Chimeric antigen receptor targeting CD5-positive cancer

Abstract

Methods and compositions relating to engineered cell receptors that target CD5 (also known as cluster of differentiation 5 or LEU1 or T1) [0010] The embodiments of the present disclosure include methods and compositions related to targeting CD5-expressing cells with specific engineered receptors. In certain embodiments, NK cells are specifically engineered to bind to the CD5 antigen with a specific chimeric antigen receptor construct. In certain embodiments, the vector expressing the CD5-targeting CAR also expresses a specific suicide gene and / or one or more specific cytokines.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 174,990, filed April 14, 2021, which is incorporated by reference in its entirety.

[0002] Embodiments of the present disclosure include at least the fields of cell biology, molecular biology, immunology, and medicine, including cancer medicine. [Background technology]

[0003] Relapsed and refractory T-cell malignancies represent a group of hematological malignancies with particularly poor prognosis, for which novel therapies are desperately needed. CD5 is a pan T-cell surface marker present in the majority of T-cell malignancies. CD5 is also expressed in some B-cell malignancies, such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) (1, 2). On the other hand, expression of CD5 by normal cells is restricted to a small proportion of thymocytes, peripheral T cells, and B lymphocytes, and is not expressed by other hematopoietic cells. Thus, CD5 is an ideal target antigen for adoptive cell therapy using CARs. However, the development of CAR-T cells targeting T-cell-derived neoplasms has been hindered because most target antigens are commonly expressed by both malignant and normal T lymphocytes, potentially promoting CAR-T cell fratricide (3). Despite the issue of fratricide, CD5 CAR-T cells have been successfully developed and have shown preclinical efficacy (4) and clinical safety, with preliminary reports of early signs of clinical efficacy in patients with T-cell malignancies. However, using alternative immune effector cells that do not express CD5 on their surface is an attractive approach, as it removes the concern of fratricide. NK cells are an attractive option as a platform for CD5-targeted CAR engineering, as they do not express the CD5 antigen on their surface. Another advantage of NK cells in CAR engineering is that, in contrast to T cells, they do not cause graft-versus-host disease (GVHD) in allogeneic transplants, opening the possibility of producing a completely off-the-shelf cell product for use at the point-of-care. A new platform has been developed that addresses potential safety concerns by genetically modifying CB-derived NK cells to express a CAR, ectopically produce IL-15, support NK cell proliferation and persistence in vivo, and express the suicide gene inducible caspase 9 (iC9). These iC9 / CAR19 / IL15-NK cells have demonstrated efficacy against B-cell malignancies in preclinical and clinical trials.(6,7) No cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), or other toxicities associated with the CAR-NK cell product have been observed.(7) The present disclosure relates to the expansion of the CAR-NK cell platform to target other cancer antigens, including CD5, thereby expanding the clinical impact of this therapy.

[0004] Multiple studies have demonstrated that the choice of costimulatory domain in CAR-T cells impacts their function, persistence, and metabolic profile (8-10). In the field of CAR-NK cells, the first constructs tested in the clinic incorporate CD28, a T cell-specific costimulatory domain borrowed from CAR-T cell design. Although there are preclinical CAR-NK cell studies that have examined the function of various NK-specific costimulatory molecules, no studies have systematically examined the impact of costimulatory domains on NK cell proliferation, transcriptome, proteome profile, multifunctionality, metabolism, and strength. In this disclosure, we aim to comprehensively study and systematically examine the impact of costimulatory domains, including those more relevant to NK cell biology. In particular, DNAX-activating protein 10 (DAP10), a major adaptor protein and the only signaling intermediate of NKG2D in human NK cells, DNAX-activating protein 12 (DAP12), an important adaptor molecule that associates with multiple activating receptors (NKG2C, NKp44, activating KIR, etc.), and NKG2D, one of the most potent NK cytotoxicity receptors essential for antitumor immunity (11). The results showed that CD5 CAR-NK cells with DAP10 costimulatory molecules performed significantly better in vitro and in vivo compared with CD5 CAR-NK cells with other costimulatory molecules, exhibiting multifunctionality, metabolic aptitude, and memory-like properties that enhance antitumor activity. Based on these preclinical data, a phase I / II clinical trial to evaluate the safety and efficacy of CD5-DAP10-CD3ζ CAR-NK cells is in preparation.

[0005] Thus, the present disclosure relates in certain embodiments to methods and compositions for the genetic engineering of cells for adoptive cell therapy, including human NK cells, to target cancers, including CD5-positive cancers. Summary of the Invention

[0006] Embodiments of the present disclosure encompass methods and compositions related to engineered cell receptors that target CD5 (e.g., also known as cluster of differentiation 5 or LEU1 or T1). In certain embodiments, the engineered receptors that target CD5 are in the form of polynucleotides, polypeptides, or are included on the surface of any type of cell, including immune cells. In certain cases, the cells are immune cells, and in certain embodiments, the immune cells are NK cells, NK T cells, invariant NKT cells, gamma delta T cells, regulatory T cells, B cells, macrophages, mesenchymal stromal cells (MSCs), dendritic cells, and the like, from any source. In certain embodiments, reprogrammed NK cells from umbilical cord blood (CB-NK) are encompassed to target cancers that express the CD5 molecule.

[0007] CD5 is utilized as a target antigen in the methods and compositions because it is expressed in a number of cancers, including chronic lymphoproliferative disorders / lymphomas, T-cell leukemias (T-ALL), T-cell lymphomas, and some B-cell malignancies such as chronic lymphocytic leukemia and mantle cell lymphoma, and is expressed in thymic carcinoma, as well as thyroid, liver, colorectal, and cervical cancers.

[0008] In certain embodiments, the adoptive cell therapy of the present disclosure, including NK cell therapy, is particularly effective in allowing eradication of cancer cells even upon re-exposure to cancer. Thus, in some embodiments, an individual may be administered any cell therapy encompassed herein in the absence of detectable cancer in the individual. In certain cases, an individual may be at risk for any type of cancer, such as a higher risk than the general population. In some cases, an individual may have undergone one or more treatments for cancer and may or may not be considered cancer-free at the time of administration of the cell therapy of the present disclosure. Thus, in various embodiments, there are methods of preventing cancer or preventing metastasis of cancer in an individual, comprising administering to the individual a therapeutically effective amount of any of the cells encompassed herein. The administration step may be performed one or more times. When performed multiple times, there may be any suitable period between administrations, including 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years between administrations.

[0009] Some embodiments of CD5-targeted CAR T cells have been tested for the treatment of T cell malignancies. However, CD5 is constitutively expressed by normal T cells and on T cell leukemia / lymphoma, and its use in CAR T cell therapy requires genetic deletion of the CD5 gene to prevent fratricide during CAR T manufacturing. In contrast, normal NK cells do not express CD5, and are therefore not exposed to fratricide. Thus, in certain embodiments of the present disclosure, NK cells, including those derived from umbilical cord blood (CB), are reprogrammed to target cancers that express CD5. The present disclosure includes several novel CAR molecules, which include fusions of scFvs targeting the human CD5 antigen with one or more activation signaling endodomains, and in some cases, include the incorporation of CD3zeta alone or in combination with a costimulatory or adaptor signaling domain, such as NKG2D, 41BB, CD28, DAP10, and / or DAP12. In certain cases, allogeneic CB-NK cells are retrovirally transduced to express the CD5 CAR.

[0010] In certain embodiments, the immune cells of the present disclosure that include a CD5 CAR molecule also express one or more proteins that support their survival and proliferation. In certain cases, the immune cells are engineered to express one or more cytokines that promote cell proliferation and persistence. In certain cases, the cytokines are interleukin 15 (IL-15), IL-2, IL-7, IL-12, IL-18, and / or IL-21, which aid in the survival and maintenance of immune cells (e.g., NK cells). In certain embodiments, the vector encoding the CAR also encodes a cytokine, each of which is ultimately produced as a separate polypeptide.

[0011]

[0013] Embodiments of the present disclosure encompass a variety of novel specific CAR constructs incorporating a CD5 scFv heterologously fused to one or more signaling domains, including, for example, the cytoplasmic portion of CD3ζ and one or more of CD28, DAP10, DAP12, and NKG2D. The scFv optionally comprises the heavy (V) domain of a murine or human antibody with specificity for the human CD5 antigen. H ) chain and light (V L ) chain. The vector may also contain one or more cytokine genes, including genes producing human IL-1, IL-2, IL-21, IL-12, IL-7, IL-23, and / or IL-18, which aid in the survival and maintenance of NK cells. As an example, CB-NK cells modified in this way contain a vector encoding a CD5 scFv within a CAR that contains one or more costimulatory domains and CD3ζ, in addition to IL15, which is produced as a molecule separate from the CAR.

[0012] Certain embodiments of the present disclosure allow for the use of pre-made immune cells, including at least NK cells, that are allogeneic to the recipient individual, target any type of CD5 positive cell, and may or may not be transduced to express one or more cytokines, such as IL15, IL-2, IL-21, IL-12, IL-7, IL-23, and / or IL-18.

[0013] In certain embodiments of the present disclosure, the expression of one or more endogenous genes in immune cells is modified, for example, expression can be partially or completely reduced.Modification can occur by any means, but in certain embodiments, the expression of one or more genes is modified, for example, by reducing expression level, which can occur by any suitable means, including CRISPR or any other gene editing technology. By way of example only, the endogenous gene may be selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, CTLA-4, TDAG8, CD38, CREM, and combinations thereof.

[0014] In one embodiment, there is an expression construct that encodes an engineered CD5-specific receptor and optionally includes a sequence that encodes one or both of the following: (a) a suicide gene; (b) a cytokine. In certain cases, the engineered CD5-specific receptor is a chimeric antigen receptor (CAR) or a T cell receptor. The CD5-specific CAR may include a scFv having a heavy chain and a light chain, and the heavy chain in the CAR-encoding sequence is upstream of the light chain in the 5' to 3' direction. In other cases, the CD5-specific CAR includes a scFv having a heavy chain and a light chain, and the heavy chain in the CAR-encoding sequence is downstream of the light chain in the 5' to 3' direction. In any case herein, the CD5-specific CAR may or may not include a codon-optimized scFv. In any case herein, the CD5-specific CAR may or may not include a humanized scFv. In any case herein, the CD5-specific CAR may or may not include a signaling peptide, such as one derived from CD8 alpha, Ig heavy chain, or granulocyte macrophage colony-stimulating factor receptor, or a signal peptide derived from one or more other surface receptors. In certain embodiments, the CD5-specific CAR includes one or more costimulatory domains, such as one or more costimulatory domains selected from the group consisting of CD28, CD27, OX-40 (CD134), DAP10, DAP12, 4-1BB (CD137), CD40L, 2B4, DNAM, CS1, CD48, NKG2D, NKp30, NKp44, NKp46, NKp80, ICOS, or a combination thereof.

[0015] Any CD5-specific CAR may or may not include CD3 zeta and / or a hinge between the scFv and the transmembrane domain. In certain cases, the hinge is a CD8-alpha hinge, the hinge includes an artificial spacer composed of Gly3, or the hinge includes the CH1, CH2, and / or CH3 domains of IgG. In certain embodiments, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, IL-7, or a combination thereof. When a suicide gene is used, the suicide gene may be a mutant TNF-alpha (such as an engineered non-secretory mutant), inducible caspase 9, HSV-thymidine kinase, CD19, CD20, CD52, or EGFRv3.

[0016] The embodiments of the present disclosure include any type of immune cell, including any expression encompassed herein. In certain embodiments, the immune cell is a NK cell, a T cell, a gamma delta T cell, an invariant NKT (iNKT) cell, a B cell, a macrophage, an MSC, a dendritic cell, or a mixture thereof. When the immune cell is a NK cell, the NK cell can be derived from umbilical cord blood (including pooled umbilical cord blood units), peripheral blood, induced pluripotent stem cells, bone marrow, and / or cell lines. In a specific embodiment, the NK cell line is the NK-92 cell line, or another NK cell line derived from a tumor, or a healthy NK cell or progenitor cell.

[0017] In certain embodiments, the immune cells are NK cells, such as those derived from cord blood, such as cord blood mononuclear cells. The NK cells may be CD56+ NK cells in certain cases. The NK cells may express one or more exogenously provided cytokines, such as IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, or combinations thereof. Certain embodiments include populations of any type of immune cells of the present disclosure, and the cells may be present in a suitable medium or any type of suitable carrier.

[0018] In one embodiment, there is a method of killing CD5 positive cells in an individual, comprising administering to the individual an effective amount of cells having any expression construct encompassed by the present disclosure. In certain embodiments, the cells are NK cells, T cells, gamma delta T cells, invariant NKT (iNKT) cells, B cells, macrophages, gamma delta T cells, or dendritic cells. The NK cells can be derived from cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, or cell lines. The NK cells can be derived from cord blood mononuclear cells. In some cases, the CD5 positive cells are cancer cells, e.g., from hematopoietic cancer or solid tumors. The cells can be allogeneic or autologous to the individual, which may or may not be human. The cells can be administered to the individual by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intrapleurally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, topically, by perfusion, into the tumor microenvironment, or a combination thereof.

[0019] In certain embodiments of the method, the cells may be administered to the individual one or more times. The period between administration of the cells to the individual may be 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or more than a year. The method may further comprise administering to the individual an effective amount of an additional therapy, such as surgery, radiation, gene therapy, immunotherapy, and / or hormone therapy. The additional therapy may optionally comprise one or more antibodies or antibody-based agents. In some embodiments of the method, the method may further comprise identifying CD5 positive cells in the individual.

[0020] Embodiments of the present disclosure include expression constructs comprising a sequence encoding an engineered CD5-specific receptor, said receptor comprising one of the following: (a) a CD28 transmembrane domain (TM) and a CD28 intracytoplasmic domain (ICD); (b) a CD28 TM, a CD28 ICD, and a CD3 zeta signaling domain; (c) a CD28 TM and a DAP12 ICD; (d) a CD28 TM, a DAP12 ICD, and a CD3 zeta signaling domain; (e) a DAP12 TM and a DAP12 ICD; (f) a DAP12 TM, a DAP12 ICD, and a CD3 zeta signaling domain; (g) a CD28 TM and a 4-1BB ICD; (h) a CD28 TM, a CD28 ICD, and a 4-1BB ICD; (i) a CD28 TM, a CD28 ICD, a 4-1BB ICD, and a CD3 zeta signaling domain; (j) a CD28 TM and a DAP10 (k) CD28 TM, DAP10 ICD, and CD3 zeta signaling domain; (l) DAP10 TM and DAP10 ICD; (m) DAP10 TM, DAP10 ICD, and CD3 zeta signaling domain; (n) NKG2D ICD; (o) NKG2D TM; (p) NKG2D TM and NKG2D ICD; (q) NKG2D TM, NKG2D ICD, and CD3 zeta signaling domain; (r) CD28 TM and NKG2D ICD; (s) NKG2D ICD and CD3 zeta signaling domain; (t) CD28 TM, NKG2D ICD, and CD3 zeta signaling domain; (u) 4-1BB TM and 4-1BB ICD; (v) CD28 TM and CD3 zeta in the absence of the costimulatory domain ICD; (w) CD8 TM and CD28 intracytoplasmic domain (ICD); (x) CD8 TM, CD28 ICD, and CD3 zeta signaling domain; (y) CD8 TM and DAP12 ICD; (z) CD8 TM, DAP12 ICD, and CD3 zeta signaling domain; (aa) CD8 TM and DAP12 ICD; (bb) CD8 TM, DAP12 ICD, and CD3 zeta signaling domain; (cc) CD8 TM and 4-1BB ICD;(dd) CD8 TM, CD28 ICD, and 4-1BB ICD; (ee) CD8 TM, CD28 ICD, 4-1BB ICD, and CD3 zeta signaling domain; (ff) CD8 TM and DAP10 ICD; (gg) CD8 TM, DAP10 ICD, and CD3 zeta signaling domain; (hh) CD8 TM and DAP10 ICD; (ii) CD8 TM, DAP10 ICD, and CD3 zeta signaling domain; (jj) CD8 ICD; (kk) CD8 TM; (II) CD8 TM and NKG2D ICD; (mm) CD8 TM, NKG2D ICD, and CD3 zeta signaling domain; (nn) CD8 TM and NKG2D ICD; (oo) CD8, NKG2D ICD, and CD3 zeta signaling domain; (pp) CD8 TM, NKG2D ICD, and CD3 zeta signaling domain; (qq) CD8 TM and 4-1BB ICD; or (rr) CD8 TM and CD3 zeta in the absence of ICD, which is a costimulatory domain. In some cases, the engineered CD5-specific receptor is a chimeric antigen receptor (CAR) or a T cell receptor. When the engineered CD5-specific receptor is a CAR, the CAR can include a CD-targeting extracellular domain that is an scFv or a ligand for CD5. The CD5-specific CAR can include an scFv having a heavy chain and a light chain, where the heavy chain in the CAR-encoding sequence is upstream of the light chain in the 5' to 3' direction. The CD5-specific CAR can include an scFv having a heavy chain and a light chain, where the heavy chain in the CAR-encoding sequence is downstream of the light chain in the 5' to 3' direction. In certain cases, the CD5-specific CAR includes a codon-optimized scFv or includes a humanized scFv. The scFv can be encoded by SEQ ID NO: 1 or SEQ ID NO: 3. The scFv can include SEQ ID NO: 2 or SEQ ID NO: 4.;

[0021] In certain embodiments, the CD5-specific CAR comprises a signaling peptide such as one derived from CD8 alpha, Ig heavy chain, granulocyte-macrophage colony-stimulating factor receptor, CD3 signaling peptide, CD4 signaling peptide, or a signal peptide derived from one or more other surface receptors.

[0022] In some embodiments of CD5-specific CARs, the TM is encoded by SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13. The TM may include SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14. If a specific ICD is utilized, it may be encoded by SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21. The ICD may include SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In certain cases, the CD5-specific CAR includes an ICD derived from CD27, OX-40 (CD134), CD40L, 2B4, DNAM, CS1, CD48, NKp30, NKp44, NKp46, NKp80, ICOS, or a combination thereof.

[0023] In a particular embodiment, the engineered CD5-specific receptor is a CD5-specific CAR that comprises a hinge between the CD5-specific scFv and the transmembrane domain. The hinge can be an IgG hinge (IgG1 hinge, IgG2 hinge, IgG3 hinge, or IgG4 hinge, CD28 hinge, CD8-alpha hinge, etc.), the hinge comprises an artificial spacer composed of Gly3, or the hinge comprises the CH1, CH2, and / or CH3 domain of IgG. The hinge can be encoded by SEQ ID NO: 23 or 25, or can comprise SEQ ID NO: 24 or SEQ ID NO: 26.

