recombinantly produced polypeptide. In certain embodiments, a polypeptide fragment can include an amino acid chain that is at least 5 to about 500 amino acids in length. In certain embodiments, it will be understood that 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 polypeptide can also be fused or conjugated in-frame to a linker or other sequence to facilitate the synthesis, purification, or identification of the polypeptide (e.g., poly-His), or to enhance the binding of the polypeptide to a solid support. As described above, the polypeptides of the present disclosure can be modified in various 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 changes in nucleotide sequences 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 the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance for appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the models of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

[0234] In certain embodiments, the variant includes conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties such that those skilled in the art of peptide chemistry would predict that the secondary structure and hydropathy characteristics of the polypeptide would not be substantially changed. Modifications can be made to the structure of the polynucleotides and polypeptides of the present disclosure, and still obtain functional molecules that encode variant or derivative polypeptides having the desired properties.

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

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

[0237] The anti-CD19 CAR may comprise an antigen-binding sequence found in the antibodies described herein (see, e.g., Table 5). In some embodiments, the anti-CD19 CAR of the present disclosure comprises an antigen-binding fragment 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 the heavy chain variable domain disclosed in Table 5. In various embodiments, the anti-CD19 binding motif comprises at least two heavy chain FRs of the heavy chain variable domain disclosed in Table 5. In various embodiments, the anti-CD19 binding motif comprises three heavy chain FRs of the 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 the light chain variable domain disclosed in Table 5. In various embodiments, the anti-CD19 binding motif comprises at least two light chain FRs of the light chain variable domain disclosed in Table 5. In various embodiments, the anti-CD19 binding motif comprises three light chain FRs of the light chain variable domain disclosed in Table 5.

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

[0244] In various embodiments, the anti-CD19 CAR comprises one, two, or three FRs having 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 the heavy chain variable domain disclosed in Table 5, either together or each individually. In various embodiments, the anti-CD19 CAR comprises one, two, or three FRs having 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 the light chain variable domain disclosed in Table 5, either together or each individually.

[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 the 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 the 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 the 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 the light chain variable domain disclosed in Table 5.

[0247] In certain embodiments, the anti-CD19 CAR comprises a binding motif comprising the heavy chain variable domain of the present disclosure, the light chain variable domain of the present disclosure, and a linker having at least 75% sequence identity to SEQ ID NO: 45 (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%). In certain embodiments, the anti-CD19 CAR comprises a binding motif comprising the heavy chain variable domain of the present disclosure, the light chain variable domain of the present disclosure, and a leader sequence having at least 75% sequence identity to SEQ ID NO: 66 (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%).

Table 5

[0248] In one embodiment, the anti-CD19 CAR 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 90. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 90). In an embodiment, 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence as follows.

[0249] In one embodiment, the anti-CD19 CAR linked to the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 92. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPGPMDWTWILFLVAAATRVHSEQKLISEEDLAGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDGGGGSGGGGSRSNLGWLCLLLLPIPLIVWVKRKEVQKT (SEQ ID NO: 92).In an embodiment, the anti-CD19 CAR linked to the 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, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having the sequence according to the following.

[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 on June 18, 2020, which is hereby specifically incorporated by reference in its entirety. In embodiments, the anti-CD20 CAR 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: 90. QVQLVQSGAEVKKPGASVKVSCKASGYTFKEYGISWVRQAPGQGLEWMGWISAYSGHTYYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGPHYDDWSGFIIWFDPWGQGTLVTVSSGSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRFPPTFGQGTKVEIKAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (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 the sequence set forth below.

[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, the 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 TRCs.

[0252] The present disclosure includes vectors comprising the nucleic acids of the present disclosure and / or encoding the membrane-bound IL-15-IL-15Rα chimeric polypeptides of the present disclosure, optionally in combination with nucleic acids encoding any of the CARs or TCRs 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 adeno-associated virus (AAV) vector, a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo large TcDNA, pBABE-hygro-hTERT, pMKO.1 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, Maryland), the pBluescript series (Stratagene, La Jolla, California), the pET series (Novagen, Madison, Wisconsin), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, California). 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 contain both a nucleic acid encoding an antigen-binding system and the inducible expression construct described herein.

