Augmenting T cell function by using proximal signaling molecules

Abstract

The present disclosure relates generally to, inter alia, isolated recombinant cells that overexpress SLP-76 polypeptides. The disclosure also provides compositions and methods useful for producing the isolated recombinant cells and the corresponding SLP-76 molecules, as well as methods of treating diseases, such as cancer, with such compositions.

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 168,624, filed March 31, 2021; the contents of which are incorporated herein in their entirety.

[0002] INCORPORATION BY REFERENCE This application contains a sequence listing, which is incorporated herein by reference in its entirety. The attached text file of the sequence listing has the file name "078430-533001WOSequenceListing_ST25.txt", was created on March 30, 2022, and is 239,808 bytes.

[0003] Field The present disclosure relates generally to the fields of oncology and immunotherapy, and in particular to compositions of polypeptides, such as SLP-76, and polynucleotides encoding these polypeptides, that can enhance the activation and function of cells, such as T cells that express a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR) in response to a target antigen. The present disclosure also provides compositions of recombinant cells comprising these polypeptides, and methods useful for making such recombinant cells, as well as methods of enhancing T cell activity for the treatment of conditions such as diseases (e.g., cancer or autoimmune diseases). [Background technology]

[0004] background Current T cell therapies, such as those involving T cells expressing chimeric antigen receptors (CARs) and / or T cell receptors (TCRs), have limitations when the target antigens recognized by the CARs or TCRs are expressed at low density. If tumor cells downregulate the amount of tumor antigens (usually designed as target antigens for the CARs or TCRs) expressed on their surface, current CARs or TCRs may not be able to activate T cells to inhibit or kill tumor cells. In another scenario, CARs or TCRs may destroy T cells even when antigen density is normal. For example, some CARs or TCRs have low affinity for their target antigens or may lead to T cell exhaustion, thus ultimately weakening or terminating the efficacy of T cell therapy. As a result, there remains a need to modulate T cell activation to overcome these obstacles to enhance and / or expand the reach of these therapies. Summary of the Invention [Problem to be solved by the invention]

[0005] summary The present disclosure generally relates to the development of immunotherapies comprising recombinant cells or lysates of recombinant cells containing polypeptides, such as lymphocyte cytoplasmic protein 2 (76 kDa SH2 domain-containing white blood cell protein), also known as LCP2 or SLP-76, in either cytoplasmic or membrane-bound form, or polynucleotides encoding such polypeptides, as well as pharmaceutical compositions containing the above for use in enhancing immunotherapies in treating various conditions, such as diseases (e.g., cancer). As described in more detail below, various SLP-76 constructs have been prepared and found to have dramatic effects in enhancing activation and increasing efficacy of immune cells (e.g., T cells) expressing CAR and / or TCR in response to an antigen, even when the antigen is expressed at low density. In some embodiments, the SLP-76 construct is bound to the membrane of T cells expressing CAR and / or TCR. [Means for solving the problem]

[0006] In one aspect, the disclosure provides compositions comprising isolated recombinant cells that have been modified to overexpress and / or contain elevated levels of an SLP-76 polypeptide. In some embodiments, the isolated recombinant cells of the compositions described herein are capable of being activated by a target antigen expressed at low density.

[0007] In some embodiments, the isolated recombinant cell further comprises a T cell receptor (TCR) polypeptide, a chimeric antigen receptor (CAR) polypeptide, and / or another receptor polypeptide. In some embodiments, the TCR or CAR polypeptide has binding affinity to a target antigen or an adaptor molecule that specifically recognizes the target antigen. In some embodiments, the isolated recombinant cell comprises a TCR polypeptide. In some embodiments, the target antigen of the TCR polypeptide is expressed by a target cell and presented to the TCR polypeptide by an antigen-presenting cell (APC). In some embodiments, the isolated recombinant cell comprises an endogenous or native TCR polypeptide. In some embodiments, the isolated recombinant cell comprises a CAR polypeptide. In some embodiments, the target antigen of the CAR polypeptide is expressed by a target cell.

[0008] In some embodiments, the target cells described herein are cells that are correlated with a condition including, for example, a proliferative disease (such as a cancer or tumor), a hematological malignancy, a solid tumor, an autoimmune disease, inflammation, an allergic disease, an infectious disease, and / or senescence / aging, etc. In some embodiments, the target cells are cancer cells.

[0009] In some embodiments, the CAR polypeptide described herein comprises: a) an extracellular ligand-binding domain that has binding affinity for a target antigen or an adaptor molecule that specifically recognizes the target antigen; b) the transmembrane domain; c) an intracellular signaling domain, including a proximal signaling molecule; and d) optionally a hinge domain and / or a costimulatory domain: Includes.

[0010] In some embodiments, the intracellular signaling domain of a CAR polypeptide described herein comprises a full-length or biologically active fragment of a protein kinase, a G protein, a GTP-binding protein, an adaptor signaling protein, or a scaffold protein capable of inducing host cell activation. In some embodiments, the intracellular signaling domain of a CAR polypeptide comprises CD3zeta, CD3-epsilon, CD3-gamma, DAP12, ZAP70, PLCG1, PKC, ITK, NCK, VAV1, GRB2, GADS, SOS1, ADAP, SYK, LYN, PI3K, or BLNK, or a biologically active fragment, mutant, or variant thereof.

[0011] In some embodiments, the TCR polypeptide and / or the CAR polypeptide induces exhaustion of the recombinant cell. In some embodiments, overexpression of an SLP-76 polypeptide described herein in an isolated recombinant cell does not enhance exhaustion of the recombinant cell. In some embodiments, overexpression of an SLP-76 polypeptide described herein in an isolated recombinant cell overcomes exhaustion of the recombinant cell.

[0012] In some embodiments, the isolated recombinant cell further expresses the TCR polypeptide and / or the CAR polypeptide at normal density. In some embodiments, the isolated recombinant cell further expresses the TCR polypeptide and / or the CAR polypeptide at high density. In some embodiments, the isolated recombinant cell further expresses the TCR polypeptide and / or the CAR polypeptide at low density.

[0013] In some embodiments, the SLP-76 polypeptide is in a free cytoplasmic form. In some embodiments, the SLP-76 polypeptide is bound to the membrane of a recombinant cell. In some embodiments, the SLP-76 polypeptide is bound to the cell membrane via a transmembrane domain. In some embodiments, the SLP-76 polypeptide is: i) interactions between SLP-76 polypeptides and membrane proteins; ii) a covalent bond between an SLP-76 polypeptide and a fatty acid in the membrane; and / or iii) Binding between the SLP-76 polypeptide and the highly polar head groups of lipids in membranes: It is attached to the cell membrane via

[0014] In some embodiments, the SLP-76 polypeptide comprises a full-length SLP-76. In some embodiments, the SLP-76 polypeptide comprises a biologically active fragment, mutant, or variant of SLP-76. In some embodiments, the SLP-76 polypeptide comprises at least one mutation (e.g., a K30R substitution) relative to a wild-type SLP-76 polypeptide. In some embodiments, the SLP-76 polypeptide comprises an amino acid sequence having at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59, or 60. In some embodiments, an SLP-76 polypeptide comprises an amino acid sequence having at least 70% or greater identity to SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59, or 60. In some embodiments, an SLP-76 polypeptide described herein comprises the amino acid sequence of SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59, or 60. In some embodiments, an SLP-76 polypeptide described herein consists of the amino acid sequence of SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59, or 60.

[0015] In some embodiments, the isolated recombinant cell is an immune cell. In some embodiments, the isolated recombinant cell is a non-immune cell. In some embodiments, the isolated recombinant cell is a T cell, a regulatory T cell (Treg), a tumor-infiltrating lymphocyte (TIL), a natural killer (NK) cell, a macrophage, a monocyte, a gamma delta T cell, a stem cell, a natural killer T (NKT) cell, an induced pluripotent stem cell (iPSC)-derived NK cell, or an induced pluripotent stem cell (iPSC)-derived T cell.

[0016] In some embodiments, the isolated recombinant cell comprises about 10, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90 In some embodiments, the antibody can be activated by less than 1,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1, million, 2, million, 3, million, 4, million, 5, million, 6, million, 7, million, 8, million, 9, million, or 10 million molecules of the target antigen.

[0017] In some embodiments, activation of the isolated recombinant cell in response to the target antigen enhances cellular proliferation, differentiation, cytokine production and / or cytotoxicity.

[0018] In another aspect, the disclosure provides an isolated polypeptide comprising an SLP-76 polypeptide comprising an amino acid sequence having at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59 or 60. In some embodiments, the isolated polypeptide comprises an SLP-76 polypeptide comprising an amino acid sequence having at least 70%, 75%, 90%, 95% or more sequence identity to SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59 or 60. In some embodiments, the isolated polypeptide comprises an SLP-76 polypeptide and a transmembrane domain as described herein. In some non-limiting embodiments, the transmembrane domain of the isolated polypeptide tethers the isolated polypeptide to a membrane (eg, a cell membrane) of the isolated recombinant cell.

[0019] In another aspect, the disclosure provides an isolated polynucleotide encoding an isolated SLP-76 polypeptide as described herein. In some embodiments, the isolated polynucleotide encodes an isolated membrane-bound SLP-76 polypeptide as described herein. Such an SLP-76 polypeptide may be bound to the membrane of an isolated recombinant cell through a transmembrane domain or other mechanisms as described herein.

[0020] In another aspect, the present disclosure provides an expression vector comprising the isolated polynucleotide described herein. In some embodiments, the expression vector comprises an expression promoter. In some embodiments, the isolated polynucleotide is conjugated to the expression promoter so as to be expressed in the isolated recombinant cell described herein.

[0021] In another aspect, the disclosure provides a host cell comprising an expression vector described herein. In some embodiments, the host cell is a recombinant cell described herein, including, for example, an immune cell or a non-immune cell.

[0022] In another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: i) the isolated polynucleotides described herein, and isolated polynucleotides encoding T cell receptor (TCR) polypeptides and / or chimeric antigen receptor (CAR) polypeptides; ii) an expression vector comprising an expression vector as described herein and an isolated polynucleotide encoding a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide; and / or iii) an expression vector comprising the isolated polynucleotide described herein and an isolated polynucleotide encoding a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide: In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutical acceptable carrier and / or excipient.

[0023] In another aspect, the present disclosure provides a method for making a recombinant cell, comprising introducing a composition containing a polynucleotide encoding an SLP-76 polypeptide into a cell. In some embodiments, the cell is a native cell. In some embodiments, the cell is a recombinant cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a non-immune cell. In some embodiments, the cell is derived from a biological sample. In some embodiments, the cell is derived from a biological sample from a human, e.g., a patient.

[0024] In some embodiments, the cell further comprises a TCR or CAR molecule. Such a TCR molecule may be native or endogenous to the cell, or exogenous.

[0025] In another aspect, the present disclosure provides a method for making a recombinant cell, comprising introducing a composition containing a polynucleotide encoding an SLP-76 polypeptide and a polynucleotide encoding a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide into a cell. In some embodiments, the cell is a native cell. In some embodiments, the cell is a recombinant cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a non-immune cell. In some embodiments, the cell is derived from a biological sample. In some embodiments, the cell is derived from a biological sample from a human, e.g., a patient.

[0026] In another aspect, the present disclosure provides a method for enhancing activation of a cell in response to a target antigen, comprising overexpressing an SLP-76 polypeptide in the cell. Such overexpression can result from introducing into the cell an expression vector comprising an SLP-76 polypeptide, a polynucleotide encoding an SLP-76 polypeptide, and / or an SLP-76 polynucleotide, as described herein. In some embodiments, the cell is a native cell. In some embodiments, the cell is a recombinant cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a non-immune cell. In some embodiments, the cell is derived from a biological sample. In some embodiments, the cell is derived from a biological sample from a human, e.g., a patient. In some embodiments, the cell further comprises a TCR or CAR molecule. Such a TCR molecule can be native or endogenous, or exogenous, in the cell.

[0027] In another aspect, the present disclosure provides a method for enhancing the activation of a cell in response to a target antigen, comprising overexpressing an SLP-76 polypeptide and a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide in a cell. In some embodiments, the cell is a native cell. In some embodiments, the cell is a recombinant cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a non-immune cell. In some embodiments, the cell is derived from a biological sample. In some embodiments, the cell is derived from a biological sample from a human, e.g., a patient.

[0028] In another aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof comprising administering to the subject a pharma- ceutical effective amount of a composition described herein. Such a composition may contain an SLP-76 polypeptide, a polynucleotide encoding an SLP-76 polypeptide, and / or an expression vector comprising an SLP-76 polynucleotide, as described herein.

[0029] In some embodiments, a disease or disorder in a subject cannot be treated, or can only be treated partially or inefficiently, by T cells expressing a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide, while the TCR or CAR polypeptide has a binding affinity to a target antigen or to an adaptor molecule that specifically recognizes a target antigen expressed by a host cell (e.g., a cancer cell or a tumor cell) associated with the disease or disorder. For example, the disease or disorder involves a host cell that expresses a low level of the target antigen (i.e., has a low density), resulting in insufficient activation of the T cell through the TCR or CAR polypeptide. Such a low density of target antigen can be caused by a mutation(s) or transcriptional or translational control(s) in the host cell associated with the disease or disorder. Such a host cell can express a low density of target antigen throughout the stages of the formation and development of the disease or disorder, or only after a certain point in the stages of the formation and development of the disease or disorder. For example, some cancer or tumor cells can employ a strategy to reduce the expression of certain cancer / tumor-associated antigens in order to evade the host immune system. In such a scenario, typical CAR T or TCR therapy by recognizing cancer / tumor-associated antigens is not effective or sufficient to treat the disease or disorder. Overexpression of SLP-76 (e.g., membrane-bound SLP-76 constructs) can enhance the activation and function of CAR T or TCR-expressing T cells to treat the disease or disorder.

[0030] In another aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a medicamentously effective amount of T cells expressing a CAR or TCR polypeptide that specifically recognizes a target antigen expressed by a host cell associated with the disease or disorder, and further expressing an SLP-76 polypeptide (e.g., a membrane-bound SLP-76 polypeptide) as described herein. Such host cells express the target antigen at low levels (i.e., have low density), such that similar T cells that express only a CAR or TCR polypeptide, but not an SLP-76 polypeptide, treat the disease or disorder insufficiently or incapably.

[0031] In another aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a medicamentically effective amount of T cells expressing a CAR or TCR polypeptide that specifically recognizes a target antigen expressed by a host cell associated with the disease or disorder, and further expressing an SLP-76 polypeptide (e.g., a membrane-bound SLP-76 polypeptide) as described herein, while the disease or disorder is not treated, or is only inefficiently or insufficiently treated, by similar T cells expressing only a CAR or TCR polypeptide, but not an SLP-76 polypeptide as described herein. Such host cells express the target antigen at low levels (i.e., have low density), such that similar T cells expressing only a CAR or TCR polypeptide, but not an SLP-76 polypeptide as described herein, are insufficient or unable to treat the disease or disorder. In some embodiments, the method further comprises a first step of detecting the expression level of the target antigen(s) in the host cell associated with the disease or disorder. In some embodiments, the expression level of such target antigen(s) is known prior to treatment.

[0032] In some embodiments, the method includes a first step of detecting the expression level of the target antigen(s) in a host cell associated with a disease or disorder in a subject in need thereof, and a second step of treating the subject with T cells expressing a CAR or TCR polypeptide that specifically recognizes the target antigen(s) expressed by the host cell associated with the disease or disorder and further expressing an SLP-76 polypeptide (e.g., a membrane-bound SLP-76 polypeptide) described herein if such expression level is lower than a control level (e.g., expression level of the target antigen(s) in a healthy subject or in a host cell not associated with the disease or disorder). In some embodiments, prior to treatment, the host cell associated with the disease or disorder in the subject is known to express a low level of the target antigen(s) recognizable by the CAR or TCR molecule used in CAR T or TCR therapy. In some embodiments, such a low density of the target antigen(s) on the host cell results in failure of the treatment of the disease or disorder by CAR T or TCR therapy alone. In some embodiments, the low density of the target antigen(s) on such host cells results in ineffective or insufficient treatment of the disease or disorder by CAR T or TCR therapy alone.

[0033] In some embodiments, the method further comprises an optional step of comparing the measured / detected expression level of the target antigen with a control level (e.g., expression level of the target antigen(s) in a healthy subject or in a host cell not associated with the disease or disorder) between the measuring / detecting step and the treatment step described herein. In some embodiments, the method further comprises an optional step of overexpressing an SLP-76 polypeptide (e.g., a membrane-bound SLP-76 polypeptide) described herein in a CAR T or TCR-expressing T cell prior to the treatment step described herein. Such overexpression can be achieved by known methods (e.g., transduction, transfection, viral infection, etc.).

[0034] In another aspect, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising: i) detecting the expression level of a target antigen expressed by a host cell associated with a disease or disorder in a subject, wherein the target antigen can be recognized by a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide; and ii) if the expression level of the target antigen is lower than the control level, administering to the subject a medicamentously effective amount of T cells that express a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide and that express an SLP-76 polypeptide: Includes.

[0035] In some embodiments, the method further comprises the optional step of comparing the level of expression of the target antigen by the host cell to a control level prior to step ii).In some embodiments, the method further comprises the optional step of overexpressing an SLP-76 polypeptide in a T cell expressing a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide prior to step ii).

[0036] In some embodiments, the SLP-76 polypeptides described herein are membrane bound.

[0037] In some embodiments, T cells that express a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide, but do not express an SLP-76 polypeptide, are unable to treat the disease or disorder, or simply do so inefficiently or insufficiently.

[0038] In another aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a medicamentically effective amount of T cells (e.g., endogenous T cells in the subject) expressing a TCR polypeptide that specifically recognizes a target antigen expressed by a host cell associated with the disease or disorder, and further expressing an SLP-76 polypeptide (e.g., a membrane-bound SLP-76 polypeptide) as described herein, while the disease or disorder is not treated, or is simply inefficiently or insufficiently treated, by similar T cells that express only a TCR polypeptide but not an SLP-76 polypeptide as described herein. Such host cells express the target antigen at low levels (i.e., have low density), such that endogenous T cells that express only a TCR polypeptide but not an SLP-76 polypeptide as described herein are insufficient or unable to treat the disease or disorder. In some embodiments, the method further comprises a first step of detecting the expression level of the target antigen(s) in the host cell associated with the disease or disorder. In some embodiments, the expression level of such target antigen(s) is known prior to treatment. In some embodiments, the method further comprises isolating T cells (e.g., endogenous T cells in the subject) from the subject. In some embodiments, the method further comprises expressing an SLP-76 polypeptide as described herein in the isolated T cells (e.g., endogenous T cells). Such T cells may be any T cells that express a TCR molecule that recognizes a target antigen, including, for example, regulatory T cells (Tregs), tumor infiltrating lymphocytes (TILs), natural killer (NK) cells, macrophages, monocytes, gamma delta T cells, stem cells, natural killer T (NKT) cells, induced pluripotent stem cell (iPSC)-derived NK cells, induced pluripotent stem cell (iPSC)-derived T cells, or a mixture of such T cells. In some embodiments, these T cells (e.g., endogenous T cells in the subject) are tumor infiltrating lymphocytes (TILs). In some embodiments, these T cells are isolated from the subject to be treated for the disease or disorder.In some embodiments, these T cells are isolated from different subjects, e.g., different subjects having the same disease or disorder, or from healthy subjects. The SLP-76 polypeptides described herein may be introduced for expression in the isolated T cells (e.g., endogenous T cells in the subject) by any of the methods described herein or known to those of skill in the art (e.g., via viral vectors).

[0039] In another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: i) detecting in a subject the expression level of a target antigen expressed by a host cell associated with the disease or disorder, wherein the target antigen can be recognized by a T cell receptor (TCR) polypeptide expressed by a T cell in the subject; ii) isolating the T cells in step i) from the subject; and iii) expressing an SLP-76 polypeptide in the T cells isolated in step ii) if the expression level of the target antigen in step i) is lower than a control level; and administering to the subject a medicamentously effective amount of isolated T cells expressing an SLP-76 polypeptide: The present invention provides a method for treating a disease or disorder in a subject in need thereof, comprising:

[0040] In another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: i) isolating at least one T cell from a subject, wherein the at least one T cell expresses a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide, wherein the TCR polypeptide and / or the CAR polypeptide specifically recognizes a target antigen expressed by a host cell associated with the disease or disorder in the subject; ii) expressing an SLP-76 polypeptide in at least one isolated T cell; and iii) administering to the subject a medicamentously effective amount of isolated T cells expressing an SLP-76 polypeptide: The present invention provides a method for treating a disease or disorder in a subject in need thereof, comprising:

[0041] In some embodiments, the TCR polypeptide is endogenous to the subject. In some embodiments, at least one T cell expressing the TCR polypeptide and / or the CAR polypeptide is endogenous to the subject. In some embodiments, the TCR polypeptide, the CAR polypeptide, and / or at least one T cell expressing the TCR polypeptide and / or the CAR polypeptide are exogenous or genetically modified.

[0042] In some embodiments, the method further comprises, prior to step i), determining the level of expression of the target antigen by the host cell.

[0043] In some embodiments, the methods described herein further comprise the optional step, prior to step i), of comparing the level of expression of the target antigen by the host cell to a control level.