[0024] The expression construct may encode a polypeptide of interest other than an engineered CD5-specific receptor, which may be separated on the construct by a 2A element (e.g., E2A (including that encoded by SEQ ID NO:31 or including SEQ ID NO:32)) or an IRES element. The polypeptide of interest may be a therapeutic protein or a protein that enhances the activity, proliferation, and / or persistence of a cell. The polypeptide of interest may be a suicide gene (e.g., non-secreted mutant TNF-alpha, inducible caspase 9, HSV-thymidine kinase, CD19, CD20, CD52, or EGFRv3), a cytokine, a human or viral protein that enhances proliferation, expansion, and / or metabolic fitness, or a combination thereof. In certain embodiments, the expression construct encodes one or more cytokines in addition to the CD5-specific CAR, which may be IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, or a combination thereof. Where IL-15 is utilized, it may be encoded by SEQ ID NO:29 or may comprise SEQ ID NO:30.

[0025] In certain cases, the expression construct encodes a CD5-specific CAR encoded by SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47. The engineered CD5-specific receptor can be a CAR comprising SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:48.

[0026] Embodiments include any vector containing any expression construct encompassed herein, including viral vectors (adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, or retroviral vectors) or non-viral vectors (plasmids, liposomes, nanoparticles, lipids, carbohydrates, or combinations thereof). Also, embodiments include any immune cell containing an expression construct of the present disclosure, where the immune cell can be a natural killer (NK) cell, a T cell, a gamma delta T cell, an invariant NKT (iNKT) cell, a B cell, a macrophage, an MSC, or a dendritic cell. When the immune cell is an NK cell, the NK cell can be derived from cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, or a cell line. The NK cell line can be an NK-92 cell line, or another NK cell line derived from a tumor, or a healthy NK cell or progenitor cell. In certain cases, the NK cell is derived from a cord blood mononuclear cell. The NK cell can be a CD56+ NK cell. NK cells can express one or more exogenously provided cytokines, including IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, IL-7, or combinations thereof. In immune cells, one or more endogenous genes can be modified in expression, for example, using CRISPR, such that expression is partially or completely reduced. The endogenous gene may be selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, CTLA-4, TDAG8, CD38, CREM, and combinations thereof.

[0027] The embodiments of the present disclosure include a population of immune cells as described herein, the cells being present in a suitable medium. The population may be stored in a storage facility, for example in a frozen state, and the medium may include one or more cryoprotectants. In a particular embodiment, the population of immune cells is a NK cell comprising an expression construct encoding a CAR and IL-15. The CAR and IL-15 may be encoded by SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47, and may be included in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:48.

[0028] Embodiments of the present disclosure include a method of killing CD5 positive cells in an individual, such as an individual with a CD5 positive cancer, comprising administering to the individual an effective amount of cells having any expression construct encompassed herein. The cells can be immune cells such as NK cells, T cells, gamma delta T cells, invariant NKT (iNKT) cells, B cells, macrophages, gamma delta T cells, dendritic cells, or mixtures thereof. The NK cells can be derived from cord blood, cord blood mononuclear cells, peripheral blood, induced pluripotent stem cells, bone marrow, cell lines, or mixtures thereof. The cells can be allogeneic or autologous to the individual. The individual can be a mammal, such as a human. The cells can be administered to the individual one or more times. The period between administration of the cells to the individual can be 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or more than a year. Any treatment method may further comprise administering to the individual an effective amount of additional therapy, such as surgery, radiation, gene therapy, immunotherapy (including one or more antibodies), or hormone therapy. The cells may be administered to the individual by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, topically, systemically, by perfusion, into the tumor microenvironment, or a combination thereof. The method may further comprise identifying a CD5-positive cancer in the individual, and / or generating cells carrying the expression construct.

[0029] It is specifically contemplated that any limitation discussed with respect to one embodiment of the present invention may apply to any other embodiment of the present invention. Moreover, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to make or utilize any composition of the present invention. Also, aspects of the embodiments described in the examples are embodiments that may be implemented in the context of embodiments discussed elsewhere in different examples or elsewhere in this application, such as the Summary of the Invention, the Detailed Description of the Invention, the Claims, and the Brief Description of the Drawings.

[0030] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure so that the detailed description of the invention that follows may be better understood. Additional features and advantages will be described hereinafter which form the subject of the claims. It should be understood by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. It should also be understood by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features believed to be characteristic of the design disclosed herein, both as to its organization and method of operation, together with further objects and advantages, will be better understood from the following description when considered in conjunction with the accompanying drawings. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.

[0031] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0032] [Figure 1A]Cord blood NK cells can be successfully transduced with CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR constructs and can target CD5+ tumor cells, but do not result in long-term cures in vivo. Map of CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR constructs. [Figure 1B] Cord blood NK cells can be successfully transduced with CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR constructs and can target CD5+ tumor cells, but do not result in long-term cures in vivo. Bar graph showing the transduction efficiency of NK cells transduced with CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR constructs compared to non-transduced (NT) NK cells (from left to right, bars represent untransduced cells, cells with CD5CAR 41BB-CD3ζ, and cells with CD5 CAR CD28-CD3ζ). [Figure 1C] Cord blood NK cells can be successfully transduced with CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR constructs and can target CD5+ tumor cells, but do not result in long-term cures in vivo. Graph showing fold change proliferation of CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR-NK cells compared to NT NK cells, where day 0 indicates the day of transduction. [Figure 1D] Cord blood NK cells can be successfully transduced with CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR constructs and can target CD5+ tumor cells, but do not result in long-term cures in vivo. Bar graphs showing the percentage of cytokine production (IFNg, TNFα) and degranulation (CD107a) of CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR-NK cells compared to NT NK cells after 6 hours of coculture with CD5+ CCRF-CEM tumors (from left to right, bars represent untransduced cells, cells with CD5CAR 41BB-CD3ζ, and cells with CD5CAR CD28-CD3ζ). [Figure 1E]Cord blood NK cells can be successfully transduced with CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR constructs and can target CD5+ tumor cells, but do not result in long-term cures in vivo. Graph showing percent lysis of CD5+ CCRF-CEM after 4 hours of co-culture with CD5-CD28-CD3ζ or CD5-41BB-CD3ζ CAR-NK cells or NT NK cells (NT at the bottom, CD5CAR CD28-CD3ζ at the top). [Figure 1F] Cord blood NK cells can be successfully transduced with CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR constructs and can target CD5+ tumor cells, but do not result in long-term cures in vivo. Bioluminescence imaging (BLI) showing tumor growth over time in CD5+ CCRF-CEM tumor-implanted mice transduced with firefly luciferase (Ffluc) and left untreated or treated with NT NK cells or CD5-CD28-CD3ζ or CD5-41BB-CD3ζ CAR-NK cells. [Figure 1G] Cord blood NK cells can be successfully transduced with CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR constructs and can target CD5+ tumor cells, but do not result in long-term cures in vivo. Graph plotting the mean radiance of BLI data comparing the various groups of mice shown in Panel 1F (CCRF-CEM alone on the left, CCRF-CEM+CD5CAR CD28-CD3ζ on the right). [Figure 1H] Cord blood NK cells can be successfully transduced with CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CAR constructs and can target CD5+ tumor cells, but do not result in long-term cures in vivo. Graph showing survival curves of various groups of mice shown in panel 1F (CCRF-CEM alone on the left, CCRF-CEM+CD5CAR CD28-CD3ζ on the right).

[0033] [Figure 2A]Optimization of CD5 CAR construct design by incorporating costimulatory molecules relevant to NK cell biology. Map of different CD5 CAR constructs with different transmembrane and costimulatory domains. [Figure 2B] Optimization of CD5 CAR construct design by incorporating costimulatory molecules relevant to NK cell biology. Bar graph showing transduction efficiency of NK cells transduced with different CD5 CAR constructs compared to non-transduced (NT) NK cells. [Figure 2C] Optimization of CD5 CAR construct design by incorporating costimulatory molecules relevant to NK cell biology. Graph showing expansion (cell numbers in millions over time) of various CD5 CAR-NK cell products compared to NT NK cells, where day 0 indicates the day of transduction. [Figure 2D] Optimization of CD5 CAR construct design by incorporating costimulatory molecules relevant to NK cell biology. Bar graph showing percentage of cytokine production (IFNg, TNFα) and degranulation (CD107a) of different CD5 CAR-NK cells compared to NT NK cells after 6 hours of co-culture with CD5+CCRF-CEM tumors. [Figure 2E] Optimization of CD5 CAR construct design by incorporating costimulatory molecules relevant to NK cell biology. Graph showing percent lysis of CD5+CCRF-CEM after 4 hours of co-culture with various CD5 CAR-NK or NT NK cells.

[0034] [Figure 3A]CD5 CAR-NK cells with DAP10 costimulatory domains significantly improve tumor control compared to CD5 CAR-NK cells with CD28 or DAP12 costimulatory domains. CD5 CAR-NK cells with DAP10 costimulatory domains significantly improve tumor control compared to CD5 CAR-NK cells with CD28 or DAP12 costimulatory domains. Bioluminescence imaging (BLI) showing tumor growth over time in CD5+CCRF-CEM tumor-implanted mice transduced with firefly luciferase (Ffluc) and left untreated or treated with NT NK cells or CD5-CD28-CD3ζ or CD5-DAP12-CD3ζ or CD5-DAP10-CD3ζ CAR-NK cells. [Figure 3B] CD5 CAR-NK cells with DAP10 costimulatory domains significantly improve tumor control compared to CD5 CAR-NK cells with CD28 or DAP12 costimulatory domains. Graph showing mean radiance plot of BLI data comparing various groups of mice shown in panel 3A, with data for each mouse plotted separately and mice belonging to the same group labeled with different colors (high CD5 CCRF-CEM alone is the left line; CCRF-CEM+CD5 CAR CD28CD3ζ is the right line). [Figure 3C] CD5 CAR-NK cells with DAP10 costimulatory domains significantly improve tumor control compared to CD5 CAR-NK cells with CD28 or DAP12 costimulatory domains. Graph showing survival curves of the various groups of mice shown in panel 3A. [Figure 3D]CD5 CAR-NK cells with DAP10 costimulatory domains significantly improve tumor control compared to CD5 CAR-NK cells with CD28 or DAP12 costimulatory domains. Graph showing the weight of mice in the various groups described in panel 3A. Data for each mouse is plotted separately, and mice belonging to the same group are labeled with different colors (NT is the vertical line on the left; CCRF-CEM+CD5 CAR CD28CD3ζ is the vertical line on the right; CCRF-CEM+CD5 CAR DAP10CD3ζ is the horizontal line on top of itself after about 40 days). [Figure 3E] CD5 CAR-NK cells with DAP10 costimulatory domains significantly improve tumor control compared with CD5 CAR-NK cells with CD28 or DAP12 costimulatory domains.CD5 CAR-NK cells with DAP10-CD3ζ signaling show superior activity against T-ALL cell lines, even with low CD5 expression. Graph showing survival curves of various groups of mice implanted with CCRF-CEM tumors with low CD5 expression and then left untreated or treated with non-transduced (NT) NK cells or CD5 CAR-NK cells of various designs (CD5-41BB-CD3ζ, CD5-DAP12-CD3ζ, CD5-CD28-CD3ζ, CD5-DAP10-CD3ζ) (Low CD5 CRF alone is the leftmost vertical line; Low CD5 CCRF-CEM+CD5CAR DAP10-CD3ζ is the top horizontal line; Low CD5 CCRF-CEM_CD5CAR CD28-CD3ζ is the short vertical line that ends at the rightmost end at a survival rate of approximately 60%).

[0035] [Figure 4A] CD5 CAR-NK cells with DAP10 signaling improve outcomes in a PDX mouse model of CD5+ mantle cell lymphoma. Schematic of mouse experimental design showing that mice were subcutaneously injected with PDX cell lines on day 0, mice were injected via tail vein with CD5-DAP10-CD3ζ CD5 CAR-NK cells with DAP10 costimulation on day 21, and mice were sacrificed on day 60 and tissues analyzed by flow cytometry. [Figure 4B] CD5 CAR-NK cells with DAP10 signaling improve outcomes in PDX mouse models of CD5+ mantle cell lymphoma. Bar graphs showing absolute numbers of CD45+CD5+ tumor cells in subcutaneous tumors, spleen, and bone marrow in untreated mice or mice treated with CD5 CAR with DAP10 costimulation (tumor alone is the left bar).

[0036] [Figure 5A] CD5 CAR-NK cells with DAP10-CD3ζ signaling show an enhanced activation profile at the proteomic level. TSNE plot showing different clusters from NT and CD5 CAR-NK cells combined together. [Figure 5B] CD5 CAR-NK cells with DAP10-CD3ζ signaling show an enhanced activation profile at the proteomic level. TSNE plot showing clusters in non-transduced (NT) NK cells and CD5 CAR-NK cells with DAP10 costimulation. Clones 8 and 11 appear to be specific for CD5 CAR-NK cells with DAP10 costimulation. [Figure 5C] CD5 CAR-NK cells with DAP10-CD3ζ signaling show an enhanced activation profile at the proteomic level. Heatmap showing different markers of NK cells on the X-axis and different clusters on the Y-axis. It shows enrichment of NK cell activation markers (DNAM, NKG2D, CD69, maturation markers (EOMES and T-BET), and cytotoxicity markers (perforin, granzyme B, and TRAIL) in clusters 8 and 11 specific for CD5 CAR-NK cells with DAP10 costimulation.

[0037] [Figure 6A]CD5 CAR-NK cells with DAP10 costimulatory domains show enhanced polyfunctionality and metabolic fitness. Bar graph showing polyfunctionality of NK cells non-transduced (NT) or transduced with various CD5 CAR-NK cells, either without costimulation (CD3ζ alone) or with various costimulatory domains following stimulation with CD5+ antigen, showing the percentage of NK cells producing either 2, 3, 4, or 5+ proteins at the single cell level in response to antigen stimulation. [Figure 6B] CD5 CAR-NK cells with DAP10 costimulatory domains show enhanced multifunctionality and metabolic fitness. Multifunctionality heatmap showing cytokine production at the single cell level. Each row represents an NK cell state and each column represents a cluster of NK cells producing a specific set of cytokines; the more clusters, the more multifunctional the NK cell. [Figure 6C] CD5 CAR-NK cells with DAP10 costimulatory domain show enhanced multifunctionality and metabolic fitness. Bar graph showing multifunctionality strength index, showing what percentage of NK cells (NT or different CD5 CAR-NK cell designs) produce effector, stimulatory, or chemoattractant cytokines at the single cell level (chemoattractant scoring is at the top of the bar, effector scoring is at the bottom of the bar). [Figure 6D] CD5 CAR-NK cells with DAP10 costimulatory domains show enhanced multi-functionality and metabolic fitness. Graph showing oxygen consumption rate (OCR) of NT NK cells or NK cells transduced with various CD5 CAR-NK cell constructs. It shows that CD5 CAR-NK cells with DAP10 costimulatory domains have the highest OCR, a measure of mitochondrial activity and metabolic fitness.

[0038] [Figure 7A]CD5 CAR-NK cells with DAP10-CD3ζ signaling domain exhibit memory-like properties and continue to kill CD5+CCRF-CEM after multiple tumor re-challenges. Schematic detailing the experimental design for the Incucyte real-time cytotoxicity assay with multiple CD5+CCRF-CEM tumor re-challenges. Tumor cells were labeled with red dye prior to real-time imaging. [Figure 7B] CD5 CAR-NK cells with DAP10-CD3ζ signaling domain show memory-like properties and continue to kill CD5+CCRF-CEM after multiple tumor rechallenges. Graph showing red counts over time correlating with live tumor cell load. This shows that initially, all NK cells, even non-transduced (NT) NK cells, can kill CD5+CCRF-CEM tumors, and then, after multiple tumor rechallenges, over time, only CD5 CAR-NK cells with DAP10 costimulation can control the tumor. CD19 CAR-NK cells and CD19 / IL-15 CAR-NK cells were used as irrelevant CAR controls. [Figure 7C] CD5 CAR-NK cells with DAP10-CD3ζ signaling domain show memory-like properties and continue to kill CD5+CCRF-CEM after multiple tumor rechallenges. Graph showing cell confluence (%) over time correlating with live tumor cell load. This shows that initially, all NK cells, even non-transduced (NT) NK cells, are able to kill CD5+CCRF-CEM tumors, and then, after multiple tumor rechallenges, over time, only CD5 CAR-NK cells with DAP10 costimulation are able to control the tumor.

[0039] [Figure 8A]CD5 CAR-NK cells can persist and initiate recall upon tumor re-challenge in vivo. Schematic detailing the experimental design of the in vivo mouse model showing timing of irradiation, timing of CD5+ CCRF-CEM tumor injection, timing of CD5 CAR-NK cell infusion, and timing of re-challenge with CD5+ CCRF-CEM tumors (transduced with GFP). [Figure 8B] CD5 CAR-NK cells are able to persist and mount recall responses to tumor rechallenge in vivo. FACS plots showing flow cytometry data before (left panel) and after (right panel) rechallenge, showing the human CD45+ gate and the NK cell gate (CD56+ and GFP-). This shows that CD5 CAR-NK cells are able to proliferate and mount recall responses to CD5+ CCRF-CEM tumors after tumor rechallenge.

[0040] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can be made by those skilled in the art without departing from the present invention. It is understood that various alternatives to the embodiments of the present disclosure described herein may be used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] 1. Definition Example Following long-standing patent law practice, the words "a" and "an" when used in conjunction with the word comprising, including in the claims, herein refer to "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

[0042] Throughout this specification, unless otherwise required by context, the words "comprise", "include", and "comprising" are understood to mean the inclusion of a recited step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and other elements may not be present. "Consisting essentially of" means including the elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0043] Throughout this specification, references to "one embodiment," "one embodiment," "particular embodiment," "related embodiment," "an embodiment," "additional embodiment," or "further embodiment," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with an embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0044] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z". It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0045] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the field of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0046] As used herein, the term "engineered" refers to entities that are produced by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, engineered entities are synthetic and comprise elements that do not occur in nature or that are constructed by the methods utilized in this disclosure.

[0047] The term "isolated" as used herein refers to a molecule or biological or cellular material that is substantially free from other materials. In one embodiment, the term "isolated" refers to a nucleic acid such as DNA or RNA, or a protein or polypeptide, or a cell or organelle, or a tissue or organ, that is separated from other DNA or RNA, or proteins or polypeptides, or cells or organelles, or tissues or organs, respectively, as they exist in a natural source. The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, culture medium, if produced by recombinant DNA technology, or chemical precursors or other chemicals, if chemically synthesized. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to a polypeptide that is isolated from other cellular proteins, and is meant to include both purified and recombinant polypeptides. The term "isolated" is also used herein to refer to a cell or tissue that is isolated from other cells or tissues, and is meant to include both cultured and engineered cells or tissues.