[0254] Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York, 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, 1994. The constructs of the expression vectors, which can be circular or linear, can be prepared to include a replication system functional in a prokaryotic host cell or a eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

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

[0256] Vectors useful in the context of the present disclosure can be "naked" nucleic acid vectors (i.e., vectors having little or no protein, sugar, and / or lipid encapsulating them), or vectors complexed with other molecules. Other molecules that can be appropriately combined with the vector include, without limitation, 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 constructed in a single multicistronic expression cassette, in multiple expression cassettes of 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 vector and the second vector are a lentiviral vector, a retroviral vector, or an adenoviral vector. In some embodiments, the second vector further comprises a promoter sequence and / or an 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 for making polycistronic expression cassettes include various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aftovirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides), but are not limited thereto. Combinations of retroviral vectors with appropriate packaging stocks are also suitable, and the capsid proteins can function to infect human cells.A variety of amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al., 1985, Mol. Cell. Biol. 5:431-437); PA317 (Miller et al., 1986, Mol. Cell. Biol. 6:2895-2902); 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 envelope and any others known in the art, are also suitable.

[0258] Vector DNA can be introduced into cells, such as immune cells, via conventional transformation, transfection, or transduction techniques. The terms "transformation" and "transfection" encompass various 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 gun, nanoparticle-mediated delivery, or electroporation. Transduction includes viral delivery of vectors into cells by vectors disclosed herein, including, but not limited to, retroviruses, lentiviruses, and AAV.

[0259] The present disclosure includes cells that contain, express, or are 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 includes (a) a CAR or TCR, and (b) a membrane-bound IL-15-IL-15Rα chimeric polypeptide. Immune cells can be transduced with a CAR or TCR and a membrane-bound IL-15-IL-15Rα chimeric polypeptide such that the cells express 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 immune cells, such as T cells, and thereby expressed. In certain embodiments, the 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 (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), such as 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, for example, Enterobacteriaceae such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, 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 cell is selected from the group consisting of T cells, B cells, tumor-infiltrating lymphocytes (TIL), TCR-expressing cells, natural killer (NK) cells, dendritic cells, granulocytes, innate lymphocytes, megakaryocytes, monocytes, macrophages, platelets, thymocytes, and myeloid cells. In one embodiment, the immune cell is a T cell. In another embodiment, the immune cell is an NK cell. In certain embodiments, the T cell is a tumor-infiltrating lymphocyte (TIL), an autologous T cell, an engineered autologous T cell (eACT™), an allogeneic T cell, a xenogeneic T cell, an iPSC cell, 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. The 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, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. Additionally, the T cells can be derived from one or more T cell lines available in the art. The T cells can also be obtained from a blood unit collected from a subject using various techniques known to those of skill in the art, such as FICOLL™ separation and / or apheresis. In some embodiments, the 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, the cells are washed with PBS. As will be appreciated, the washing step can be performed using, for example, a semi-automated flow-through centrifuge, such as the Cobe™ 2991 cell processor, the Baxter CytoMate™, etc. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers or other saline solutions with or without buffer. In some embodiments, unwanted components of the apheresis sample are removed. Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, International Patent Application Publication Nos. 2015 / 120096, and 2017 / 070395, all of which are incorporated herein by reference in their entirety for the purpose of describing those methods.

[0264] In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, using centrifugation with 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 specific 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 population of cells 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 T lymphocytes and helper T lymphocytes are sorted into subpopulations of naive T cells, memory T cells, and effector T cells, either before or after genetic modification and / or expansion. In some embodiments, CD8+ cells are further sorted into naive cells, central memory cells, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127 and is 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 having cell surface antigens.

[0266] In some embodiments, immune cells, such as NK cells or T cells, are genetically modified after isolation using known methods, or the immune cells are activated and expanded in vitro (or differentiated in the case of progenitor cells) before being genetically modified. In another embodiment, immune cells, such as 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), and 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 the 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. Pat. Nos. 6,905,874, 6,867,041, and 6,797,514, and 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 in a culture medium containing an appropriate cytokine, such as IL-2, with stimulators and co-stimulators, such as anti-CD3 and anti-CD28 antibodies, generally bound to beads or other surfaces. 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, for example, methods described in U.S. Pat. Nos. 6,040,177 and 5,827,642 and International Patent Application Publication No. 2012 / 129514, the entire contents of which are incorporated herein by reference, are used to activate and stimulate T cells to proliferate with feeder cells and appropriate antibodies and cytokines.

