Reverse fate receptors to enhance the efficacy of engineered t cells

Abstract

Aspects of the present disclosure include a therapeutic immune cell comprising an Antigen Receptor for Immune Cell Activation (i.e. CAR, TCR, or BiTE) and a Reverse Fate Receptor (RFR). The RFR comprises an extracellular Fas receptor domain, a transmembrane domain and an intracellular domain that transmits cytokine signaling.

Description

REVERSE FATE RECEPTORS TO ENHANCE THE EFFICACY OF ENGINEERED T CELLSCLAIM OF PRIORITYThis Application claims the benefit of U.S. Provisional Patent Application No. 63 / 595,178, filed on November 1, 2023, which is incorporated by reference in full herein.BACKGROUND1. Field

[0001] The present disclosure relates generally to the fields of cancer treatment. More particularly, it concerns chimeric antigen receptor (CAR)-based cancer therapy and methods of use thereof.2. Description of Related Art

[0002] Chimeric antigen receptor (CAR) cell therapy is generally regarded as an effective solution to treat cancer, particularly for hematological malignancies. Vy9V52 T cells (hereafter V52 T cells) are an attractive cell platform in CAR-modified cell therapy, as their invariable T cell receptor recognizes a common phosphoantigen-induced alteration in a major histocompatibility complex (MHC)-independent manner. This specificity aids in tumor recognition and minimizes the risk of graft-versus-host disease, making V52 T cells suitable for allogeneic cell therapy. However, V82 T cells are susceptible to activation-induced cell death (AICD) through the Fas receptor expressed on their cell surface and require exogenous cytokines for expansion and persistence. These attributes of V82 T cells are shared with other common cell platforms, such as iPSC-derived T-cells and overall limit the clinical potency of these cell platforms.

[0003] Gamma delta T cells are a small subset of T cells that express a unique T cell receptor (TCR). Among them, V,y9V82 T cells are the major population found in the peripheral blood. This subset expresses the V32 TCR chain exclusively paired with the Vy9 TCR chain (Vy9V82 TCR), which recognizes the conformational change of BTN3A1 expressed on cell surface induced by phosphoantigens such as isopentenyl pyrophosphate (IPP). Since cancer cells show enhanced mevalonate pathway activity for their growth, leading to the accumulation of IPP intracellularly, Vy9V62 T cells are able to recognize and kill cancer cell through BTN3A1. Importantly, due to their invariant TCR expression, Vy9V62 T cells do not cause alloreactions such as graft-versus-host disease (GVHD) in the allogeneic setting, enabling use as an allogeneicoff-the-shelf cell product. In addition, V52 T cell can directly kill tumor cells via NKG2D by recognizing stress-induced ligands such as MHC class I polypeptide-related sequence A and B (MICA / B) and UL16 binding proteins (ULBPs) upregulated on tumor cells. V52 T cells also mediate antibody-dependent cellular cytotoxicity (ADCC) via the Fc receptor CD16 (FcyRIII) expressed on a subpopulation of V82 T cells.

[0004] However, V52 T cells have been demonstrated to be vulnerable to apoptosis triggered by Fas signaling (Fig. 1). Upon engagement of the Fas ligand (FasL), Fas forms a trimer, resulting in the formation of the death-inducing signaling complex (DISC) via the recruitment of the FADD adapter protein to the death domain of the Fas cytoplasmic tail. This catalyzes the activation of caspase-8 and caspase- 10, inducing the activation of downstream effector caspases, resulting in apoptosis.

[0005] It has been shown that T cells require 3 signals for full activation; signal 1 - TCR signaling, signal 2 - costimulation, and signal 3 - cytokine signaling. While both signal 1 and 2 can be provided through the CAR; however, the lack of survival cytokine secretion in V62 T cells necessitates an alternate source of cytokine signaling to provide signal 3 to V82 T cells. One method of delivering cytokine signaling involves the administration of exogenous cytokines alongside the T cell product. Other methods include engineering T cells to produce either secreted or membrane-bound cytokines of interest. However, the above methods have the risk of causing systemic toxicides attributable to the stimulation of endogenous immune system, as has indeed been reported in clinical trials utilizing such methods. Alternative approaches include the overexpression of native cytokine receptors which utilize cytokines provided by other cell type at the tumor site, or engineered cytokine receptors which spontaneously transmit cytokine signaling in the absence of cytokines. However, the former requires the presence of a cytokine milieu, and the latter might cause the autonomous growth of infused cells. Hence, ideal cytokine signaling should be transient and restricted to infused effector cells..