[0048] As used herein, "prevent" and similar terms such as "protect," "prevented," and the like refer to an approach to prevent, inhibit, or reduce the likelihood of the onset or recurrence of a disease or condition, such as cancer. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0049] The term "sample" as used herein generally refers to a biological sample. A sample can be taken from an individual's tissue or cells. In some examples, a sample consists of or is derived from a tissue biopsy, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spot, cultured cells, discarded tissue. A sample may be separated from its source prior to collection. Non-limiting examples include blood, cerebrospinal fluid, pleural fluid, amniotic fluid, lymphatic fluid, saliva, urine, stool, tears, sweat, or mucosal excretions, and other bodily fluids separated from a primary source prior to collection. In some examples, a sample is separated from its primary source (cells, tissues, bodily fluids such as blood, environmental samples, etc.) during sample preparation. A sample may or may not be purified or concentrated from its primary source. In some cases, the primary source is homogenized before further processing. The sample may be filtered or centrifuged to remove buffy coat, lipids, or particulate matter. The sample may also be purified or concentrated for nucleic acids or treated with RNase. The sample may include intact, fragmented, or partially disaggregated tissues or cells.

[0050] The term "subject" as used herein generally refers to an individual having a biological sample being processed or analyzed, and in certain cases, an individual having or suspected of having cancer. A subject may be any living organism or animal subject that is the subject of a method or material, including mammals, such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), household pets (e.g., dogs, cats, rodents), horses, and transgenic non-human animals. A subject may be, for example, a patient having or suspected of having a disease (sometimes referred to as a pathology), such as a benign or malignant neoplasm, or cancer. A subject may be undergoing or having been treated. A subject may be asymptomatic. A subject may be a healthy individual, but may wish to prevent cancer. The terms "individual" are used interchangeably, at least in some cases. A "subject" or "individual" as used herein may or may not be housed in a medical facility, or may be treated as an outpatient in a medical facility. An individual may be administered one or more medical compositions via the Internet.An individual may include any age of human or non-human animal, and thus includes both adults and juveniles (i.e., children) and infants, including individuals in utero.The term does not imply the need for medical treatment, so an individual may participate in an experiment, whether clinical or in support of basic science research, whether voluntary or involuntary.

[0051] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathological condition of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment may optionally include either the reduction or amelioration of symptoms of a disease or condition, or the delay in the progression of a disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of a disease or condition, or symptoms associated therewith.

[0052] The present disclosure relates to methods and compositions directed to the treatment of CD5-positive cancers, particularly utilizing adoptive cell therapy targeting CD5-positive cancer cells. In certain embodiments, any kind of genetically engineered mammalian immune cells are generated that target CD5-positive cancers, including at least human NK cells. The present disclosure encompasses any kind of genetically engineered receptors for CD5, including chimeric antigen receptors (CARs). In certain embodiments, several novel expression constructs are provided, including retroviral constructs that express CD5-targeting single-chain variable fragments (scFvs) used in CARs, and in some cases, also express one or more cytokines, such as IL-15, to support cell survival and proliferation.

[0053] Provided herein is a designed CAR that targets CD5, which is expressed on at least T-cell malignancies, such as T-cell leukemia (T-ALL) and T-cell lymphoma. Targeting CD5 is particularly advantageous for immunotherapy of T-cell malignancies, since NK cells, unlike normal T cells, do not express CD5, a molecule expressed on all T-lymphoblastic leukemia cells as well as normal T cells. Thus, unlike CAR T cells, CAR NK cells are not targeted by CAR.CD5-mediated fratricide. Thus, the present disclosure provides several novel retroviral constructs that express single-chain variable fragments (scFv) against CD5, and in some cases, also express IL-15 to support NK cell survival and proliferation. In a series of in vitro and in vivo studies, the inventors confirmed the activity of CAR5 / IL-15-transduced CB-NK cells against T-cell malignancies. I. Engineered Receptors

[0054] The immune cells of the present disclosure can be engineered to express one or more antigen-binding receptors that target CD5, such as engineered CARs or other engineered TCRs.For example, the immune cells can be immune cells that are modified to express CARs and / or TCRs with antigen specificity for CD5.Other CARs and / or TCRs can be expressed by the same cell as the CD5 antigen receptor-expressing cell and can be directed to different antigens.In some embodiments, the immune cells are engineered to express CD5-specific CARs or CD5-specific TCRs by knocking in CARs or TCRs using CRISPR.

[0055] Suitable cell modification methods are known in the art. For example, see Sambrook and Ausubel above. For example, using the transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009, cells can be transduced to express CAR or TCR with antigen specificity against cancer antigen.

[0056] In some embodiments, the cell contains one or more nucleic acids introduced via genetic engineering that code for one or more antigen targeting receptors (at least one of which is for CD5), and the genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or sample obtained from the cell, e.g., obtained from another organism or cell that is not normally found, e.g., in the cell to be engineered and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, e.g., a nucleic acid that is not found in nature (e.g., a chimera).

[0057] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods of engineering and introducing such receptors into cells are described, for example, in WO 2000 / 14257, WO 2013 / 126726, WO 2012 / 129514, WO 2014 / 031687, WO 2013 / 166321, WO 2013 / 071154, WO 2013 / 123061, U.S. Patent Application Publication No. 2002 / 131960, U.S. Patent Application Publication No. 2013 / 287748, U.S. Patent Application Publication No. 2013 / 0149337, and the like. , U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, as well as in EP 2 537 416 A1, and / or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some embodiments, engineered antigen receptors include CARs described in U.S. Pat. No. 7,446,190 and those described in WO 2014 / 055668. A. Chimeric Antigen Receptor

[0058] In certain embodiments, a CD5-specific CAR is utilized that includes at least an extracellular domain that includes a) one or more intracellular signaling domains, b) a transmembrane domain, and c) at least one antigen-binding region that targets CD5, including specifically binding to CD5. In certain embodiments, the antigen-binding region is an antibody or a functional fragment thereof, while in other cases, the antigen-binding region of the CAR is not an antibody or a functional fragment thereof (such as a ligand for CD5). In some embodiments, the CD5-specific CAR binds only to CD5, while in other cases, the CAR as a single polypeptide is bispecific by including two or more antigen-binding domains, one of which binds to CD5 and the other of which binds to another non-identical antigen.

[0059] In some embodiments, engineered antigen receptors include CARs, including activating or stimulating CARs, or costimulatory CARs (see WO2014 / 055668). CARs generally comprise an extracellular antigen (or ligand) binding domain that is linked to one or more intracellular signaling components, in some embodiments, via a linker and / or a transmembrane domain. Such molecules typically mimic or approximate the signaling through a natural antigen receptor, the signaling through such receptor in combination with a costimulatory receptor, and / or the signaling through a costimulatory receptor alone.

[0060] It is believed that chimeric constructs can be introduced into immune cells as naked DNA or in suitable vectors.Methods for stable transfection of cells by electroporation with naked DNA are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding chimeric receptor contained in a plasmid expression vector in a suitable orientation for expression.

[0061] Alternatively, chimeric CAR constructs can be introduced into immune cells using viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors).Vector suitable for use according to the method of the present disclosure is non-replicative in immune cells.Many virus-based vectors are known, such as HIV, SV40, EBV, HSV, or BPV-based vectors, whose virus copy number maintained in cells is low enough to maintain cell viability.

[0062] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding CD5-specific CAR polypeptides, including CARs that are humanized to reduce immunogenicity (hCARs), and that include at least one intracellular signaling domain, a transmembrane domain, and an extracellular domain that includes one or more signaling motifs. In certain embodiments, CD5-specific CARs can recognize epitopes that include shared spaces between one or more antigens. In certain embodiments, the binding region can include the complementarity determining region of a monoclonal antibody, the variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0063] It is believed that the human CD5 CAR nucleic acid can be a human gene used to enhance cellular immunotherapy for human patients. In certain embodiments, the present disclosure includes a full-length CD5-specific CAR cDNA or coding region. The antigen-binding region or domain is the V of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody. H Chain and V L The fragments may include fragments of the chains, such as those described in U.S. Patent No. 7,109,304, which is incorporated herein by reference. The fragments may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is a CD5-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0064] The configuration can be a multimer, such as a diabody or a multimer. The multimer is most likely formed by cross-pairing of the variable parts of the light and heavy chains into a diabody. The hinge part of the construct can have a number of options, from being completely deleted, to maintaining the first cysteine, to a proline instead of a serine substitution, to being truncated to the first cysteine. The Fc part can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. Only one of the Fc domains can be used, for example, either the CH2 or CH3 domain from a human immunoglobulin. The hinge, CH2 and CH3 regions of a human immunoglobulin that have been modified to improve dimerization can also be used. Only the hinge part of an immunoglobulin can also be used. A portion of CD8 alpha can also be used.

[0065] In some embodiments, a CD5-specific CAR is constructed with specificity for CD5, such as CD5 expressed on disease cell types. Thus, a CAR typically comprises one or more CD5 binding molecules, such as one or more antigen-binding fragments, domains, antibody variable domains, and / or any type of antibody molecule, in its extracellular portion. An example of human CD5 nucleic acid is in the GenBank® database of the National Center for Biotechnology Information under accession number NM_014207. An example of human CD5 polypeptide is in GenBank® under accession number NP_055022. A person skilled in the art can generate antibodies, including scFvs against CD5, based at least on knowledge of polypeptides and routine practice, but many anti-CD5 scFvs and monoclonal antibodies already exist in the art.

[0066] In some embodiments, a CD5-specific CAR comprises an antigen-binding portion of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb). In certain embodiments, the antibody or functional fragment thereof is or is derived from H65 (Santa Cruz Biotechnology; Santa Cruz, Calif.); clone CRIS1 or clone 4C7 (Abnova Corporation; Walnut, Calif.); OX-19 (Santa Cruz Biotechnology; Santa Cruz, Calif.); Leu-1 (Becton-Dickinson; Mountain View, Calif.) and UCHT2 (Accurate Scientific; Westbury, NY); 53-7.3 (Affymetrix; Santa Clara, Calif.); 4H8E6 (Life Technologies; Grand Island, NY); T101; EP2952 (Abcam, Cambridge Mass.); or L17F12. In other embodiments, the antibody, e.g., scFv, is or is derived from the anti-CD5 antibodies D-9, H-3, HK231, N-20, Y2 / 178, H-300, L17F12, CD5 / 54 / F6, Q-20, or CC17, all available from Santa Cruz Biotechnology, Dallas Tex. Antibodies may also be generated de novo against CD5, and scFv sequences may be obtained or derived from such de novo antibodies.

[0067] In certain embodiments, the anti-CD5 CAR comprises an extracellular domain that is or comprises a ligand for CD5. In certain embodiments, the anti-CD5 CAR comprises an extracellular domain that comprises CD72 (Lyb-2), gp40-80, gp150, gp200, an IgVH framework region, IL-6, and fragments and mimetics thereof.

[0068] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or can be synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns have been shown to stabilize mRNA. It may also be more advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0069] In some embodiments, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally binds to one of the domains in the CAR is used. In some examples, the transmembrane domain is selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or different surface membrane protein, and to minimize interaction with other members of the receptor complex. The transmembrane domain in some embodiments is derived from either natural or synthetic sources. When the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. The transmembrane region may be derived from (i.e., at least includes) the alpha, beta, or zeta chain of the T cell receptor, CD28, DAP12, DAP10, NKG2D, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, and the like. Additionally, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises primarily hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0070] In some embodiments, the CD5 CAR nucleic acid includes sequences encoding other costimulatory receptors, such as transmembrane domains and one or more intracellular signaling domains. In addition to the primary T cell activation signal, such as may be initiated by CD3ζ and / or FcεRIγ, additional stimulatory signals for immune effector cell proliferation and effector function after chimeric receptor binding to target antigen may be utilized. For example, some or all of the human costimulatory receptors for enhanced activation of cells may be utilized, which may help to improve in vivo persistence and improve the therapeutic success of adoptive immunotherapy. Examples include costimulatory domains from molecules such as DAP12, DAP10, NKG2D, CD2, CD28, CD27, 4-1BB, (CD137), OX40, ICOS, (CD278), CD30, HVEM, CD40, LFA-1 (CD11a / CD18), and / or ICAM-1, although in alternative specific embodiments, any one of these listed may be excluded from use in the CAR.

[0071] In certain embodiments, the platform technology disclosed herein for genetically modifying immune cells, such as NK cells, includes (i) non-viral gene transfer using an electroporation device (e.g., nucleofector), (ii) CARs that signal through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations), (iii) CARs with variable length extracellular domains linking the CD5 recognition domain to the cell surface, and, in some cases, (iv) CARs. + It contains K562-derived artificial antigen-presenting cells (aAPCs) to enable robust and numerical expansion of immune cells ( Singh et al., 2008 ; Singh et al., 2011 ). B. Examples of Specific CAR Embodiments

[0072] In certain embodiments, specific CD5 CAR molecules are encompassed herein. In some cases, the CD5 binding domain of the CAR is an scFv, and any scFv that binds to CD5 and / or a ligand that binds to CD5 can be utilized herein. When an anti-CD5 scFv is utilized in the extracellular domain of the CAR, the variable heavy and variable light chains of the scFv can be in any order from N-terminus to C-terminus. For example, the variable heavy chain can be N-terminal to the variable light chain, or vice versa. The scFv and / or ligand that binds to CD5 in the CAR may or may not be codon-optimized. In certain embodiments, a vector encodes a CD5-specific CAR and also encodes one or more other molecules. For example, a vector may encode a CD5-specific CAR and also encode another protein of interest, such as another engineered antigen receptor, a suicide gene, and / or a specific cytokine.

[0073] On the same molecule, a CD5-specific CAR may include one or more antigen-specific extracellular domains, a specific hinge, a specific transmembrane domain, one or more specific costimulatory domains, and one or more specific activation signals. For example, when multiple antigen-specific extracellular domains are utilized to target two different antigens, one of which is CD5, there may be a linker between the two antigen-specific extracellular domains.

[0074] In certain embodiments of certain CAR molecules, CARs may utilize DAP10, DAP12, 4-1BB, NKG2D, or other costimulatory domains (which may be referred to herein as cytoplasmic domains). In some cases, CD3 zeta is utilized without any costimulatory domain. In certain embodiments of certain CAR molecules, CARs may utilize any suitable transmembrane domain, for example, from DAP12, DAP10, NKG2D, or CD28.

[0075] In certain embodiments, there is an expression construct that includes a sequence encoding a particular engineered CD5-specific receptor. In certain embodiments, any CD5 CAR comprises, in the absence of a costimulatory domain, an ICD, one of the following: (a) a CD28 transmembrane domain (TM) and a CD28 intracytoplasmic domain (ICD); (b) a CD28 TM, a CD28 ICD, and a CD3 zeta signaling domain; (c) a CD28 TM and a DAP12 ICD; (d) a CD28 TM, a DAP12 ICD, and a CD3 zeta signaling domain; (e) a DAP12 TM and a DAP12 ICD; (f) a DAP12 TM, a DAP12 ICD, and a CD3 zeta signaling domain; (g) a CD28 TM and a 4-1BB ICD; (h) a CD28 TM, a CD28 ICD, and a 4-1BB ICD; (i) a CD28 TM, a CD28 ICD, a 4-1BB ICD, and a CD3 zeta signaling domain; (j) a CD28 TM and a DAP10 ICD; (k) a CD28 (l) DAP10 TM and DAP10 ICD; (m) DAP10 TM, DAP10 ICD, and CD3 zeta signaling domain; (n) NKG2D ICD; (o) NKG2D TM; (p) NKG2D TM and NKG2D ICD; (q) NKG2D TM, NKG2D ICD, and CD3 zeta signaling domain; (r) CD28 TM and NKG2D ICD; (s) NKG2D ICD and CD3 zeta signaling domain; (t) CD28 TM, NKG2D ICD, and CD3 zeta signaling domain; or (u) CD28 TM and CD3 zeta.