[0044] In some embodiments, the level of the target antigen expressed by the host cell is lower than the control level. Such a control level may refer to the expression level of the target antigen by different host cells that are not related to the disease or disorder (e.g., in the same or different tissues or organs of the same subject, or in the same or different tissues or organs of different hosts, either with the same disease or disorder or without the disease or disorder, such as healthy individuals). Such a control level may already be known to the physician or clinician before this treatment.

[0045] In some embodiments, an SLP-76 polypeptide described herein may be introduced for expression in an isolated T cell by any of the methods described herein or known to one of skill in the art (e.g., via a viral vector).

[0046] In some embodiments, the SLP-76 polypeptide is membrane bound.

[0047] In some embodiments, the T cells in the subject are regulatory T cells (Tregs), tumor infiltrating lymphocytes (TILs), natural killer (NK) cells, macrophages, monocytes, gamma delta T cells, stem cells, natural killer T (NKT) cells, induced pluripotent stem cell (iPSC)-derived NK cells, or induced pluripotent stem cell (iPSC)-derived T cells. In some embodiments, the T cells in the subject are tumor infiltrating lymphocytes (TILs).

[0048] In some embodiments, expression of an SLP-76 polypeptide enhances the activity of at least one T cell to inhibit or kill a host cell.

[0049] In some embodiments, the host cell is a cancer cell or a tumor cell.

[0050] Under non-limiting mechanisms, the SLP-76 polypeptides described herein can enhance the activity of another CAR or TCR molecule, either endogenous or exogenous, and / or can lower the activation threshold of cells expressing a CAR or TCR molecule in response to a target antigen, particularly when the antigen is expressed at low density. Expression of the SLP-76 polypeptides described herein can enhance current T cell therapies that use T cells expressing a CAR or TCR molecule.

[0051] The SLP-76 polypeptides described herein may include full-length SLP-76 or biologically active fragments, mutants, or variants of SLP-76. In some embodiments, the SLP-76 polypeptides include at least one mutation relative to the wild-type SLP-76 polypeptide. Such a mutation may be any mutation known to those skilled in the art, such as a mutation at the K30 position of wild-type SLP-76 (e.g., K30R substitution). Under non-limiting mechanisms, any mutation that does not decrease the expression level, stability, and / or biological function of SLP-76 may be used herein. Some mutations, such as K30R substitution, may enhance the expression level, stability, and / or biological function of SLP-76. SLP-76 mutants with such mutations may further enhance T cell activation upon binding to a target antigen, thereby further enhancing the therapeutic effect of CAR T or TCR therapy for a disease or disorder described herein. [Brief description of the drawings]

[0052] [Figure 1]FIG. 1 is a set of graphs showing T cell activity in response to tumor cells. Histograms of FACS analysis show expression of CD19-targeting CAR constructs ("CD19-CD8TM-BBZ", top left panel) or HER2-targeting CAR constructs ("HER-2-CD28TM-BBZ", top right panel) in T cells that also overexpress LCK (SEQ ID NO:9, top trace), LAT (SEQ ID NO:8, second trace from top), SLP-76 (SEQ ID NO:7, third trace from top), or a control without proximal signaling molecules (bottom trace). T cells expressing different CAR constructs with or without proximal signaling molecules were incubated with a control ("No Tumor"), wild type NALM6 cells expressing CD19 but not HER2 ("WT NALM6"), or NALM6 cells expressing HER2 but not CD19 ("HER2+NALM6"). The levels of IL-2 production by T cells were measured and compared (lower panel). The CAR constructs used in each incubation condition, from left to right, are CD19-CD8TM-BBZ, CD19-CD8TM-BBZ+SLP-76, CD19-CD8TM-BBZ+LAT, CD19-CD8TM-BBZ+LCK, HER2-CD28TM-BBZ, HER2-CD28TM-BBZ+SLP-76, HER2-CD28TM-BBZ+LAT, and HER2-CD28TM-BBZ+LCK. [Figure 1-1]FIG. 1 is a set of graphs showing T cell activity in response to tumor cells. Histograms of FACS analysis show expression of CD19-targeting CAR constructs ("CD19-CD8TM-BBZ", top left panel) or HER2-targeting CAR constructs ("HER-2-CD28TM-BBZ", top right panel) in T cells that also overexpress LCK (SEQ ID NO:9, top trace), LAT (SEQ ID NO:8, second trace from top), SLP-76 (SEQ ID NO:7, third trace from top), or a control without proximal signaling molecules (bottom trace). T cells expressing different CAR constructs with or without proximal signaling molecules were incubated with a control ("No Tumor"), wild type NALM6 cells expressing CD19 but not HER2 ("WT NALM6"), or NALM6 cells expressing HER2 but not CD19 ("HER2+NALM6"). The levels of IL-2 production by T cells were measured and compared (lower panel). The CAR constructs used in each incubation condition, from left to right, are CD19-CD8TM-BBZ, CD19-CD8TM-BBZ+SLP-76, CD19-CD8TM-BBZ+LAT, CD19-CD8TM-BBZ+LCK, HER2-CD28TM-BBZ, HER2-CD28TM-BBZ+SLP-76, HER2-CD28TM-BBZ+LAT, and HER2-CD28TM-BBZ+LCK. [Figure 1-2]FIG. 1 is a set of graphs showing T cell activity in response to tumor cells. Histograms of FACS analysis show expression of CD19-targeting CAR constructs ("CD19-CD8TM-BBZ", top left panel) or HER2-targeting CAR constructs ("HER-2-CD28TM-BBZ", top right panel) in T cells that also overexpress LCK (SEQ ID NO:9, top trace), LAT (SEQ ID NO:8, second trace from top), SLP-76 (SEQ ID NO:7, third trace from top), or a control without proximal signaling molecules (bottom trace). T cells expressing different CAR constructs with or without proximal signaling molecules were incubated with a control ("No Tumor"), wild type NALM6 cells expressing CD19 but not HER2 ("WT NALM6"), or NALM6 cells expressing HER2 but not CD19 ("HER2+NALM6"). The levels of IL-2 production by T cells were measured and compared (lower panel). The CAR constructs used in each incubation condition, from left to right, are CD19-CD8TM-BBZ, CD19-CD8TM-BBZ+SLP-76, CD19-CD8TM-BBZ+LAT, CD19-CD8TM-BBZ+LCK, HER2-CD28TM-BBZ, HER2-CD28TM-BBZ+SLP-76, HER2-CD28TM-BBZ+LAT, and HER2-CD28TM-BBZ+LCK.

[0053] [Diagram 2] Figure 2 is a bar graph of IL-2 production levels of T cells in response to control ("Alone"), wild-type NALM6 cells expressing high levels of CD19 ("WT N6"), or NALM6 cells expressing low levels of CD19 ("CD19 low N6"). These T cells also express the same CD19-targeting CAR construct as in Figure 1 ("CD19-CD8TM-BBZ") with or without SLP-76. The CAR constructs used in each incubation condition, from left to right, are CD19-CD8TM-BBZ, and CD19-CD8TM-BBZ+SLP-76.

[0054] [Figure 3A] Figures 3A-3C are a set of graphs showing T cell activity in response to tumor cells. Figure 3A shows FACS histograms detecting expression of a CD19-targeting CAR construct ("CD19-CD8TM-BBZ") on T cells, which are also overexpressing LCK (SEQ ID NO:9; top trace), a membrane-bound LCK construct (SEQ ID NO:17; second trace from the top), SLP-76 (SEQ ID NO:7; third trace from the top), membrane-bound SLP-76 (SEQ ID NO:16; fourth trace from the top), or a control (bottom trace). [Figure 3B] Figure 3B is a bar graph comparing the levels of IL-2 production by these T cells in response to control ("Alone"), wild-type NALM6 cells ("WT N6"), or NALM6 cells expressing low levels of CD19 ("CD19 low N6"). The CAR constructs used in each incubation condition, from left to right, are CD19-CD8TM-BBZ, CD19-CD8TM-BBZ+SLP-76, CD19-CD8TM-BBZ+membrane-bound SLP-76, CD19-CD8TM-BBZ+membrane-bound LCK, and CD19-CD8TM-BBZ+LCK. [Figure 3C] Figure 3C is a graph comparing IL-2 production levels of T cells expressing a CD19-targeting CAR construct ("CD19-CD8TM-BBZ") with or without membrane-bound SLP-76. For the experiment in Figure 3C, anti-idiotypic antibodies that specifically recognize the extracellular domain of the CD19-targeting CAR were coated onto plates in different amounts (x-axis) by serial dilution. T cells were incubated with each amount of coated antibody and the corresponding IL-2 production levels were measured to indicate the degree of T cell activation.

[0055] [Figure 4A]

[0023] Figures 4A-4C are a set of graphs showing T cell activity in response to tumor cells, similar to Figures 3A-3C. Figure 4A shows FACS histograms detecting expression of a HER2-targeting CAR construct ("HER-2-CD28TM-BBZ") on T cells, which are also overexpressing LCK (SEQ ID NO:9; top trace), a membrane-bound LCK construct (SEQ ID NO:17; second trace from the top), SLP-76 (SEQ ID NO:7; third trace from the top), membrane-bound SLP-76 (SEQ ID NO:16; fourth trace from the top), or a control (bottom trace). [Figure 4B] FIG. 4B is a bar graph comparing the IL-2 production levels of these T cells in response to control ("No Tumor"), NALM6 cells expressing very low levels of HER2 ("Very Low HER2+N6"), NALM6 cells expressing low levels of HER2 ("Low HER2+N6"), NALM6 cells expressing high levels of HER2 ("High HER2+N6"), or wild-type 143B cells (which express low levels of HER2). The CAR constructs used in each incubation condition, from left to right, are HER2-CD28TM-BBZ, HER2-CD28TM-BBZ+SLP-76, HER2-CD28TM-BBZ+membrane-bound SLP-76, HER2-CD28TM-BBZ+LCK, and HER2-CD28TM-BBZ+membrane-bound LCK. [Figure 4C] Figure 4C is a graph comparing the IL-2 production levels of T cells expressing the same HER2-targeting CAR construct with or without overexpression of membrane-bound SLP-76. Recombinant Her-2 antigen was coated onto the plate in different amounts (x-axis) by serial dilution. T cells were incubated with each amount of coated recombinant antigen, and the corresponding IL-2 production levels were measured to indicate the degree of T cell activation.

[0056] [Figure 5A]Figures 5A-5F are a set of graphs showing the ability of membrane-bound SLP-76 (SEQ ID NO: 16) to enhance CAR T cell activity in response to tumor cells. Figure 5A shows FACS histograms detecting the expression of a CD19-targeting CAR construct ("CD19-CD28TM-CD28Z", left panel) and membrane-bound SLP-76 (through detection of a small extracellular tag conjugated to membrane-bound SLP-76) (right panel) on T cells. [Figure 5B] Figure 5B is a bar graph comparing the levels of IL-2 production by these T cells in response to control ("Alone"), wild-type NALM6 cells ("WT N6"), or NALM6 cells expressing low levels of CD19 ("CD19 low N6"). The CAR constructs used in each incubation condition, from left to right, are mock, mock + membrane-bound SLP-76, CD19-CD28TM-CD28Z, and CD19-CD28TM-CD28Z + membrane-bound SLP-76. [Figure 5C] Figure 5C is a graph comparing the cytotoxicity of these T cells against CD19 low N6 cells over time. Specifically, T cells expressing CD19-targeting CAR constructs with or without membrane-bound SLP-76 were incubated with CD19 low N6 cells expressing GFP, and the number of remaining N6 cells was counted based on their GFP expression after 3 to 72 hours of incubation. The cytotoxicity index (% GFP signal normalized to baseline measurements) was calculated, which reflected the ability of T cells to inhibit or kill target cells. [Figure 5D] Figure 5D shows the effect of membrane-bound SLP-76 in an in vivo NALM6 stress test model. Specifically, T cells expressing CD19-targeting CAR constructs with or without membrane-bound SLP-76 were injected at sub-curative doses into animals bearing wild-type NALM-6 tumors. Tumor burden (shown as total flux as the y-axis) was measured and compared at various time points after treatment. [Figure 5E]Figure 5E shows survival of mice from the experiment described in Figure 5D. Lines represent, from left to right, survival of mice expressing mock+membrane-bound SLP-76, CD19 28Z, or CD19 28Z+membrane-bound SLP-76. [Figure 5F] FIG. 5F shows the results of FIG. 5D after correction for miscalculated data points (excluding mock+membrane SLP-76 treatment).

[0057] [Figure 6A] Figures 6A-6F are a set of graphs showing the ability of membrane-bound SLP-76 to enhance CAR T cell activity in response to tumor cells. Figure 6A shows FACS histograms detecting the expression of a CD22-targeting CAR construct ("CD22-CD8TM-BBZ", left panel) and membrane-bound SLP-76 (through detection of a small extracellular tag conjugated to membrane-bound SLP-76) (right panel) on T cells. [Figure 6B] Figure 6B is a bar graph comparing IL-2 production levels of these T cells in response to control ("Alone"), or NALM6 cells expressing low levels of CD22 ("CD22 low N6"), or mock controls. The CAR constructs used in each incubation condition, from left to right, are mock, mock + membrane-bound SLP-76, CD22-CD8TM-BBZ, and CD22-CD8TM-BBZ + membrane-bound SLP-76. [Figure 6C] Figure 6C is a graph comparing IL-2 production levels of T cells expressing a CD22-targeting CAR construct ("CD22-CD8TM-BBZ") with or without membrane-bound SLP-76. Recombinant CD22 was coated onto plates in different amounts (x-axis) by serial dilution. T cells were incubated with each amount of coated CD22 and the production levels of IL-2 were measured to indicate T cell activation. [Figure 6D]Figures 6D-6F show the effect of membrane-bound SLP-76 in an in vivo CD22 low NALM6 (cells expressing low density CD22) model. Specifically, T cells expressing CD22-targeting CAR constructs with or without overexpression of membrane-bound SLP-76 were injected into animals bearing CD22 low NALM-6 tumors. Tumor burden (shown as total flux as the y-axis) was measured and compared 22 days after treatment (Figure 6D). [Figure 6E] In a separate experiment, tumor burden (shown as total luminescence as the y-axis) was measured and compared at different time points after treatment (FIG. 6E). [Figure 6F] FIG. 6F shows mouse survival from the experiment described in FIG. 6E.

[0058] [Figure 7A] Figures 7A-7D are a set of graphs showing the ability of membrane-bound SLP-76 to enhance CAR T cell activity in response to tumor cells. Figure 7A shows FACS histograms detecting the expression of a B7-H3-targeting CAR construct ("B7-H3-CD28TM-BBZ", left panel) and membrane-bound SLP-76 (through detection of a small extracellular tag conjugated to membrane-bound SLP-76) (right panel) on T cells. [Figure 7B] Figure 7B is a graph comparing the cytotoxicity of these CAR T cells against the neuroblastoma cell line CHLA-255, which expresses low density B7-H3. Specifically, T cells expressing B7-H3-CD28TM-BBZ with or without overexpression of membrane-bound SLP-76 were incubated with CHLA-255 cells expressing GFP. The number of remaining CHLA-255 cells was counted 3 to 78 hours after incubation based on their expression levels of GFP. The cytotoxicity index was calculated, which reflected the ability of T cells to inhibit or kill target cells. [Figure 7C]Figure 7C shows tumor burden in an in vivo CHLA-255 (which expresses low density B7-H3) metastasis model. T cells expressing B7-H3-CD28TM-BBZ with or without overexpression of membrane-bound SLP-76 were injected into animals bearing CHLA-255 tumors. Tumor burden (shown as total flux as the y-axis) was measured at different time points after treatment. [Figure 7D] FIG. 7D compares animal survival after each treatment at different time points.

[0059] [Figure 8A] Figures 8A-8D are a set of graphs showing the ability of membrane-bound SLP-76 to enhance CAR T cell activity in response to tumor cells. Figure 8A shows FACS histograms detecting expression on T cells of a CD19-targeting CAR construct (containing a ZAP70 signaling domain) containing a CD3-zeta signaling domain ("CD19-CD28TM-ZAP70", left panel) and a ZAP70255-600 fragment, but not membrane-bound SLP-76 (through detection of a small extracellular tag conjugated to membrane-bound SLP-76) (right panel). [Figure 8B] FIG. 8B compares the levels of IL-2 production by these T cells in response to control ("tumor-free") or wild-type NALM6 cells, or mock controls. [Figure 8C] Figure 8C shows tumor burden in an in vivo NALM6 xenograft model. T cells expressing CD19-CD28TM-ZAP70 with or without overexpression of membrane-bound SLP-76 were injected into animals bearing NALM6 tumors. Tumor burden (shown as total flux as the y-axis) was measured and compared at different time points after treatment. [Figure 8D] FIG. 8D compares animal survival after each treatment at different time points.

[0060] [Figure 9A]Figures 9A-9B are a set of graphs showing the ability of membrane-bound SLP-76 to enhance CAR T cell activity in response to tumor cells. Figure 9A shows FACS histograms detecting the expression on T cells of a high affinity (HA) GD2-targeting CAR construct ("HA 28Z", left panel) as well as two membrane-bound SLP-76 constructs (through detection of an illustrative anti-HER2 ECD conjugated to the membrane-bound SLP-76 construct), each containing a different hinge-transmembrane domain (CD8 vs. CD28) (right panel). [Figure 9B] Figure 9B compares the IL-2 production levels (after baseline subtraction) of these T cells in response to GD2-expressing NALM6 cells ("GD2 N6"), wild-type 143B cells, or CHLA-255 cells. The CAR constructs used in each incubation condition, from left to right, are HA-28Z, HA-28Z+CD8TM-SLP-76, and HA-28Z+CD28TM-SLP-76.

[0061] [Figure 10] Figure 10 is a set of graphs showing the expression of cell exhaustion biomarkers on CAR T cells. FACS analysis was performed to detect the expression of TIM-3 (left panel), PD-1 (middle panel), and LAG-3 (right panel) on T cells expressing the B7-H3-CD28TM-BBZ CAR construct with or without overexpression of membrane-bound SLP-76. [Figure 10-1] Figure 10 is a set of graphs showing the expression of cell exhaustion biomarkers on CAR T cells. FACS analysis was performed to detect the expression of TIM-3 (left panel), PD-1 (middle panel), and LAG-3 (right panel) on T cells expressing the B7-H3-CD28TM-BBZ CAR construct with or without overexpression of membrane-bound SLP-76. [Figure 10-2]Figure 10 is a set of graphs showing the expression of cell exhaustion biomarkers on CAR T cells. FACS analysis was performed to detect the expression of TIM-3 (left panel), PD-1 (middle panel), and LAG-3 (right panel) on T cells expressing the B7-H3-CD28TM-BBZ CAR construct with or without overexpression of membrane-bound SLP-76.

[0062] [Figure 11] Figure 11 is a set of graphs showing the expression of cell exhaustion biomarkers on CAR T cells. FACS analysis was performed to detect the expression of TIM-3 (left panel), PD-1 (middle panel), and LAG-3 (right panel) on T cells expressing the HA 28Z CAR construct with or without overexpression of membrane-bound SLP-76. [Figure 11-1] Figure 11 is a set of graphs showing the expression of cell exhaustion biomarkers on CAR T cells. FACS analysis was performed to detect the expression of TIM-3 (left panel), PD-1 (middle panel), and LAG-3 (right panel) on T cells expressing the HA 28Z CAR construct with or without overexpression of membrane-bound SLP-76. [Figure 11-2] Figure 11 is a set of graphs showing the expression of cell exhaustion biomarkers on CAR T cells. FACS analysis was performed to detect the expression of TIM-3 (left panel), PD-1 (middle panel), and LAG-3 (right panel) on T cells expressing the HA 28Z CAR construct with or without overexpression of membrane-bound SLP-76.

[0063] [Figure 12A]Figures 12A-12B are a set of graphs comparing the ability of membrane-bound SLP-76 versus LAT / SLP-76 chimera (transmembrane LAT fused to SLP-76) to enhance CAR T cell activity in response to tumor cells. Figure 12A shows FACS histograms detecting expression of CD19 28Z CAR construct (left panel), overexpressing membrane-bound SLP-76 construct (middle panel, right trace), or LAT / SLP-76 chimeric construct (right panel, right trace) in T cells. In the middle and right panels, the left trace represents the control without overexpression of any SLP-76 construct. [Figure 12A-1] Figures 12A-12B are a set of graphs comparing the ability of membrane-bound SLP-76 versus LAT / SLP-76 chimera (transmembrane LAT fused to SLP-76) to enhance CAR T cell activity in response to tumor cells. Figure 12A shows FACS histograms detecting expression of CD19 28Z CAR construct (left panel), overexpressing membrane-bound SLP-76 construct (middle panel, right trace), or LAT / SLP-76 chimeric construct (right panel, right trace) in T cells. In the middle and right panels, the left trace represents the control without overexpression of any SLP-76 construct. [Figure 12A-2] Figures 12A-12B are a set of graphs comparing the ability of membrane-bound SLP-76 versus LAT / SLP-76 chimera (transmembrane LAT fused to SLP-76) to enhance CAR T cell activity in response to tumor cells. Figure 12A shows FACS histograms detecting expression of CD19 28Z CAR construct (left panel), overexpressing membrane-bound SLP-76 construct (middle panel, right trace), or LAT / SLP-76 chimeric construct (right panel, right trace) in T cells. In the middle and right panels, the left trace represents the control without overexpression of any SLP-76 construct. [Figure 12B]Figure 12B compares the levels of IL-2 production by these T cells in response to control ("Alone"), wild-type NALM6 cells expressing high antigen (i.e., CD19) density ("WT N6"), or either of two clones of NALM6 cells expressing low antigen density ("Clone F N6" and "Clone Z N6"). The CAR constructs used in each incubation condition, from left to right, are mock, mock+LAT / SLP-76 chimera, mock+membrane-bound SLP-76, CD19-28Z, CD19-28Z+LAT / SLP-76 chimera, and CD19-28Z+membrane-bound SLP-76.