[0006] While autologous CAR T cell therapy has shown efficacy against hematological malignancies, the high cost of autologous scale-out manufacture, and the manufacturing time (from leukapheresis to infusion) limits the pool of patients that can benefit from such an approach. Hence, there is a need for the development of efficacious allogeneic cell products which can be used immediately in multiple patients and which can overcome the limitations of high susceptibility to AICD and additional cytokine requirements needed for full activation.SUMMARY

[0007] The present disclosure concerns methods and compositions related to a composition comprising a first polypeptide comprising an Antigen Receptor for Immune Cell Activation (AgRICA) and a second polypeptide comprising an extracellular domain of a death receptor, a transmembrane domain, and an intracellular domain that transmits cytokine signaling In embodiments, the AgRICA comprises a extracellular receptor domain, a transmembrane domain, and internal Signal 1 domain, a Signal 2 domain, or a combination of a Signal 1 domain and a Signal 2 domain. In embodiments, the AgRICA is a CAR, TCR, or BiTE. In specific embodiments, the intracellular domain that transmits cytokine signaling is cMPL, myD88, gpl30, gp 130(51), TLR2, TLR4, or a combination thereof. In embodiments, the intracellular domain that transmits cytokine signaling is any one of SEQ ID NOs: 8-13 In specific embodiments, the extracellular domain of a death receptor is Fas, TNFR1, DR3, TRAIL-R1, TRAIL-R2, DR6, or ED AR. In embodiments, the extracellular domain of a death receptor comprises any one of SEQ ID NOs. 1-7.

[0008] Another embodiment of the disclosure is a composition comprising a first polynucleotide that encodes a first polypeptide comprising an AgRICA and a second polynucleotide that encodes a second polypeptide comprising an extracellular domain of a death receptor, a transmembrane domain, and an intracellular domain that transmits cytokine signaling, optionally wherein the first polynucleotide and the second polynucleotide is comprised on one or more vectors. In embodiments the first polynucleotide and the second polynucleotide are attached. In embodiments, the AgRICA comprises a extracellular receptor domain, a transmembrane domain, and interacelluar Signal 1 domain, a Signal 2 domain, or a combination of a Signal 1 domain and a Signal 2 domain. In embodiments, the AgRICA is a CAR, TCR, or BiTE. In specific embodiments, the intracellular domain that transmits cytokine signaling is cMPL, myD88, gpl30, gp 130(51), TLR2, TLR4, or a combination thereof. In embodiments, the intracellular domain that transmits cytokine signaling is any one of SEQ ID NOs: 8-13. In specific embodiments, the extracellular domain of a death receptor is Fas, TNFR1, DR3, TRAIL-R1, TRAIL-R2, DR6, or ED AR. In embodiments, the extracellular domain of a death receptor comprises any one of SEQ ID NOs. 1-7.

[0009] Another embodiment of the disclosure is a cell comprising a first polypeptide comprising a AgRICA and a second polypeptide comprising an extracellular domain of a death receptor, a transmembrane domain, and an intracellular domain that transmits cytokine signaling. In embodiments, the AgRICA comprises a extracellular receptor domain, a transmembranedomain, and internal Signal 1 domain, a Signal 2 domain, or a combination of a Signal 1 domain and a Signal 2 domain. In embodiments, the AgRICA is a CAR, TCR, or BiTE. In specific embodiments, the intracellular domain that transmits cytokine signaling is cMPL, myD88, gpl3O, gp 130(51), TLR2, TLR4, or a combination thereof. In embodiments, the intracellular domain that transmits cytokine signaling is any one of SEQ ID NOs: 8-13. In specific embodiments, the extracellular domain of a death receptor is Fas, TNFR1, DR3, TRAIL-R1, TRAIL-R2, DR6, or ED AR. In embodiments, the extracellular domain of a death receptor comprises any one of SEQ ID NOs. 1-7. In embodiments, the cell does not natively express Jakl or Jak3. In some embodiments the cell is a T cell. In specific embodiments the cell is gamma-delta T cell, NK cell, IPS cell, or a mixture thereof. As used herein, cell can refer to a single cell or a plurality of cells. In some embodiments, the cell is frozen.