[0076] Examples of specific sequence embodiments are provided below. 1. Antigen-specific extracellular domain

[0077] In a specific embodiment, the following anti-CD5 scFv (humanized) sequence is utilized: ATGGAGTTCGGTCTCAGTTGGCTGTTTTTGGTTGCTATCTTGAAGGGCGTCCAATGCAGCCGGGACATCCAGATGACCCAGTCTCCCTCTAGCATGTCAGCGAGTCTTGGTGATCGAGTGACGATTACCTGCAGAGCCTCTCAAGATATAAACAGCTATCTTTCATGGTTCCAACAGAAGCCGGGGAAGTCCCCAAAA ACTCTCATATACAGGGCGAATCGACTCGTAGACGGTGTGCCTTCAAGGTTTTCCGGGAGTGGTAGTGGCACAGATTACACACTTACAATCTCTTCATTGCAGTATGAGGATTTCGGGATCTACTACTGTCAACAGTACGACGAATCCCCATGGACGTTTGGGGGCGGGACCAAACTTGAGATAAAAGGGAGCACATCT GGAAGTGGTAAACCTGGGTCAGGGGAGGTTCCACAAAAGGACAAATTCAACTTGTCCAAAGCGGTCCTGGTCTTAAGAAGCCTGGAGGGTCTGTCAGGATAAGTTGTGCGGCATCCGGCTACACCTTCACCAACTATGGGATGAACTGGGTGAAACAAGCGCCTGGGAAAGGTCTTCGATGGATGGGCTGGATTAAT ACCCACACTGGAGAGCCCACTTACGCTGATGATTTCAAAGGACGATTTACCTTCTCCTTGGATACTTCCAAGAGTACCGCGTACTTGCAAATCAACAGTCTCCGGGCTGAAGACACGGCCACATACTTCTGTACGCGGAGAGGTATGACTGGTATTTTGATGTGTGGGGTCAGGGAACAACCGTGACTGTTTCAAGC (Sequence number in the sequence listing: 1)

[0078] The amino acid sequence of the translated scFv (humanized) (translated from SEQ ID NO: 1 in the sequence listing) is as follows: MEFGLSWLFLVAILKGVQCSRDIQMTQSPSSMSASLGDRVTITCRASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPGLKKPGGSVRISCAASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFTFSLDTSKSTAYLQINSLRAEDTATYFCTRRGYDWYFDVWGQGTTVTVSS (Sequence number: 2 in the sequence listing)

[0079] In a specific embodiment, the anti-CD5 scFv [mouse] sequence is utilized as follows: ATGGAGTTCGGCTTGAGTTGGTTGTTCCTTGTGGCGATACTCAAAGGCGTTCAATGTAGCCGAGACATAAAAAATGACCCAGTCTCCGTCATCTATGTATGCAAGCCTCGGCGAGCGAGTGACCATCACGTGCAAGGCGAGTCAAGATATAAACAGCTACTTGTCATGGTTCCAACAAAAACCAGGGAAATCACCTAAG ACCCTGATCTATAGAGCCAATCGCCTGGTTGACGGTGTCCCCTCCCGCTTTAGCGGCTCCGGAAGCGGTCAAGATTACTCTCTCACAATTTCTTCCTTGGATTATGAAGACATGGGGATCTACTATTGTCAACAGTATGACGAATCCCCGTGGACTTTCGGTGGCGGTACCAAATTGGAAATAAAGGGCTCTACAAGC GGCTCAGGAAAACCTGGATCAGGCGAAGGGTCTACGAAGGGCCAGATACAACTCGTTCAAAGTGGGCCAGAACTCAAAAAACCGGGAGAAACAGTGAAAATTTCTTGTAAGGCATCAGGATACACATTCACAAACTACGGGATGAATTGGGTCAAACAAGCACCCGGAAAGGGGCTGCGCTGGATGGGGTGGATCAAC ACACATACTGGGGAACCTACTTACGCAGACGATTTCAAGGGCAGATTCGCCTTTTCTTTGGAGACCTCCGCCTCTACTGCATACTTGCAGATAAACAACCTGAAGAATGAAGATACCGCCACCTACTTCTGTACGCGCAGGGGCTACGATTGGTATTTTGATGTATGGGGGGCAGGCACCACTGTTACTGTGTCAAGC (Sequence number in sequence list: 3)

[0080] The amino acid sequence of the translated scFv (mouse) (translated from SEQ ID NO:3 in the Sequence Listing) is as follows: MEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSS (Sequence number: 4 in the sequence listing) 2. Transmembrane domain

[0081] CD5-specific CAR can utilize any suitable transmembrane domain. Examples include at least DAP10, DAP12, CD28, NKG2D, CD3ε, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, T cell receptor a or b chain, CD3 zeta chain, ICOS, their functional derivatives, and combinations thereof. Specifically, DAP10, DAP12, CD28, or NKG2D transmembrane domain is utilized. Examples of specific transmembrane domain sequences are shown below.

[0082] The nucleotide sequence of the 4-1BB transmembrane domain: ATCATCTCCTTCTTTCTTGCGCTGACGTCGACTGCGTTGCTCTTCCTGCTGTTCTTCCTCACGCTCCGTTTCTCTGTTGTT (Sequence number: 5 in the sequence listing)

[0083] Amino acid sequence of the 4-1BB transmembrane domain: IISFFLALTSTALLFLLFFLTLRFSVV (Sequence Listing SEQ ID NO: 6)

[0084] CD28 transmembrane domain sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (Sequence number: 7 in the sequence listing)

[0085] The amino acid sequence of the CD28 transmembrane domain: FWVLVVVGGVLACYSLLVTVAFIIFWV (Sequence Listing SEQ ID NO: 8)

[0086] Nucleotide sequence of DAP10 transmembrane domain: CTCCTGGCAGGCCTCGTGGCTGCTGATGCGGTGGCATCGCTGCTCATCGTGGGGGCGGTGTTC (Sequence number: 9 in the sequence listing)

[0087] Amino acid sequence of DAP10 transmembrane domain: LLAGLVAADAVASLLIVGAVF (Sequence number: 10 in the sequence listing)

[0088] Nucleotide sequence of DAP12 transmembrane domain: GGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTG (Sequence Number: 11 in the Sequence Listing)

[0089] Amino acid sequence of DAP12 transmembrane domain: GVLAGIVMGDLVLTVLIALAV (Sequence Listing SEQ ID NO: 12)

[0090] NKG2D transmembrane domain sequence: GCGGTGATGATTATTTTTCGCATTGGCATGGCGGTGGCGATTTTTTGCTGCTTTTTTTTTCCG (Sequence number: 13 in the sequence listing)

[0091] Amino acid sequence of the NKG2D transmembrane domain: AVMIIFRIGMAVAIFCCFFFP (Sequence Listing SEQ ID NO: 14) 3. Intracytoplasmic domain (ICD)

[0092] One or more cytoplasmic domains (which may also be referred to herein as costimulatory domains, where appropriate) may or may not be utilized in a particular anti-CD5 CAR of the present disclosure. Specific examples include ICDs from 4-1BB, NKG2D, DAP10, DAP12, B7-1 / CD80, CD28, 4-1BBL, B7-2 / CD86, CTLA-4, B7-H1 / PD-L1, ICOS, B7-H2, PD-1, B7-H3, PD-L2, B7-H4, PDCD6, BTLA; or combinations thereof.

[0093] Examples of specific ICD sequences that can be used in a CAR include:

[0094] 4-1BB cytoplasmic domain sequence AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (Sequence number: 15 in the sequence listing)

[0095] 4-1BB cytoplasmic domain amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (Sequence Listing SEQ ID NO: 16)

[0096] DAP10 cytoplasmic domain sequence: CTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (Sequence Number: 17 in the Sequence Listing)

[0097] Amino acid sequence of the DAP10 cytoplasmic domain: LCARPRRSPAQEDGKVYINMPGRG (Sequence Listing SEQ ID NO: 18)

[0098] DAP12 cytoplasmic domain sequence: TACTTCCTGGGCCGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAA (Sequence number in the sequence listing: 19)

[0099] Amino acid sequence of the DAP12 cytoplasmic domain: YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (Sequence number: 20 in the sequence listing)

[0100] NKG2D cytoplasmic domain sequence: AGCGCGAACGAACGCTGCAAAAGCAAAGTGGTGCCGTGCCGCCAGAAACAGTGGCGCACCAGCTTTGATAGCAAAAAACTGGATCTGAACTATAACCATTTTGAAAGCATGGAATGGAGCCATCGCAGCCGCCGCGGCCGCATTTGGGGCATG (Sequence number: 21 in the sequence listing)

[0101] NKG2D cytoplasmic domain amino acid sequence: SANERCKSKVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGM (Sequence number: 22 in the sequence listing) 4. Hinge

[0102] In some embodiments of the CAR, there is a hinge region between one or more extracellular antigen binding domains and the transmembrane domain. In specific embodiments, the hinge is of a particular length, such as, for example, 10-20, 10-15, 11-20, 11-15, 12-20, 12-15, or 15-20 amino acids. The hinge can be any suitable hinge, and optionally includes a hinge from IgG, or CD28. In specific embodiments, the hinge is a small flexible polypeptide that connects the CH2-CH3 domain and the CH1 domain of IgG Fc. For example, CH2-CH3 hinges (partial or complete) of various IgG subclasses (IgG1-4, modified or unmodified) can be utilized. However, in some cases, rather than the entire CH2-CH3 hinge, a portion of the hinge is utilized (such as the CH3 alone or a portion of the CH3 alone). In certain embodiments, the CH2-CH3 hinge from IgG1 is utilized, and in some cases, either the entire CH2-CH3 hinge is utilized (all 229 amino acids), or only the CH3 hinge (119 amino acids), or a short hinge (12 amino acids).

[0103] In specific embodiments, the identity or length of the spacer and / or hinge can be altered to optimize the efficiency of the CAR. See, for example, Hudecek et al. (2014) and Jonnalagadda et al. (2015). In specific embodiments, the CD5 CAR utilizes, for example, an IgG4 hinge + CH3 or utilizes a CD8a stalk.

[0104] Thus, in certain embodiments, the IgG hinge region utilized is typically IgG1 or IgG4, and in some cases, the CAR comprises the CH2-CH3 domain of IgG Fc. The use of IgG Fc domains can provide flexibility to the CAR, making it less immunogenic, easier to detect CAR expression with anti-Fc reagents, and allowing for the removal of one or more CH2 or CH3 modules to accommodate different spacer lengths. However, in one embodiment, mutations of certain spacers to avoid FcγR binding, for example to avoid binding of soluble and cell surface Fc gamma receptors, may improve CAR+ T cell engraftment and anti-tumor efficacy, yet maintain activity to mediate antigen-specific lysis. For example, an IgG4-Fc spacer with modified CH2 region can be used. For example, the CH2 region may be mutated, including point mutations and / or deletions. Certain modifications have been demonstrated at two sites within the CH2 region (L235E; N297Q) and / or incorporate CH2 deletions (Jonnalagadda et al, 2015). In certain embodiments, the IgG4 hinge-CH2-CH3 domain (229 aa in length) or only the hinge domain (12 aa in length) can be employed (Hudececk et al., 2015).

[0105] In specific embodiments, the hinge is any of IgG, CD28, CD-8α, 4-1BB, 0X40, CD3-zeta, T cell receptor a chain or b chain, CD3 zeta chain, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, or CD154.

[0106] Examples of specific hinge arrangements that may be utilized include at least the following:

[0107] IgG hinge sequence: GTACGGTCACTGTCTCTTCACAGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATT (Sequence number in the sequence listing: 23)

[0108] Amino acid sequence of the IgG hinge: TVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK (Sequence number: 24 in the sequence listing)

[0109] CD28 hinge sequence: ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (Sequence Number: 25 in the Sequence Listing)

[0110] CD28 hinge amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (Sequence Listing SEQ ID NO: 26) 5. Signal Activation Domain

[0111] In a specific embodiment, the anti-CD5 CAR molecule comprises a signal activation domain, such as from CD3zeta or FcεRIγ.

[0112] An example of a CD3 zeta sequence that can be utilized is as follows: CGCGTgtGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG GCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAC (Sequence number in sequence listing: 27)

[0113] An example of a CD3 zeta amino acid sequence that can be used is as follows: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG (Sequence number: 28 in the sequence listing) 6. Other proteins

[0114] In some embodiments, one or more other proteins are utilized with the anti-CD5 CAR. The one or more other proteins can be utilized for any reason, including promoting the efficacy of the CAR itself and / or any type of cell expressing the CAR. In some cases, the other protein promotes the treatment of an individual receiving a cell expressing the CAR as a treatment, regardless of whether the other protein(s) directly or indirectly affect the activity of the CAR or the cell. In some cases, the other protein is a suicide gene, one or more cytokines, or both. In specific embodiments, the one or more other proteins are produced from a vector and are ultimately produced as two separate polypeptides. For example, the anti-CD5 CAR and the other protein(s) can be separated, for example, by a 2A sequence or an IRES.

[0115] In a specific embodiment, a cytokine such as IL-15 is utilized in conjunction with an anti-CD5 CAR.

[0116] An example of the IL-15 sequence is as follows:

[0117] IL-15 sequence: GCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGA CTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGC AAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGACAATT (Sequence number: 29 in the sequence listing)

[0118] The amino acid sequence of IL-15: ISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence number: 30 in the sequence listing)

[0119] If it is intended that the CAR and another protein in the same vector be produced into two distinct polypeptides, a specific 2A sequence may be utilized.

[0120] In one example, the E2A sequence is utilized as follows: CAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCC (Sequence number: 31 in the sequence listing)

[0121] The amino acid sequence of E2A is available as follows: QCTNYALLKLAGDVESNPGP (Sequence Listing SEQ ID NO: 32)

[0122] Other 2A examples are available, including: T2A: EGRGSLLTCGDVEENPGP (Sequence Number: 49 in the Sequence Listing) P2A: ATNFSLLKQAGDVEENPGP (Sequence Listing SEQ ID NO: 50) F2A: VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 51 in the sequence listing)

[0123] The present disclosure also encompasses specific CAR molecules, including expression in any type of immune effector cell.

[0124] In one example, an anti-CD5 CAR is utilized that includes an IgG1 hinge, a CD28 transmembrane domain, and CD3zeta. In the vector, the CAR may be expressed together with IL-15, and may be separated from the CAR by, for example, a 2A sequence. In a specific example, such a CAR and IL-15 construct may have the following nucleotide sequence:

[0125] The amino acid sequence corresponding to CD5-IgG1-CD28TMD-CD3zeta-2A-IL15 is as follows: MLEMEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYE DMGIYYCQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYA DDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSSRTVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWFWVLVVVGG VLACYSLLVTVAFIIFWVTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence number: 34 in the sequence listing)

[0126] In one example, an anti-CD5 CAR is utilized that includes an IgG1 hinge, DAP10 transmembrane and cytoplasmic domains, and CD3 zeta. In the vector, the CAR may be expressed together with IL-15, and may be separated from the CAR by, for example, a 2A sequence. In a specific example, such a CAR and IL-15 construct may have the following nucleotide sequence: CD5-IgG1-DAP10TMD-DAP10 ICD.CD3zeta-2A-IL15

[0127] The amino acid sequence corresponding to CD5-IgG1-DAP10TMD-DAP10 ICD.CD3zeta-2A-IL15 is as follows: MLEMEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM GIYYCQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFK GRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSSRTVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWLLAGLVAADAVASLLIVGAV FLCARPRRSPAQEDGKVYINMPGRGTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence number: 36 in the sequence listing)

[0128] In one example, an anti-CD5 CAR is utilized that includes an IgG1 hinge, a CD28 transmembrane domain and a 4-1BB cytoplasmic domain, and CD3 zeta. In the vector, the CAR may be expressed together with IL-15, and may be separated from the CAR by, for example, a 2A sequence. In a specific example, such a CAR and IL-15 construct may have the following nucleotide sequence:

[0129] The amino acid sequence corresponding to CD5-IgG1-CD28TMD-41BB-CD3zeta-2A-IL15 is as follows: MLEMEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIY YCQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFS LETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSSRTVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence Number: 38 in the Sequence Listing)

[0130] In one example, an anti-CD5 CAR is utilized that includes an IgG1 hinge, a CD28 transmembrane domain and a DAP10 cytoplasmic domain, and CD3zeta. In the vector, the CAR may be expressed together with IL-15, and may be separated from the CAR by, for example, a 2A sequence. In a specific example, such a CAR and IL-15 construct may have the following nucleotide sequence: CD5-IgG1-CD28TMD-DAP10 ICD.CD3zeta-2A-IL15

[0131] The amino acid sequence corresponding to CD5-IgG1-CD28TMD-DAP10 ICD.CD3zeta-2A-IL15 is as follows: MLEMEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMG IYYCQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGR FAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSSRTVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWFWVLVVVGGVLACYSLLVTVAFII FWVLCARPRRSPAQEDGKVYINMPGRGTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence Number: 40 in the Sequence Listing)

[0132] In one example, an anti-CD5 CAR is utilized that includes an IgG1 hinge, a DAP12 transmembrane domain and a DAP12 cytoplasmic domain, and CD3 zeta. In the vector, the CAR may be expressed together with IL-15, and may be separated from the CAR by, for example, a 2A sequence. In a specific example, such a CAR and IL-15 construct may have the following nucleotide sequence: CD5-IgG1-DAP12TMD-DAP12 ICD.CD3zeta-2A-IL15

[0133] The amino acid sequence corresponding to CD5-IgG1-DAP12TMD-DAP12 ICD.CD3zeta-2A-IL15 is as follows: MLEMEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIY YCQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSL ETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSSRTVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEA ATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence number: 42 in the sequence listing)

[0134] In one example, an anti-CD5 CAR consisting of an IgG1 hinge, a CD28 transmembrane domain and a DAP12 cytoplasmic domain, and CD3zeta is utilized. In the vector, the CAR may be expressed together with IL-15, and may be separated from the CAR by, for example, a 2A sequence. In a specific example, such a CAR and IL-15 construct may have the following nucleotide sequence: CD5-IgG1-CD28TMD-DAP12 ICD.CD3zeta-2A-IL15

[0135] The amino acid sequence corresponding to CD5-IgG1-CD28TMD-DAP12 ICD.CD3zeta-2A-IL15 is as follows: MLEMEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYY CQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLET SASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSSRTVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWFWVLVVVGGVLACYSLLVTVAFIIFWVYFLGRLVPRGRGAA EAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence number: 44 in the sequence listing)

[0136] In one example, an anti-CD5 CAR is utilized that includes an IgG1 hinge, an NKG2D transmembrane domain and an NKG2D cytoplasmic domain, and CD3zeta. In the vector, the CAR can be expressed with IL-15, which can be separated from the CAR by a 2A sequence. In a specific example, such a CAR and IL-15 construct can have the following nucleotide sequence: CD5-IgG1-NKG2DTMD-NKG2D ICD.CD3zeta-2A-IL15

[0137] The amino acid sequence corresponding to CD5-IgG1-NKG2DTMD-NKG2D ICD.CD3zeta-2A-IL15 is as follows: MLEMEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIY YCQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSL ETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSSRTVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWAVMIIFRIGMAVAIFCCFFFPSANERCKSKVVPCRQ KQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence number: 46 in the sequence listing)

[0138] In one example, an anti-CD5 CAR is utilized that includes an IgG1 hinge, a CD28 transmembrane domain and an NKG2D cytoplasmic domain, and CD3 zeta. In the vector, the CAR may be expressed together with IL-15, and may be separated from the CAR by, for example, a 2A sequence. In a specific example, such a CAR and IL-15 construct may have the following nucleotide sequence: CD5-IgG1-CD28TMD-NKG2D ICD.CD3zeta-2A-IL15

[0139] The amino acid sequence corresponding to CD5-IgG1-CD28TMD-NKG2D ICD.CD3zeta-2A-IL15 is as follows: MLEMEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYY CQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLE TSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSSRTVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWFWVLVVVGGVLACYSLLVTVAFIIFWVSANERCKSKVVPC RQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence number: 48 in the sequence listing)

[0140] In one example, an anti-CD5 CAR consisting of CD28 hinge, CD28 transmembrane domain and DAP10 cytoplasmic domain, and CD3 zeta is utilized. In the vector, the CAR may or may not be expressed together with IL-15, for example, it may be separated from the CAR by a 2A sequence, for example. In a specific example, such a CAR and IL-15 construct may have the following nucleotide sequence: iC9.CD5CARCD28hinge.tmdDAP103z.IL15

[0141] The amino acid sequence corresponding to iC9.CD5CARCD28hinge.tmdDAP103z.IL15 is as follows:

[0142] In one example, an anti-CD5 CAR is utilized, which includes a CD28 hinge, a CD28 transmembrane domain, and a CD28 cytoplasmic domain, and CD3 zeta. In the vector, the CAR may or may not be expressed together with IL-15, and may be separated from the CAR by, for example, a 2A sequence. In a specific example, such a CAR and IL-15 construct may have the following nucleotide sequence: iC9.CD5CARCD28hinge.28tmd3z.IL15

[0143] The amino acid sequence corresponding to iC9.CD5CARCD28hinge.28tmd3z.IL15 is as follows: CT cell receptor (TCR)

[0144] In some embodiments, the engineered CD5-targeted antigen receptor comprises a recombinant TCR and / or a TCR cloned from a naturally occurring T cell. "T cell receptor" or "TCR" refers to a molecule that contains variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively) and can specifically bind to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is an αβ form.