[0064] [Figure 13A] Figures 13A-13B are graphs showing the ability of membrane-bound SLP-76 (SEQ ID NO: 16) to enhance CAR T cell activity in response to tumor cells. Figure 13A includes histograms of FACS analysis showing expression of a CD19-targeting CAR construct ("CD19-CD8TM-BBZ") in T cells. These T cells were further transduced to overexpress a membrane-bound SLP-76 construct (top trace), a membrane-bound PLCG construct (second trace from the top; SEQ ID NO: 50), a membrane-bound ZAP-70255-600 construct (third trace from the top; SEQ ID NO: 46), a free PLCG construct (fourth trace from the top; SEQ ID NO: 48), a free ZAP-70 construct (fifth trace from the top; SEQ ID NO: 44), or a mock control (bottom trace). [Figure 13B] Figure 13B includes a set of graphs showing cytokine production (IL-2 in the upper panels and IFNγ in the lower panels) by CD19-CD8TM-BBZ CAR T cells overexpressing, from left to right, mock control, ZAP-70, PLCg, membrane-bound ZAP-70255-600, membrane-bound PLCg, or membrane-bound SLP-76 when co-cultured with tumor cells (wild-type NALM-6).

[0065] [Figure 14A]Figures 14A-14E are graphs showing the ability of membrane-bound SLP-76 (SEQ ID NO: 16) to enhance CAR T cell activity in response to tumor cells expressing different levels of an antigen recognizable by the CAR molecule. Figure 14A includes histograms of FACS analysis showing expression of an anaplastic lymphoma kinase (ALK)-targeting CAR construct ("ALK-CD8TM-BBZ") in T cells. These T cells were further transduced to overexpress the membrane-bound SLP-76 construct (top trace) or a mock control (second trace from the top). The bottom trace refers to control T cells that do not express the CAR construct or the membrane-bound SLP-76 construct. [Figure 14B] FIG. 14B contains histograms of FACS analysis showing expression (right trace) of membrane-bound SLP-76 (by detection of its surface markers described herein) in T cells. [Figure 14C] Figure 14C includes histograms of FACS analysis showing expression levels of ALK in different Nalm-6 cells, including cells with high levels of ALK expression (ALKhigh, top trace), cells with medium levels of ALK expression (ALKmed, second trace from the top), cells with low levels of ALK expression (ALKlow, third trace from the top), or cells that do not express ALK (ALK-, bottom trace). [Figure 14D] Figure 14D includes graphs showing cytokine (IL-2 in upper panel, IFNγ in lower panel) production by ALK-CD8TM-BBZ CAR T cells with or without membrane-bound SLP-76 overexpression when co-cultured with tumor cells at different antigen densities. [Figure 14E] Figure 14E includes graphs showing the killing of ALK Nalm-6 cells with different levels of ALK expression by ALK-CD8TM-BBZ CAR T cells without (upper line in each panel) or with (lower line in each panel) membrane-bound SLP-76 overexpression. The cell number ratio between NALM-6 cells and T cells is indicated at the top of each panel.

[0066] [Figure 15A]Figures 15A-15C are graphs showing the ability of membrane-bound SLP-76 (SEQ ID NO: 16) to enhance T cell activity in response to tumor cells. Figure 15A includes histograms of FACS analysis showing expression of NY-ESO-1 TCR construct (left panel) or membrane-bound SLP-76 construct (detected by its surface markers as described herein; right panel) in T cells. T cells tested were transduced to overexpress the TCR construct and membrane-bound SLP-76 construct (top trace), TCR construct only (second trace from the top), mock control and membrane-bound SLP-76 construct (third trace from the top), or mock control only (bottom trace). [Figure 15B] FIG. 15B includes a set of graphs showing IL-2 production by NY-ESO-1 TCR T cells when co-cultured with antigen-positive tumor cells (A2+ / NY-ESO-1+Nalm-6 cells, upper panel, or A375 cells, lower panel) or antigen-negative tumor cells Mel888 (lower panel). [Figure 15C] FIG. 15C illustrates the killing of A2+ / NY-ESO-1+Nalm-6 cells by NY-ESO-1 TCR T cells with or without overexpression of membrane-bound SLP-76, as represented by the cytotoxicity index (y-axis) over a time period following co-culture (x-axis, hours).

[0067] [Figure 16A] Figures 16A-16C are graphs showing the ability of membrane-bound SLP-76 (SEQ ID NO: 16) to enhance T cell activity in response to tumor cells. Figure 16A includes histograms of FACS analysis showing expression of B-cell maturation antigen (BCMA)-targeting CAR constructs (left panel) or membrane-bound SLP-76 constructs (by detecting its surface marker VSV-G as described herein; right panel) in T cells. Tested T cells were transduced to overexpress the CAR construct and membrane-bound SLP-76 construct (top trace), the CAR construct only (second trace from the top), or a mock control (bottom trace). [Figure 16B]Figure 16B includes a set of graphs comparing the production of IL-2 (upper panel) or IFNγ (lower panel) by T cells expressing both the CAR construct and the membrane-bound SLP-76 construct, or the CAR construct alone, in response to MM1.S tumor cells. [Figure 16C] FIG. 16C compares in vivo tumor control by these T cells with or without membrane-bound SLP-76 overexpression in mice inoculated with luciferase-expressing MM1.S tumor cells and treated with these T cells.

[0068] [Figure 17A] Figures 17A-17B are graphs showing the ability of membrane-bound SLP-76 (SEQ ID NO: 16) to enhance T cell activity in response to tumor cells. Figure 17A includes histograms of FACS analysis showing expression of a B7-H3-targeting CAR construct ("B7-H3-CD28TM-BBZ") in T cells. T cells tested were transduced to overexpress the CAR construct and the membrane-bound SLP-76 construct (top trace), the CAR construct only (second trace from the top), or a mock control (bottom trace). [Figure 17B] FIG. 17B compares in vivo tumor control by these T cells with or without membrane-bound SLP-76 overexpression in mice inoculated with luciferase-expressing diffuse pontine glioma 6 xenografts (DIPG-6).

[0069] [Figure 18A]Figures 18A-18B are graphs showing the ability of membrane-bound SLP-76 (SEQ ID NO: 16) to enhance T cell proliferation in response to tumor cells. Figure 18A includes histograms of FACS analysis showing expression of a CD19-targeting CAR construct ("CD19-CD28TM-CD28Z"; left panel) or a membrane-bound SLP-76 construct (as detected by its surface marker VSV-G as described herein; right panel) in T cells. Tested T cells were transduced to overexpress both the CAR construct and the membrane-bound SLP-76 construct (top trace), the CAR construct only (second trace from the top), or a mock control (bottom trace). [Figure 18B] Figure 18B compares the amount of CD19-CD28TM-CD28Z CAR T cells with or without membrane-bound SLP-76 overexpression harvested from the spleen (top) or bone marrow (bottom) of mice inoculated with luciferase-expressing WT Nalm-6 at different time points.

[0070] [Figure 19A] Figures 19A-19C are graphs showing the ability of membrane-bound SLP-76 (SEQ ID NO: 16) to enhance T cell proliferation in response to tumor cells. Figure 19A includes histograms of FACS analysis showing expression of a CD19-targeting CAR construct ("CD19-CD28TM-BBZ"; left panel) or a membrane-bound SLP-76 construct (as detected by its surface marker VSV-G as described herein; right panel) in T cells. Tested T cells were transduced to overexpress both the CAR construct and the membrane-bound SLP-76 construct (top trace), or the CAR construct alone (bottom trace). [Figure 19B] FIG. 19B compares in vivo tumor control by these T cells with or without membrane-bound SLP-76 overexpression in mice inoculated with luciferase-expressing leukemia xenografts (WT Nalm-6). [Figure 19C] FIG. 19C compares survival of mice after each treatment over time periods.

[0071] [Figure 20A] Figures 20A-20C are graphs showing the ability of membrane-bound SLP-76 mutant (SEQ ID NO:60) to enhance T cell activation in response to tumor cells. Figures 20A-20B include histograms of FACS analysis showing the expression of a CD19-targeting CAR construct ("CD19-CD28TM-BBZ"; Figure 20A) or various SLP-76 constructs (as detected by its surface marker VSV-G as described herein; Figure 20B) in T cells. Tested T cells were transduced to overexpress both the CAR construct and the membrane-bound SLP-76 K30R mutant construct (top trace), the membrane-bound SLP-76 (wild type) construct (middle trace), or the CAR construct alone (bottom trace). [Figure 20B] Figures 20A-20B include histograms of FACS analyses showing expression of a CD19-targeting CAR construct ("CD19-CD28TM-BBZ"; Figure 20A) or various SLP-76 constructs (as detected by its surface marker VSV-G as described herein; Figure 20B) in T cells. Tested T cells were transduced to overexpress both the CAR construct and the membrane-bound SLP-76 K30R mutant construct (top trace), the membrane-bound SLP-76 (wild-type) construct (middle trace), or the CAR construct alone (bottom trace). [Figure 20C] Figure 20C compares IL-2 expression levels by these T cells when co-cultured with NALM-6 cells expressing low levels of CD19 or with a control ("non-tumor"). The CAR constructs used in each incubation condition, from left to right, were CD19BBZ CAR (i.e., CD19-CD28TM-BBZ) only, CD19BBZ CAR + membrane-bound SLP-76, and CD19BBZ CAR + membrane-bound SLP-76 K30R mutant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] Detailed Description of the Disclosure The present disclosure generally relates to compositions of polypeptides, e.g., SLP-76, and polynucleotides encoding the polypeptides, that can enhance activation and function of cells (e.g., immune cells expressing a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), e.g., T cells) in response to a target antigen, such as to target, inhibit, and / or eliminate specific target cells (e.g., cancer or tumor cells) that express the target antigen. Also provided herein are compositions of recombinant cells comprising the polypeptides and methods useful for making such recombinant cells, as well as methods of enhancing cellular (e.g., immune cell) activity to treat conditions such as disease (e.g., proliferative diseases (such as cancer), hematological malignancies, solid tumors, autoimmune diseases, inflammation, allergic diseases, infectious diseases, senescence / aging, etc.).

[0073] Illustrative experimental results presented herein demonstrate that SLP-76 can enhance cell (e.g., CAR T cell) activation, resulting in increased T cell effector function and anti-tumor efficacy (e.g., increased cytokine production and cytotoxicity). Furthermore, SLP-76, such as membrane-bound SLP-76, has been shown to be capable of enhancing CAR T cell activity in response to low antigen density, resulting in increased T cell effector function and anti-tumor efficacy (e.g., increased cytokine production and cytotoxicity). Thus, SLP-76 can lower the T cell activation threshold, improve TCR or CAR function in immune cells, and improve immune cell function by enhancing proximal signaling. Domain swapping has been used to engineer various CAR molecules or SLP-76 constructs to confirm that such capabilities of SLP-76 are independent of CAR specificity or the structure of SLP-76 (e.g., not dependent on the transmembrane domain used to tether SLP-76 to the membrane). Other advantages of SLP-76, e.g., membrane-bound SLP-76, include not exacerbating cell exhaustion, e.g., in immune cells (e.g., T cells), e.g., cells expressing CAR, TCR, or other molecules that cause tonic signaling. In particular, SLP-76 molecules can significantly enhance the activation of CAR T cells, which otherwise exhibit characteristics of T cell exhaustion. While SLP-76, e.g., membrane-bound SLP-76, can promote CAR, TCR, or other receptor function or other immune cell function in most situations, it is particularly advantageous when the target antigen is expressed at low density, when the CAR, TCR, or other receptor construct has low affinity for the target antigen, when the CAR, TCR, or other receptor is expressed at low density, and / or when the CAR, TCR, or other receptor construct induces cell exhaustion.

[0074] Nucleic acid molecules encoding these polypeptides and recombinant cells expressing these polypeptides are also provided. The disclosure also provides compositions and methods useful for making such recombinant cells containing SLP-76 polypeptides, as well as methods for enhancing cell activation and preventing and / or treating conditions such as proliferative diseases (e.g., cancer), hematological malignancies, solid tumors, autoimmune diseases, inflammation, allergic diseases, infectious diseases, senescence / aging, and the like.

[0075] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0076] General Experimental Procedures The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those of skill in the art. Such techniques are described in Sambrook, J., and Russell, DW (2012), Molecular Cloning: A Laboratory Manual (4th ed.), Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, and Sambrook, J. and Russell, DW (2001), Molecular Cloning: A Laboratory Manual (3rd ed.), Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as "Sambrook"); Ausubel, FM (1987), Current Protocols in Molecular Biology, New York, NY: Wiley (with supplements through 2014); Bollag, DM et al., (1996), Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al., (2005), Nonviral Vectors for Gene Therapy, San Diego: Academic Press; Kaplitt, MG et al., (1995), Viral Vectors: Gene Therapy and Neuroscience Applications, San Diego, CA: Academic Press; Lefkovits, I., (1997), The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, San Diego, CA: Academic Press; Doyle, A. et al., (1998), Cell and Tissue Culture: Laboratory Procedures in Biotechnology, New York, NY: Wiley; Mullis, K.B., Ferre, F. and Gibbs, R.(1994), PCR: The Polymerase Chain Reaction, Boston: Birkhauser Publisher; Greenfield, EA, (2014), Antibodies: A Laboratory Manual (2nd ed.), New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al., (2000), Current Protocols in Nucleic Acid Chemistry, New York, NY: Wiley, (including supplements up to 2014); and Makrides, SC (2003), Gene Transfer and Expression in Mammalian Cells, Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference. Where appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to manufacturer-defined protocols and / or parameters unless otherwise noted.

[0077] definition Unless otherwise specified, all technical terms, notes, and other scientific or technical terms used herein are intended to have the meanings commonly understood by those skilled in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein is not necessarily to be construed as representing a substantial difference beyond that commonly understood in the art. Many of the techniques and procedures described or referred to herein are well understood and commonly employed by those skilled in the art using conventional methodology.

[0078] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all of the following options: "A," "B," "A or B," and "A and B."

[0079] The term "about" as used herein has its usual meaning of approximately. If the degree of approximation is not otherwise clear from the context, "about" means either within plus or minus 10% of the value provided, or rounded to the nearest meaningful figure, inclusive of the provided value in all cases. When ranges are provided, these include the boundaries.

[0080] The term "derived from" refers to the origin or source of a molecule and may include natural, recombinant, unpurified, or purified molecules. Nucleic acid or polypeptide molecules are considered to be "derived from" when they contain parts or elements that are assembled in a manner that results in a functional unit. These parts or elements may be assembled from multiple sources so long as they retain their evolutionarily conserved function. In some embodiments, these derived nucleic acid or polypeptide molecules contain substantially the same sequence as the source nucleic acid or polypeptide molecule. For example, the derived nucleic acid or polypeptide molecules of the present disclosure may have at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the source nucleic acid or polypeptide molecule.

[0081] The term "recombinant" nucleic acid molecules, polypeptides, and / or cells as used herein refers to nucleic acid molecules, polypeptides, and / or cells that have been altered through human intervention. In non-limiting examples, recombinant nucleic acid molecules can be: 1) synthesized or modified in vitro, for example, using chemical or enzymatic techniques, or recombination of nucleic acid molecules; 2) containing linked nucleotide sequences that are not linked in nature; 3) genetically engineered, using molecular cloning techniques, to lack one or more nucleotides relative to the naturally occurring nucleic acid molecule sequence; and / or 4) engineered, using molecular cloning techniques, to have one or more sequence changes or rearrangements relative to the naturally occurring nucleic acid sequence. Non-limiting examples of recombinant proteins are the SLP-76 polypeptides, chimeric antigen receptors (CARs), T cell receptors (TCRs), or other receptors provided herein.

[0082] The terms "cell," "cell culture," and "cell line" refer not only to a particular subject cell, cell culture, or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, regardless of the number of transplants or passages in culture. It should be understood that all progeny are not exactly identical to the parent cell. This is because certain modifications may occur in subsequent generations, either due to mutations (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that the progeny are not in fact identical to the parent cell, but are still within the scope of the term as used herein, so long as the progeny retain the same functionality as the original cell, cell culture, or cell line.

[0083] As used herein, a "host cell" refers to a cell for introducing a nucleic acid and / or a polypeptide (e.g., SLP-76, CAR, and / or TCR molecule) described herein and / or for expressing a nucleic acid or polypeptide described herein. A host cell can be either an untransformed cell or a cell into which at least one nucleic acid molecule (e.g., SLP-76, CAR, and / or TCR molecule) described herein has already been introduced. A "recombinant cell" refers to a cell that has a genetic modification and / or has an introduced nucleic acid and / or polypeptide described herein.

[0084] As used herein, a "subject" or "individual" includes animals, such as humans (e.g., human subjects) and non-human animals. In some embodiments, a "subject" or "individual" is a patient under the care of a physician. Thus, a subject can be a human patient or individual who has, is at risk of, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. A subject can also be an individual who has been diagnosed at or after diagnosis as being at risk for a condition of interest. The term "non-human animals" includes all vertebrates, such as mammals, such as rodents, such as mice, and non-mammals, such as non-human primates, such as sheep, dogs, cows, poultry, amphibians, reptiles, and the like.

[0085] The term "vector" is used herein to refer to a nucleic acid molecule or sequence that can transfer or transport another nucleic acid molecule. For example, a vector can be used as a gene delivery vehicle to transfer a gene into a cell. The transferred nucleic acid molecule is generally linked, e.g., inserted, into the vector nucleic acid molecule. Generally, a vector is capable of replication when associated with appropriate control elements. The term "vector" includes cloning vectors and expression vectors, as well as viral vectors and integrative vectors. An "expression vector" is a vector that includes a control region, which allows DNA sequences and fragments to be expressed in vitro and / or in vivo. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses. In some embodiments, the vector is a gene delivery vector.

[0086] Aspects and embodiments of the disclosure described herein are understood to include "comprising," "consisting," and "consisting essentially of" aspects and embodiments. As used herein, "comprising" is synonymous with "including," "comprising," or "characterized by," and is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps. As used herein, "consisting" excludes any element, step, or ingredient not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic characteristics of the claimed composition or method. Recitation herein of the term "comprising," particularly in the description of a component of a composition or in the description of a step of a method, is understood to include those compositions and methods that consist essentially of and consist of the recited components or steps.

[0087] For example, headings such as (a), (b), (i), etc. are provided solely for ease of reading of the specification and claims. The use of headings in the specification and claims does not require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0088] As would be understood by one of ordinary skill in the art, for any and all purposes, including with respect to providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any recited range can be readily recognized as fully describing the same range, as well as allowing for subdivision into at least equal, half, third, quarter, fifth, tenth, etc., of the same range. As a non-limiting example, each range discussed herein can be readily subdivided into a lower third, middle third, upper third, etc. As would also be understood by one of ordinary skill in the art, all phrases such as "up to," "at least," "greater than," "less than," and the like, refer to ranges that include the recited numerical values ​​and that can be substantially subdivided into subranges as discussed above. Finally, as would be understood by one of ordinary skill in the art, a range includes each of the individual numerical values. Thus, for example, a group having 1 to 3 articles refers to a group having 1, 2, or 3 articles. Similarly, a group having 1-5 items refers to groups having 1, 2, 3, 4 or 5 items, and so on.

[0089] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact numerical value it precedes, as well as numerical values ​​that are approximations or estimates of the numerical value preceded by the term. In determining whether a numerical value is an approximation or estimate of a specifically recited numerical value, the approximation or estimate unrecited numerical value may be a numerical value that, in the context in which it is presented, provides a substantial equivalent to the specifically recited numerical value.

[0090] It is understood that certain features of the present disclosure that are described for clarity in the context of a single embodiment may also be provided in combination in a single embodiment. In contrast, various features of the present disclosure that are described for brevity in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of the embodiments related to the present disclosure are specifically embraced by the present disclosure and are disclosed herein exactly as if each and every combination were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein exactly as if each and every such subcombination were individually and expressly disclosed.