[0267] The methods described herein may further include concentrating a population of lymphocytes obtained from a donor. Concentration of a population of lymphocytes, such as a population of one or more T cells, can be achieved by separation media (e.g., FICOLL-PAQUE™, ROSETTESEP™ HLA Total Lymphocyte Enrichment cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical catalog number 0850494X), etc.), 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 a population of lymphocytes with one or more T cell stimulants to produce a population of activated T cells under appropriate conditions. An antibody or a functional fragment thereof that targets a T cell stimulating molecule or a costimulatory molecule (e.g., anti-CD2 antibody, anti-CD3 antibody, anti-CD28 antibody, or a functional fragment thereof), or any other suitable mitogen (e.g., tetradecanoyl phorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), or any combination of one or more suitable T cell stimulants including, but not limited to, natural ligands for T cell stimulating molecules or costimulatory molecules can be used to produce a population of activated T cells.

[0269] Suitable conditions for stimulating the population of lymphocytes described herein include temperature, duration, and / or a level of CO 2It may include in the presence of. 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 the population of lymphocytes described herein may include the period for stimulation. In some embodiments, the period for stimulation is about 24 - 72 hours. In some embodiments, the period for stimulation is about 24 - 36 hours, about 30 - 42 hours, about 36 - 48 hours, about 40 - 52 hours, about 42 - 54 hours, about 44 - 56 hours, about 46 - 58 hours, about 48 - 60 hours, about 54 - 66 hours, about 60 - 72 hours. In one particular embodiment, the period for stimulation is about 48 hours or at least about 48 hours. In other embodiments, the period for stimulation is about 44 - 52 hours. In certain embodiments, the period for stimulation is about 40 - 44 hours, about 40 - 48 hours, about 40 - 52 hours, or about 40 - 56 hours.

[0271] Another condition for stimulating the population of lymphocytes described herein may include the CO 2 level. In some embodiments, the CO 2 level for stimulation is about 1.0 - 10% CO 2 . In some embodiments, the CO 2 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% CO 2 . In one embodiment, the CO 2 level for stimulation is about 3 - 7% CO 2 . In other embodiments, the CO 2 level for stimulation is about 4 - 6% CO 2 . In one embodiment, the CO 2The level is about 4.5 - 5.5% CO 2 In one particular embodiment, the CO 2 level for stimulation is about 5% CO 2 In one particular embodiment, the CO

[0272] The conditions for stimulating the population of lymphocytes may further include any combination of temperature, duration for stimulation, and / or the presence of a certain level of CO 2 For example, the step of stimulating the population of lymphocytes may include stimulating the population of lymphocytes with one or more T cell stimulants at a temperature of about 36 - 38°C, for about 44 - 52 hours, in the presence of a level of CO 2 at a level of about 4.5 - 5.5% CO 2 In one particular embodiment, the CO