[0145] Typically, TCRs, which exist in αβ and γδ forms, are generally structurally similar, but the T cells expressing them may have different anatomical locations or functions. TCRs may be found on the surface of cells or in soluble forms. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where they are generally responsible for the recognition of antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al, 1997). For example, in some aspects, each chain of a TCR may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, TCRs are associated with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise specified, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full length TCRs, including αβ or γδ forms of the TCR.

[0146] Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR that binds to a particular antigenic peptide bound to an MHC molecule, i.e., an MHC-peptide complex. An "antigen-binding portion" or "antigen-binding fragment" of a TCR, which may be used interchangeably, refers to a molecule that contains a portion of the structural domain of a TCR but binds to the antigen (e.g., an MHC-peptide complex) that the complete TCR binds. In some cases, the antigen-binding portion contains sufficient variable domains of the TCR, such as the variable α and β chains of the TCR, to form a binding site for binding to a particular MHC-peptide complex, e.g., where each chain generally contains three complementarity determining regions.

[0147] In some embodiments, the variable domains of the TCR chains combine to form loops, or complementarity determining regions (CDRs) similar to immunoglobulins. This confers antigen recognition and determines peptide specificity by forming the binding site of the TCR molecule, and determines peptide specificity. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, for example, Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR responsible for recognition of processed antigens, but CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, and CDR1 of the beta chain interacts with the C-terminal portion of peptides. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the beta chain may contain an additional hypervariable (HV4) region.

[0148] In some embodiments, the TCR chain contains a constant domain. For example, similar to an immunoglobulin, the extracellular portion of a TCR chain (e.g., α chain, β chain) contains two immunoglobulin domains, an N-terminal variable domain (e.g., V aor Vp; typically, Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.), and one constant domain adjacent to the cell membrane (e.g., the α chain constant domain or C a , typically amino acids 117-259 based on Kabat, and a beta chain constant domain or Cp, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane proximal constant domains, and two membrane distal variable domains that contain the CDRs. The constant domain of the TCR domain contains a short linking sequence in which cysteine ​​residues form disulfide bonds to link the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the alpha and beta chains such that the TCR contains two disulfide bonds within the constant domains.

[0149] In some embodiments, TCR chains may contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, TCR chains contain a cytoplasmic tail. In some cases, this structure allows TCR to bind to other molecules such as CD3. For example, TCRs that contain a constant domain with a transmembrane region may anchor the protein in the cell membrane and bind to the invariant subunit of the CD3 signaling device or complex.

[0150] In general, CD3 is a multiprotein complex that may have three different chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, the complex may contain a homodimer of CD3γ, CD3δ, two CD3ε, and CD3ζ chains. CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that allows these chains to bind to the positively charged T cell receptor chains. The intracellular tails of CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, although each CD3ζ chain has three. In general, ITAMs are involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and are responsible for transmitting signals from the TCR into the cell. The CD3 chain and the ζ chain, together with the TCR, form what is known as the T cell receptor complex.

[0151] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β or γ and δ chains) linked by a disulfide bond or the like. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, a nucleic acid encoding the TCR may be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, for example, from cells derived from T cells (e.g., cytotoxic T cells), T cell hybridomas, or other publicly available sources. In some embodiments, the T cells may be obtained from in vivo isolated cells. In some embodiments, high affinity T cell clones may be isolated from a patient and the TCRs isolated. In some embodiments, the T cells may be cultured T cell hybridomas or clones. In some embodiments, TCR clones against target antigens are generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against target antigens (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, TCRs or antigen-binding portions thereof can be synthetically generated from knowledge of the sequence of the TCR. II. Cytokines

[0152] One or more cytokines may be utilized with one or more engineered CD5-targeting receptors, such as CD5-specific CARs. In some cases, one or more cytokines are present on the same vector molecule as the engineered receptor, while in other cases, they are present on separate vector molecules. In certain embodiments, one or more cytokines are co-expressed from the same vector as the engineered receptor. One or more cytokines may be generated as separate polypeptides from the CD5-specific receptor. As an example, interleukin-15 (IL-15) is utilized. IL-15 may be used because, for example, it is tissue-restricted and is only observed in serum or systemically at some level under pathological conditions. IL-15 has several attributes that are desirable for adoptive therapy. IL-15 is a homeostatic cytokine that induces the development and cell proliferation of natural killer cells, promotes the eradication of established tumors through the relief of functional inhibition of tumor-resident cells, and inhibits activation-induced cell death. In addition to IL-15, other cytokines are envisioned. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. By way of example, the cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-7, or combinations thereof. NK cells that express IL-15 can be utilized and are capable of continued supportive cytokine signaling, which is useful for survival after infusion.

[0153] In certain embodiments, the NK cells express one or more exogenously provided cytokines. The cytokines can be exogenously provided to the NK cells because they are expressed from an expression vector in the cells and / or provided in the culture medium of the cells. Alternatively, the endogenous cytokines in the cells are upregulated by regulatory engineering of the expression of the endogenous cytokines, such as genetic engineering at the promoter site of the cytokine. When the cytokines are provided to the cells on an expression construct, they can be encoded from the same vector as the suicide. The cytokines can be expressed as separate polypeptide molecules as the suicide gene and as separate polypeptides from the engineered receptor of the cells. In some embodiments, the present disclosure relates to the co-use of CAR and / or TCR vectors with IL-15, particularly in NK cells. III. Suicide Genes

[0154] In certain embodiments, suicide genes are utilized with any type of cell therapy to control its use and allow for the termination of cell therapy at a desired event and / or time. Suicide genes are used in transduced cells to induce the death of the transduced cells as needed. The CD5 targeted cells of the present disclosure modified to have a vector encompassed by the present disclosure may contain one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that results in the transfer of the gene product to a compound that kills the host cell upon administration of a prodrug or other agent. In other embodiments, the suicide gene encodes a gene product that is targeted by an agent (such as an antibody) that targets the suicide gene product, if desired.

[0155] Examples of suicide gene / prodrug combinations that can be used include Herpes Simplex Virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. E. coli purine nucleoside phosphorylase, a suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, can be used. Other examples of suicide genes used in prodrug therapy include the E. coli cytosine deaminase gene and the HSV thymidine kinase gene.

[0156] Exemplary suicide genes also include CD20, CD52, EGFRv3, or inducible caspase 9. In one embodiment, a truncated version of EGFR variant III (EGFRv3) can be used as a suicide antigen that can be removed by cetuximab. Additional suicide genes known in the art that can be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-alpha, gamma-lyase (MET), and thymidine phosphorylase (TP).

[0157] In certain embodiments, the vector encoding CD5-targeted CAR or any vector in the NK cell encompassed herein comprises one or more suicide genes.The suicide gene may or may not be on the same vector as the CD5-targeted CAR.When the suicide gene is on the same vector as the CD5-targeted CAR, the suicide gene and the CAR may be separated by, for example, an IRES or a 2A element. IV. Vectors

[0158] CD5 targeting CAR can be delivered to recipient immune cells by any suitable vector, including viral vector or non-viral vector.Examples of viral vector include at least retrovirus, lentivirus, adenovirus, or adeno-associated virus vector.Examples of non-viral vector include at least plasmid, transposon, lipid, nanoparticle, etc.

[0159] When immune cells are transduced with a vector encoding a CD5-targeted receptor and also require the transduction of another gene, such as a suicide gene and / or a cytokine and / or an optional therapeutic gene product, into the cells, the CD5-targeted receptor, the suicide gene, the cytokine, and the optional therapeutic gene may or may not be included in the same vector. In some cases, the CD5-targeted CAR, the suicide gene, the cytokine, and the optional therapeutic gene are expressed from the same vector molecule, such as the same viral vector molecule. In such cases, the expression of the CD5-targeted CAR, the suicide gene, the cytokine, and the optional therapeutic gene may or may not be regulated by the same regulatory element. When the CD5-targeted CAR, the suicide gene, the cytokine, and the optional therapeutic gene are on the same vector, they may or may not be expressed as separate polypeptides. When they are expressed as separate polypeptides, they may be separated on the vector by, for example, a 2A element or an IRES element (or both types may be used on the same vector, one or more times). A. General embodiment

[0160] Those of skill in the art would be well-versed in constructing vectors by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference) for expression of the antigen receptors of the present disclosure. 1. Adjustment element

[0161] The expression cassette contained in the vector useful in the present disclosure contains, inter alia, a eukaryotic transcriptional promoter operably linked to a protein coding sequence, a splice signal with intervening sequences, and a transcription termination / polyadenylation sequence (5' to 3' direction). The promoters and enhancers that control the transcription of protein coding genes in eukaryotic cells can be composed of multiple genetic elements. The cellular machinery can collect and incorporate the regulatory information transmitted by each element, allowing different genes to evolve different, often complex patterns of transcriptional regulation. Promoters used in connection with the present disclosure include, for example, constitutive promoters, inducible promoters, and tissue-specific promoters. When the vector is utilized for the production of cancer treatment, the promoter can be effective under conditions of hypoxia. 2. Promoter / Enhancer

[0162] The expression constructs provided herein include promoters for driving the expression of antigen receptors and other cistron gene products. Promoters generally contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, although in some promoters that lack a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late gene, separate elements that cover the start site itself serve to fix the location of initiation. Additional promoter elements regulate the frequency of transcription initiation. Typically, this is located in the region upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA to promote expression of the encoded RNA.

[0163] Spacing between promoter elements is frequently flexible enough that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, for example, spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "elements," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0164] A promoter may be one that is naturally associated with a nucleic acid sequence, as may be obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of said sequence. Besides this, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. Also, a recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR™, in conjunction with the compositions disclosed herein. Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be used as well.

[0165] Of course, it will be important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, which is incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, such as being advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0166] Additionally, expression can be driven using any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Data Base EPDB, via the World Wide Web at epd.isb-sibi.ch / ). Use of the T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0167] Non-limiting examples of promoters include early or late viral promoters, such as SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter, eukaryotic promoters, such as beta actin promoter, GADPH promoter, metallothionein promoter, and linked response element promoters, such as cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA), and response element promoter near minimal TATA box (tre). It is also possible to use a human growth hormone promoter sequence (e.g., human growth hormone minimal promoter described in GenBank®, Accession No. X05244, nucleotides 283-341) or a mouse mammary tumor promoter (available from the ATCC, Cat No. ATCC 45007). In certain embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I, or MHC class II promoter. However, any other promoter useful for driving expression of therapeutic genes can be used in the practice of the present disclosure.

[0168] In certain embodiments, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that have the potential to increase the activity of a promoter and act in cis and regardless of its orientation, even over relatively long distances (up to several kilobases away from the target promoter). However, enhancer function is not necessarily limited to such long distances, since it may function in close proximity to a given promoter. 3. Initiation Signals and Linked Expression

[0169] Also, specific initiation signals can be used in the expression constructs provided in this disclosure for efficient translation of the coding sequence. Such signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translation control signals including the ATG initiation codon. Those skilled in the art can easily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. Exogenous translation control signals and initiation codons can be either natural or synthetic. The efficiency of expression can be enhanced by including appropriate transcription enhancer elements.

[0170] In certain embodiments, an internal ribosome entry site (IRES) element is used to generate multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and initiate translation at an internal site. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) as well as IRES from mammalian messages have been described. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES resulting in a polycistronic message. Thanks to the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed by transcribing a single message using a single promoter / enhancer.

[0171] As detailed elsewhere herein, certain 2A sequence elements can be used to provide for linked or co-expression of genes in constructs provided in this disclosure. For example, a truncation sequence can be used to link open reading frames to form a single cistron, thereby allowing genes to be co-expressed. Exemplary truncation sequences include Equine Rhinitis A Virus (E2A) or F2A (Foot and Mouth Disease Virus 2A) or "2A-like" sequences (e.g., Thosea asigna Virus 2A; T2A) or Porcine Teschovirus-1 (P2A). In certain embodiments, in a single vector, multiple 2A sequences are not identical, but in alternative embodiments, the same vector utilizes two or more identical 2A sequences. Examples of 2A sequences are described in U.S. Patent Application Publication No. 2011 / 0065779, which is incorporated herein by reference in its entirety. 4. Origin of replication

[0172] To propagate the vector in a host cell, the vector may contain one or more origin of replication sites (often referred to as "ori"), such as a nucleic acid sequence corresponding to the EBV oriP described above, or a genetically engineered oriP with similar or enhanced function in programming, which is a specific nucleic acid sequence from which replication is initiated. Alternatively, the origin of replication of other extrachromosomally replicating viruses described above, or an autonomously replicating sequence (ARS), may be used. 5. Selectable and Screenable Markers

[0173] In some embodiments, NK cells containing the CD5 targeting receptor construct of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker will confer an identifiable change to the cell that allows easy identification of cells containing the expression vector. In general, a selection marker is one that confers a property that allows selection. A positive selection marker is one whose presence allows selection, and a negative selection marker is one whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0174] In general, the inclusion of a drug selection marker aids in cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows the identification of transformants based on the implementation of conditions, other types of markers are also contemplated, including screenable markers such as GFP, whose basis is colorimetric analysis. Other screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be utilized as negative selection markers. Also, those skilled in the art will likely know how to use immunological markers in combination with FACS analysis. The marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection and screenable markers are well known to those skilled in the art. B. Multicistronic Vectors

[0175] In certain embodiments, the CD5 targeting receptor, optional suicide gene, optional cytokine, and / or optional therapeutic gene are expressed from a multicistronic vector (the term "cistron" as used herein refers to a nucleic acid sequence from which a gene product can be generated). In certain embodiments, the multicistronic vector encodes the CD5 targeting receptor, suicide gene, and at least one cytokine and / or engineered receptor, such as a T cell receptor, and / or an additional non-CD5 targeting CAR. Optionally, the multicistronic vector encodes at least one CD5 targeting CAR, at least one TNF-alpha mutant, and at least one cytokine. The cytokine can be a specific type of cytokine, such as human or mouse, or any species. In certain cases, the cytokine is IL15, IL12, IL2, IL18, and / or IL21.

[0176] In certain embodiments, the present disclosure provides a flexible modular system utilizing polycistronic vectors capable of expressing multiple cistrons at substantially the same level (the term "module" as used herein refers to a cistron or a component of a cistron that allows its interchangeability, such as by removal and replacement of the entire cistron or each of the components of the cistron, such as by using standard recombinant techniques). The system can be used for cell engineering to allow combinatorial expression (including overexpression) of multiple genes. In certain embodiments, one or more of the genes expressed by the vector includes one, two, or more antigen receptors. Multiple genes can include, but are not limited to, CARs, TCRs, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vector can further include: (1) one or more reporters, such as fluorescent or enzymatic reporters for cell assays and animal imaging, and the like; (2) one or more cytokines or other signaling molecules; and / or (3) a suicide gene.

[0177] In certain cases, the vector may contain at least four cistrons separated by any kind of cleavage site, such as 2A cleavage sites. The vector may or may not be based on Moloney Murine Leukemia Virus (MoMLV or MMLV) containing 3' and 5' LTR with psi packaging sequence in pUC19 backbone. The vector may contain four or more cistrons with three or more 2A cleavage sites and multiple ORFs for gene exchange. The system allows for combinatorial overexpression of multiple genes (seven or more) in some embodiments flanked by restriction sites for rapid integration by subcloning, and also contains at least three 2A self-cleavage sites. Thus, the system allows for the expression of multiple CARs, TCRs, signaling molecules, cytokines, cytokine receptors, and / or homing receptors. The system may also be used for other viral and non-viral vectors, including but not limited to lentivirus, adenovirus AAV, and non-viral plasmids.

[0178] The modularity of the system also allows for efficient subcloning of genes into each of the four cistrons within the polycistronic expression vector, and swapping of genes for rapid testing, etc. Strategically positioned restriction sites within the polycistronic expression vector allow genes to be swapped efficiently.

[0179] Embodiments of the present disclosure encompass systems that utilize polycistronic vectors in which at least a portion of the vector is modular, for example, by allowing for the removal and replacement of one or more cistrons (or components of one or more cistrons), such as by utilizing one or more restriction enzyme sites whose identities and locations are specifically selected to facilitate modular use of the vector. Vectors also have embodiments that confer the advantage that the vector expresses separate gene products in substantially equimolar concentrations, with multiple cistrons being translated into a single polypeptide and processed into separate polypeptides.

[0180] The vectors of the present disclosure are configured such that modularity can be altered for one or more cistrons of the vector and / or one or more components of one or more particular cistrons. The vectors can be designed to take advantage of unique restriction enzyme sites flanking the ends of one or more cistrons and / or flanking the ends of one or more components of a particular cistron.

[0181] The embodiments of the present disclosure include polycistronic vectors that include at least two, at least three, or at least four cistrons, each flanked by one or more restriction enzyme sites, with at least one cistron encoding at least one antigen receptor. In some cases, two, three, four, or more cistrons are translated into a single polypeptide and cleaved into separate polypeptides, while in other cases, multiple cistrons are translated into a single polypeptide and cleaved into separate polypeptides. Adjacent cistrons on a vector can be separated by a self-cleaving site, such as a 2A self-cleaving site. In some cases, each cistron expresses a separate polypeptide from the vector. In certain cases, adjacent cistrons on a vector are separated by an IRES element.

[0182] In certain embodiments, the present disclosure provides a system for cell engineering that allows for combinatorial expression, including overexpression, of multiple cistrons, which may include, for example, one, two, or more antigen receptors. In certain embodiments, the use of polycistronic vectors described herein allows the vectors to generate equimolar levels of multigene products from the same mRNA. Multigenes may include, but are not limited to, CARs, TCRs, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vectors may further include one or more fluorescent or enzymatic reporters for cell assays, animal imaging, and the like. The vectors may also include suicide gene products for the termination of cells carrying the vector when the cells are no longer needed or become harmful to the host to which it is provided.