[0091] Compositions of the Disclosure As described in more detail below, the present disclosure provides, inter alia, compositions of SLP-76 polypeptides, either cell membrane bound or unbound, that are capable of enhancing cell (e.g., immune cell such as T cells) activation. This cell activation can be induced by a natural / endogenous or recombinant T cell receptor (TCR), a recombinant chimeric antigen receptor (CAR), or another natural / endogenous or recombinant receptor that is expressed on the surface of a cell and specifically binds to a target antigen expressed by a target cell (e.g., a cancer cell or tumor cell). For example, a CAR can specifically bind to a target antigen expressed on the surface of a target cell, whereas a TCR can specifically bind to a target antigen (expressed by a target cell) presented by the major histocompatibility complex (MHC) on the surface of an antigen-presenting cell (APC). Furthermore, the SLP-76 polypeptides described herein can lower the T cell activation threshold by enhancing activation in response to low antigen density (e.g., through a TCR or CAR). In some embodiments, the present disclosure provides compositions of SLP-76 polypeptides, e.g., membrane-bound SLP-76 polypeptides, in combination with recombinant CARs or native or recombinant TCRs. In some embodiments, the present disclosure provides compositions of polynucleotides and expression vectors for expressing SLP-76 polypeptides, e.g., membrane-bound SLP-76 polypeptides. In some embodiments, the present disclosure provides compositions of polynucleotides and expression vectors for expressing SLP-76 polypeptides, e.g., membrane-bound SLP-76 polypeptides, in combination with polynucleotides and expression vectors for expressing recombinant CARs or native or recombinant TCRs. In some embodiments, the present disclosure provides compositions of recombinant cells comprising polynucleotides and / or expression vectors for SLP-76. In some embodiments, the present disclosure provides compositions of recombinant cells comprising polynucleotides and / or expression vectors for SLP-76 in combination with polynucleotides and / or expression vectors for recombinant CARs or native or recombinant TCRs.In some embodiments, the present disclosure provides a system comprising a composition for SLP-76 described herein, alone or in combination with a composition for CAR or TCR. In some embodiments, the present disclosure provides a composition for SLP-76, alone or in combination with a composition for CAR or TCR, to enhance T cell activation in response to a target antigen, particularly an antigen expressed at low density, to lower the T cell activation threshold, and / or not exacerbate T cell exhaustion. In some embodiments, the present disclosure provides a composition for SLP-76, alone or in combination with a composition for CAR or TCR, to enhance T cell activation in response to a target antigen, particularly when the CAR or TCR has low affinity for the target antigen. In some embodiments, the present disclosure provides a composition for SLP-76, alone or in combination with a composition for CAR or TCR, to prevent or treat a condition, such as a disease.

[0092] SLP-76 Polypeptides of the present disclosure include SLP-76 polypeptides, as described herein. SLP-76 was originally identified as a substrate of ZAP-70 protein tyrosine kinase following T cell receptor (TCR) ligation in the leukemic T cell line Jurkat. The SLP-76 locus is located on human chromosome 5q33, and its gene structure has been partially characterized in mice. The encoded SLP-76 protein is thought to associate with growth factor receptor-bound protein 2 and play a role in TCR-mediated intracellular signaling. The similar protein in mice plays a role in normal T cell development and activation. Mice lacking this gene exhibit subcutaneous and intraperitoneal fetal bleeding, dysfunctional platelets, and impaired viability. Both human and mouse SLP-76 cDNAs encode a 533 amino acid protein, are 72% identical, and are composed of three modular domains. The human SLP-76 protein can be obtained by its NCBI reference sequence: NP_005556.1. The coding polynucleotide (e.g., mRNA) sequence is available at NCBI reference sequence: NM_005565.5. 2 The -terminus contains a PEST domain and an acidic region containing several tyrosine residues that are phosphorylated after TCR ligation. SLP-76 also contains a central proline-rich domain and a COOH-terminal SH2 domain. Numerous additional proteins have been identified that associate with SLP-76 both constitutively and inducibly after receptor ligation, supporting the notion that SLP-76 functions as an adaptor or scaffold protein. Studies using SLP-76-deficient T cell lines or mice have provided strong evidence that SLP-76 plays a positive role in promoting T cell development and activation, as well as mast cell and platelet function. SLP-76 serves as an integration site for signals upon activation of the NK cell receptor. In NK cells, SLP-76 can be phosphorylated by SYK or ZAP70 after ligation to activate the receptor.

[0093] The polypeptides herein may include the full-length sequence of SLP-76, or a biologically functional fragment thereof, as well as any mutant, variant, ortholog, fusion, or other modified construct. In some embodiments, the SLP-76 polypeptides described herein include at least one mutation (e.g., a mutation at the K30 position, such as a K30R substitution) relative to the wild-type SLP-76 polypeptide. For example, the SLP-76 polypeptides described herein may have an amino acid sequence that is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59, or 60. In some embodiments, the SLP-76 polypeptides described herein comprise the amino acid sequence of SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59, or 60. In some embodiments, the SLP-76 polypeptides described herein consist of the amino acid sequence of SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59, or 60.

[0094] The polypeptides herein may include SLP-76 constructs in a free cytoplasmic form without binding to a cell membrane. Examples of cytoplasmic forms of SLP-76 that are not membrane bound may include polypeptides that include full-length SLP-76 (e.g., SEQ ID NO:1), a biologically active fragment, mutant, or variant of SLP-76, or a full-length, biologically active fragment, mutant, or variant of SLP-76 fused or conjugated to another molecule (e.g., 2A-tNGFR polypeptide and / or HA tag, having any one of the sequences of SEQ ID NOs:4 to 6). In some embodiments, the SLP-76 polypeptide may be genetically engineered to be membrane bound. For example, SLP-76 may be fused or conjugated to another polypeptide or polynucleotide or other substance to be tethered to the membrane. In some embodiments, the SLP-76 polypeptides herein may include a transmembrane domain conjugated to the full-length or fragment of SLP-76. Such transmembrane domains may be or be derived from a CAR molecule, a transmembrane domain of a TCR molecule, or other transmembrane proteins. Exemplary transmembrane domains (TMDs) from CAR molecules can be found in the following discussion section on CAR structures. Examples of suitable TMDs include, but are not limited to, CD28 TMD, CD8 TMD, CD4 TMD, CD3 TMD, CTLA-4 TMD, OX40 TMD, 4-1BB TMD, CD2 TMD, and PD-1 TMD.Additional examples of TMDs include CD3D, CD3E, CD3G, CD3 zeta, CD8a, CD8b, CD16, CD25, CD27, CD40, CD79A, CD79B, CD80, CD84, CD86, CD95, CD150 (SLAMF1), CD166, CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD300, CD357 (GITR), A2aR, ICAM-1, 2B4, BT The TMDs include those derived from LA, DAP10, FcRα, FcRβ, Fyn, GAL9, IL7, IL12, IL15, KIR, KIR2DL4, KIR2DS1, LAG-3, Lck, LAT, LPA5, LRP, NKp30, NKp44, NKp46, NKG2C, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, SLP-76, SIRPα, pTα, T cell receptor polypeptides (e.g., TCRα and TCRβ), TIM3, TRIM, and ZAP70. Thus, in some embodiments, the TMDs are derived from CD28 TMD, CD8 TMD, CD4 TMD, CD3 TMD, CTLA4 TMD, OX40 TMD, 4-1BB TMD, CD2 TMD, and PD-1 TMD. In some embodiments, the TMD comprises a CD28 TMD, a CD4 TMD, a CD8 TMD, a CD3 TMD, a CTLA4 TMD, an OX40 TMD, a 4-1BB TMD, a CD2 TMD, and a PD-1 TMD. In some embodiments, the SLP-76 polypeptide disclosed herein comprises a TMD derived from CD8. In some embodiments, the SLP-76 polypeptide disclosed herein comprises a CD8 TMD. In some embodiments, the SLP-76 polypeptide disclosed herein comprises a TMD derived from CD28. In some embodiments, the SLP-76 polypeptide disclosed herein comprises a CD28 TMD.In some embodiments, the SLP-76 polypeptides disclosed herein comprise a TMD comprising an amino acid sequence having at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 12 or 13. In some embodiments, the SLP-76 polypeptides disclosed herein may further comprise an additional domain derived from a CAR or TCR molecule. For example, the SLP-76 polypeptide may comprise an extracellular domain or an extracellular tag further conjugated to the transmembrane domain. In some embodiments, any extracellular domain or tag in the SLP-76 polypeptides described herein may facilitate anchoring of SLP-76 to the cell membrane. In some embodiments, any extracellular domain or tag in the SLP-76 polypeptides described herein may provide a means to detect the SLP-76 polypeptide (e.g., by FACS or other immunological methods). In some embodiments, any extracellular domain may be an ECD as described in detail in the following section regarding CAR molecules. In some embodiments, any extracellular domain comprises an antigen-binding domain. In some embodiments, any extracellular tag comprises an amino acid sequence having at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 11 or 39. In some embodiments, any extracellular tag may be a molecule such as a polypeptide or polynucleotide. In some embodiments, any extracellular tag comprises a VSV-G polypeptide. In some embodiments, any extracellular tag comprises an amino acid sequence having at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:10.Under non-limiting mechanisms, the SLP-76 polypeptides described herein, with or without any extracellular domain or tag, do not recognize target antigens and / or activate cells expressing the SLP-76 polypeptides themselves. In contrast, they may enhance the activity of another CAR or TCR molecule, either endogenous or exogenous, and / or lower the activation threshold of cells expressing a CAR or TCR molecule in response to a target antigen, especially when the antigen is expressed at low density.

[0095] In some embodiments, the SLP-76 polypeptides described herein may comprise a protein domain or adapter sequence conjugated to a full-length or fragment of SLP-76. In some embodiments, the protein domain or adapter sequence may interact (e.g., via binding) with a membrane protein, thereby tethering SLP-76 to the membrane.

[0096] In some embodiments, full length or fragments of SLP-76 may be tethered to a membrane via a covalent bond between SLP-76 and a membrane fatty acid, hi some embodiments, full length or fragments of SLP-76 may be tethered to a membrane via binding to the polar head group of a membrane lipid.

[0097] In some embodiments, the SLP-76 polypeptides described herein may comprise a membrane-binding domain conjugated to a full-length or fragment of SLP-76. In some embodiments, the membrane-binding domain may tether SLP-76 to a membrane. Exemplary membrane-binding domains may include the C1, C2, PH, FYVE, PX, ENTH, and BAR domains, or other domains described in Hurley, Biochim Biophys Acta, 2006; 1761:805-811, the contents of which are incorporated herein by reference in their entirety.

[0098] In some embodiments, the SLP-76 polypeptides described herein may be crosslinked or chemically conjugated to any membrane protein or membrane fatty acid or lipid.

[0099] Chimeric antigen receptors (CARs) As explained above, the SLP-76 polypeptide of the present disclosure can enhance activation of cells (e.g., immune cells, such as T cells expressing CAR or TCR) by a target antigen. In principle, there are no particular limitations on the CAR molecules suitable for the enhancement function of SLP-76 described herein. In some embodiments, the CAR molecules described herein may comprise (i) an extracellular ligand-binding domain (also known as an extracellular antigen-binding domain, or ECD) having binding affinity to the target antigen; (ii) a transmembrane domain (TMD); and (iii) an intracellular signaling domain (also known as a cytosolic signaling domain). In some embodiments, binding of the target antigen to the extracellular ligand-binding domain activates the intracellular signaling domain of the CAR polypeptide. In some embodiments, the disclosed CAR has the domains listed above in the N-terminal to C-terminal direction (i)-(iii). In some embodiments, the disclosed CARs are described in the format XYZ, where X represents a ligand / antigen recognizable by an extracellular ligand-binding domain, Y represents a hinge / transmembrane (H / TM) domain, and Z represents an intracellular signaling domain. For example, "CD19-CD28H / TM-CD28-CD3 zeta," "CD19-CD28TM-CD28Z," or "CD19 28Z" refers to a CAR molecule having an extracellular ligand-binding domain that specifically binds CD19, a CD28 hinge / transmembrane domain, a CD28 intracellular region, and a CD3 zeta intracellular signaling domain. Unless otherwise specified, a "TM" domain in this application includes a TMD, with or without any hinge domain.

[0100] In some embodiments, the disclosed CAR in the cell further comprises one or more hinge domains and / or costimulatory domains.

[0101] In some embodiments, the disclosed CARs comprise at least one intracellular (i.e., cytosolic) signaling domain as described herein, including but not limited to, a proximal signaling molecule, such as CD3 zeta, ZAP70, PLCG1, PKC, ITK, NCK, VAV1, GRB2, GADS, SOS1, ADAP, SYK, LYN, PI3K, BLNK, or the like, or a biologically active fragment, mutant, or variant thereof. In some embodiments, the disclosed CARs comprise two or more intracellular (i.e., cytosolic) signaling domains as described herein, including but not limited to, a proximal signaling molecule, such as CD3 zeta, ZAP70, PLCG1, PKC, ITK, NCK, VAV1, GRB2, GADS, SOS1, ADAP, SYK, LYN, PI3K, BLNK, or a biologically active fragment, mutant, or variant thereof.

[0102] Extracellular ligand (antigen)-binding domain (ECD) The CAR molecules described herein have at least one ECD that has binding affinity for one or more target ligands (or antigens, which are used interchangeably in this application). In some embodiments, the target antigen is expressed on the cell surface or is otherwise tethered, fixed, or constrained to the cell surface. Non-limiting examples of suitable target antigen types include cell surface receptors, adhesion proteins, carbohydrates, lipids, glycolipids, lipoproteins, and surface-bound lipopolysaccharides, integrins, mucins, and lectins. In some embodiments, the antigen is a protein. In some embodiments, the antigen is a carbohydrate. In some embodiments, the antigen is expressed by a target cell (e.g., a cancer cell / tumor cell). In some embodiments, the antigen is an adaptor molecule that specifically recognizes a target cell (e.g., a cancer cell / tumor cell). In some embodiments, the antigen is a biomarker for a specific disease, disorder, or condition (e.g., a cancer / tumor). Non-limiting examples of suitable antigens include CD19, HER2, ROR1, B7-H3 (CD276), CD22, CD2, CD5, CD6, 4-1BB, GD2, FcγR1, and integrins, as well as those described in the section below entitled "Antigens."

[0103] In some embodiments, the ECD of the CAR polypeptide disclosed herein comprises an antigen-binding portion that binds to one or more target antigens. In some embodiments, the antigen-binding portion comprises one or more antigen-binding determinants of an antibody, or a functional antigen-binding fragment thereof, including at least the ligand-binding domain of an antibody, an antigen-binding fragment, an antibody mimetic, a receptor, or a ligand of a targeted receptor. Upon reading this disclosure, the skilled artisan can readily understand that the term "functional fragment thereof" or "functional variant thereof" refers to a molecule that has a qualitative and / or quantitative biological activity in common with the wild-type molecule from which the fragment or variant is derived. For example, a functional fragment or functional variant of an antibody is one that essentially retains the same ability to bind to the same epitope as the antibody from which the functional fragment or functional variant is derived. For example, an antibody capable of binding to an epitope of a cell surface receptor may be truncated at the N-terminus and / or C-terminus, and retention of the epitope binding activity is assessed using assays known to those skilled in the art. In some embodiments, the antigen-binding moiety is an antibody, a monoclonal antibody, an antigen-binding fragment (Fab), a nanobody, a diabody, a triabody, a minibody, a F(ab') 2 Fragment, F(ab)v fragment, single chain variable region (scFv), single domain antibody (sdAb), V H Domain, V L Domains, Fv fragments, VNAR domains, and V HH In some embodiments, the antigen-binding portion comprises a heavy chain variable region and a light chain variable region. In some embodiments, the antigen-binding portion comprises an scFv. In some embodiments, the antibody mimetic is selected from the group consisting of an affibody molecule, an affilin, an affimer, an alphabody, an avimer, a DARPins, a finomer, a Kunitz domain peptide, a monobody, a nanoCLAMP, and a biologically active fragment thereof.

[0104] The antigen-binding portion can comprise a naturally occurring amino acid sequence, or can be engineered, designed, or modified to provide desired and / or improved properties, such as binding affinity. In general, the binding affinity of an antibody or antigen-binding portion for a target antigen (e.g., CD19 antigen or HER2 antigen) can be calculated by the Scatchard method described by Frankel et al., Mol. Immunol, 16: 101-106, 1979. In some embodiments, binding affinity can be measured by antigen / antibody dissociation rate. In some embodiments, high binding affinity can be measured by competitive radioimmunoassay. In some embodiments, binding affinity can be measured by ELISA. In some embodiments, antibody affinity can be measured by flow cytometry. An antibody that "selectively binds" to a target antigen (such as CD19 or HER2) is an antibody that binds to the target antigen with high affinity but does not bind significantly to other unrelated antigens, but binds to the antigen with high affinity, e.g., an equilibrium constant (K D ) binds at 100 nM or less, such as 60 nM or less, such as 30 nM or less, such as 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less, etc.

[0105] One skilled in the art can select an ECD based on the desired localization or function of the cell to be genetically modified to express the CAR polypeptide of the present disclosure. For example, a CAR polypeptide with an ECD that includes an antibody specific for the HER2 antigen can target cells to HER2-expressing breast cancer cells. In some embodiments, the ECD of the CAR polypeptide disclosed herein is capable of binding a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). One skilled in the art can understand that a TAA includes a molecule, such as a protein that is present on tumor cells and normal cells, or a protein that is present on many normal cells but at a much lower concentration on tumor cells. In contrast, a TSA generally includes a molecule, such as a protein that is present on tumor cells but not on normal cells.

[0106] antigen In the present application, the terms "ligand(s)" and "antigen(s)" are used interchangeably to mean the target molecule(s) specifically recognized by the extracellular antigen-binding domain of the CAR molecule described in this section or by the TCR molecule described in the following section. In principle, there are no special limitations regarding suitable target antigens. In some embodiments of the present disclosure, the antigen-binding portion of the ECD is specific for an epitope present in an antigen expressed or recognized by a target cell. In some embodiments, the target cell is correlated with a disease or disorder. Illustrative diseases or disorders may include, for example, proliferative diseases (e.g., cancer), hematological malignancies, solid tumors, autoimmune diseases, inflammation, allergic diseases, infectious diseases, senescence / aging, etc. In some embodiments of the present disclosure, the antigen-binding portion of the ECD of the CAR is specific for an epitope present in an antigen expressed by a tumor cell, i.e., a tumor-associated antigen. The tumor-associated antigen can be, for example, an antigen associated with leukemia cells, neuroblastoma cells, osteosarcoma cells, pancreatic cancer cells, colon cancer cells, ovarian cancer cells, prostate cancer cells, lung cancer cells, mesothelioma cells, breast cancer cells, urothelial cancer cells, liver cancer cells, head and neck cancer cells, sarcoma cells, cervical cancer cells, gastric cancer cells, gastric cancer cells, melanoma cells, uveal melanoma cells, cholangiocarcinoma cells, multiple myeloma cells, lymphoma cells, glioblastoma cells, or other cancer cells described in this disclosure. In some embodiments, the antigen-binding moiety is specific for an epitope present in a tissue-specific antigen. In some embodiments, the antigen-binding moiety is specific for an epitope present in a disease-associated antigen. Tumors are often referred to as a subtype of cancer when uncontrolled growth of cells occurs in solid tissues such as organs, muscles, or bones. In this application, the terms "tumor" and "cancer" are used interchangeably, unless otherwise specified, to generally refer to cells with uncontrolled growth.

[0107] In some embodiments, the antigen is selected from the group consisting of CD19, HER2, ROR1, B7-H3 (CD276), CD22, GD2, CD2, CD5, CD6, 4-1BB, FcγR1, and integrins. In some embodiments, the antigen is selected from the group consisting of CD19, HER2, ROR1, B7-H3 (CD276), influenza hemagglutinin (HA), CD22, CD2, CD5, CD6, 4-1BB, FcγR1, GD2, CD22, CD10, CD20, GPC2, GD3, GM2, GM3, and integrins.

[0108] Non-limiting examples of suitable target antigens also include glypican 2 (GPC2), human epidermal growth factor receptor 2 (Her2 / neu), CD276 (B7-H3), PSMA, IL-13-receptor alpha 1, IL-13-receptor alpha 2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA). Other suitable target antigens include, but are not limited to, tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD123, CD93, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), total cystic disease fluid protein (GCDFP-15), ALK, DLK1, FAP, NY-ESO, WT1, HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, CD138, FolR1, LeY, MCSP, and TYRP1.

[0109] Additional antigens that may be suitable for the CARs disclosed herein include, but are not limited to, the dimeric form of pyruvate kinase isoform enzyme M2 (tumor M2-PK), CD19, CD20, CD5, CD7, CD3, TRBC1, TRBC2, BCMA, CD38, CD123, CD93, CD34, CD1a, SLAMF7 / CS1, FLT3, CD33, CD123, TALLA-1, CSPG4, DLL3, kappa light chain, lambda light chain, CD16 / FcγRIII, CD64, FITC, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), GD3, EGFRvIII (epidermal growth factor variant III), EGFR and its isovariants, TEM-8, sperm protein 17 (Sp17), and mesothelin. Further non-limiting examples of suitable antigens include PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma chain alternative reading frame protein), Trp-p8, STEAP1 (prostate six transmembrane epithelial antigen 1), abnormal ras protein, abnormal p53 protein, integrin β3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), and Ral-B. In some embodiments, the antigen is CD19, human epidermal growth factor receptor 2 (Her2 / neu), CD276 (B7-H3), or GD-2.