[0010] An embodiment of the disclosure is a method of treating a cancer in an individual comprising administering to the individual a therapeutically effective amount of a plurality of cells comprising a first polypeptide comprising a AgRICA and a second polypeptide comprising an extracellular domain of a death receptor, a transmembrane domain, and a intracellular domain that transmits cytokine signaling. In embodiments, the AgRICA comprises a extracellular receptor domain, a transmembrane domain, and intracellular Signal 1 domain, a Signal 2 domain, or a combination of a Signal 1 domain and a Signal 2 domain. In embodiments, the AgRICA is a CAR, TCR, or BiTE. In specific embodiments, the intracellular domain that transmits cytokine signaling is cMPL, myD88, gpl30, gpl30(51), TLR2, TLR4, or a combination thereof. In embodiments, the intracellular domain that transmits cytokine signaling is any one of SEQ ID NOs: 8-13. In specific embodiments, the extracellular domain of a death receptor is Fas, TNFR1, DR3, TRAIL- Rl, TRAIL-R2, DR6, or ED AR. In embodiments, the extracellular domain of a death receptor comprises any one of SEQ ID NOs. 1-7. In embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a solid tumor. The method can further comprise administering at least a second therapeutic agent to the subject. In some embodiments, the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiotherapy, drug therapy, targeted therapy, hormone therapy, biotherapy, or a combination thereof.

[0011] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications withinthe spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0013] Fig. 1 illustrates Fas signaling via the physiological model and by soluble Fas ligand.

[0014] Figs. 2A-B. Fig. 2A is a diagram of an example reverse fate receptor (RFR) construct. Fig. 2B is an illustration of an expected mechanism of action for RFRs.

[0015] Figs. 3A-F. Fig. 3A is a diagram of an exemplary RFR. Fig. 3B show expression of RFRs and 19BBz after transduction into V52 T cells. Fig. 3C shows resistance of RFRs to sFasL-induced apoptosis. Fig. 3D shows coculture of RFR-engineered V52 T cells with target NALM6 cells. Fig. 3E shows residual target cells of RFR-expressing 19BBz-V82 T cells cocultures. Fig. 3F shows T cell expansion of 19BBz-V32 T cells cocultures.

[0016] Figs. 4A-E. Fig. 4A illustrates an exemplary xenograft experimental design. Fig. 4B quantifies tumor expansion in vivo. Fig. 4C shows T cell persistence in vivo. Fig. 4D is a graph of RFR+CAR+ (NGFR+ / FLAG+) fraction of human CD3+CD45+ cells in mouse peripheral blood. Fig. 4E shows Mouse overall survival.

[0017] Fig. 5 shows a coculture of RFR-engineered iT cells with target NALM6 cells.DESCRIPTION OF ILLUSTRATIVE EMBODIMENTSIL Definitions

[0018] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0019] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0020] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more. The terms “about”, “substantially” and “approximately” mean, in general, the stated value plus or minus 5%.

[0021] “Treating” or treatment of a disease or condition refers to executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0022] The term “therapeutic benefit” or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0023] “Subject” and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.

[0024] As used herein a “RFR” or a “reverse fate receptor” refers to a polypeptide comprising at least an extracellular Fas receptor domain, a transmembrane domain, and an intracellular domain that transmits cytokine signaling.

[0025] As used herein a “CAR” or a “chimeric antigen receptor” refers to an engineered polypeptide comprising at least an extracellular antigen receptor domain which targets naturally occurring antigens, a transmembrane domain, and an intracellular signaling domain. Once a target engages the extracellular receptor domain, the intracellular signaling domains elicits effector T- cell functions including enhanced proliferation, cytokine release, cytotoxicity, or a combination thereof.

[0026] As used herein a “TCR” or a “T-cell receptor” refers to an engineered polypeptide comprising at least an extracellular receptor domain which targets intracellular antigens presented on surface MHC molecules. Once a target engages the extracellular receptor domain, the intracellular signaling domains elicits effector T-cell functions including enhanced proliferation, cytokine release, cytotoxicity, or a combination thereof.

[0027] As used herein a “Antigen Receptor for Immune Cell Activation (AgRICA)” refers to an engineered polypeptide comprising an antigen binding domain that targets an antigen on the exterior of a target cell and which leads to activation of immune effector cells. The AgRICA may include a transmembrane domain and an intracellular signaling domain, for example as found in CARs and TCRs. The AgRICA may also be a BiTE polypeptide that creates a connection between a target cell comprising the targeted antigen and an immune effector cell, and which leads to the immune effector cell’s activation. That is, the antigen binder can be a receptor or an engager, as long as binding to a target cell’s antigen leads to immune effector cell activation. CAR, TCR and BiTE polypeptides are all AgRICAs.