[0183] In certain embodiments, the vector is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector) or a non-viral vector. The vector may include Moloney Murine Leukemia Virus (MMLV) 5'LTR, 3'LTR, and / or psi packaging elements. In certain cases, the psi packaging is integrated between the 5'LTR and the antigen receptor coding sequence. The vector may or may not include a pUC19 sequence. In some embodiments of the vector, at least one cistron encodes a cytokine (e.g., interleukin 15 (IL-15), IL-7, IL-21, IL-18, IL-12, or IL-2), a chemokine, a cytokine receptor, and / or a homing receptor.

[0184] If 2A cleavage sites are utilized in the vector, the 2A cleavage sites may include a P2A, T2A, E2A, and / or F2A site.

[0185] The restriction enzyme site may be of any type and may contain any number of bases in its recognition site, for example, 4-8 bases; the number of bases in the recognition site may be at least 4, 5, 6, 7, 8, or more. The site when cleaved may generate blunt cut ends or sticky ends. The restriction enzyme may be, for example, type I, type II, type III, or type IV. Restriction enzyme sites may be obtained from available databases, for example, the Integrated relational Enzyme database (IntEnz) or BRENDA (The Comprehensive Enzyme Information System).

[0186] An exemplary vector may be circular, and by convention, position 1 (the 12 o'clock position at the top of the circle, with the remaining sequences in a clockwise direction) is set at the start of the 5'LTR.

[0187] In embodiments in which a self-cleaving 2A peptide is utilized, the 2A peptide can be a viral oligopeptide 18-22 amino acids (aa) long that mediates the "cleavage" of the polypeptide during translation in eukaryotic cells. The designation "2A" refers to a specific region of the viral genome, and various viral 2As are commonly named after the viruses from which they are derived. The first 2A discovered was F2A (foot and mouth disease virus), and subsequently E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (Thosea asigna virus 2A) were also identified. The mechanism of 2A-mediated "self-cleavage" was discovered to be ribosomal skipping, which skips the formation of a glycyl-prolyl peptide bond at the C-terminus of 2A.

[0188] In certain cases, the vector can be a gamma-retroviral transfer vector. Retroviral transfer vectors can include a backbone based on a plasmid, such as pUC19 plasmid (large fragment (2.63 kb) between the restriction enzyme sites of HindIII and EcoRI). The backbone can have viral components derived from Moloney Murine Leukemia Virus (MoMLV), including 5'LTR, psi packaging sequence, and 3'LTR. LTRs are long terminal repeat sequences found on both sides of retroviral proviruses, and in the case of transfer vectors, they enclose the genetic cargo of interest, such as CD5-targeted CAR and related components. Also, the psi packaging sequence, which is the target site for packaging by the nucleocapsid, is integrated in cis and sandwiched between the 5'LTR and the CAR coding sequence. Thus, the basic structure of an example transfer vector can be constructed as follows: pUC19 sequence-5'LTR-psi packaging sequence-genetic cargo of interest-3'LTR-pUC19 sequence. The system can also be used with other viral and non-viral vectors, including but not limited to lentiviruses, adenoviruses AAV, and non-viral plasmids. V. Cell

[0189] The present disclosure encompasses any kind of immune cell or stem cell with at least one vector that encodes a CD5 targeting receptor and may also encode at least one cytokine and / or at least one suicide gene. In some cases, various vectors encode CARs versus encoding suicide genes and / or cytokines. Immune cells, including NK cells, can be derived from cord blood (including pooled cord blood from multiple sources), peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), bone marrow, or mixtures thereof. NK cells can be derived from cell lines, such as, for example, but not limited to, NK-92 cells. NK cells can be cord blood mononuclear cells, such as CD56+ NK cells.

[0190] The present disclosure encompasses any type of immune cell or other cell, including conventional T cells, gamma-delta T cells, NK T and invariant NK T cells, regulatory T cells, macrophages, B cells, dendritic cells, mesenchymal stromal cells (MSCs), or mixtures thereof.

[0191] In some cases, the cells are grown in the presence of an effective amount of universal antigen presenting cells (UAPC), including any suitable ratio. The cells can be cultured with UAPC at a ratio of 10:1 to 1:10; 9:1 to 1:9; 8:1 to 1:8; 7:1 to 1:7; 6:1 to 1:6; 5:1 to 1:5; 4:1 to 1:4; 3:1 to 1:3; 2:1 to 1:2; or 1:1, including, for example, a ratio of 1:2. In some cases, the NK cells were expanded in the presence of IL-2 at a concentration of, for example, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400, or 400-500 U / mL.

[0192] After genetic modification with the vector, the NK cells may be infused immediately or stored. In certain embodiments, after genetic modification, the cells may be expanded ex vivo as a bulk population for days, weeks, or months within about 1, 2, 3, 4, 5 days or more after gene introduction into the cells. In further embodiments, the transfectants are cloned (clones showing the presence of a single integrated or episomally maintained expression cassette or plasmid) and the expression of the CD5-targeted CAR is expanded ex vivo. Clones selected for expansion demonstrate the ability to specifically recognize and lyse CD5-expressing target cells. The recombinant immune cells may be expanded by stimulation with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, etc.). The recombinant immune cells may be expanded by stimulation with artificial antigen-presenting cells. In further embodiments, the genetically modified cells may be cryopreserved.

[0193] The embodiments of the present disclosure include cells that express one or more CD5-targeted CARs and one or more suicide genes as encompassed herein. In certain embodiments, NK cells contain recombinant nucleic acids that encode one or more CD5-targeted CARs and one or more engineered, non-secretable membrane-bound TNF-alpha mutant polypeptides. In certain embodiments, in addition to expressing one or more CD5-targeted CARs and TNF-alpha mutant polypeptides, the cells also contain nucleic acids that encode one or more therapeutic gene products.

[0194] The cells may be obtained directly from an individual or from a repository or other storage facility. Cells as a therapy may be autologous or allogeneic to the individual to whom the cells are provided as a therapy.

[0195] The cells may be derived from an individual in need of treatment for a medical condition and, after engineering to express the CD5-targeted CAR, optional suicide gene, optional cytokine, and optional therapeutic gene product (e.g., using standard techniques for transduction and expansion for adoptive cell therapy), may be returned to the individual from whom they were originally sourced. In some cases, the cells are stored for later use in the individual or another individual.

[0196] The immune cells may be included within a population of cells that may be predominantly transduced with one or more CD5 targeting receptors and / or one or more suicide genes and / or one or more cytokines. The cell population may include 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of immune cells transduced with one or more CD5 targeting receptors and / or one or more suicide genes and / or one or more cytokines. The one or more CD5 targeting receptors and / or the one or more suicide genes and / or the one or more cytokines may be separate polypeptides.

[0197] Immune cells can be generated with one or more CD5-targeted receptors and / or one or more suicide genes and / or one or more cytokines for the purpose of being modular with respect to a particular purpose. For example, cells expressing CD5-targeted CARs and / or one or more suicide genes and / or one or more cytokines (or nucleic acids encoding variants are distributed for subsequent transduction) can be generated, for example, for commercial distribution, and users can modify the cells to express one or more other genes of interest (including therapeutic genes) depending on the intended purpose. For example, an individual interested in treating CD5-positive cells, including CD5-positive cancer, can obtain or generate suicide gene-expressing cells (or heterologous cytokine-expressing cells) and modify them to express receptors including CD5-specific scFvs, or vice versa.

[0198] In certain embodiments, NK cells can be utilized to modify the genome of transduced NK cells that express one or more CD5 targeting CARs and / or one or more suicide genes and / or one or more cytokines.Genome can be modified in any manner, but in certain embodiments, genome is modified by, for example, CRISPR gene editing.Genome of cell can be modified to enhance the effectiveness of cell for any purpose. VI. Gene editing of CD5-specific CAR cells

[0199] In certain embodiments, at least the cell that comprises the engineered CD5-specific receptor is genetically edited to modify the expression of one or more endogenous genes in the cell.In certain cases, the CD5-specific CAR cell is modified to reduce the expression level of one or more endogenous genes, including the inhibition (which may be referred to as knockout) of the expression of one or more endogenous genes.Such cells may or may not be proliferated.

[0200] In certain cases, one or more endogenous genes of CD5-specific CAR cells are modified, for example, expression is disrupted, expression is partially or completely reduced. In certain cases, one or more genes are knocked down or knocked out using the process of the present disclosure. In certain cases, multiple genes are knocked down or knocked out, which may or may not occur in the same step of their generation. The gene edited in CD5-specific CAR cells can be of any type, but in a specific embodiment, the gene is a gene whose gene product inhibits the activity and / or proliferation of CD5-specific CAR cells (one example is CD5-specific CAR NK cells, such as those derived from umbilical cord blood). In certain cases, the gene edited in CD5-specific CAR cells allows CD5-specific CAR cells to act more effectively in the tumor microenvironment. In certain cases, the gene is one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, CD38, CREM, IL10R, CD5, and CD7. In certain embodiments, the TGFBR2 gene is knocked out or knocked down in CD5-specific CAR cells.

[0201] In some embodiments, gene editing is performed using one or more DNA-binding nucleic acids, such as modification via RNA-guided endonucleases (RGENs). For example, modification can be performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In some embodiments, CpF1 is utilized instead of Cas9. In general, "CRISPR system" refers to the transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active portion tracrRNA), tracr-mate sequences ("direct repeats", encompassing tracrRNA-processed portion direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from CRISPR loci.

[0202] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner, and a Cas protein (e.g., Cas9) that has nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0203] In some embodiments, Cas nuclease and gRNA (including a fusion of a crRNA specific for a target sequence and a fixed tracrRNA) are introduced into cells. In general, a target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene, using complementary base pairing. The target site can be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, typically NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems feature elements that promote the formation of a CRISPR complex at the site of the target sequence. Typically, a "target sequence" generally refers to a sequence that the guide sequence is designed to have complementarity with, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.

[0204] The CRISPR system can induce a double-strand break (DSB) at the target site, followed by destruction or modification, as discussed herein. In other embodiments, a Cas9 variant considered a "nickase" is used to nick a single strand at the target site. Paired nickases can be used, for example, to improve specificity, each guided by a pair of different gRNA targeting sequences, such that a 5' overhang is introduced when a nick is introduced simultaneously. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0205] The target sequence can comprise any polynucleotide, for example, DNA or RNA polynucleotide. The target sequence can be located in the nucleus or cytoplasm of a cell, for example, in an organelle of a cell. In general, the sequence or template that can be used for recombination into the target locus that comprises the target sequence is referred to as "editing template" or "editing polynucleotide" or "editing sequence". In some embodiments, the exogenous template polynucleotide may be referred to as editing template. In some embodiments, the recombination is homologous recombination.

[0206] Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in the cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence). Also, the tracr sequence may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence), but may form part of a CRISPR complex, for example, by hybridization with all or a portion of a tracr mate sequence operably linked to the guide sequence along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence (such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned) to hybridize and participate in the formation of a CRISPR complex.

[0207] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into a cell, such that the expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. Also, the components can be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector with one or more additional vectors that provide any components of the CRISPR system that are not included in the first vector. The vector can include one or more insertion sites (also referred to as "cloning sites"), such as restriction endonuclease recognition sequences. In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in a cell.

[0208] The vector can include regulatory elements operably linked to an enzyme coding sequence that encodes a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known, for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under the accession number Q99ZW2.

[0209] The CRISPR enzyme may be Cas9 (e.g., from S. pyogenes or S. pneumonia). In some cases, CpF1 may be used as an endonuclease instead of Cas9. The CRISPR enzyme may direct the cleavage of one or both strands at the location of the target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector may encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvCI catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). In some embodiments, Cas9 nickases can be used in combination with guide sequences, such as two guide sequences, that target the sense and antisense strands of a DNA target, respectively, allowing both strands to be nicked and used to induce NHEJ or HDR.

[0210] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Different species show a particular bias for a particular codon of a particular amino acid. Codon bias (the difference in codon usage between organisms) often correlates with the translation efficiency of messenger RNA (mRNA), which is believed to depend, among other things, on the properties of the codon to be translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of a selected tRNA in a cell is generally a reflection of the codon that is most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be tailored for optimal gene expression in a given organism.

[0211] In general, a guide sequence is any polybase sequence that has sufficient complementarity with a target polybase sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0212] Optimal alignment can be determined by use of any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0213] CRISPR enzyme can be part of a fusion protein that contains one or more heterologous protein domains. CRISPR enzyme fusion protein can contain any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences that code for proteins or fragments of proteins that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of fusion proteins that contain CRISPR enzymes are described in US Patent Publication No. 2011 / 0059502, which is incorporated herein by reference. VII. Treatment Methods

[0214] In various embodiments, diseased cells or other cells that express endogenous CD5 on their surface are targeted to improve a medical condition in an individual with a medical condition, or to reduce the risk or delay the severity and / or onset of a medical condition in an individual. In certain cases, cancer cells that express endogenous CD5 are targeted to kill the cancer cells.

[0215] The CD5-targeting CAR constructs, nucleic acid sequences, vectors, immune cells, etc. contemplated herein, and / or pharmaceutical compositions comprising them, are used to prevent, treat, or ameliorate cancerous diseases, such as neoplastic diseases. In certain embodiments, the pharmaceutical compositions of the present disclosure may be particularly useful for preventing, ameliorating, and / or treating cancers, including, for example, cancers that express CD5 and may or may not be solid tumors.

[0216] The immune cells in which the CD5 targeting receptor is utilized may be, in certain embodiments, NK, T cells, gamma delta T cells, or NKT or invariant NKT (iNKT), or invariant NKT cells engineered for cell therapy for mammals. When the cells are NK cells, the NK cell therapy may be of any type, and the NK cells may be of any type. In certain embodiments, the cells are NK cells engineered to express one or more CD5-targeted CARs and / or one or more suicide genes and / or one or more cytokines. In certain embodiments, the cells are NK cells transduced with CD5-targeted CARs.

[0217] In certain embodiments, the present disclosure contemplates, in part, CD5 CAR expressing cells, CD5 targeted CAR constructs, CD5 targeted CAR nucleic acid molecules, and CD5 targeted CAR vectors that can be administered alone or in any combination using standard vectors and / or gene delivery systems, and at least in some embodiments, with pharma- ceutically acceptable carriers or excipients. In certain embodiments, after administration, the nucleic acid molecules or vectors can be stably integrated into the genome of the subject.

[0218] In certain embodiments, viral vectors can be used that are specific to certain cells or tissues and persist in NK cells.Suitable pharmaceutical carriers and excipients are well known in the art.The composition prepared according to the present disclosure can be used for the prevention or treatment or delay of the above-mentioned specified diseases.

[0219] Furthermore, the present disclosure relates to a method for the prevention, treatment, or amelioration of a neoplastic disease, comprising administering to a subject in need thereof an effective amount of cells expressing a CD5-targeted CAR, nucleic acid sequence, vector as contemplated herein and / or produced by a process as contemplated herein.

[0220] Exemplary possible indications for administration of the composition of CD5-targeted CAR cells are cancerous diseases, including, for example, tumor diseases, including B-cell malignancies, multiple myeloma, breast cancer, glioblastoma, renal cancer, pancreatic cancer, or lung cancer. Exemplary indications for administration of the composition of CD5-targeted CAR cells are cancerous diseases, including any malignant tumor that expresses CD5. Administration of the composition of the present disclosure is useful for all stages (I, II, III, or IV) and types of cancer, including, for example, minimal residual disease, early cancer, advanced cancer, and / or metastatic cancer and / or refractory cancer.

[0221] The present disclosure further includes co-administration protocols with other compounds, such as bispecific antibody constructs, targeted toxins, or other compounds that act through immune cells.The clinical regimen for co-administration of the compounds of the present invention may include co-administration at the same time, before, or after administration of other components.Specific combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy.

[0222] The embodiments relate to kits comprising a CD5-targeted CAR construct as defined herein, a nucleic acid sequence as defined herein, a vector as defined herein, and / or a host cell (such as an immune cell) as defined herein. It is also contemplated that the kits of the present disclosure comprise a pharmaceutical composition as described herein above, alone or in combination with additional agents to be administered to an individual in need of medical treatment or intervention. A. Pharmaceutical Compositions

[0223] Also provided herein are pharmaceutical compositions and formulations comprising the transduced NK cells and a pharma- ceutically acceptable carrier. The transduced cells may be contained in a medium suitable for transfer into an individual and / or a medium suitable for storage, such as cryopreservation, including prior to transfer into an individual.

[0224] The pharmaceutical compositions and formulations described herein comprise an active ingredient (such as cells) having a desired purity in the form of a lyophilized formulation or an aqueous solution, optionally in one or more pharma- ceutical acceptable carriers (see Remington's Pharmaceutical Sciences 22). ndPharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) salts; (full) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases. B. Combination Therapy

[0225] In certain embodiments, the compositions and methods of the present embodiment comprise immune cell populations (including NK cell populations) combined with at least one additional therapy.Additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, virus therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, oncolytic virus, or combinations of the above.Additional therapy can be in the form of adjuvant therapy or neoadjuvant therapy.

[0226] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an antiemetic agent, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents known in the art.

[0227] In certain embodiments, in addition to the inventive cell therapy of the present disclosure, the individual may have been, is being, and / or will be receiving certain additional therapies for cancer, including one or more of surgery, radiation, immunotherapy (other than the cell therapy of the present disclosure), hormone therapy, gene therapy, chemotherapy, and the like.

[0228] Immune cell therapy may be administered before, during, after, or in various combinations with additional cancer therapy. Administration may be at intervals ranging from simultaneous to minutes to days to weeks. In embodiments where immune cell therapy is provided to a patient separately from additional therapeutic agents, one skilled in the art would generally ensure that a significant period does not lapse between each delivery time so that the two compounds can still exert their beneficially combined effect on the patient. In such cases, it is believed that the antibody therapy and the anti-cancer therapy may be administered to the patient within about 12-24 or 72 hours of each other, more specifically within about 6-12 hours of each other. In some situations, it may be desirable to extend the treatment period significantly when several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between the respective administrations.