[0110] Antigens that may be suitable for the CARs disclosed herein include, but are not limited to, one of the following or any combination thereof: CD1a, CD1b, CD1c, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD13, CD14, CD15 (SSEA-1), CD16 (Fc γRIII), CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32( FcγRII), CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD43, CD44, CD44V6, CD45, CD45R / B220, CD 45RO, CD49b, CD49d, CD49f, CD52, CD53, CD54, CD56 (NCAM), CD57, CD61 (integrin β3), CD62L, CD63, CD64, CD 66b, CD68, CD69, CD70, CD73, CD74, CD79a(Igα), CD79b(Igβ), CD80, CD83, CD85k(ILT3), CD86, CD88, CD93( C1Rqp), CD94, CD95, CD99, CD103, CD105 (endoglin), CD107a, CD107b, CD114 (G-CSFR), CD115, CD117, CD122, CD123, CD129, CD133, CD134, CD138 (syndecan-1), CD141, CD146, CD152 (CTLA-4), CD158 (Kir), CD161 (NK-1.1), CD163, CD183, CD191, CD193(CCR3), CD194(CCR4), CD195(CCR5), CD197(CCR7), CD203c, CD205(DEC-205), CD207(Langerin), CD209(DC-S IGN), CD223, CD235, CD244(2B4), CD252(OX40L), CD267, CD268(BAFF-R), CD273(B7-DC, PD-L2), CD276(B7-H3), CD279(PD1), CD282(TLR2) ), CD284 (TLR4), CD294, CD304 (neuropilin-1), CD305, CD314 (NKG2D), CD319 (CRACC), CD326, CD328 (Siglec-7), CD335 (NKp46), fetal acetylcholine receptor (AChR), ADGRE2, alpha-fetoprotein (AFP), ALK, BCMA, BDCA3, C3AR, Lewis A (CA19.9), carbonic anhydrase IX (CA1X), calretinin, cancer antigen-125 (CA-125), CCR1, CCR4, CDS, carcinoembryonic antigen (CEA), chromogranin, CLEC12A, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), CS-1, CSPG4, cytokeratin, desmin, DLK1, DLL3, EGFRvIII (epidermal growth factor variant III), EGFR and its isovariants, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial membrane protein (EMA), ERBB, epithelial tumor antigen (ETA), FA P, folate binding protein (FBP), FcγR1, FcεRIα, FITC, FLT3, FOLR1, FOLR3, galactin, ganglioside, total cystic disease fluid protein (GCDFP-15), GD2 (ganglioside G2), GD3, GM2, GM3, glial fibrillary acidic protein (GFAP), gpA33, glycopeptide, glypican 2 (GPC2), carcinoembryonic antigen (h5T4), influenza hemagglutinin (HA), human epidermal growth factor receptor 2 (Her2 / neu), HLA-DR, HM1.24, HMB-45 antigen, HPV E6, HPV E7, ICAM-1, IgG, IgD, IgE, IgM, IL-13-receptor alpha 1, integrin, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa light chain, kinase insert domain receptor (KDR), lambda light chain, LILRB2, Lewis Y (LeY), LGR5, Ly49, Ly108, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE), melanoma antigen family A1 (MAGE-A1), melanoma protein A (melanoma antigen recognized by T lymphocytes; MART-1), MCSP, c-Met, MICA / B, mesothelin, muscle-specific actin (MSA), mesothelin (MSLN), dimeric form of pyruvate kinase isoform enzyme M2 (tumor M2-PK), mucin 1 (Muc-1), mucin 16 (Muc-16), myo-D1, Necl-2, neurofilament, NKCSI, NKG2D, neuron-specific enolase (NSE), NY-ESO, testis cancer antigen NY-ESO-1, abnormal p53 protein, PAP (prostate cancer antigen), glandular acid phosphatase), PAMA, P-cadherin, placental alkaline phosphatase, PRAIVIE, prostein, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), Ral-B, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), abnormal ras proteins, ROR1, SLAMF7 / CS1, receptor tyrosine-protein kinase erb-B2,3,4, sperm protein 17 (Sp17), STEAP1 (six-transmembrane epithelial antigen of the prostate 1), synaptonemal protein 1 (STEAP1), Physin, tumor-associated glycoprotein 72 (TAG-72), TALLA-1, TARP (T cell receptor gamma chain alternative reading frame protein), TEM-8, human telomerase reverse transcriptase (hTERT), TIM-3, TLR4, TRBC1, TRBC2, Trp-p8, thyroglobulin, thyroid transcription factor-1, TYRP1, tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), Vα24, Wilms tumor protein (WT-1), and various pathogenic antigens known in the art. In some embodiments, the antigens described herein are tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs).

[0111] In some embodiments, the CAR polypeptides disclosed herein comprise an ECD that comprises an antigen-binding portion that binds to CD19. In some embodiments, the CAR polypeptides disclosed herein comprise an ECD that comprises an antigen-binding portion that binds to HER2. In some embodiments, the CAR polypeptides disclosed herein comprise an ECD that comprises an antigen-binding portion that binds to B7-H3. In some embodiments, the CAR polypeptides disclosed herein comprise an ECD that comprises an antigen-binding portion that binds to GD2. In some embodiments, the CAR polypeptides disclosed herein comprise an ECD that comprises an antigen-binding portion that binds to CD22. In some embodiments, the CAR polypeptides disclosed herein comprise an ECD that comprises an antigen-binding portion having an amino acid sequence that exhibits at least 50% sequence identity to any ECD described herein. In some embodiments, the antigen-binding portion has an amino acid sequence that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any ECD described herein. Percent identity as used herein refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same over a specified region.Two or more sequences or subsequences can be compared and aligned to maximize correspondence over a comparison window or specified region, for example, as measured by BLAST or BLAST2.0 sequence comparison algorithms, or by manual alignment and visual inspection.See, for example, the NCBI website ncbi.nlm.nih.gov / BLAST.This definition can also refer to or apply to the complement of a sequence.This definition also includes sequences that have deletions and / or additions, and sequences that have substitutions.Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990), etc. Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with its default parameters. The amino acid substitution(s) may be conservative amino acid substitutions, for example, at non-essential amino acid residue(s) in the CDR sequence. A "conservative amino acid substitution" is understood to be one in which the original amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0112] One advantage of the SLP-76 polypeptides described herein is that they can enhance cell activation in response to a target antigen. In some embodiments, such activation is induced by a CAR or TCR molecule expressed on the surface of a cell that specifically binds to the target antigen. Furthermore, SLP-76 polypeptides, such as the membrane-bound SLP-76 polypeptides described herein, can enhance cell activation when the target antigen is expressed at low density and / or lowers the cell activation threshold via a CAR or TCR molecule. The term "low density of antigen", unless otherwise specified in this disclosure, generally refers to a situation in which in a microenvironment comprising at least one cell expressing a CAR or TCR molecule and a target antigen (e.g., a tumor antigen expressed by a tumor cell or a tumor antigen expressed by an antigen-presenting cell), the amount of target antigen molecules per cell is insufficient to activate (through binding) a sufficient number of CAR or TCR molecules required for activation of the cell expressing the CAR or TCR molecule. The density of a target antigen is a relative term that depends on the antigen, the CAR or TCR structure, and / or the target cell that expresses or presents the target antigen. An exemplary low antigen density of CD19 may refer to about 1000 or less molecules for TCR or CAR in its microenvironment (e.g., 1000 molecules on each target cell for CAR activation and on each antigen-presenting cell for TCR activation). Another exemplary low density of antigen may refer to about 100 or less to about 10 million or less antigen molecules.In some embodiments, low antigen density is about 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 700 In some embodiments, the number of target antigen molecules may refer to up to 00, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, or 10 million target antigen molecules. In some embodiments, low antigen density may refer to 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less of target antigen molecules compared to normal or high antigen density (e.g., density of target antigen expressed on wild-type target cells). The density or amount of target antigen may be measured by various methods, such as semi-quantitative FACS, immunohistochemistry, or other immunological methods. SLP-76 polypeptides, such as membrane-bound SLP-76 polypeptides described herein, can also enhance cell activation when target antigen is at normal or high density. In a non-limiting example, overexpression of CAR or TCR molecules in cells can result in cell exhaustion. SLP-76 overexpression can enhance CAR or TCR capacity without exacerbating T cell exhaustion. In some embodiments, a CAR or TCR or other receptor molecule expressed on a cell may have low affinity for its target antigen, resulting in weak activation even when multiple target antigens are present in the microenvironment.In some embodiments, SLP-76 overexpression can enhance the ability of these CARs or TCRs or other receptors that have low affinity for target antigens, thereby enhancing cell activation. In some non-limiting embodiments, the CARs or TCRs or other receptor molecules expressed on cells can be expressed at low density, resulting in weak activation of the cells even when there are many target antigens in the microenvironment. In some non-limiting embodiments, SLP-76 overexpression can enhance the ability of these CARs or TCRs or other receptors that have low affinity for target antigens, thereby enhancing cell activation. In some non-limiting embodiments, cells associated with a disease or disorder (e.g., cancer cells or tumor cells or other target cells) can express low levels of antigens (i.e., have low antigen density), which is insufficient to activate CAR or TCR molecules that recognize the antigen, and therefore general CAR T or TCR therapy using such CAR or TCR molecules is insufficient or ineffective to treat the disease or disorder. In some non-limiting embodiments, overexpression of SLP-76 (e.g., a membrane-bound SLP-76 construct) can enhance the activation and function of these CARs or TCRs or other receptors, thereby allowing CAR T or TCR therapy to treat the disease or disorder.

[0113] Hinge Domain As explained above, the CAR polypeptides described herein may have any hinge domain. Within a chimeric antigen receptor, the term "hinge domain" generally refers to a flexible polypeptide connector region located between the targeting moiety (ECD) and the TMD. These sequences are generally derived from IgG subclasses (such as IgG1 and IgG4), IgD and CD8 domains, of which IgG1 is the most widely used.

[0114] In some embodiments, the hinge domain provides structural flexibility to adjacent polypeptide regions. The hinge domain may be composed of natural or synthetic polypeptides. In recent years, some hinge domain research has mainly focused on the following aspects: (1) reducing the binding affinity to Fcγ receptors, thereby eliminating certain types of non-target activation; (2) enhancing the flexibility of single chain variable fragments (scFv), thereby relieving the spatial constraint between a specific epitope targeted to a tumor antigen and the antigen-targeting portion of the CAR; (3) reducing the distance between the scFv and the target epitope(s); and (4) facilitating the detection of CAR expression using anti-Fc reagents. Nevertheless, the impact of the hinge domain on the physics of CAR T cells is not well understood.

[0115] It can be understood by those skilled in the art that the hinge domain can improve the function of the CAR polypeptide described herein by promoting optimal positioning of the antigen-binding portion in relation to the portion of the antigen recognized thereby. It can be understood that in some embodiments, the hinge domain may not be required for optimal CAR activity. In some embodiments, a beneficial hinge domain having a short amino acid sequence promotes CAR activity by promoting antigen binding, for example, by relieving any steric hindrance that may otherwise alter antibody binding kinetics. The sequence encoding the hinge domain may be located between the antigen recognition portion and the TMD. In some embodiments, the hinge domain is operably linked downstream of the antigen-binding portion and upstream of the TMD. When the CAR polypeptide described herein has an optional hinge domain, the format describing the hinge domain and the TMD domain may be "H / TM" or "H-TM".

[0116] The hinge sequence can generally be any portion or sequence derived from or derived from any suitable molecule. For example, in some embodiments, the hinge sequence can be derived from the human CD8 molecule or the CD28 molecule, as well as any other receptor that provides similar functionality in providing mobility to adjacent regions. The hinge domain can have a length of about 4 amino acids (aa) to about 50 aa, for example, about 4 aa to about 10 aa, about 10 aa to about 15 aa, about aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa. Suitable hinge domains can be readily selected and can be any of a number of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 aa, 2 aa to 15 aa, 3 aa to 12 aa, including 4 aa to 10 aa, 5 aa to 9 aa, 6 aa to 8 aa, or 7 aa to 8 aa, and can be 1, 2, 3, 4, 5, 6, or 7 aa. Non-limiting examples of suitable hinge domains include a CD8 hinge domain, a CD28 hinge domain, a CD4 hinge domain, a PD-1 hinge domain, a CD2 hinge domain, a CTLA4 hinge domain, or an IgG4 hinge domain. In some embodiments, the hinge domain can include a region derived from the human CD8α (also known as CD8a) molecule or any other receptor that provides a similar function of providing flexibility to the CD28 molecule and adjacent regions. Additional illustrative hinge domains are derived from or include the hinge domains of LFA-1 (CD11a / CD18), CD5, CD27 (TNFRSF7), CD70, 4-1BB, OX40 (CD134), ICOS (CD278), IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgE Fc region, IgM Fc region, IgA Fc region, or combinations thereof. In some embodiments, the CARs disclosed herein comprise a hinge domain derived from a CD8 hinge domain. In some embodiments, the hinge domain can comprise one or more copies of a CD8 hinge domain. In some embodiments, the CARs disclosed herein comprise a hinge domain derived from a CD28 hinge domain.In some embodiments, the hinge domain can comprise one or more copies of a CD28 hinge domain. In some embodiments, the CARs disclosed herein comprise a hinge domain derived from a CD4 hinge domain. In some embodiments, the hinge domain comprises one or more copies of a CD4 hinge domain. In some embodiments, the CARs disclosed herein comprise a hinge domain derived from an IgG4 hinge domain. In some embodiments, the hinge domain can comprise one or more copies of an IgG4 hinge domain.

[0117] Costimulatory domain As explained above, the CAR polypeptides described herein may have any costimulatory domain. Generally, the costimulatory domain suitable for the CAR polypeptides disclosed herein can be any one of the costimulatory domains known in the art. Examples of suitable costimulatory domains can enhance cytokine production and include, but are not limited to, costimulatory polypeptide sequences derived from 4-1BB (CD137), CD27, CD28, OX40 (CD134), and costimulatory-inducible T cell costimulatory molecule (ICOS) polypeptide sequences. Additional exemplary costimulatory polypeptide sequences may be or be derived from the following costimulatory domains: CD28, ICOS (CD278), CD27, 4-1BB (CD137), OX40 (CD134), CD2, CD4, CD5, CD7, CD8, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD4 0L(CD154), CD48, CD49a, CD49D, CD49f, CD58, CD53, ICAM-1(CD54), CD69, CD70, CD80(B7-1), CD82, CD83, CD84, CD 86(B7-2), CD90, CD96, CD100, CD103, CD122, CD132, CD150(SLAMF1), CD160(BY55), CD162(DNAM1), CD223(LAG3), C D226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278, LAT, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptors (TLRs, e.g., TLR2, etc.) etc.), DAP10, DAP12, LAG-3, 2B4, CARD1, CTLA-4 (CD152), TRIM, ZAP70, FcERIγ, 4-1BBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TAC1, BLAME, CRACC, CD2F-10, NTB-A, integrin α4, integrin α4β1, integrin α4β7, IA4, ICAM-1,IL2Rβ, IL2Rγ, IL7Rα, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LTBR, ​​PAG / Cbp, PSGL1, SLAMF6(NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, BTLA, Ikaros, LAG-3, LMIR, CEACAM1, CRTAM, TCL1A, DAP12, TIM-1, Dectin-1, PDCD6, PD-1, TIM-4, TSLP, EphB6, TSLP-R, HLA-DR, or any combination thereof.

[0118] Thus, in some embodiments, the costimulatory domain of the CAR disclosed herein is selected from the group consisting of a costimulatory 4-1BB (CD137) polypeptide sequence, a costimulatory CD27 polypeptide sequence, a costimulatory CD28 polypeptide sequence, a costimulatory OX40 (CD134) polypeptide sequence, and a costimulatory inducible T cell costimulatory molecule (ICOS) polypeptide sequence. In some embodiments, the CAR disclosed herein comprises a costimulatory domain derived from a costimulatory 4-1BB (CD137) polypeptide sequence. In some embodiments, the CAR disclosed herein comprises a costimulatory domain derived from a costimulatory CD28 polypeptide sequence. In some embodiments, the CAR disclosed herein comprises a costimulatory CD28 polypeptide sequence. In some embodiments, the CAR disclosed herein comprises a costimulatory domain having an amino acid sequence that exhibits at least 50% sequence identity to the sequence of any of the costimulatory domains described herein. In some embodiments, the costimulatory domain comprises an amino acid sequence that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any costimulatory domain described herein.

[0119] Transmembrane domain (TMD) In general, a suitable transmembrane domain (also referred to as a transmembrane region) for the CAR polypeptide disclosed herein can be any one of the TMDs known in the art. Without being bound by theory, it is believed that the TMD crosses the cell membrane, anchors the CAR to the cell surface, and connects the ECD to the intracellular signaling domain, thereby influencing the expression of the CAR at the cell surface. Examples of suitable TMDs include, but are not limited to, CD28 TMD, CD8 TMD, CD4 TMD, CD3 TMD, CTLA-4 TMD, OX40 TMD, 4-1BB TMD, CD2 TMD, and PD-1 TMD. Examples of additional TMDs include CD3D, CD3E, CD3G, CD3 zeta, CD8a, CD8b, CD16, CD25, CD27, CD40, CD79A, CD79B, CD80, CD84, CD86, CD95, CD150 (SLAMF1), CD166, CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD300, CD357 (GITR), A2aR, ICAM-1, 2B4, BT The TMDs include those derived from LA, DAP10, FcRα, FcRβ, Fyn, GAL9, IL7, IL12, IL15, KIR, KIR2DL4, KIR2DS1, LAG-3, Lck, LAT, LPA5, LRP, NKp30, NKp44, NKp46, NKG2C, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, SLP-76, SIRPα, pTα, T cell receptor polypeptides (e.g., TCRα and TCRβ), TIM3, TRIM, and ZAP70. Thus, in some embodiments, the TMDs are derived from CD28 TMD, CD8 TMD, CD4 TMD, CD3 TMD, CTLA4 TMD, OX40 TMD, 4-1BB TMD, CD2 TMD, and PD-1 TMD. In some embodiments, the TMD comprises a CD28 TMD, a CD4 TMD, a CD8 TMD, a CD3 TMD, a CTLA4 TMD, an OX40 TMD, a 4-1BB TMD, a CD2 TMD, and a PD-1 TMD. In some embodiments, the CAR disclosed herein comprises a TMD from CD8.In some embodiments, the CAR polypeptides disclosed herein comprise a CD8 TMD. In some embodiments, the CARs disclosed herein comprise a TMD derived from CD28. In some embodiments, the CARs disclosed herein comprise a CD28 TMD. In some embodiments, the CARs disclosed herein comprise a TMD derived from CD4. In some embodiments, the CARs disclosed herein comprise a CD4 TMD.

[0120] Exemplary CAR molecules as described herein include a hinge domain and a TMD domain adjacent to each other. Sequences for illustrative hinge / transmembrane (H / TM or hinge / TM) domains for various CAR molecules are disclosed herein. In some embodiments, the CAR molecules disclosed herein include an H / TM domain having an amino acid sequence that exhibits at least 50% sequence identity to any TMD or hinge-TMD described herein. In some embodiments, the CAR molecules disclosed herein include an H / TM domain having an amino acid sequence that exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any TMD or hinge-TMD described herein.

[0121] Extracellular spacer The CAR disclosed herein may further comprise an optional extracellular spacer domain comprising one or more intervening amino acid residues disposed between the ECD and any hinge domain. In some embodiments, the extracellular spacer domain is operably linked downstream of the ECD and upstream of the optional hinge domain. In principle, there is no particular restriction on the length and / or amino acid composition of the extracellular spacer. In some embodiments, any single-chain peptide comprising about 1-100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as an extracellular spacer. In some embodiments, the extracellular spacer comprises about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, or about 30 to 90 amino acid residues. In some embodiments, the extracellular spacer comprises about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, or about 50 to 70 amino acid residues. In some embodiments, the extracellular spacer comprises about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the extracellular spacer comprises about 1-10, about 5-15, about 10-20, about 15-25 amino acid residues. In some embodiments, the length and amino acid composition of the extracellular spacer can be optimized to vary the orientation and / or proximity of the ECD and any hinge domain to each other to achieve a desired activity of the CAR. In some embodiments, the orientation and / or proximity of the ECD and any hinge domain to each other can be varied and / or optimized as a "tuning" tool or effect to enhance or decrease the efficacy of the CAR. In some embodiments, the orientation and / or proximity between the ECD and the optimal hinge domain to each other can be varied and / or optimized to produce fully functional and / or partially functional forms of the CAR. In some embodiments, the extracellular spacer domain comprises amino acid sequences corresponding to an IgG4 hinge domain and an IgG4 CH2-CH3 domain.Additional illustrative extracellular spacer domains are derived from or include immunoglobulin hinge regions (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD), all or a portion of an immunoglobulin Fc domain (e.g., the CH1 domain, the CH2 domain, the CH3 domain, or a combination thereof), the stalk region of a type II C-lectin (an extracellular domain disposed between a C-type lectin domain and a transmembrane domain). Type II C-lectins include, for example, CD23, CD69, CD72, CD94, NKG2A, and NKG2D. In yet further embodiments, the extracellular spacer domain may be derived from or include a toll-like receptor (TLR) juxtamembrane domain. The TLR juxtamembrane domain includes acidic amino acids that are present between the leucine-rich repeat (LRR) and the transmembrane domain of the TLR. In some embodiments, the TLR juxtamembrane domain is a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 juxtamembrane domain.

[0122] One skilled in the art can understand that the complete amino acid sequence of a CAR polypeptide of the present disclosure can be used to construct a reverse-translated gene. For example, a DNA oligomer containing a nucleotide sequence encoding a given CAR can be synthesized. For example, several small oligonucleotides encoding portions of the desired CAR or antibody can be synthesized and then ligated. The individual oligonucleotides typically have 5' or 3' overhangs for complementary assembly.

[0123] In addition to producing the desired CAR through expression of a nucleic acid molecule modified by recombinant molecular biology techniques, the subject CARs according to the present disclosure can be chemically synthesized. Chemically synthesized polypeptides are routinely produced by those skilled in the art.