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

[0274] An anti-CD3 antibody (or a functional fragment thereof), an anti-CD28 antibody (or a functional fragment thereof), or a combination of an anti-CD3 antibody and an anti-CD28 antibody can be used according to the step of stimulating a population of lymphocytes. Any soluble or immobilized anti-CD2, anti-CD3, and / or anti-CD28 antibody or a functional fragment 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, the antibody can be commercially purchased from vendors known in the art including, but not limited to, Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 pure 1mg / mL, Part No.170-076-116), and eBioscience, Inc. Furthermore, those skilled in the art will understand methods for producing anti-CD3 antibodies and / or anti-CD28 antibodies by standard methods. In some embodiments, one or more T cell stimulants used according to the step of stimulating a population of lymphocytes include an antibody or a functional fragment thereof that targets a T cell stimulatory or co-stimulatory molecule in the presence of a T cell cytokine. In one aspect, one or more T cell stimulants include an anti-CD3 antibody and IL-2. In certain embodiments, the T cell stimulant includes 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 a population of lymphocytes to produce a population of activated T cells is omitted from this method, and the population of lymphocytes from which T lymphocytes can be enriched 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 nucleic acid molecule encoding a membrane-bound IL-15-IL-15Rα chimeric polypeptide and / or a CAR or TCR, using a single-cycle transduction, to produce a population of transduced T cells. In embodiments utilizing a viral vector having a membrane-bound IL-15-IL-15Rα chimeric polypeptide, the viral vector may be separate from the viral vector encoding the CAR or TCR, or the viral vector may encode both the membrane-bound IL-15-IL-15Rα chimeric polypeptide and the CAR or TCR. Transduction of the population of activated immune cells described herein can be performed in any combination in the presence of a period, a specific temperature, and / or a specific level of CO 2 at any combination of: at a temperature of about 36 to 38 °C for about 16 to 24 hours in the presence of about 4.5 to 5.5% CO 2 at a level of CO 2 . Immune cells can be prepared by any combination of any one of the methods of the present application and any manufacturing method for preparing T cells for immunotherapy, and any such manufacturing method is incorporated herein by reference in its entirety for the purpose of describing these methods: International Patent Application Publication No. WO 2015 / 120096 and International Patent Application Publication No. WO 2017 / 070395; 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 and all methods used in the preparation of "off-the-shelf" T cells for immunotherapy; and any other method for preparing lymphocytes for administration to a human, including but not limited to these. The manufacturing process can be adapted to remove circulating tumor cells from cells obtained from a patient.

[0276] To deliver genetic material to cells, several recombinant viruses have been used as viral vectors. Viral vectors that can be used according to the transduction process include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors, and can be any ecotropic or amphotropic viral vector. In some embodiments, the method further comprises transducing one or more NK cells or T cells with a retrovirus. In one embodiment, the viral vector used to transduce a population of NK cells or activated T cells is the MSGV1γ retroviral vector. In certain embodiments, the viral vector used to transduce a 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 grown in suspension culture in a medium specific for the production of viral vectors, herein referred to as "viral vector inoculum material". Any suitable growth medium and / or supplement for growing the viral vector can be used in the viral vector inoculum material according to the methods described herein. According to some aspects, the viral vector inoculum material is then added to the serum-free culture medium described below during the transduction process.

[0277] The conditions for transducing the population of NK cells or activated T cells described herein may include the presence of a specific period, a specific temperature, and / or 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 period for transduction is about 12 to 36 hours. In some embodiments, the 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 period for transduction is about 20 hours or at least about 20 hours. In one embodiment, the period for transduction is about 16 to 24 hours. In other embodiments, the 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 CO 2 level for transduction is about 1.0 to 10% CO 2 In other embodiments, the CO 2 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% CO 2 In one embodiment, the CO 2 level for transduction is about 3 to 7% CO 2 In another embodiment, the CO 2 level for transduction can be about 4 to 6% CO 2 In another embodiment, the CO 2 level for transduction is about 4.5 to 5.5% CO 2 In one embodiment, the CO 2 level for transduction is about 5% CO 2

[0280] In some embodiments, transduction of the population of activated T cells described herein is at any combination of a particular period, a particular temperature, and / or the presence of a particular level of CO 2 in the presence of: at a temperature of about 36 to 38° C., for about 16 to 24 hours, at a level of about 4.5 to 5.5% CO 2 2 can be carried out.​​

[0281] The methods described herein can include expanding a population of one or more transduced NK cells or T cells over a specified period to produce a population of engineered NK cells or T cells. The specified period for expansion can be (i) a sufficient number of cells in at least one dose of the population of engineered NK cells or T cells for administration to a patient, (ii) a population of engineered T cells having a preferred percentage of naive cells as compared to a typical longer process, or (iii) any suitable period that enables the production of both (i) and (ii). This period depends on 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 specified 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 specified period for expansion can 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% shorter, or more than 75% shorter. In one aspect, the period for expansion is about 3 days, and the period from enrichment of the lymphocyte population to production of engineered NK cells or T cells is about 6 days.