[0229] Various combinations can be used. In the following examples, the immune cell therapy is "A" and the anti-cancer therapy is "B". A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0230] Administration of any compound or cell therapy of the present embodiments to a patient will follow standard protocols for administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from combination therapy. 1.Chemotherapy

[0231] A wide variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered during the treatment of cancer. The agents or drugs are classified according to their mode of activity within the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, to intercalate into DNA, or to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0232] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylameramines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine); acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 2). liptophysin 8; dolastatins; duocarmycins (including synthetic analogs, KW-2189, and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongiostatins; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma II (calicheamicin gammaII, and calicheamicin omegaII; dynemicins (including dynemicin A); bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autarmicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxo rubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, promycin, and folic acid analogs. teropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenals such as mitotane and trilo Stans; folic acid supplements, such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol;Nitraerine; pentostatin; phenamet; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2´-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; Arabinosides ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g. CPT-11); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicamycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.; 2. Radiation therapy

[0233] Other agents that cause DNA damage and have been widely used include gamma radiation, X-rays, and / or what is commonly known as the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam irradiation (US Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells. 3. Immunotherapy

[0234] Those skilled in the art will understand that additional immunotherapy can be used in conjunction or in combination with the method of the embodiment. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody can function alone as an effector of therapy or can recruit other cells to actually affect cell death. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) to function as a targeting agent. Alternatively, the effector can be a lymphocyte carrying a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0235] Antibody-drug conjugates have emerged as a revolutionary approach to the development of cancer therapeutics. Cancer is one of the leading causes of death in the world. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cell-killing drug. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells enriched with antigen levels. Targeted delivery of the drug also minimizes exposure in normal tissues, resulting in reduced toxicity and improved therapeutic index. The FDA's approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, has validated the approach. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become more and more mature, the discovery and development of new ADCs increasingly depends on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are up-regulation / high levels of expression in tumor cells and robust internalization.

[0236] In one aspect of immunotherapy, the tumor cells must have some marker suitable for targeting, i.e., not present on the majority of other cells. There are many tumor markers, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0237] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, such as interferon alpha, beta, and gamma, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0238] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints make signals (e.g., costimulatory molecules) stronger or weaker. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0239] The immune checkpoint inhibitor may be a drug, such as a small molecule, a recombinant form of a ligand or receptor, or an antibody, such as a human antibody (e.g., WO2015016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, particularly chimeric, humanized, or human forms of antibodies. Alternative and / or equivalent names known to those skilled in the art may be used for the specific antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0240] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain embodiments, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain embodiments, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain embodiments, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Pat. Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Application Publication No. 2014 / 0294898, U.S. Patent Application Publication No. 2014 / 022021, and U.S. Patent Application Publication No. 2011 / 0008369, all of which are incorporated herein by reference.

[0241] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO 2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342.

[0242] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0243] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0244] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, any art-recognized anti-CTLA-4 antibody can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Pat. No. 8,119,129, WO 01 / 14424, WO 98 / 42752, WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004) J Clin Oncology 22(145):Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 may be used in the methods disclosed herein. The teachings of each of the above publications are incorporated herein by reference.Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used.For example, humanized CTLA-4 antibodies are described in WO 2001 / 014424, WO 2000 / 037504, and U.S. Patent No. 8,017,114 (all of which are incorporated herein by reference).

[0245] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0246] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as those described in U.S. Pat. No. 5,844,905, U.S. Pat. No. 5,885,796, and WO 1995 / 001994 and WO 1998 / 042752 (all of which are incorporated by reference herein), and immunoadhesins such as those described in U.S. Pat. No. 8,329,867 (which is incorporated by reference herein). 4.Surgery

[0247] Approximately 60% of people with cancer will undergo some kind of surgery, including preventive surgery, diagnostic surgery, or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of cancerous tissue is physically removed, excised, and / or destroyed, and may be used in combination with other therapies, such as the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0248] Removal of part or all of cancerous cells, tissues, or tumors may result in the formation of a cavity in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with additional anticancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also vary in dosage. 5. Other drugs

[0249] It is believed that other agents can be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative effect of the treatment. Inhibitors of cell adhesion are believed to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. In addition, it is believed that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment. VIII. Kits of the Present Disclosure

[0250] Any of the compositions described herein may be included in the kit. In a non-limiting example, the kit may include cells, reagents for generating the cells, vectors, and reagents for generating the vectors and / or components thereof. In certain embodiments, the kit may include NK cells, which may or may not express a CD5 targeting receptor, an optional cytokine, or an optional suicide gene. Such kits may or may not have one or more reagents for the manipulation of cells. Such reagents include, for example, small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and / or combinations thereof. Nucleotides encoding one or more CD5 targeting CARs, suicide gene products, and / or cytokines may be included in the kit. Proteins such as cytokines or antibodies, including monoclonal antibodies, may be included in the kit. Nucleotides encoding components of engineered CAR receptors may be included in the kit, including reagents for generating said components.

[0251] In certain embodiments, the kit also includes the NK cell therapy of the present disclosure and another cancer therapy. In some cases, the kit also includes a second cancer therapy, such as, for example, chemotherapy, hormone therapy, and / or immunotherapy, in addition to the cell therapy embodiment. The kit can be tailored to the specific cancer of the individual and can include the respective second cancer therapy for the individual.

[0252] The kit may include a properly aliquoted composition of the present disclosure. The components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed and preferably properly aliquoted. If there are multiple components in the kit, the kit may also generally contain a second, third, or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in a vial. The kit of the present invention also typically includes a means for containing the composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are held. IX. Working Examples EXAMPLES

[0253] The following examples are included to demonstrate certain non-limiting aspects of the present disclosure.It should be understood by those skilled in the art that the techniques disclosed in the following examples represent the techniques that the inventors have discovered to work well in implementing the disclosed subject matter.However, it should be understood by those skilled in the art in light of this disclosure that many changes can be made in the disclosed specific embodiments and still obtain the same or similar results without departing from the spirit and scope of the disclosed subject matter. Example 1 DAP10 costimulation confers memory-like characteristics to CD5-targeted cord blood-derived CAR-NK cells

[0254] This example relates to the analysis of various CD5-chimeric antigen receptor (CAR)-related constructs used in adoptive cell therapy for the treatment of CD5+ cancer cells.

[0255] Testing the T cell-centric costimulatory domains CD28 or 4-1BB in the design of CD5 CAR-NK cells. We characterized the efficacy of CD5 CAR-NK cells incorporating CD28 or 4-1BB as costimulatory domains in the CAR construct. Two CD5 CAR constructs were generated with scFv against the CD5 antigen, with either the CD28 or 4-1BB costimulatory domain, a CD3ζ signaling domain, and IL-15 as a cytokine to enhance in vivo proliferation and persistence (Figure 1A). Cord blood (CB)-derived NK cells were successfully transduced with these constructs (Figure 1B) and showed good proliferation with no evidence of fratricide (Figure 1C). These CD5 CAR-NK cells also showed enhanced cytokine production (IFNγ, TNFα), degranulation (CD107a), and cytotoxicity compared to non-transduced (NT) NK cells against the CD5+ T-cell acute lymphoblastic leukemia (T-ALL) cell line, CCRF-CEM (Figure 1D,E). In an in vivo mouse model of T-ALL, mice implanted with firefly luciferase-transduced CCRF-CEM (CCRF-CEM-FFluc) and treated with CD5-CD28-CD3ζ-IL-15 or CD5-41BB-CD3ζ-IL-15 CAR-NK cells showed improved tumor control and survival compared to mice implanted with tumor alone or mice implanted with tumor and treated with NT NK cells (Figure 1F-1H). However, all mice eventually succumbed to tumors.

[0256] Evaluation of the role of NK-associated costimulatory domains in the in vitro function of CD5 CAR-NK cells. Given that CD28 and 4-1BB are T cell-centric costimulatory domains and the fact that CD28 is not expressed on non-engineered NK cells, in certain embodiments, it was believed that the incorporation of costimulatory molecules more relevant to NK cell biology and signaling would enhance the efficacy of CD5 CAR-NK cells. Three additional retroviral constructs were designed; the constructs shared the same scFv against CD5, CD28 transmembrane (TM) domain, CD3ζ signaling domain, and IL-15 as a cytokine to enhance in vivo persistence and proliferation, but differed in the choice of costimulatory domain (NKG2D vs. DAP10 vs. DAP12) (Figure 2A). For constructs with DAP10 and DAP12 costimulatory domains, designs with DAP10 and DAP12 TM domains were also included (Figure 2A). A first-generation CAR construct without a costimulatory domain was used as a negative control to investigate the role of costimulation in the function of CAR-NK cells. CD5-CD28-CD3ζ-IL-15 CAR-NK cells served as a positive control with T cell-centric costimulation, as they showed in vitro and in vivo efficacy against CD5+ T-ALL targets. By adjusting the multiplicity of infection (MOI), similar transduction efficiencies were obtained for all constructs tested (50-60%) (Figure 2B). Proliferation of the various CD5 CAR-NK cells was performed as previously described. 12 , were very similar in vitro since the expansion protocols included universal antigen presenting cells (uAPCs) and IL-2 and optimized NK cell expansion regardless of their manipulation (Figure 2C). Regardless of CAR design, all CD5 CAR-NK cells showed enhanced cytokine production (IFNγ and TNFα) and degranulation (CD107a) when co-cultured with CD5+CCRF-CEM tumor cells for 6 hours compared to control NT NK cells and CD19 CAR-NK cells (with or without IL-15) used as irrelevant target CAR controls (Figure 2D). 51In Cr release cytotoxicity assays, all CD5 CAR-NK cells killed CD5+ tumor targets better than NT NK cells, however, no significant differences in cytotoxicity were observed among the different CAR-NK cell designs (Figure 2E).

[0257] CD5 CAR-NK cells with DAP10 costimulation resulted in enhanced tumor control and survival in a mouse model of CD5+ malignancies. To investigate the in vivo antitumor activity of different CD5 CAR-NK cell designs, the same NSG mouse model of CD5+ CCRF-CEM used previously was used to test the in vivo efficacy of CD5-CD28-CD3ζ and CD5-41BB-CD3ζ CD5 CAR-NK cells. Mice implanted with tumor alone and mice implanted with tumor and treated with NT NK cells served as negative controls. Mice were irradiated (225cGy) on day -1 and then injected intravenously with 100,000 CCRF-CEM-FFluc tumor cells per mouse on day 0. On day 2, treatment groups were divided into 2x10 6 NT NK cells or 2 x 10 6 Mice received either one of various preparations of CAR-NK cells. CD5 CAR-NK cells with DAP10 signaling showed clearly superior tumor control and significantly improved survival compared to control mice and mice that received CD5 CAR-NK cells incorporating other costimulatory domains (Figure 3A-C). Furthermore, animals treated with these CAR-NK cells showed no signs of toxicity (no weight loss) as evidenced by weight monitoring (Figure 3D). Interestingly, the survival advantage conferred by CD5 CAR-NK cells with DAP10 costimulation was maintained even in mice implanted with CCRF-CEM, which has low CD5 expression (Figure 3E). Furthermore, in an in vivo PDX mouse model of subcutaneously injected CD5+ mantle cell lymphoma, CD5 CAR-NK cells with DAP10 costimulation led to improved tumor control, accompanied by a significant reduction in the absolute numbers of CD45+CD5+ lymphoma cells from subcutaneous tumors, spleen, and bone marrow in CAR-NK cell-treated mice compared to untreated mice implanted with tumor alone (Figure 4A, Figure 4B).

[0258] DAP10 costimulation confers an activated phenotype to CD5 CAR-NK cells. To characterize why CD5 CAR-NK cells with DAP10 costimulatory domains perform significantly better in vivo compared to other CAR designs, we looked more deeply into their proteomic profile. To investigate the phenotype of the different CD5 CAR-NK cells with respect to NT NK cells, we used mass cytometry and a panel of 47 antibodies against activating and inhibitory NK cell receptors, as well as proliferation, differentiation, and maturation markers. Using viSNE, a t-distributed stochastic neighborhood embedding (t-SNE) algorithm, we observed that CD5 CAR-NK cells with DAP10 costimulatory domains exhibited two specific clusters (clusters 8 and 11) compared to NT NK cells (Figure 5A, Figure 5B). By examining in more detail the combination of markers defining each cluster, these clusters 8 and 11 were enriched in activation markers (NKG2D, DNAM, CD69, CD3ζ), cytotoxicity markers (perforin, granzyme B, TRAIL), and maturation markers (eomesodermin (eomes) and T-bet) (Figure 5C).

[0259] CD5 CAR-NK cells harboring a DAP10 costimulatory domain exhibit enhanced multifunctionality and metabolic fitness. To gain insight into why CAR-NK cells with DAP10 signaling exhibit less functional exhaustion in vivo compared to other CAR-NK cell designs, we performed a series of experiments to evaluate the polyfunctionality and metabolism of CAR-NK cells with various costimulatory domains. To evaluate cytokine secretion at the single-cell level, we used the IsoPlexis single-cell secretome assay, a well-validated tool to evaluate polyfunctionality that correlates with immune cell potency. After 4 hours of stimulation with plate-bound CD5 antigen, CAR-NK cells with DAP10 signaling exhibited the highest polyfunctionality, with a higher percentage of cells secreting 3, 4, or 5+ cytokines at the single-cell level (Figure 6A, Figure 6B). A higher percentage of these secreted cytokines in DAP10-containing CD5 CAR-NK cells belonged to the effector, stimulatory, or chemoattractant categories (Figure 6C). From a metabolic standpoint, CD5 CAR-NK cells with DAP10 signaling exhibited significantly higher oxidative phosphorylation and oxygen consumption rate (OCR), a measure of mitochondrial fitness, compared to other constructs and control NT NK cells (Figure 6D).

[0260] CD5 CAR-NK cells bearing the DAP10 costimulatory domain exhibit memory-like characteristics. We next investigated whether the favorable transcriptomic, proteomic, and metabolic changes associated with DAP10 costimulation in CD5 CAR-NK cells translated into memory-like features that may explain the remarkable in vivo efficacy. Thus, we simulated an in vivo setting in which CAR-NK cells are prone to exhaustion, since they must exhibit continuous killing capacity to adequately control tumors. In long-term Incucyte® cytotoxicity assays with multiple tumor rechallenges performed every 2-3 days (Figure 7A), CD5 CAR-NK cells with DAP10 signaling showed superior cytotoxicity against CD5+CCRF-CEM compared to other CAR-NK cell designs with sustained ability to kill after multiple tumor rechallenges, whereas NT NK cells, NK cells transduced with irrelevant targeting CD19 or CD19 / IL-15 CARs, and NK cells transduced with other CD5 CAR constructs lost their killing capacity over time (Figure 7B,C). Similarly, mice treated with DAP10-containing CD5 CAR-NK cells, which survived long term and appeared cured of disease in an in vivo model, showed no evidence of tumor development upon re-challenge with CD5+CCRF-CEM, suggesting that these CD5 CAR-NK cells possess memory characteristics and are capable of eradicating tumors following re-exposure (Figures 7D-7F). Examples of Materials and Methods Cell lines and culture media

[0261] CCRF-CEM (T-ALL cell line) was purchased from American Type Culture Collection (Manassa, VA, USA). CCRF-CEM cells were transduced with firefly luciferase (CCRF-CEM-FFluc) for in vivo experiments. For low CD5 model, shRNA was used to knock down the expression of CD5 in CCRF-CEM. K562-based feeder cells were retrovirally transduced to co-express 4-1BBL, CD48, and membrane-bound IL-21, which are referred to as uAPC. All cell lines were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine. NK cells were cultured in stem cell growth medium (SCGM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine. Cord blood NK cell proliferation

[0262] CB units for the study were provided by the MD Anderson Cancer Center CB Bank. CB were isolated by density gradient technique (Ficoll-Histopaque; Sigma, St Louis, MO, USA). CD56+ NK cells purified by NK isolation kit (Miltenyi Biotec, Inc., San Diego, CA, USA) were stimulated with irradiated (100 Gy) uAPC (feeder cell:NK ratio 2:1) and recombinant human IL-2 (Proleukin, 200 U / ml; Chiron, Emeryville, CA, USA) in complete stem cell growth medium (CellGenix GmbH, Freiburg, Germany) on day 0. Activated NK cells were transduced with retroviral supernatant on day +6 in human fibronectin-coated plates (Clontech Laboratories, Inc., Mountain View, CA, USA). On days +7 and +14, NK cells were restimulated with irradiated uAPC and IL-2. On day +21, CAR-transduced NK cells were harvested for use in the experiments described above. Retroviral transfection and transduction

[0263] Retroviral vectors encoding various CD5 constructs were designed in-house and plasmids were ordered from GeneArt™. The CD19-CD28-CD3ζ and CD19-CD28-CD3ζ-IL-15 vectors have been previously described. 6 As previously described 14 Transient retroviral supernatants were generated. Activated NK cells were transduced with retroviral supernatants on day +6 in human fibronectin-coated plates (Clontech Laboratories, Inc., Mountain View, CA, USA). On day +8, CAR transduction efficiency was measured by flow cytometry, and NK cells were restimulated with irradiated uAPC and IL-2. Functional assays

[0264] 100x10e3 cells / well of CAR19 or CAR19 / IL-15 NK cells, or CD5 CAR-NK cells were co-cultured with CCR-CEM cells or K562 targets (positive control) at an effector:target cell ratio (E:T) of 2:1 in round-bottom 96-well plates for 6 hours in the presence of brefeldin A. To measure degranulation, CD107a antibody (Brilliant Violet 785™ anti-human CD107a (LAMP-1) antibody, Biolegend, San Diego, CA, USA) was added to the wells at the beginning of the co-culture. After cell harvest and surface staining for CD56, CD3, and CAR, cells were fixed and intracellular staining was performed for TNFα (TNF alpha monoclonal antibody (MAb11), Alexa Fluor 700, eBioscience Inc., San Diego, CA, USA) and IFNγ (BD Horizon™ V450 mouse anti-human IFNγ, BD Biosciences, San Jose, CA, USA). Cells were cultured as previously described. 15 , and assessed by flow cytometry. Cytotoxicity assay Chromium release assay

[0265] To assess cytotoxicity, ex vivo expanded NT or CAR-transduced NK cells were 51 Cr-labeled CCRF-CEM targets were co-cultured at multiple E:T ratios. Cytotoxicity was assessed as previously described. 15 , 51 Cr release was measured. Incucyte live cell imaging assay

[0266] Ex vivo expanded NT and CAR-transduced NK cells were co-cultured with lentivirally transduced CCRF-CEM tumor targets in 96-well or 48-well plates at a 1:1 E:T ratio to express red dye (PE-Texas red). Each well contained 5×104 CCRF-CEM cells and 5×104 NK cells. Apoptosis was detected using CellEvent™ Caspase-3 / 7 Green Detection Reagent (ThermoFisher). Wells containing tumor alone or NK cell alone served as negative controls. Four separate 1.75×1.29 mm per well were analyzed with a 10× objective using an IncuCyte S3 live cell analysis system (Sartorius). 2 Frames were captured from the regions at 1 hour intervals over time. Values ​​from all four regions of each well were pooled and averaged across all three replicates. Results were graphed as percent cytotoxicity by calculating the ratio of overlapping red and green signals (counts per image) divided by red signal (counts per image). For re-challenge experiments, CCRF-CEM was added to all wells (except for control wells containing NK cell alone group) every 2-3 days. Red counts and confluence were plotted over time to track tumor growth or elimination by NK cells. Mass Cytometry Antibody Conjugation