[0124] Once assembled (by synthesis, recombinant methods, site-directed mutagenesis, or other suitable techniques), the DNA sequence encoding the CAR disclosed herein can be inserted into an expression vector or operably linked to expression control sequences suitable for expression of the CAR in a desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is known in the art, to obtain high expression levels of a transfected gene in a host, one should ensure that the gene is operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

[0125] Intracellular signaling domains The CARs disclosed herein may comprise an intracellular signaling domain that transmits signaling from the ECD upon binding to a target antigen and induces cell activation.

[0126] In a chimeric antigen receptor (CAR), the intracellular signaling domain (also known as the cytosolic signaling domain) generally refers to the cytosolic domain that transmits an activation signal to the cell expressing the CAR molecule after binding the extracellular domain to the corresponding ligand or antigen. In some cases, the intracellular signaling domain includes a functional signaling domain derived from a stimulatory molecule. Conventional intracellular signaling domains almost always include CD3 zeta, the prototypic "master switch" that triggers T cell activity (Irving and Weiss, Cell 1991;64:891-901; Letourneur and Klausner, Science 1992;255:79-82), as well as a variety of optional co-stimulatory domains that enhance efficacy and persistence.

[0127] In some embodiments, the CARs disclosed herein have an intracellular signaling domain derived from or derived from a proximal signaling molecule. In some embodiments, the CARs disclosed herein have an intracellular signaling domain with an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the CARs disclosed herein have an intracellular signaling domain without an immunoreceptor tyrosine-based activation motif (ITAM). Exemplary proximal signaling molecules include, for example, CD3 zeta (CD3ζ), CD3-epsilon, DAP12, ZAP70, PLCG1, PKC, ITK, NCK, VAV1, GRB2, GADS, SOS1, ADAP, SYK, LYN, PI3K, BLNK, or biologically active fragments, mutants, or variants thereof.

[0128] T cell receptor (TCR) The T cell receptor (TCR) is a protein complex found on the surface of T cells, or T lymphocytes, that is involved in recognizing fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules. The binding between the TCR and antigenic peptides is relatively low affinity and degenerate. The TCR is composed of two different protein chains (i.e., as a heterodimer). In humans, in 95% of T cells, the TCR consists of alpha (α) and beta (β) chains (encoded by TRA and TRB, respectively), whereas in 5% of T cells, the TCR consists of gamma and delta (γ / δ) chains (encoded by TRG and TRD, respectively). Within these chains are complementarity determining regions (CDRs) that determine which antigens the TCR will bind to. Antigen presenting cells (APCs) digest pathogens and present their fragments on major histocompatibility complex (MHC) molecules. This MHC / antigen complex binds to the TCR while other costimulatory molecules (e.g. CD28) are activated, leading to T cell activation, proliferation, differentiation, apoptosis, or cytokine release. However, the MHC / antigen complex is not the only molecule capable of interacting with the TCR. Non-peptide antigens such as lipids can interact with the TCR through some of the five isoforms of CD1, and several studies have described TCR binding to metabolic intermediates bound to the MHC-like molecule MR1.

[0129] Stimulation of T cell function may be initiated upon interaction of the TCR with short peptides presented by MHC class I or II molecules (MHC1 for CD8 T cells and MHC II for CD4 T cells). However, the TCR heterodimer itself cannot activate downstream pathways to initiate T cell activation. Initiation of TCR signaling requires co-receptors, such as CD4 for helper T cells and CD8 for cytotoxic T cells. These co-receptors act as cell adhesion molecules that bind their respective MHC molecules and stabilize the interaction of the T cell and the antigen-presenting cell.

[0130] The TCR is also located in close proximity to the CD3 family of proteins (CD3δ, CD3ε, and CD3γ) and the TCR zeta (ζ) chain. Once the TCR is properly engaged with a peptide-MHC complex, a conformational change is induced in the associated CD3 chains, which leads to their phosphorylation and signaling by proximal signaling molecules, as well as signaling by CAR molecules.

[0131] As explained above, the SLP-76 polypeptides of the present disclosure are capable of enhancing the activation of cells expressing TCR (either native or recombinant) (e.g., immune cells such as T cells) in response to a target antigen presented by an MHC molecule on an antigen-presenting cell. In principle, there are no particular limitations regarding the suitable TCR molecule for the enhancing function of the LP-76 polypeptides described herein. In non-limiting embodiments, the SLP-76 polypeptides described herein are capable of enhancing the activation of TCR-expressing cells in response to a target antigen presented, for example, by an antigen-presenting cell (APC). In some embodiments, the SLP-76 polypeptides, for example, the membrane-bound SLP-76 polypeptides described herein, are capable of activating or enhancing the activation of TCR-expressing cells in response to a target antigen at low density. In some embodiments, the activation of TCR-expressing cells may enhance the proliferation, differentiation, cytokine production and / or cytotoxicity of the cells. For example, in adoptive cell therapy (ACT), the patient's white blood cells are collected in a process called leukopheresis. Isolation of the most cancer-fighting potent specific T cells is followed by in vitro culture of these cells. This large expanded T cell population may then be reinfused into the patient to act against the cancer. In some embodiments, even if the target antigen is expressed at low density on the antigen-presenting cells, SLP-76 polypeptides, such as the membrane-bound SLP-76 polypeptides described herein, may be capable of activating or enhancing activation and / or proliferation of TCR-expressing cells in response to a presented target antigen (e.g., a tumor antigen). Thus, the SLP-76 polypeptides described herein may be used to selectively enhance proliferation and / or isolation of TCR-expressing cells with specificity and / or potency for a target antigen (e.g., a tumor antigen).

[0132] Chimeric antigen receptor (CAR) or T cell receptor (TCR) mutations Multiple mutations can be introduced into the CAR or TCR polypeptide of the present disclosure. Mutations include at least substitutions, deletions, insertions, or other methods known in the art. The purpose of these mutations includes, for example, to enhance the efficacy of the CAR or TCR polypeptide, to enhance the stability (e.g., half-life) of the CAR or TCR polypeptide, to enhance expression of the CAR or TCR polypeptide, to enhance the solubility and / or reduce aggregation of the CAR or TCR polypeptide, to engineer the modification of the CAR or TCR polypeptide upon expression, to engineer the binding of the CAR or TCR polypeptide to its binding partner(s), to enhance purification of the CAR or TCR polypeptide, to reduce ubiquitination and / or degradation of the CAR or TCR polypeptide, etc.

[0133] nucleic acid molecule In one aspect, provided herein are various nucleic acid molecules comprising nucleotide sequences encoding the SLP-76, CAR, or TCR polypeptides of the present disclosure, including expression cassettes and expression vectors that contain these nucleic acid molecules operably linked to heterologous nucleic acid sequences, such as control sequences that enable in vivo expression of the SLP-76, CAR, or TCR polypeptides in a host cell or ex vivo cell-free expression system.

[0134] The nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, and generally include nucleic acid molecules of from about 0.5 Kb to about 50 Kb, e.g., from about 0.5 Kb to about 20 Kb, from about 1 Kb to about 15 Kb, from about 2 Kb to about 10 Kb, or from about 5 Kb to about 25 Kb, e.g., from about 10 Kb to 15 Kb, from about 15 Kb to about 20 Kb, from about 5 Kb to about 20 Kb, from about 5 Kb to about 10 Kb, or from about 10 Kb to about 25 Kb. In some embodiments, the nucleic acid molecule of the present disclosure is from about 1.5 Kb to about 50 Kb, from about 5 Kb to about 40 Kb, from about 5 Kb to about 30 Kb, from about 5 Kb to about 20 Kb, or from about 10 Kb to about 50 Kb, e.g., from about 15 Kb to 30 Kb, from about 20 Kb to about 50 Kb, from about 20 Kb to about 40 Kb, from about 5 Kb to about 25 Kb, or from about 30 Kb to about 50 Kb.

[0135] In some embodiments, the recombinant nucleic acid comprises a nucleic acid sequence encoding an SLP-76 polypeptide, a CAR polypeptide, or a TCR polypeptide.

[0136] In some embodiments, the composition has at least two recombinant nucleic acids, each comprising a nucleic acid sequence encoding an SLP-76 polypeptide as described herein and a CAR or TCR polypeptide as described herein, to form a combination. In some embodiments, these at least two recombinant nucleic acids are conjugated together. In some embodiments, these at least two recombinant nucleic acids are in a single strand of recombinant nucleic acid.

[0137] In some embodiments, the recombinant nucleic acid encodes an amino acid sequence having at least 50% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59, or 60. In some embodiments, the recombinant nucleic acid encodes an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59, or 60. In some embodiments, the recombinant nucleic acid comprises a nucleic acid sequence having at least 50% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 20, 26, 33, 35, 37, 38, 42, 61, and 62. In some embodiments, the recombinant nucleic acid comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 20, 26, 33, 35, 37, 38, 42, 61, and 62.

[0138] In some embodiments, the recombinant nucleic acid molecule is operably linked to a heterologous nucleic acid sequence.

[0139] In some embodiments, the recombinant nucleic acid molecule is further defined as an expression cassette or vector. An expression cassette is generally understood to include a construct of genetic material that includes a coding sequence and sufficient control information to direct the appropriate transcription and / or translation of the coding sequence in a recipient cell, in vivo and / or ex vivo. In general, the expression cassette may be inserted into a vector for targeting a desired host cell and / or individual. Thus, in some embodiments, the expression cassette of the present disclosure includes a coding sequence for the SLP-76, CAR, or TCR polypeptide disclosed herein, which is operably linked to an expression control element, such as a promoter, and optionally any other sequence or combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0140] In some embodiments, the nucleotide sequence is incorporated into an expression vector.Those skilled in the art can understand that the term "vector" generally refers to a recombinant polynucleotide construct designed for introduction between host cells, and may be used for transformation purposes, such as the introduction of heterologous DNA into host cells.Thus, in some embodiments, the vector can be a replicon, such as a plasmid, a phage, or a cosmid, into which another DNA segment can be inserted to cause the replication of the inserted segment.In some embodiments, the expression vector can be an integrative vector.

[0141] In some embodiments, the expression vector can be a viral vector. As can be understood by those skilled in the art, the term "viral vector" is used broadly to refer to either a nucleic acid molecule (e.g., a transferable plasmid) that generally contains a virus-derived nucleic acid element that facilitates the introduction or integration of a nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid introduction. A viral particle generally contains a nucleic acid(s) as well as various viral components, and sometimes host cell components. The term viral vector can refer to either a virus or viral particle that allows the introduction of a nucleic acid into a cell, or the introduced nucleic acid itself. Viral vectors and transferable plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. In some embodiments, the vector is a vector derived from a lentivirus, adenovirus, adeno-associated virus, baculovirus, or retrovirus. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, derived primarily from lentiviruses, a genus of retroviruses.

[0142] In some embodiments, provided herein are nucleic acid molecules encoding a polypeptide with an amino acid sequence having at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to a CAR, TCR, or another receptor polypeptide disclosed herein.

[0143] The nucleic acid sequence encoding the CAR polypeptide can be optimized for expression in a host cell of interest. For example, the GC content of this sequence can be adjusted to an average level for a given cell host, calculated with reference to known genes expressed in the host cell. Methods for optimizing codon usage are known in the art. The codon usage in the coding sequence of the chimeric receptor disclosed herein can be optimized to enhance expression in a host cell, so that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons in the coding sequence are optimized for expression in a particular host cell.

[0144] The nucleic acid molecules provided can include naturally occurring sequences or sequences that differ from naturally occurring sequences but, due to the degeneracy of the genetic code, code for the same polypeptide, e.g., an antibody of ECD. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within such nucleic acids. In addition, these nucleic acid molecules can be double-stranded or single-stranded (e.g., either the sense strand or the antisense strand).

[0145] These nucleic acid molecules are not limited to sequences encoding a polypeptide (e.g., an antibody of an ECD); they may also include some or all of the non-coding sequences located upstream or downstream of a coding sequence (e.g., a coding sequence of a chimeric receptor). Those skilled in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They may be produced, for example, by treating genomic DNA with restriction endonucleases or by performing the polymerase chain reaction (PCR). In the event that the nucleic acid molecule is a ribonucleic acid (RNA), the molecule may be produced, for example, by in vitro transcription.

[0146] Recombinant Cells and Cell Cultures The nucleic acid molecule of the present disclosure can be introduced into a cell (i.e., a host cell), such as an immune cell, including a T cell, to generate a recombinant cell containing the nucleic acid molecule. In principle, there is no particular limitation regarding the cells suitable for expressing the nucleic acid molecule of the present disclosure. Thus, some embodiments of the present disclosure relate to a method of generating a recombinant cell, including: (a) providing a host cell capable of protein expression; and transducing the provided host cell with a recombinant nucleic acid of the present disclosure to generate a recombinant cell. Introduction of the nucleic acid molecule of the present disclosure into a cell can be achieved by methods known to those skilled in the art, such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, biolistic technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0147] In one aspect, the disclosure provides compositions having isolated recombinant cells modified to overexpress and / or contain elevated levels of SLP-76 polypeptide. In some embodiments, the recombinant cells are capable of being activated by target antigens expressed at low density. In some embodiments, the recombinant cells are cells expressing SLP-76 polypeptides as described herein. In some embodiments, the recombinant cells are cells expressing SLP-76 polypeptides as described herein and further expressing a CAR or TCR as described in the previous section. For example, SLP-76 constructs may be introduced into cells with or without expression of a CAR or TCR to generate recombinant cells that overexpress SLP-76 (or further overexpress a CAR or TCR), thereby enhancing cell activation by promoting proximal signaling and / or by enhancing the ability of the CAR or TCR to activate cells. In some embodiments, the recombinant cells are cells expressing a CAR or TCR. In some embodiments, the SLP-76 construct may be introduced into a cell expressing a CAR or TCR to overexpress SLP-76. In some embodiments, the recombinant cell is a cell that does not express a CAR or TCR. In some embodiments, the SLP-76 construct may be introduced into a cell that does not express a CAR or TCR to enhance cell activation. In a non-limiting embodiment, the SLP-76 construct can enhance the activation of a cell that does not express a CAR or TCR by promoting proximal signaling in the cell. In some embodiments, the cell that does not express a CAR or TCR is a NK cell. In some embodiments, the SLP-76 construct may be introduced into a cell that does not express a CAR or TCR in combination with a CAR or TCR construct (either sequentially or together) to generate a recombinant cell that overexpresses SLP-76 and a CAR or TCR, thereby enhancing the ability of the CAR or TCR to activate the cell. In a non-limiting embodiment, SLP-76 may be introduced into a native host cell or a recombinant cell for overexpression.In some embodiments, SLP-76 can be introduced into native host cells or recombinant cells that express native or endogenous TCR.In some embodiments, overexpressed SLP-76 can enhance cell activation through native or endogenous TCR.In some embodiments, SLP-76 can be introduced into recombinant cells that express recombinant TCR or CAR.In some embodiments, overexpressed SLP-76 can enhance cell activation through recombinant TCR or CAR.

[0148] Thus, in some embodiments, the nucleic acid molecule can be introduced into a host cell by a viral or non-viral delivery vehicle known in the art to generate a genetically engineered cell. For example, the nucleic acid molecule can be stably integrated into the host genome, or replicated as an episome, or present in the recombinant host cell as a small circular expression vector for stable or transient expression. Thus, in some embodiments disclosed herein, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be accomplished using classical random genome recombination techniques, or more precise genome editing techniques, such as by using zinc-finger proteins (ZNFs), guide RNA-directed CRISPR / Cas9, DNA-guided endonuclease genome editing NgAgo (Natronobacterium gregorii Argonaute), or TALEN genome editing (transcription activator-like effector nuclease).

[0149] These nucleic acid molecules can be encapsulated in viral capsids or lipid nanoparticles, or can be delivered by viral or non-viral delivery means, and methods known in the art, such as electroporation. For example, introduction of nucleic acid into cells can be achieved by viral transduction. In a non-limiting example, baculovirus-based viruses or adeno-associated viruses (AAV) can be engineered to deliver nucleic acid to target cells by viral transduction. Several AAV serotypes have been described, and all known serotypes are capable of infecting cells from multiple diverse tissue types. AAV can transduce a wide range of species and tissues in vivo without evidence of toxicity, which produces a relatively mild innate or adaptive immune response.

[0150] Lentivirus-derived vector systems are also useful for nucleic acid delivery and gene therapy by viral transduction. Lentivirus vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into the host genome; (ii) ability to infect both dividing and non-dividing cells; (iii) broad tissue tropism, including important gene and cell therapy target cell types; (iv) no expression of viral proteins after vector transduction; (v) ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) a relatively easy system for vector engineering and construction.

[0151] In some embodiments, the host cell may be genetically engineered (e.g., transduced or transformed or transfected) with a vector construct of the present application, which may be, for example, a viral vector, or a vector for homologous recombination that includes a nucleic acid sequence that is homologous to a portion of the genome of the host cell, or may be an expression vector for expression of the SLP-76, CAR, and / or TCR polypeptide of interest.

[0152] In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a mouse cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the recombinant cell is an immune system cell, e.g., a B cell, a monocyte, a NK cell, a tumor-infiltrating lymphocyte (TIL), a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell (Treg), a helper T cell (T H ), cytotoxic T cells (T CTL ), a memory T cell, a gamma delta (γδ) T cell, another T cell, a stem cell (e.g., a hematopoietic stem cell), a stem cell precursor (e.g., a hematopoietic stem cell precursor), an induced pluripotent stem cell (iPSC)-derived NK cell, or an induced pluripotent stem cell (iPSC)-derived T cell.

[0153] In some embodiments, the immune system cells are lymphocytes. In some embodiments, the lymphocytes are T lymphocytes. In some embodiments, the lymphocytes are T lymphocyte precursors. In some embodiments, the T lymphocytes are CD4+ T cells or CD8+ T cells. In some embodiments, the T lymphocytes are CD8+ cytotoxic T lymphocyte cells. Non-limiting examples of CD8+ cytotoxic T lymphocyte cells suitable for the compositions and methods disclosed herein include naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, and bulk CD8+ T cells. In some embodiments, the T lymphocytes are CD4+ helper T lymphocyte cells. Suitable CD4+ helper T lymphocyte cells include, but are not limited to, naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

[0154] In some embodiments, the cell described herein is a non-immune cell.For example, the SLP-76, CAR or TCR molecule described herein provides a method to regulate (e.g., activate) the activity of the cell expressing such molecule when it recognizes corresponding extracellular ligand(s) / antigen(s).In this sense, there is no particular limitation on suitable host cell.

[0155] The cells described herein may be of any type and / or of any origin, natural or artificial. In principle, any type of cell that an investigator wishes to investigate activation may be used for expression of the SLP-76, CAR, and / or TCR polypeptides described herein. For the prevention or treatment of the diseases described herein, cells with cytotoxic or other inhibitory functions (e.g., producing and secreting cytokines) against target cells that are correlated with the disease may be used. For example, immune cells such as T cells may be used to express these polypeptides because they have direct cytotoxicity against tumor cells expressing tumor antigens and / or they produce at least one cytokine that inhibits or kills target cells (e.g., tumor cells).

[0156] As outlined above, some embodiments of the present disclosure relate to various methods of making recombinant cells, including: (a) providing a host cell capable of protein expression; and transducing the provided host cell with a recombinant nucleic acid of the present disclosure to make a recombinant cell. Non-limiting examples of embodiments of the disclosed methods of making recombinant cells may further include one or more of the following features: In some embodiments, the host cell is obtained by leukopheresis performed on a sample obtained from a subject, and the cell is transduced ex vivo. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or lipid nanoparticle. In some embodiments, these methods further include isolating and / or purifying the produced cells. Thus, these recombinant cells produced by the methods disclosed herein are also within the scope of the present disclosure.

[0157] Techniques for transforming a wide variety of the aforementioned host cells and species are known in the art and described in the technical and scientific literature. For example, DNA vectors can be introduced into eukaryotic cells by conventional transformation or transfection techniques. Suitable methods for transforming or transfecting cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic transduction, nucleoporation, hydrodynamic shock, and infection. In some embodiments, the nucleic acid molecule is transduced into the host cell by transduction, electroporation, or gene gun techniques. Thus, cell cultures comprising at least one recombinant cell disclosed herein are also within the scope of this application. Suitable methods and systems for the creation and maintenance of cell cultures are known in the art.

[0158] In some embodiments, the recombinant cell comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an SLP-76 polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to an SLP-76 polypeptide disclosed herein. In some embodiments, the recombinant cell comprises a nucleic acid molecule encoding a polypeptide with an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs:1, 7, 14, 16, 18, 19, 40, 59, or 60. In some embodiments, the recombinant cell comprises a nucleic acid molecule having a sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs:20, 26, 33, 35, 37, 38, 42, 61, and 62.

[0159] In a related aspect, some embodiments of the present disclosure relate to a cell culture comprising at least one recombinant cell disclosed herein and a culture medium. In general, the culture medium can be any one of the culture media suitable for the cell culture described herein. In some embodiments, the recombinant cell expresses SLP-76, CAR, or TCR described herein. Thus, cell cultures comprising at least one recombinant cell disclosed herein are also within the scope of the present application. Methods and systems suitable for producing and maintaining cell cultures are known in the art.

[0160] Pharmaceutical Compositions The SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, and / or cell cultures of the present disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include the polypeptides, nucleic acids, recombinant cells, and / or cell cultures described herein and a pharma- ceutical acceptable carrier. Thus, in one aspect, some embodiments of the present disclosure relate to pharmaceutical compositions for treating, preventing, ameliorating, alleviating, or delaying the onset of a health condition, such as a proliferative disease (e.g., cancer). Other illustrative health conditions include, for example, hematological malignancies, solid tumors, autoimmune diseases, inflammation, allergic diseases, infectious diseases, senescence / aging, and the like.