[0282] The conditions for expanding a population of transduced NK cells or T cells are temperature and / or a level of CO 2may include being in the presence of. 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 level of CO2 is 1.0 - 10% CO 2 is. In other embodiments, CO 2 levels are 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% CO 2 is. In one embodiment, the level of CO 2 is about 4.5 - 5.5% CO2. In another embodiment, the level of CO 2 is about 5% CO 2 is. 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% CO 2 is. In some embodiments, the conditions for expanding the population of transduced NK cells or T cells may include any combination in the presence of temperature and / or a certain level of CO 2 . For example, the conditions for expanding the population of transduced T cells include being in the presence of a temperature of about 36 - 38 °C and a level of about 4.5 - 5.5% CO 2 in the presence of CO 2 .

[0283] Each step of the production described in this specification can be carried out 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 step. The recombinant human fibronectin fragment can contain three functional domains, namely, a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. By using the recombinant human fibronectin fragment to assist in the co-localization of target cells and viral vectors, the gene efficiency of retroviral transduction of immune cells can be increased. In certain embodiments, the recombinant human fibronectin fragment is RETRONECTIN® (Takara Bio, Japan). In certain embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1 - 60 μg / mL or about 1 - 40 μg / mL. In other embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1 - 20 μg / mL, 20 - 40 μg / mL, or 40 - 60 μg / mL. In some embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at 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 a recombinant human fibronectin fragment at 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. In certain embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at 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. In one particular embodiment, the cell culture bag is coated with a recombinant human fibronectin fragment at at least about 10 μg / mL. The cell culture bag used in a closed bag culture system can optionally be 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 method can optionally be cryopreserved so that the cells can be used later. Methods for cryopreserving a population of engineered immune cells are also provided herein. Such methods can include the step of 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. Also, HSA can be added to the washed and concentrated cells to improve cell viability and cell recovery after thawing. In another embodiment, the wash solution is saline and the washed and concentrated cells are supplemented with HSA (5%). The method can also include the step of generating a cryopreservation mixture, where the cryopreservation mixture includes the diluted cell population in the diluent and a suitable cryopreservation solution. The cryopreservation solution can be any suitable cryopreservation solution including, but not limited to, CryoStor 10 (BioLife Solutions), and is mixed with the diluent of the engineered immune cells at a ratio of 1:1 or 2:1. HSA can be added in the cryopreserved mixture 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. Cryopreservation of the population of engineered immune cells 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 the step of freezing the cryopreservation mixture. Also, the cryopreservation mixture can have about 1×10 6 ~ about 1.5×10 7At a cell concentration of cells / mL, it is frozen in a controlled-rate freezer using a defined freezing cycle. This method may also include the step of storing the cryopreservation mixture in vapor-phase liquid nitrogen.

[0285] A population of engineered immune cells produced by the methods described herein can be cryopreserved at a predetermined dose. The predetermined dose can be a therapeutically effective dose, which can be any therapeutically effective dose provided below. The predetermined dose of engineered immune cells can depend on the binding motif expressed by the immune cells (e.g., the affinity and density of the binding motif expressed on the cell surface), 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 can be any antigen or molecule targeted by a CAR or TCR. In certain embodiments, the predetermined dose of engineered immune cells expressing a CAR or TCR can 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 can be about 1 million to about 2 million transduced engineered NK cells or T cells (cells / kg) per kilogram of body weight. The predetermined dose of engineered NK cells or T cells expressing a CAR or TCR can be 1 million to about 2 million, at least about 2 million to less than about 3 million transduced engineered NK cells or T cells (cells / kg) per kilogram of body weight. In one embodiment, the 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, the 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 the predetermined dose 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 (cells / kg) per kilogram of body weight.In certain embodiments, a population of engineered NK cells or T cells can be cryopreserved at a predetermined dose of about 500,000 to about 1 million engineered NK cells or T cells / kg. In certain embodiments, a population of engineered NK cells or T cells can be cryopreserved at a predetermined dose 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, or at least about 10 million engineered NK cells or T cells / kg. In other aspects, a population of engineered NK cells or T cells can 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, greater than 10 million cells / kg, greater than 20 million cells / kg, greater than 30 million cells / kg, greater than 40 million cells / kg, greater than 50 million cells / kg, greater than 60 million cells / kg, greater than 70 million cells / kg, greater than 80 million cells / kg, greater than 90 million cells / kg, or greater than 100 million cells / kg. In certain aspects, a population of engineered NK cells or T cells can be cryopreserved at a predetermined dose of about 1 million to about 2 million engineered NK cells or T cells / kg. A population of engineered NK cells or T cells can be cryopreserved at a predetermined volume of about 1 million cells to about 2 million cells / kg, about 1 million cells to about 3 million cells / kg, about 1 million cells to about 4 million cells / kg, about 1 million cells to about 5 million cells / kg, about 1 million cells to about 6 million cells / kg, about 1 million cells to about 7 million cells / kg, about 1 million cells to about 8 million cells / kg, about 1 million cells to about 9 million cells / kg, or about 1 million cells to about 10 million cells / kg. The predetermined dose of a population of engineered NK cells or T cells can be calculated based on the body weight of the subject. In one example, a 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 can be cryopreserved in a cryopreservation medium of 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. In certain embodiments, the population of engineered NK cells or T cells can be cryopreserved in a cryopreservation medium of about 50 - 70 mL.