[0267] A panel containing 38 metal-tagged antibodies was used for detailed characterization of NK cells. A list of antibodies with corresponding metal tag isotopes is shown in the table. All unlabeled antibodies were purchased in carrier-free form and conjugated in-house with the corresponding metal tag using Maxpar X8 or MCP9 polymers according to the manufacturer's instructions (Fluidigm). All metal isotopes were obtained from Fluidigm, except for indium(III) chloride (Sigma-Aldrich, St. Louis, MO). Antibody concentrations were determined by measuring the amount of A280 protein using a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA). The conjugated antibodies were then diluted to a final concentration of 0.5 mg / ml in PBS-based antibody stabilization solution or LowCross-Buffer (Candor Bioscience GmbH, Wangen, Germany) supplemented with 0.05% sodium azide (Sigma-Aldrich, St. Louis, MO). Step titration experiments were performed to determine the concentration with the optimal signal-to-noise ratio for each antibody. [Table 1] Sample preparation and acquisition:

[0268] NT NK cells or various CD5 CAR-transduced NK cells were collected, washed twice with cell staining buffer (0.5% bovine serum albumin, 0.02% sodium azide in PBS) at 4°C for 5 minutes at 400g, and incubated with 2.5 μM cisplatin-198Pt for 2 minutes in the dark. The cells were then washed once with FACS buffer (2% fetal bovine serum in PBS) and once with cell staining buffer. 10 μl of human Fc receptor blocking solution (Milteny Biotec, 130-059-901) for 10 minutes at room temperature. The cells were then stained with freshly prepared antibody mixtures against cell surface markers diluted in 1% bovine serum albumin in PBS for 40 minutes at room temperature on a shaker (130 rpm). The cells were washed twice with cell staining buffer and fixed with 1.8% paraformaldehyde for 10 minutes at room temperature in the dark. The cells were washed twice with cell staining buffer at 4° C. for 5 min at 800 g and stored in 1 ml of methanol at −80° C. overnight. The next day, the samples were washed twice with cell staining buffer and stained with freshly prepared antibody mixtures against intracellular markers diluted in 1% bovine serum albumin + 0.2% saponin in PBS. The cells were washed twice with cell staining buffer and incubated in 1 ml intercalator iridium (1 μl of 125 μM Ir in 1 ml PBS) for 20 min at room temperature. The cells were washed twice with cell staining buffer and resuspended in 1 ml of 0.1% bovine serum albumin in Milli-Q® dH2O, filtered through a 35 μm nylon mesh (cell strainer cap tube, BD, San Jose, CA) and counted. Prior to analysis, samples were suspended in Milli-Q® dH2O supplemented with EQ™ four element calibration beads at a concentration of 0.5×105 / ml. Samples were acquired at 300 events / sec on a Helios instrument (Fluidigm) using Helios 6.5.358 acquisition software (Fluidigm). 4. Data Analysis

[0269] Mass cytometry data were normalized based on EQ™ 4 element signal shift over time using Fluidigm normalization software 2. Initial data quality control was performed using Flowjo version 10.2. Calibration beads were gated out and singlets were selected based on Iridium 193 staining and event length. Dead cells were excluded by the Pt195 channel and further gating was performed to select the NK cell population of interest (CD3-CD56+) after CD45+ cells. A total of 320,000 cells were proportionally sampled from all samples to perform automated clustering. Previously published 17 FlowSOM for clustering for deep phenotyping of immune cells like 16 Data were analyzed using automated dimensionality reduction including (viSNE) combined with Isoplexis single-cell secretome assay

[0270] NT NK cells or various CD5 CAR-NK cells were labeled with a fluorescent dye (Isoplexis stained cell membrane 405) and stimulated with plate-bound CD5 antigen for 4 h or co-cultured with CCRF-CEM cells for 4 h, and then purified using positive selection with CD56+ microbeads. From the purified population, 3 × 10 4 NK cells were loaded onto individual Iso-coded chips according to the manufacturer's protocol (Isoplexis, Branford, CT, USA) and subjected to single-cell 32-plex cytokine secretome profiling using a fully validated panel of cytokines on the IsoPlexis single-cell platform as previously described. 18、19, IsoSpeak software was used to quantitate the number of polyfunctional NK cells, measure the percentage of cells in each sample secreting multiple cytokines (2+, 3+, 4+, or 5+), assign the category of cytokine being secreted (effector, stimulatory, regulatory, or chemoattractant), and measure the polyfunctional intensity index (PSI), defined as the percentage of polyfunctional cells multiplied by the mean fluorescence intensity (MFI) of proteins secreted by NK cells. Seahorse assay

[0271] NT-NK cells or various CD5 CAR-transduced NK cells were assayed using the Seahorse XF Glycolysis Stress Test Kit and Mito Stress Test Kit (Agilent). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using an Agilent Seahorse XFe96 Analyzer (Agilent) according to the manufacturer's instructions. Xenogeneic Mouse Models

[0272] To evaluate the antitumor efficacy of CD5 CAR-transduced CB-NK cells in vivo, we used a NOD / SCID IL-2 Rγnull (NSG) xenograft model implanted with CCRF-CEM cell line. Mouse experiments were performed in accordance with NIH recommendations following protocols approved by the Institutional Animal Care and Use Committee. NSG mice (10–12 weeks old; Jackson Laboratories, Bar Harbor, ME, USA) were irradiated with 300 cGy on day -1 and inoculated intravenously with firefly luciferase-labeled CCRF-CEM cells (1 × 10e5 cells) on day 0. Where indicated, fresh, expanded, NT or CD5 CAR-transduced CB-NK cells were injected via the tail vein on day 2. Mice were subjected to weekly bioluminescence imaging (Xenogen-IVIS 200 Imaging system; Caliper, Waltham, MA, USA). Signal quantification in photons / second was performed using Living Image software (Caliper). 20 This was performed by determining the photon flux rate in a standardized region of interest using a flow cytometer. NK cell trafficking, persistence, and proliferation were measured by flow cytometry. Mice were also monitored for survival and toxicity by recording body weight and scoring. Antibodies used to examine in vivo trafficking, persistence, and proliferation of NK cells

[0273] The following antibodies were used for flow cytometry staining: APC / Cy7 anti-human CD3 antibody (clone HIT3a, Biolegend), Brilliant Violet 605™ anti-human CD56 (NCAM) antibody (clone HCD56, Biolegend), anti-CAR antibody goat F(ab')2 anti-human IgG(H+L)-Alexa Fluor 647 (Jackson), PE mouse anti-human CD7 clone M-T701 (BD biosciences), BV711 mouse anti-human CD5 clone UCHT2 (BD biosciences), BV650 mouse anti-human CD16 clone 3G8 (BD biosciences), PerCP anti-human CD45 antibody clone HI30 (Biolegend), Live / Dead Aqua Dead Cell Stain (ThermoFisher). statistics

[0274] Quantitative differences (mean ± sd) between groups were compared using two-way Anova tests. P values ​​were two-sided and P < 0.05 was considered significant. For all bioluminescence experiments, intensity signals were summarized by group of mice as mean ± sd at baseline and multiple subsequent time points. 20 Survival probabilities were calculated using the Kaplan-Meier method. Statistical tests shown were performed using Prism software (GraphPad version 7.0c). Importance of specific embodiments

[0275] The effect of different costimulatory domains on CAR-T cell function and fitness has been well studied. Few studies have been reported in the CAR-NK cell field investigating the role of various costimulatory molecules, but none have systematically examined the impact of costimulatory choice on proliferation, transcriptomic profile, phenotype, polyfunctionality, metabolism, cytotoxicity, and memory formation. This study delves into these specific questions and shows that the NK-centric costimulatory domain, DAP10, provides favorable phenotypic changes and synergistic signaling characteristic of optimal overall antitumor activity and fitness, as well as memory formation, of CD5 CAR-NK cells.

[0276] In CAR-T cells, the impact of 4-1BB versus CD28 costimulation on T cell signaling and metabolism has been thoroughly investigated and correlated with clinical outcomes and data on the persistence and attrition of cell therapy products. 21 DAP10 has previously been shown to enhance the function of CAR-T cells and NKG2D-based CAR-NK cells. 22、23 However, no mechanistic insights have been provided to explain its advantages over other costimulatory molecules. Moreover, signaling pathways downstream of DAP10 costimulation have not been analyzed in the context of CAR-T or CAR-NK cells.

[0277] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. 1.Tsuyama N, Ennishi D, Yokoyama M, et al. Clinical and prognostic significance of aberrant T-cell marker expression in 225 cases of de novo diffuse large B-cell lymphoma and 276 cases of other B-cell lymphomas. Oncotarget. 2017;8(20):33487-33500. 2.Bashford-Rogers RJ, Palser AL, Hodkinson C, et al. Dynamic variation of CD5 surface expression levels within individual chronic lymphocytic leukemia clones. Exp Hematol. 2017;46:31-37 e10. 3.Scherer LD, Brenner MK, Mamonkin M. Chimeric Antigen Receptors for T-Cell Malignancies. Front Oncol. 2019;9:126. 4.Mamonkin M, Rouce RH, Tashiro H, Brenner MK. A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies. Blood. 2015;126(8):983-992. 5.Hill L RR, Smith T et al. Safety and Anti-Tumor Activity of CD5 CAR T-Cells in Patients with Relapsed / Refractory T-Cell Malignancies. Blood. 2019;134 (Supplement_1): 199. https: / / doi.org / 10.1182 / blood-2019-129559. 6.Liu E, Tong Y, Dotti G, et al. Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity. Leukemia. 2018;32(2):520-531. 7.Liu E, Marin D, Banerjee P, et al. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N Engl J Med. 2020;382(6):545-553. 8.Kawalekar OU, O'Connor RS, Fraietta JA, et al. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells. Immunity. 2016;44(2):380-390. 9.Guedan S, Posey AD, Jr., Shaw C, et al. Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight. 2018;3(1). 10.Zhang H, Snyder KM, Suhoski MM, et al. 4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy. J Immunol. 2007;179(7):4910-4918. 11.Guerra N, Tan YX, Joncker NT, et al. NKG2D-deficient mice are defective in tumor surveillance in models of spontaneous malignancy. Immunity. 2008;28(4):571-580. 12.Liu E, Ang SOT, Kerbauy L, et al. GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells. Front Immunol. 2021;12:626098. 13.Lanier LL. Up on the tightrope: natural killer cell activation and inhibition. Nat Immunol. 2008;9(5):495-502. 14.Vera J, Savoldo B, Vigouroux S, et al. T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells. Blood. 2006;108(12):3890-3897. 15.Rouce RH, Shaim H, Sekine T, et al. The TGF-beta / SMAD pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia. Leukemia. 2016;30(4):800-811. 16.Van Gassen S, Callebaut B, Van Helden MJ, et al. FlowSOM: Using self-organizing maps for visualization and interpretation of cytometry data. Cytometry A. 2015;87(7):636-645. 17.Kordasti S, Costantini B, Seidl T, et al. Deep-phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment. Blood. 2016. 18.Rossi J, Paczkowski P, Shen YW, et al. Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL. Blood. 2018;132(8):804-814. 19.Xue Q, Bettini E, Paczkowski P, et al. Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response. J Immunother Cancer. 2017;5(1):85. 20.Shah N, Martin-Antonio B, Yang H, et al. Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity. PLoS One. 2013;8(10):e76781. 21.Kawalekar OU, RS OC, Fraietta JA, et al. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells. Immunity. 2016;44(3):712. 22.Chang YH, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D. A chimeric receptor with NKG2D specificity enhances natural killer cell activation and killing of tumor cells. Cancer Res. 2013;73(6):1777-1786. 23.Zhao R, Cheng L, Jiang Z, et al. DNAX-activating protein 10 co-stimulation enhances the anti-tumor efficacy of chimeric antigen receptor T cells. Oncoimmunology. 2019;8(1):e1509173.

[0278] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described herein. As will be readily understood by those skilled in the art from the present disclosure, any currently existing or later developed process, machine, manufacture, composition of matter, means, methods or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the present disclosure. Thus, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods or steps.

Claims

1. 1. An expression construct comprising a sequence encoding an engineered CD5-specific receptor, said receptor comprising: (a) CD28 transmembrane domain (TM) and CD28 intracytoplasmic domain (ICD); (b) the CD28 TM, CD28 ICD, and CD3 zeta signaling domains; (c) CD28 TM and DAP12 ICD; (d) CD28 TM, DAP12 ICD, and CD3 zeta signaling domains; (e) DAP12 TM and DAP12 ICD; (f) DAP12 TM, DAP12 ICD, and CD3 zeta signaling domain; (g) CD28 TM and 4-1BB ICD; (h) CD28 TM, 4-1BB ICD, and the CD3 zeta signaling domain; (h) CD28 TM, CD28 ICD, and 4-1BB ICD; (i) CD28 TM, CD28 ICD, 4-1BB ICD, and CD3 zeta signaling domains; (j) CD28 TM and DAP10 ICD; (k) CD28 TM, DAP10 ICD, and CD3 zeta signaling domain; (l) DAP10 TM and DAP10 ICD; (m) DAP10 TM, DAP10 ICD, and CD3 zeta signaling domain; (n) NKG2D ICD; (o) NKG2DTM; (p) NKG2D TM and NKG2D ICD; (q) the NKG2D TM, NKG2D ICD, and CD3 zeta signaling domains; (r) CD28 TM and NKG2D ICD; (s) NKG2D ICD and CD3 zeta signaling domain; (t) CD28 TM, NKG2D ICD, and CD3 zeta signaling domains; (u) 4-1BB TM and 4-1BB ICD; (v) CD28 TM and CD3 zeta in the absence of the costimulatory domain, ICD; (w) CD8 TM and CD28 intracytoplasmic domain (ICD); (x) CD8 TM, CD28 ICD, and CD3 zeta signaling domains; (y) CD8 TM and DAP12 ICD; (z) CD8 TM, DAP12 ICD, and CD3 zeta signaling domain; (aa) CD8 TM and DAP12 ICD; (bb) CD8 TM, DAP12 ICD, and CD3 zeta signaling domain; (cc) CD8 TM and 4-1BB ICD; (ee) CD8 TM, 4-1BB ICD, and the CD3 zeta signaling domain; (dd) CD8 TM, CD28 ICD, and 4-1BB ICD; (ee) CD8 TM, CD28 ICD, 4-1BB ICD, and CD3 zeta signaling domains; (ff) CD8 TM and DAP10 ICD; (gg) CD8 TM, DAP10 ICD, and CD3 zeta signaling domain; (hh) CD8 TM and DAP10 ICD; (ii) CD8 ICD; (jj)CD8TM; (kk) CD8 TM and NKG2D ICD; (ll) CD8 TM, NKG2D ICD, and CD3 zeta signaling domains; (mm) CD8 TM and CD3 zeta in the absence of the costimulatory domain ICD (nn) CD27 TM and CD28 intracytoplasmic domain (ICD); (oo) CD27 TM, CD28 ICD, and CD3 zeta signaling domains; (pp) CD27 TM and DAP12 ICD; (qq) CD27 TM, DAP12 ICD, and CD3 zeta signaling domain; (rr) CD27 TM and DAP12 ICD; (ss) CD27 TM, DAP12 ICD, and CD3 zeta signaling domain; (tt) CD27 TM and 4-1BB ICD; (uu) CD27 TM, 4-1BB ICD, and CD3 zeta signaling domains; (vv) CD27 TM, CD28 ICD, and 4-1BB ICD; (ww) CD27 TM, CD28 ICD, 4-1BB ICD, and CD3 zeta signaling domain; (xx) CD27 TM and DAP10 ICD; (yy) CD27 TM, DAP10 ICD, and CD3 zeta signaling domain; (zz) CD27 TM and DAP10 ICD; (aaa)CD27TM; (bbb) CD27 TM and NKG2D ICD; (ccc) the CD27 TM, NKG2D ICD, and CD3 zeta signaling domains; or (ddd) CD27TM and CD3zeta in the absence of the costimulatory domain, ICD An expression construct comprising one of the following:

2. 2. The expression construct of claim 1, wherein the engineered CD5-specific receptor is a chimeric antigen receptor (CAR) or a T cell receptor.

3. 3. An expression construct according to claim 1 or 2, encoding a polypeptide of interest other than the engineered CD5-specific receptor.

4. 4. The expression construct of claim 3, wherein the polypeptide of interest is a therapeutic protein or a protein that enhances cellular activity, proliferation, and / or persistence of the cells contained therein.

5. The expression construct of claim 3, wherein the polypeptide of interest is a suicide gene; a cytokine; a human or viral protein that enhances the proliferation, expansion, and / or metabolic fitness of the contained cell; or a combination thereof.

6. 6. The expression construct of claim 5, wherein the cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, IL-7, or a combination thereof.

7. A vector comprising an expression construct according to any one of claims 1 to 6.

8. An immune cell comprising an expression construct according to any one of claims 1 to 6 or a vector according to any one of claims 7.

9. The immune cell of claim 8, wherein the immune cell is a natural killer (NK) cell, a T cell, a gamma delta T cell, an invariant NKT (iNKT) cell, a B cell, a macrophage, an MSC, or a dendritic cell.

10. The immune cell of claim 8 or 9, wherein the NK cell expresses one or more exogenously provided cytokines.

11. The immune cell of claim 10, wherein the cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, IL-23, or a combination thereof.

12. The immune cell of any one of claims 8 to 11, wherein expression of one or more endogenous genes in the immune cell is modified.

13. The population of immune cells according to any one of claims 8 to 12, wherein the cells are present in a suitable culture medium.

14. The population of claim 13 , wherein the immune cells are NK cells.

15. 15. The population of claim 13 or 14, wherein the immune cells are NK cells comprising an expression construct encoding a CAR and a cytokine.

16. The population of any one of claims 13 to 15, wherein the immune cells are NK cells comprising an expression construct encoding a CAR and IL-15.

17. A composition for killing CD5 positive cells in an individual, the composition comprising cells carrying an expression construct according to any one of claims 1 to 6.

18. The composition of claim 17 , wherein the individual has cancer.

19. The composition of claim 18, wherein the cancer is a solid tumor.

20. 20. The composition of claim 18, wherein the cancer is a hematological cancer.

21. A composition for preventing cancer or preventing metastasis of cancer in an individual, comprising cells carrying an expression construct according to any one of claims 1 to 6.

22. 22. The composition of claim 21, wherein the individual is at risk for or has previously had cancer.

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