[0161] Thus, one aspect of the present disclosure relates to a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and one or more of the following: (a) an SLP-76, CAR, and / or TCR polypeptide of the present disclosure; (b) a nucleic acid molecule of the present disclosure; and / or (c) a recombinant cell of the present disclosure. In some embodiments, the composition comprises (a) a recombinant nucleic acid of the present disclosure, and (b) a pharma- ceutically acceptable carrier. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or lipid nanoparticle. In some embodiments, the composition comprises (a) a recombinant cell of the present disclosure, and (b) a pharma- ceutically acceptable carrier.

[0162] In certain embodiments, pharmaceutical compositions according to some embodiments disclosed herein include cell cultures that can be washed, treated, mixed, supplemented, or otherwise modified prior to administration to an individual in need thereof. Furthermore, administration can be at varying dosages, time intervals, or repeated administrations.

[0163] The pharmaceutical compositions provided herein can be in any form that allows the composition to be administered to an individual. In some specific embodiments, the pharmaceutical compositions are suitable for human administration. As used herein, the term "pharmaceutical acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, more particularly in humans. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline and aqueous dextrose and glycerol solutions can also be utilized as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. In some embodiments, the pharmaceutical composition is formulated aseptically for administration to an individual. In some embodiments, the individual is a human. One skilled in the art can appreciate that the formulation must be suitable for the mode of administration.

[0164] In some embodiments, the pharmaceutical composition of the present disclosure is formulated to be suitable for the intended route of administration to an individual.For example, the pharmaceutical composition may be formulated to be suitable for parenteral, intraperitoneal, transrectal, intraperitoneal, and intratumoral administration.In some embodiments, the pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intratracheal, subcutaneous, intramuscular, local, or intratumoral administration.

[0165] Use of the composition The compositions described herein, e.g., SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, can be used as systems in response to extracellular signals (e.g., ligands / antigens) for a variety of conditions.

[0166] The compositions described herein may also be used as an activation system to manipulate cell function in response to certain ligands / antigens. In some embodiments, the host is activated by sensing a specific ligand / antigen or a specific profile of ligands / antigens through the ECD(s) of the CAR or TCR molecule. Such activation can enhance or inhibit the normal biological function of the host cell and / or provide exogenous signaling function to manipulate cell function. In some embodiments, the SLP-76 compositions described herein can enhance such activation through the CAR or TCR molecule.

[0167] The compositions described herein may also be used to manipulate the function of cells in response to certain ligands / antigens. In some embodiments, the host is activated by sensing a specific ligand / antigen through the ECD(s) of the CAR or TCR molecule. Such activation can enhance or inhibit the function of the host cell to alter the function of the target cell that expresses such ligand(s) / antigen(s) or is specifically recognized by a certain ligand(s) / antigen(s). In some embodiments, the SLP-76 compositions described herein can enhance such activation through the CAR or TCR molecule. For example, a cancer cell that expresses a certain ligand(s) / antigen(s) may be recognized by the CAR molecule(s) or CAR molecule combination(s) described herein either through an ECD that binds to the ligand(s) / antigen(s) on the surface of the cancer cell or an ECD that binds to some ligand(s) / antigen(s) that specifically bind to the ligand(s) / antigen(s) expressed on the cancer cell. The activated host cells can then manipulate the function of the cancer cells. For example, the compositions described herein can be used to activate host cells (e.g., immune system cells such as T cells) to inhibit or kill (e.g., by secreting cytokines or directly killing) the cancer cells. In some embodiments, such cancer cells are obtained from a biological sample from a subject. In some embodiments, such cancer cells are in the biological environment (e.g., cancer microenvironment) of the subject. In some embodiments, the target cells are correlated with a disease or disorder. Illustrative diseases or disorders can include, for example, proliferative diseases (e.g., cancer), hematological malignancies, solid tumors, autoimmune diseases, inflammation, allergic diseases, infectious diseases, senescence / aging, etc.

[0168] Manufacturing method In one aspect, the disclosure provides a method of making a recombinant cell as described herein. The compositions described herein, such as SLP-75, CAR, and / or TCR polypeptides or nucleic acids, may be introduced into the cells described herein. For example, a composition comprising a polynucleotide encoding an SLP-76 polypeptide may be introduced into a cell, either alone or in combination with a polynucleotide encoding a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide into the cell.

[0169] Treatment method In one aspect, the present disclosure provides a method of enhancing activation of a cell in response to a target antigen, comprising overexpressing an SLP-76 polypeptide in a cell. In some embodiments, the cell further comprises a TCR and / or a CAR molecule.

[0170] In another aspect, the disclosure provides a method of enhancing activation of a cell in response to a target antigen comprising overexpressing an SLP-76 polypeptide and a T cell receptor (TCR) polypeptide and / or a chimeric antigen receptor (CAR) polypeptide in a cell.

[0171] In another aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a medicamentously effective amount of a recombinant cell, polypeptide, polynucleotide, expression vector, or other composition described herein. Administration of any one of the therapeutic compositions described herein, e.g., SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, can be used in the diagnosis, prevention, and / or treatment of conditions associated with, such as, proliferative diseases (e.g., cancer), hematological malignancies, solid tumors, autoimmune diseases, inflammation, allergic diseases, infectious diseases, senescence / aging, and the like. In some embodiments, the SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be incorporated into therapies and therapeutics for use in methods of preventing and / or treating individuals having, suspected of having, or at high risk of developing one or more health conditions, such as a proliferative disease (e.g., cancer, such as leukemia, neuroblastoma, or osteosarcoma). In some embodiments, the individual is a patient under the supervision of a clinician.

[0172] Exemplary proliferative diseases can include, but are not limited to, angiogenic diseases, metastatic diseases, tumorigenic diseases, neoplastic diseases, and cancer. In some embodiments, the proliferative disease is cancer. In some embodiments, the cancer is childhood cancer. In some embodiments, the cancer is pancreatic cancer, colon cancer, ovarian cancer, prostate cancer, lung cancer, mesothelioma, breast cancer, urothelial cancer, liver cancer, head and neck cancer, sarcoma, cervical cancer, gastric cancer, gastric cancer, melanoma, uveal melanoma, cholangiocarcinoma, multiple myeloma, leukemia, lymphoma, blood cancer, bladder cancer, neuroblastoma, malignant pleural mesothelioma, sarcoma, and glioblastoma. Exemplary cancer types also include: acute myeloid leukemia, hematologic immunoblastic T-cell lymphoma, B-cell acute lymphoblastic leukemia, Sweet's syndrome, T-cell non-Hodgkin's lymphoma (including natural killer / T-cell lymphoma, adult T-cell leukemia / lymphoma, intestinal T-cell lymphoma, hepatosplenic T-cell lymphoma, and cutaneous T-cell lymphoma), T-cell acute lymphoblastic leukemia, B-cell non-Hodgkin's lymphoma (including Burkitt's lymphoma, diffuse large B-cell ... lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, etc.), hairy cell leukemia, Hodgkin's lymphoma, lymphoblastic lymphoma, lymphoplasmacytic lymphoma, mucosa-associated lymphoid tissue lymphoma, multiple myeloma, myelodysplastic syndromes, plasma cell myeloma, primary mediastinal large B-cell lymphoma, chronic myeloproliferative syndromes (chronic myelogenous leukemia, primary myelofibrosis, essential thrombocythemia, polycythemia vera, etc.) and chronic lymphocytic leukemia. Illustrative cancer types also include: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancer, Kaposi's sarcoma (a sarcoma of soft tissue), AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, childhood brain tumors, atypical teratoma / rhabdoid tumor, central nervous system cancer, skin cancer (e.g., basal cell carcinoma), bile duct cancer, bladder cancer, bone cancer (Ewing's sarcoma, osteosarcoma and malignant fibrous histiocytoma), brain tumors, breast cancer, bronchial tumors, Burkitt's lymphoma, non-Hodgkin's lymphoma, carcinoid tumors, cardiac (heart) tumors, medulloblastoma and other CNS embryonal tumors, germ cell tumors, primary CNS lymphoma, cervical cancer, childhood cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma,Cutaneous T-cell lymphoma, mycosis fungoides, Sézary syndrome, ductal carcinoma in situ (DCIS), embryonal tumors, medulloblastoma, endometrial cancer (uterine cancer), ependymoma, esophageal cancer, nasal neuroblastoma (head and neck cancer), Ewing's sarcoma, extracranial germ cell tumors, childhood extragonadal germ cell tumors, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST) (soft tissue sarcomas), germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head Cervical cancer, cardiac tumors, childhood hepatocellular (liver) carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer (head and neck cancer), intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney (renal cell) cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, cancer of the lip and oral cavity, liver cancer, lung cancer (e.g., non-small cell, small cell, pleuropulmonary blastoma, and tracheobronchial tumors), lymphoma, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, melanoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer of unknown primary, midline tract) carcinoma, oral cavity cancer, multiple endocrine neoplasia, multiple myeloma / plasma cell neoplasm, mycosis fungoides (lymphoma), myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloid leukemia, chronic myeloproliferative neoplasm, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, cancer of the lip and oral cavity, and oropharyngeal cancer, osteosarcoma, undifferentiated pleomorphic sarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumor (inslet cell tumor), papillomatosis (pediatric larynx), paraganglioma, cancer of the paranasal sinuses and nasal cavity, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma (lung cancer), pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, childhood rhabdomyosarcoma, childhood hemangioma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer of unknown primary, gastric (stomach) cancer, T-cell lymphoma, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, tracheobronchial tumors, transitional cell carcinoma of the renal pelvis and ureter, carcinoma of the ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, Wilms' tumor,and other pediatric kidney tumors.

[0173] In some embodiments, the cancer is a multidrug resistant cancer or a recurrent cancer. It is contemplated that the compositions and methods disclosed herein are suitable for both non-metastatic and metastatic cancers. Thus, in some embodiments, the cancer is a non-metastatic cancer. In some other embodiments, the cancer is a metastatic cancer. In some embodiments, the composition administered to the subject inhibits metastasis of the cancer in the subject. In some embodiments, the composition administered inhibits tumor growth in the subject.

[0174] Accordingly, in one aspect, some embodiments of the present disclosure relate to methods of preventing and / or treating a condition in a subject in need thereof, wherein these methods comprise administering to the subject a composition containing one or more of the following: an SLP-76, CAR, or TCR polypeptide of the present disclosure, a recombinant nucleic acid of the present disclosure, a recombinant cell of the present disclosure, and / or a pharmaceutical composition of the present disclosure.

[0175] In some embodiments, the administered composition inhibits the proliferation of the target cancer cells and / or inhibits the tumor growth of the cancer in the subject. For example, the target cells may be inhibited if their proliferation is decreased, if their pathological or pathogenic behavior is alleviated, if they are destroyed or killed, etc. Inhibition includes at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% reduction in the measured pathological or pathogenic behavior. In some embodiments, the methods include administering to an individual an effective number of recombinant cells disclosed herein, wherein the recombinant cells inhibit the proliferation of the target cancer cells and / or inhibit the tumor growth of the target cancer in the subject compared to the proliferation of the target cells and / or the tumor growth of the target cancer in a subject to which the recombinant cells are not administered. In some embodiments, the target cancer cell is a leukemia cancer cell, a cell derived from a leukemia cancer cell, or a cell in the microenvironment of a leukemia.In some embodiments, the target cancer cell is a neuroblastoma cell, a cell derived from a neuroblastoma cell, or a cell in the microenvironment of a neuroblastoma.In some embodiments, the target cancer cell is an osteosarcoma cell, a cell derived from an osteosarcoma cell, or a cell in the microenvironment of an osteosarcoma.

[0176] As used herein, the terms "administration" and "administering" refer to the delivery of a bioactive composition or formulation by a route of administration including, but not limited to, oral, intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or a combination thereof. This term includes, but is not limited to, administration by a medical professional and self-administration.

[0177] Administration of compositions described herein, such as SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, can be used in stimulating immune responses. In some embodiments, SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein are administered to an individual after induction of cancer remission by chemotherapy or after autologous or allogeneic hematopoietic stem cell transplantation. In some embodiments, compositions described herein are administered to an individual in need of increased production of interferon gamma (IFNγ), TNF-α, and / or interleukin-2 (IL-2) in a treated subject compared to the production of these molecules in a subject not administered one of the therapeutic compositions disclosed herein.

[0178] The effective amount of the compositions described herein, e.g., SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, is determined based on the intended goal, e.g., tumor regression. For example, when an existing cancer is being treated, the amount of the compositions disclosed herein to be administered may be greater than when the administration of the composition is directed to the prevention of cancer. One of skill in the art will be able to determine the amount of the composition to be administered and the frequency of administration in light of this disclosure. The amount to be administered will also depend on the individual being treated, the condition of the individual, and the protection desired, according to both the number and dosage of treatments. The exact amount of the composition will also depend on the judgment of the practitioner, and will be particularly individual. The frequency of administration can range from 1-2 days, to 2-6 hours, to 6-10 hours, to 1-2 weeks, or longer, depending on the judgment of the practitioner.

[0179] In some embodiments, administration is by bolus injection. In some embodiments, administration is by intravenous infusion. In some embodiments, the composition containing the recombinant cells described herein is administered to the subject at a dose of about 1 million cells / kg body weight per day to about 100 million cells / kg body weight per day. In some embodiments, the composition containing the recombinant cells disclosed herein is administered at a dose of about 1000 cells / kg to about 100 million cells / kg body weight per day. In some embodiments, these therapeutic agents are administered in a single dose. In some embodiments, the therapeutic agent is administered in repeated doses (e.g., once or more per week for one or more weeks).

[0180] Those of skill in the art will be familiar with the techniques for administering the compositions of the present disclosure to an individual, and further, will be familiar with the techniques and pharmaceutical agents necessary for the preparation of these compositions prior to administration to an individual.

[0181] In certain embodiments of the present disclosure, the compositions of the present disclosure include aqueous compositions comprising one or more of the SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein. The aqueous compositions of the present disclosure include an effective amount of the compositions disclosed herein in a pharma- ceutical acceptable carrier or aqueous medium. Thus, the "pharmaceutical preparation" or "pharmaceutical composition" of the present disclosure can include any or all of the following: solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the recombinant cells disclosed herein, its use in the manufacture of pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into these compositions. For human administration, preparations must meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Center for Biologics Evaluation and Control.

[0182] Those skilled in the art will understand that the biological material may be dialyzed extensively, if appropriate, to remove undesirable small molecular weight molecules, and / or lyophilized for easier formulation into the desired vehicle. The compositions described herein, such as SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, may then generally be formulated for administration by any known route, such as parenteral administration. The determination of the amount of the composition to be administered may be determined by one of skill in the art and may depend in part on the extent and severity of the cancer, and on whether the recombinant cells are administered to treat an existing cancer or to prevent cancer. The preparation of compositions containing the SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions of the present disclosure may be known to one of skill in the art in view of the present disclosure.

[0183] Upon formulation, the compositions of the present disclosure can be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The compositions can be administered in a variety of dosage forms, such as the type of injectable solutions described above.

[0184] In some embodiments, the SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be used to overcome T cell exhaustion in treated subjects compared to corresponding T cells or in subjects not administered one of the therapeutic compositions disclosed herein. In some embodiments, the SLP-76, CAR, and / or TCR polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be used to stimulate the proliferation and / or killing capacity of CAR T cells in treated subjects compared to the production of these molecules in subjects not administered one of the therapeutic compositions disclosed herein. Production of interferon gamma (IFNγ), tumor necrosis factor-α (TNF-α), and / or interleukin-2 (IL-2) can be stimulated to be up to about 20-fold, such as about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold or more, relative to interferon gamma (IFNγ), tumor necrosis factor-α (TNF-α), and / or interleukin-2 (IL-2) production in a subject not administered one of the therapeutic compositions disclosed herein.

[0185] Administration of Recombinant Cells to a Subject In some embodiments, the methods of the present disclosure involve administering an effective amount or number of recombinant cells provided herein to a subject in need thereof. This administering step can be accomplished using any method of implant delivery in the art. For example, the recombinant cells can be directly injected into the subject's bloodstream or otherwise administered to the subject.

[0186] In some embodiments, the methods disclosed herein include administering recombinant cells to an individual by a method or route that results in at least partial localization of the introduced cells at the desired site so that the desired effect(s) occur, which terms are used interchangeably with "introducing," "implanting," and "implanting." These recombinant cells or their differentiated progeny can be administered by any suitable route that results in delivery to a desired location in the individual where at least a portion of the administered cells or components of the cells remain viable. The period of survival of the cells after administration to a subject can be as short as a few hours, e.g., 24 hours, a few days, or as long as several years, or even the lifetime of the individual, i.e., long-term engraftment.

[0187] In some embodiments, delivery of a recombinant cell composition (e.g., a composition comprising a plurality of recombinant cells according to any of the cells described herein) to a subject by a method or route results in at least partial localization of the cell composition at a desired site. Modes of administration include, for example, injection, infusion, and infusion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For cell delivery, delivery by injection or infusion is the standard mode of administration.

[0188] In some embodiments, the recombinant cells are administered systemically, for example, by infusion or injection.

[0189] kit Various kits for practicing the methods described herein are also provided herein. In particular, some embodiments of the present disclosure provide kits for the diagnosis of a condition in a subject. Some other embodiments relate to kits for the prevention of a condition in a subject in need thereof. Some other embodiments relate to kits for methods of treating a condition in a subject in need thereof. For example, in some embodiments, kits are provided herein that include one or more SLP-76, CAR, and / or TCR polypeptides, recombinant nucleic acids, engineered cells, or pharmaceutical compositions provided and described herein, as well as written instructions for their manufacture and use.

[0190] In some embodiments, the kits of the present disclosure further comprise one or more means useful for administering any one of the provided SLP-76, CAR, and / or TCR polypeptides, recombinant nucleic acids, engineered cells, or pharmaceutical compositions to an individual. For example, in some embodiments, the kits of the present disclosure further comprise one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any one of the provided SLP-76, CAR, and / or TCR polypeptides, recombinant nucleic acids, engineered cells, or pharmaceutical compositions to an individual. In some embodiments, the kits can comprise one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, e.g., to diagnose, prevent, or treat a condition in a subject in need thereof.

[0191] In some embodiments, the kits can further include instructions for using the components of the kit to practice the methods disclosed herein.

[0192] No admission is made that any reference cited herein constitutes prior art. The discussion of these references refers to what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. Many sources of information, including scientific journal articles, patent documents, and texts, are referred to herein; however, it should be clearly understood that this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0193] The discussion of general methods provided herein is intended for illustrative purposes only: other alternative methods and substitutes may be apparent to those of skill in the art upon consideration of this disclosure, and are within the spirit and scope of the present application. EXAMPLES

[0194] Working Example Additional embodiments are disclosed in further detail in the following examples, which are provided for illustrative purposes and are in no way intended to limit the scope of the disclosure or claims.

[0195] Example 1 Augmenting CAR T cell activation by proximal signaling molecules This example describes experiments performed to demonstrate the ability of proximal signaling molecules to enhance CAR T cell activation when overexpressed. Exemplary CAR molecules that recognize different extracellular ligands were engineered and tested for their ability to activate these molecules and cells expressing various proximal signaling molecules.

[0196] Proximal signaling molecules were tested to see whether any of them were sufficient to enhance T cell activation by CAR molecules. CAR constructs were prepared by directly linking scFv (recognizing, for example, CD19 or HER2), CD8 or CD28 hinge-transmembrane domain (H / TM), and 4-1BB costimulatory domain to CD3 zeta domain. These engineered CARs were co-expressed in primary T cells with constructs of proximal signaling molecules. Cytokine production by these T cells in the presence of tumor cells expressing CD19 or HER2 antigens was measured and compared. Figure 1 shows FACS histograms that determine the extracellular ligand binding domain of each CAR molecule, such as CD19 (upper left panel) or HER2 (upper right panel), which represents the expression of CAR molecules in T cells. When T cells expressing CD19 CAR molecules are incubated with wild-type NALM6 cells expressing CD19 at high density, or when T cells expressing HER2 CAR molecules are incubated with NALM6 cells expressing high density HER2, the CAR molecules activate the corresponding T cells to produce cytokines (e.g., IL-2). As shown in the lower panel of FIG. 1, such activation (represented by the level of IL-2 production) by any of the CAR constructs is enhanced only by overexpression of SLP-76 (e.g., SEQ ID NO:7) in the T cells, but not by other proximal signaling molecules, such as LAT (e.g., SEQ ID NO:8) or LCK (e.g., SEQ ID NO:9).

[0197] One limitation of current CAR T therapy is that tumors can evade CAR T cells by downregulating the amount of expressed tumor antigens that are normally used as target antigens for recognition by CAR molecules on CAR T cells. CAR T cell activity can be limited when antigen density is low. The above co-expression system was then tested under an experimental setting of low antigen density. Specifically, T cells expressing CD19 CAR molecules were incubated with wild-type NALM6 cells or NALM6 cells expressing low density CD19 on the cell surface with or without overexpression of SLP-76 (SEQ ID NO: 7). T cell activation was measured by IL-2 production as before. As shown in Figure 2, overexpression of SLP-76 (SEQ ID NO: 7; not bound to the cell membrane) enhanced T cell activation via CD19 CAR molecules in response to only wild-type NALM6 cells, but not to cells with low antigen density.