[0286] The present disclosure also provides a composition (e.g., a pharmaceutical composition) 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 an NK cell or T cell comprising a membrane-bound IL-15-IL-15Rα chimeric polypeptide.

[0287] In other embodiments, the composition is selected for parenteral delivery, inhalation, or delivery via the digestive tract such as oral. Preparation of such pharmaceutically acceptable compositions is within the ability of one of ordinary skill in the art. In certain embodiments, a buffer is used to maintain the composition within a physiological pH or slightly lower pH, typically within a pH range of about 5 to about 8. In certain embodiments, when parenteral administration is contemplated, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution containing the composition described herein, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, the parenteral injection vehicle is sterile distilled water in which the composition described herein is formulated as a properly preserved sterile isotonic solution, with or without at least one additional therapeutic agent. In certain embodiments, the preparation involves formulating the desired molecule using beads or liposomes that 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 previously obtained from a subject, e.g., a subject identified or diagnosed as having cancer). In one embodiment, the cell contains a nucleic acid encoding a membrane-bound IL-15-IL-15Rα chimeric polypeptide and / or any of the CARs or TCRs described herein. In a composition containing any of the cells described herein, the composition can further include a cell culture medium or a pharmaceutically acceptable buffer (e.g., phosphate buffered saline).

[0289] The pharmaceutical composition may comprise the CAR-expressing cells or TCR-expressing cells described herein, for example, 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 may further comprise a buffer, such as neutral buffered saline, phosphate buffered saline, etc., carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, etc.; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (such as 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, and at least one non-limiting example thereof is intravenous administration. The composition may be formulated for intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, and / or subcutaneous routes of administration. In embodiments, the composition is formulated for a parenteral route of administration. Compositions suitable for parenteral administration may be aqueous or non-aqueous isotonic sterile injection solutions, which may contain, for example, antioxidants, buffers, bacteriostatic agents and solutes to render the composition isotonic with the blood of the intended recipient. Aqueous or non-aqueous sterile suspensions may contain one or more suspending, solubilizing, thickening, stabilizing, and preserving agents. The pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease being treated (or prevented).

[0291] In various embodiments, the engineered NK cells or T cells described herein may be incorporated into a pharmaceutical composition. As disclosed herein, the pharmaceutical composition comprising the engineered T cells may be in any form. Such forms include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (such as injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.

[0292] The pharmaceutical composition containing the binder of the present disclosure can be formulated by known methods (e.g., Remington’s Pharmaceutical Sciences, 17th Edition, edited by Alfonso R. Gennaro, Mack Publishing Company, Easton, Pennsylvania (1985)). In various examples, the pharmaceutical composition containing the binder 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 agents, and absorption delaying agents that are physiologically compatible. The composition containing engineered T cells can include pharmaceutically acceptable salts, such as acid addition salts or base addition salts.

[0293] The sterile injectable composition can be formulated according to conventional pharmaceutical practices using distilled water for injection as the vehicle. For example, aqueous solutions for injection can include physiological saline or isotonic solutions containing other adjuvants such as glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc., and can be used in combination with appropriate solubilizing agents such as alcohols like ethanol, polyhydric alcohols such as propylene glycol and polyethylene glycol, and nonionic surfactants such as polysorbate 80 (trademark) and HCO-50 as needed.

[0294] Non-limiting examples of oily liquids include sesame oil and soybean oil, and can be combined with benzyl benzoate or benzyl alcohol as solubilizing agents. Other articles that can be included in the composition are 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 injection can be packaged in appropriate 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 cells or plasmid components, bacteria, and fungi. In one embodiment, the bacteria are at least one 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) prior to injection into a cancer patient, and the cancer patient is treated with IL-2 after injection. Further, in some embodiments, the cancer patient may undergo preconditioning lymphodepletion (temporary removal of the immune system) prior to administration of the conjugate. The combination of IL-2 treatment and preconditioning lymphodepletion may enhance the persistence of the conjugate. 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), and the nucleic acid can 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 contraction, which may facilitate the formation and survival of memory T lymphocytes.

[0297] In some embodiments, the dosage administered to a subject can 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 a composition to be administered to a human or other subject in order to treat or prevent a medical condition. The exact amount of the composition required to be therapeutically effective can depend on a number of factors, such as the activity of the binder and the route of administration.

[0298] An appropriate number of engineered cells comprising a CAR or TCR can be administered to a subject. A single engineered cell as described herein can be expanded and provide a therapeutic benefit, but in some embodiments, 10 2 or more, for example, 10 3 or more, 10 4 or more, 10 5 or more, or 10 8 or more engineered cells are administered. In some embodiments, 10 12 or fewer, for example, 10 11 or fewer, 10 9 or fewer, 10 7 or fewer, or 105 or fewer of the engineered cells described herein are administered to a subject. In some embodiments, 10 2 to 10 5 cells, 10 4 to 10 7 cells, 10 3 to 10 9 cells, or 10 5 to 10 10 cells of the engineered cells are administered. A pharmaceutical composition comprising cells comprising a CAR or TCR can be administered, for example, at a dosage of 10 4 to 10 9 cells / kg body weight (e.g., 10 5 to 10 6 cells / kg body weight). In another embodiment, a therapeutically effective amount of T cells is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or about 10 8It is a cell. The pharmaceutical composition may be administered, for example, at a dose of about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7 cells / kg, about 7×10 7 cells / kg, about 8×10 7 cells / kg, or about 9×10 7 cells / kg.

[0299] The dosage of the engineered T cells or NK cells described herein can be administered to a mammal, as needed, in a single dose or as a series of sub-doses administered over a suitable period, for example, daily, every other week, weekly, every other week, every two weeks, every other month, every six months, or annually. A dosage unit containing an effective amount of the binder may be administered once daily, or the total daily dosage may be administered in divided doses, if necessary, two, three, four or more times a day.

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

[0301] The choice or use of any form may depend in part on the intended mode of administration and therapeutic application. For example, a composition comprising the engineered cells of the present disclosure intended for systemic or local delivery may be in the form of an injectable or infusible solution. Thus, a composition comprising the engineered product of the present disclosure may be formulated for administration by a parenteral mode (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection). Parenteral administration refers to a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracavernous, and intrasternal injections and infusions.

[0302] In various embodiments, a pharmaceutical composition comprising the engineered cells of the present disclosure may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. A sterile injectable solution can be prepared by incorporating the required amount of the composition comprising the engineered cells of the present disclosure into a suitable solvent, optionally containing one or a combination of the ingredients listed above, followed by filtration sterilization. In general, dispersions are prepared by incorporating the composition comprising the 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 a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants. Sustained absorption of an injectable composition comprising a binder of the present disclosure can be brought about by including in the composition comprising the binder of the present disclosure a reagent that delays absorption, such as monostearate and gelatin.

[0303] The pharm...