[0198] Most proximal signaling molecules are cytosolic and do not have a transmembrane domain. To improve the function of SLP-76 to enhance CAR T activity in response to low antigen density, membrane-bound constructs were prepared and tested. One illustrative construct (VSV-G-CD8TM-SLP-76; SEQ ID NO: 16) contains a CD8 hinge-transmembrane (H / TM) domain conjugated to the N-terminus of SLP-76, plus a short extracellular tag (VSV-G polypeptide) for easy detection by FACS. A similar membrane-bound LCK construct (VSV-G-CD8TM-LCK; SEQ ID NO: 17) was prepared as a control. Compared to the cytosolic SLP-76 (SEQ ID NO: 7) shown in Figures 1 and 2, the exemplary membrane-bound SLP-76 not only further enhanced T cell activity in response to wild-type NALM6 cells (which express high density CD19), but also enhanced T cell activity in response to low density antigens on the target NALM6 cells (Figures 3A and 3B). Anti-idiotypic antibodies that recognize the extracellular domain of the CD19 CAR were used to activate or stimulate T cells, and lower amounts of such antibodies were sufficient to activate T cells overexpressing membrane-bound SLP-76 (Figure 3C). These data indicate that overexpression of membrane-bound SLP-76 lowers the CAR T cell activation threshold.

[0199] Experiments were also performed with T cells expressing a HER2-targeting CAR molecule (HER2-CD28TM-BBZ). Similar to Figures 3A-3C, membrane-bound SLP-76 (SEQ ID NO: 16) not only further enhanced T cell activity in response to wild-type NALM6 cells expressing high levels of HER2 or wild-type 143B osteosarcoma cells expressing HER2, but also in response to target cell clones with low or very low antigen density (Figures 4A and 4B). Plates were coated with recombinant Her2 antigen, and a lower amount of the antigen coated was sufficient to activate T cells when membrane-bound SLP-76 was overexpressed (Figure 4C).

[0200] In a separate experiment, T cells were transduced to overexpress an illustrative CD19-targeting CAR construct ("CD19-CD8-BBZ") with or without a free ZAP-70 construct (ZAP-70-2A-tNGFR, SEQ ID NO:44), a free PLCγ1 (also known as PLCg1 or PLCG) construct (PLCg1-2A-tNGFR, SEQ ID NO:48), a membrane-bound ZAP-70 construct [VSV-G-CD8-ZAP-70 KIDB(255-600), comprising a fragment of ZAP-70 interdomain B and kinase domain; SEQ ID NO:46], a membrane-bound PLCγ1 construct (VSV-G-CD8-PLCg1, SEQ ID NO:50), or the membrane-bound SLP-76 construct previously described (SEQ ID NO:16) (FIG. 13A). As shown in Figure 13B, only membrane-bound SLP-76, but not free or membrane-bound ZAP-70 or PLCγ1 constructs, significantly increased cytokine (e.g., IL-2 and IFNγ) production by T cells expressing CD19-targeting CAR constructs when contacted with wild-type NALM-6 cells.

[0201] Example 2 Membrane-bound SLP-76 enhances CAR T activity independent of SLP-76 or CAR structure This example describes experiments performed to confirm the ability of membrane-bound SLP-76 to enhance CAR T activation by various CAR molecules.

[0202] One illustrative CAR construct contains an extracellular domain for recognizing CD19, a CD28 transmembrane domain and an intracellular region, and a CD3 zeta signaling domain ("CD19-CD28TM-CD28Z" or "CD19-28Z"). T cells expressing such a CAR construct were incubated with various tumor cells with or without overexpression of a membrane-bound SLP-76 construct (SEQ ID NO: 16) (Figure 5A). IL-2 production levels were measured by ELISA for these T cells after incubation with tumor cells, indicating that membrane-bound SLP-76 significantly enhanced T cell activity in response to either normal or low antigen density (Figure 5B). Cytotoxicity index also confirmed such ability of membrane-bound SLP-76 to enhance T cell activation to kill tumor cells at low antigen density (Figure 5C). In an in vivo wild-type NALM6 stress model, membrane-bound SLP-76, when expressed in CAR T cells, significantly reduced tumor burden compared to treatment with CAR T cells that did not express membrane-bound SLP-76, particularly at 14 days after treatment (Figures 5D and 5F). Correspondingly, membrane-bound SLP-76 significantly enhanced CAR T cell function and increased animal survival (Figure 5E).

[0203] Similar experiments were performed with another illustrative CAR construct ("CD22-CD8TM-BBZ"), which includes an extracellular domain recognizing CD22, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. Downregulation of CD22 is a known mechanism of antigen escape from CD22 CAR T cell therapy in patients (Fry et al., Nature Medicine, 2017). T cells expressing such a CAR construct (Figure 6A) were incubated with tumor cells with or without overexpression of a membrane-bound SLP-76 construct (SEQ ID NO: 16). IL-2 production levels were measured by ELISA for these T cells after incubation with tumor cells, showing that membrane-bound SLP-76 significantly enhanced T cell activity in response to low antigen density (Figure 6B). Recombinant CD22 was used as the target antigen to activate T cells by recombinant antigen stimulation assay as described above. Lesser amounts of such antigens coated were sufficient to activate T cells when membrane-bound SLP-76 was overexpressed (Figure 6C). In an in vivo CD22 low NALM6 model, membrane-bound SLP-76, when expressed on CAR T cells, significantly reduced tumor burden (e.g., 22 days after treatment as shown in Figure 6D) compared to CAR T cells that did not express membrane-bound SLP-76 (Figures 6D-6F).

[0204] Similar experiments were performed with another illustrative CAR construct ("B7-H3-CD28TM-BBZ"), which includes an extracellular domain that recognizes B7-H3, a CD28 transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3 zeta signaling domain. T cells expressing such a CAR construct were incubated with tumor cells with or without overexpression of a membrane-bound SLP-76 construct (SEQ ID NO: 16) (Figure 7A). The cytotoxicity index was calculated by detecting surviving target tumor cells (neuroblastoma cell line CHLA-255, which expresses low density of B7-H3) after treatment, indicating that membrane-bound SLP-76 significantly enhances the activity of T cells expressing B7-H3-CD28TM-BBZ to inhibit or kill target tumor cells (Figure 7B). In an in vivo CHLA-255 (which express low density B7-H3) metastatic model, membrane-bound SLP-76, when expressed on CAR T cells, significantly reduced tumor burden throughout the experimental period, whereas CAR T cells not expressing membrane-bound SLP-76 led to an increase in tumor burden 7-14 days after treatment (Figure 7C). As a result, animals treated with CAR T cells overexpressing membrane-bound SLP-76 survived for more than 100 days, which was much longer than animals treated with CAR T cells not expressing membrane-bound SLP-76 (Figure 7D).

[0205] Thus, the ability of membrane-bound SLP-76 to enhance CAR T activity is not limited to a particular CAR construct.

[0206] CAR constructs with different proximal signaling molecules were further used to test the function of membrane-bound SLP-76. For example, an illustrative CAR construct was made to include ZAP70 as the intercellular signaling domain rather than the CAR molecule with CD3 zeta signaling domain described above (CD19-CD28TM-ZAP70). T cells expressing such CD19-targeting CAR molecules were incubated with tumor cells with or without overexpression of membrane-bound SLP-76 construct (SEQ ID NO: 16) (Figure 8A). IL-2 production levels were measured by ELISA for these T cells after incubation with tumor cells, indicating that membrane-bound SLP-76 significantly enhanced T cell activity via this CAR molecule (Figure 8B). In an in vivo NALM6 xenograft model, membrane-bound SLP-76, when expressed on CAR T cells, significantly reduced tumor burden compared to CAR T cells that did not express membrane-bound SLP-76 (Figure 8C). As a result, animals treated with CAR T cells overexpressing membrane-bound SLP-76 had a higher survival rate than animals treated with CAR T cells that did not express membrane-bound SLP-76 (Figure 8D).

[0207] Similar experiments were performed using T cells expressing an exemplary CAR construct ("HA 28Z") comprising a high affinity (HA) GD2-targeting extracellular domain, a CD28 transmembrane and costimulatory domain, and a CD3 zeta signaling domain, with or without different membrane-bound SLP-76 constructs. For example, an exemplary membrane-bound SLP-76 construct comprises a CD28 transmembrane domain (SEQ ID NO: 19; "CD28TM SLP-76"). Another exemplary membrane-bound SLP-76 construct comprises a CD8 transmembrane domain (SEQ ID NO: 18; "CD8TM SLP-76"). T cells expressing such high affinity GD2-targeting CAR molecules were incubated with tumor cells with or without overexpression of an exemplary membrane-bound SLP-76 construct (Figure 9A). IL-2 production levels were measured by ELISA in these T cells after incubation with various tumor cells, indicating that membrane-bound SLP-76 significantly enhanced CAR T cell activity, regardless of the transmembrane domain utilized in the membrane-bound SLP-76 construct (Figure 9B).

[0208] This example demonstrates that membrane-bound SLP-76 enhances the activity of a CAR whether its scFv (antigen-specific), signaling domain (costimulatory or other), or transmembrane domain is included in the CAR construct or the transmembrane domain is utilized in a membrane-bound SLP-76 construct.

[0209] Example 3 Further advantages of membrane-bound SLP-76 This example describes experiments that were performed to test the effect of membrane-bound SLP-76 on T cell exhaustion and to demonstrate its advantages over reported SLP-76 fusion proteins.

[0210] Persistent activation of T cells expressing TCR or CAR can lead to the gradual development of an exhausted phenotype, usually due to chronic infection or tumor progression. Various CAR constructs described in the previous examples were tested for their effect on T cell exhaustion when overexpressed. Specifically, FACS analysis was performed to detect T cell exhaustion biomarkers expressed on the surface of T cells, including, for example, TIM-3, PD-1, and LAG-3, while the expression levels of such biomarkers correlated with the degree of T cell exhaustion. As shown in Figure 10, overexpression of the B7-H3-CD28TM-BBZ CAR construct described in the previous examples induced moderate expression of all three biomarkers, indicating that the B7-H3-CD28TM-BBZ CAR construct can induce moderate T cell exhaustion. Surprisingly, although membrane-bound SLP-76 (CD19-CD8TM-SLP-76, SEQ ID NO: 40) can amplify CAR T activity and lower the activation threshold, it did not exacerbate CAR T cell exhaustion because co-expression of membrane-bound SLP-76 did not increase biomarker expression levels (Figure 10). Figure 11 shows more severe CAR T exhaustion by overexpression of HA 28Z CAR construct. Overexpression of membrane-bound SLP-76 (Her2-CD8H / TM-SLP-76, SEQ ID NO: 18) in CAR T cells did not exacerbate cell exhaustion either (Figure 11). Therefore, with regard to the issue of T cell exhaustion, the use of membrane-bound SLP-76 to boost other CAR T therapies is safe.

[0211] LAT / SLP-76 chimeric proteins (LAT transmembrane fused to SLP-76) restore TCR signaling in T cells with the endogenous LAT gene knocked out (Boerth et al., J. Exp. Med. 2000, 192:1047-1058). Such chimeras were compared to the membrane-bound SLP-76 constructs described in the previous examples. T cells expressing the CD19 28Z CAR construct described above, T cells overexpressing the membrane-bound SLP-76 construct (VSV-G-CD8TM-SLP-76, SEQ ID NO: 16) or the LAT / SLP-76 chimeric construct were incubated with tumor cells (Figure 12A). IL-2 production levels were measured by ELISA for these T cells after incubation with tumor cells, which showed that membrane-bound SLP-76 has a higher ability to enhance T cell activity in response to high antigen density than the chimera (Figure 12B, WT N6). More importantly, membrane-bound SLP-76 can enhance CAR T activity in response to low antigen density, but the chimera cannot (Figure 12B, clone F N6). Thus, membrane-bound SLP-76 has an advantage over LAT / SLP-76 chimera, especially when antigen density is low, which is used by tumor cells to avoid current T cell therapy with CAR or TCR.

[0212] Example 4 Further investigation of membrane-bound SLP-76 in various cell or animal models This example describes experiments that were performed to test the effect of membrane-bound SLP-76 in enhancing T cell activation and function in various cellular and animal models.

[0213] In an illustrative experiment, T cells were transduced to overexpress an anaplastic lymphoma kinase (ALK)-targeting CAR construct ("ALK-CD8TM-BBZ"). This ALK-targeting CAR construct was previously found to be poorly expressed (at low levels) on the surface of T cells, limiting its therapeutic utility and its ability to fully activate T cells (Walker et al., Molecular Therapy, 2017: 25(9):2189-2201; PMID 28676342). ALK-CD8TM-BBZ was expressed on T cells with or without a membrane-bound SLP-76 construct (Figures 14A-14B) and then co-cultured with NALM-6 cancer cells expressing different levels of the ALK antigen (Figure 14C). This membrane-bound SLP-76 construct significantly increased both IL-2 and IFNγ production by T cells when contacted with NALM-6 cells expressing high levels of ALK, and significantly increased IFNγ but not IL-2 production by T cells when contacted with NALM-6 cells expressing intermediate or low levels of ALK (Figure 14D). The cytotoxicity of T cells against target NALM-6 cells was measured. Figure 14E illustrates that this membrane-bound SLP-76 construct significantly increased the cytotoxicity of T cells against all three lines of target NALM-6 cells with different ALK densities.

[0214] In another illustrative experiment, T cells engineered to express the NY-ESO-1 transgenic TCR construct (A2+ / NY-ESO-1) were transduced with or without a membrane-bound SLP-76 construct (Figure 15A) and co-cultured with either antigen-positive tumor cells (A2+ / NY-ESO-1+Nalm-6 or A375) or antigen-negative tumor cells Mel888. Similarly, the membrane-bound SLP-76 construct significantly increased IL-2 production by T cells when contacted with antigen-positive NALM-6 or A375 cells, but not with antigen-negative Mel888 cells (Figure 15B). The membrane-bound SLP-76 construct also increased the cytotoxicity of T cells when co-cultured with antigen-positive NALM-6 cells (Figure 15C).

[0215] In another illustrative experiment, T cells expressing a B-cell maturation antigen (BCMA)-targeting CAR construct were transduced with or without a membrane-bound SLP-76 construct (Figure 16A) and co-cultured with MM1.S tumor cells. Similarly, the membrane-bound SLP-76 construct significantly increased cytokine (e.g., IL-2 and IFNγ) production by T cells (Figure 16B). In mice inoculated with luciferase-expressing MM1.S tumor cells and treated with these T cells, the membrane-bound SLP-76 construct enhanced the ability of T cells to control tumor growth (Figure 16C).

[0216] In another illustrative experiment, T cells expressing a B7-H3-targeting CAR construct ("B7-H3-CD28TM-BBZ") were transduced with or without a membrane-bound SLP-76 construct (Figure 17A). In mice inoculated with luciferase-expressing diffuse intrinsic pontine glioma 6 xenografts (DIPG-6) and treated with these T cells, the membrane-bound SLP-76 construct enhanced the ability of the T cells to control tumor growth (Figure 17B).

[0217] In another illustrative experiment, T cells expressing a CD19-targeting CAR construct ("CD19-CD28TM-CD28Z") were transduced with or without a membrane-bound SLP-76 construct (Figure 18A). In a B-cell acute lymphoblastic leukemia (B-ALL) model, mice were inoculated with luciferase-expressing wild-type Nalm-6 cells and treated with these T cells. The membrane-bound SLP-76 construct increased T cell proliferation in either the spleen or bone marrow (Figure 18B).

[0218] In another illustrative experiment, T cells expressing a CD19-targeting CAR construct ("CD19-CD28TM-BBZ") were transduced with or without a membrane-bound SLP-76 construct (Figure 19A). In a stress test model (split-dose of CAR T cells), mice were inoculated with luciferase-expressing leukemia xenografts (WT Nalm-6) and treated with these T cells. The membrane-bound SLP-76 construct increased the ability of T cells to control tumor growth (Figure 19B) and improved mouse survival (Figure 19C).

[0219] Further experiments were performed to test the function of SLP-76 mutants. In an illustrative experiment, T cells were transduced to express a CD19-targeting CAR construct ("CD19-CD8TM-BBZ") alone or together with a membrane-bound SLP-76 construct (SEQ ID NO: 16) or a membrane-bound SLP-76 mutant with a K30R substitution (SEQ ID NO: 60) described herein (Figures 20A-20B). This K30R mutation was previously reported to be capable of reducing ubiquitination downregulation of SLP-76 and thus enhancing downstream T cell activation. The resulting T cells were incubated with NALM-6 tumor cells expressing low levels of CD19 (target antigen) while the levels of IL-2 produced by these T cells were measured. As shown in FIG. 20C, the membrane-bound SLP-76 K30R mutant enhanced T cell activity in response to target antigen on tumor cells (as indicated by IL-2 expression levels), and was more potent in this scenario with low levels of target antigen on tumor cells than the membrane-bound SLP-76 wild-type construct.

[0220] Sequence Listing The table below lists exemplary sequences of various molecules. [Table 1-1] [Table 1-2]

Claims

1. 1. A composition comprising an isolated recombinant cell modified to overexpress and / or contain elevated levels of an SLP-76 polypeptide, wherein said isolated recombinant cell comprises a chimeric antigen receptor (CAR) polypeptide having binding affinity for a target antigen expressed by a target cell or for an adapter molecule that specifically recognizes said target antigen, and wherein said isolated recombinant cell is capable of being activated by said target antigen expressed at low density by said target cell.

2. 2. The composition of claim 1, wherein the target cell is a cell correlated with a proliferative disease, a hematological malignancy, a solid tumor, an autoimmune disease, inflammation, an allergic disease, an infectious disease, and / or senescence / aging.

3. The composition of claim 2 , wherein the target cell is a cancer cell.

4. 34. The CAR polypeptide comprising: a) an extracellular ligand-binding domain that has binding affinity for a target antigen or an adaptor molecule that specifically recognizes the target antigen; b) a transmembrane domain; c) an intracellular signaling domain, including a proximal signaling molecule; and d) optionally a hinge domain and / or a costimulatory domain: The composition of claim 1 , comprising:

5. A composition described in any one of claims 1 to 4, wherein the CAR polypeptide induces attrition of the isolated recombinant cell, and overexpression of the SLP-76 polypeptide does not enhance attrition of the recombinant cell.

6. The composition of any one of claims 1 to 5, wherein the isolated recombinant cell expresses the CAR polypeptide at low density.

7. The composition of any one of claims 1 to 6, wherein said SLP-76 polypeptide is bound to the cell membrane of said isolated recombinant cell.

8. The SLP-76 polypeptide comprises: i) a transmembrane domain; ii) the interaction between the SLP-76 polypeptide and a membrane protein; iii) a covalent bond between an SLP-76 polypeptide and a fatty acid in the membrane; and / or iv) Binding between the SLP-76 polypeptide and the highly polar head groups of lipids in membranes: The composition of claim 7, which is attached to a cell membrane via

9. The composition of any one of claims 1 to 8, wherein the SLP-76 polypeptide comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59 or 60.

10. The composition of any one of claims 1 to 9, wherein the isolated recombinant cell is an immune cell.

11. The composition of any one of claims 1 to 10, wherein activation of the isolated recombinant cell in response to the target antigen enhances cellular proliferation, differentiation, cytokine production and / or cytotoxicity.

12. A composition described in any one of claims 1 to 11, wherein the target antigen is expressed at a concentration of 20% or less relative to the target antigen expressed by a wild-type target cell.

13. A composition comprising an isolated polynucleotide encoding an SLP-76 polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1, 7, 14, 16, 18, 19, 40, 59, or 60, as well as a chimeric antigen receptor (CAR) polypeptide.

14. A method for producing the isolated recombinant cell described in claim 1, comprising introducing into the cell a composition containing a polynucleotide encoding the SLP-76 polypeptide and a polynucleotide encoding a chimeric antigen receptor (CAR) polypeptide.

15. A method for enhancing activation of the isolated recombinant cell of claim 1 in response to said target antigen, comprising overexpressing said SLP-76 polypeptide in said isolated recombinant cell.

16. A method for enhancing activation of a recombinant cell in response to a target antigen, comprising overexpressing an SLP-76 polypeptide and a chimeric antigen receptor (CAR) polypeptide in the recombinant cell.

17. The composition according to any one of claims 1 to 12 for use as a medicine.

18. Use of one or more T cells in the manufacture of a medicament for treating a disease or condition in a subject, wherein the one or more T cells express a chimeric antigen receptor (CAR) polypeptide, the CAR polypeptide specifically recognizes a target antigen expressed by a host cell associated with the disease or condition in the subject, and the one or more T cells overexpress and / or maintain enhanced levels of an SLP-76 polypeptide.

19. 20. The use of claim 18, wherein the level of the target antigen expressed by the host cell is lower than a control level.

20. 20. The use according to any of claims 18 or 19, wherein the SLP-76 polypeptide is membrane bound.

21. The use according to any one of claims 18 to 20, wherein at least one T cell in the subject is a tumor infiltrating lymphocyte (TIL).

22. 22. The use according to any one of claims 18 to 21, wherein overexpression and / or enhancing the level of SLP-76 polypeptide in said at least one T cell enhances the activity of said at least one T cell to inhibit or kill a host cell.

23. The use according to any one of claims 18 to 22, wherein the host cell is a cancer cell or a tumor cell.

Images