Claims

1. An antibody comprising an IL-15 polypeptide, an IL-15Rα sushi domain polypeptide, and a transmembrane domain comprising an IL-7Rα transmembrane domain or a FAS transmembrane domain, A membrane-bound interleukin 15 (IL-15)-IL-15Rα sushi domain chimeric receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 29 and 94.

2. The transmembrane domain comprises a FAS transmembrane domain, 2. The membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric receptor of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 and 94.

3. A nucleic acid encoding the membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor of claim 1 or 2.

4. 4. The nucleic acid of claim 3, wherein the nucleic acid comprises a nucleic acid sequence set forth in a sequence selected from the group consisting of SEQ ID NO:31 and SEQ ID NO:

32.

5. 5. The nucleic acid of claim 3 or 4, further comprising a nucleic acid encoding a chimeric antigen receptor or a T cell receptor.

6. The nucleic acid of claim 5, wherein the chimeric antigen receptor or T cell receptor binds to a tumor antigen.

7. The tumor antigen is 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenergic receptor beta 3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), beta2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (a cancer gene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CTCF (zinc finger protein)-like (BORIS or Brother of the Regulator of ImprintedSites), 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, 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, 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, CRACC, CD319, and also called 19A24), cyclin B1, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 (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), endoglin, epithelial cell adhesion molecule (EpCAM), Ephrin type A receptor 2 (EphA2), Ephrin B2, receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), ERBB, ERBB2 (Her2 / neu), ERG (membrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS-introduced variant 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)bDGlc p(1-1)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF18), GM1, ganglioside GM3, hexasaccharide moiety of Globoh glyco sphingolipid (Globoh), glycoprotein 75, glypican-3 (GPC3), glycoprotein 100 (gp100), 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 variant (muthsp70-2), 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, κ light chain, KCS1, kinase insert domain receptor (KDR), KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KG2D ligand, KIT (CD117), KLRGI, LAGE-1a, 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 recognized by T cells (MelanA or MART1), MelanA / MART1, mesothelin, MAGE A3, melanoma inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSP), MORAb-009, MS4A1, Mucin1 (MUC1), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian duct inhibitor (MIS) receptor type II, v-myc avian myelocytomatosis virus oncogene neuroblastoma-derived homolog (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKRPIA, NPC-1C, NTBA, breast differentiation antigen (NY-BR-1), NY-ESO-1, cancer fetal antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, PolySA, proacrosin-binding protein sp32 (OY-TES 1), p53, p53 variant, pannexin 3 (PANX3), prostate 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 variant, 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 3 recognized by T cells (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), TCR5γ, TCRα, TCRβ, TCRγ alternative reading frame protein (TARP), telomerase, TIGIT, TNF-alpha precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGF-beta1, TGF-beta2, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TACI, TIM1, TIM2, TIM3, Tn7. The nucleic acid of claim 6, wherein the nucleic acid is selected from the group consisting of 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).

8. A recombinant vector comprising a nucleic acid described in any one of claims 3 to 7.

9. An immune cell comprising a membrane-bound IL-15-IL-15Rα sushi domain chimeric receptor according to claim 1 or 2, a nucleic acid according to any one of claims 3 to 7, or a recombinant vector according to claim 8.

10. The immune cell of claim 9 , which is a T cell or a natural killer (NK) cell.

11. A pharmaceutical composition comprising the immune cell according to claim 9 or 10.

12. Use of an immune cell according to claim 9 or 10 or a pharmaceutical composition according to claim 11 in the manufacture of a medicament for treating a cancer associated with expression of a tumor antigen in a subject.

13. Use of an immune cell according to claim 9 or 10 or a pharmaceutical composition according to claim 11 in the manufacture of a medicament for inducing an immune response in a subject or immunizing a subject against cancer.

14. 3. A method for improving immune cell function comprising engineering immune cells in vitro or ex vivo to express the membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric receptor of claim 1 or 2.

15. 10. A method for increasing phospho-STAT5 levels in an immune cell, comprising engineering said immune cell in vitro or ex vivo to express a membrane-bound interleukin-15 (IL-15)-IL-15Rα sushi domain chimeric receptor according to claim 1 or 2.

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