[0028] An “AgRICA immune cell” refers to an immune effector cell, such as a T-cell, that express an AgRICA. The AgRICA immune cell may comprise a AgRICA with extracellular receptor, a transmembrane domain, and an intracellular signaling domain, or the AgRICA immune cell may secrete a BiTE, for example.

[0029] As used herein “BiTE” refers to a bispecific T-cell engager that simultaneously bind to a target antigen on a tumor cell and a TCR on a T-cell triggering T-cell effector function. BiTEs can be recombinant or secreted by engineered cells.

[0030] As used herein “target cell” refers to a cell comprising antigens on the target cell’s exterior in which an immune effector cell can bind to and trigger cell death in the target cell.

[0031] As used herein a “intracellular signaling domain” refers to a polypeptide which elicits signaling cascades. In embodiments, the intracellular signaling domain elicits immuneeffector cell functions including enhanced proliferation, cytokine release, cytotoxicity, or a combination thereof.

[0032] As used herein “immune effector cells” refers to cells which create an immune response.

[0033] As used herein “Signal 1” in T-cells refers to antigen stimulation. As used herein “Signal 2” in T-cells refers to costimulatory signaling through costimulatory molecules expressed on T-cells. As used herein “Signal 3” in T-cells refers to cytokine signaling driving T cell proliferation.III. Overview

[0034] Embodiments of the disclosure comprise adding an additional synthetic receptor, RFR, to CAR, TCR, or BiTE immune effector cells. RFRs are synthesized constructs consisting of a extracellular domain of a death receptor fused with the intracellular domain of an immune stimulatory molecule. The RFR enhances the efficacy of immune effector cell (e.g. CAR T cells) by converting the death signal (e.g. Fas) to a survival signal upon ligation of a death ligand. Thus, once immune cells are engineered to express an RFR, RFR-expressing cells are resistant to AICD and receive immune stimulatory signaling, enabling them to proliferate and mediate tumor control.

[0035] RFRs consist of the extracellular and transmembrane domains of the Fas receptor fused with an intracellular signaling domain capable of transmitting immunostimulatory signaling (Fig. 2A). This synthetic receptor has two functions; 1) to function as a dominant-negative receptor - competing with the native Fas receptor and therefore diminishing the death signal, 2) to function as an immunostimulatory receptor - binding of FasL to the RFR causes the multimerization of the intracellular immunostimulatory domain, thereby inducing the transmission of signal 3 (Fig. 2B). In the context of CAR cell therapy, being able to provide and control signal 3 can enhance therapeutic effect. Importantly, although Fas is constitutively expressed on the surface of V52 T cells after initial stimulation, the express! on / secreti on of FasL is transiently induced by V52 T cells upon activation, controlling the activity of the RFR and thus preventing continuous signaling of the RFR.IV. Chimeric Antigen Receptors and T-Cell Receptors, Generally

[0036] Disclosed herein are chimeric antigen receptors (CARs) that can be used in combination with RFRs. The CARs generally include an extracellular antigen (or ligand) binding(receptor) domain linked to one or more intracellular signaling components, in some aspects via hinges, linkers, and / or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone.

[0037] Also disclosed herein are T-Cell receptors (TCRs) that can be used in combination with RFRs. The TCRs generally include variable and constant region as an extracellular domain linked to transmembrane and short intracellular domain. TCRs form complex with CD3 molecules and transmit signal upon recognition of intracellular antigens that are presented on surface of a target cell by MHC molecules.

[0038] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and one or more intracellular signaling domains. In addition to a primary T cell activation signal, such as may be initiated by CD3^ and / or FceRIy, an additional stimulatory signal for immune effector cell proliferation and effector function following engagement of the chimeric receptor with the target antigen may be utilized. For example, part or all of a human costimulatory receptor for enhanced activation of cells may be utilized that could help improve in vivo persistence and improve the therapeutic success of the adoptive immunotherapy. Examples include costimulatory domains from molecules such as DAP 12, DAP 10, NKG2D, CD2, CD28, CD27, 4- IBB (CD 137), OX-40 (CD 134), ICOS, (CD278), CD30, HVEM, CD40, LFA-1 (CD1 la / CD18), and ICAM-1, although in specific alternative embodiments any one of these listed may be excluded from use in a CAR.

[0039] In particular embodiments, specific CAR and TCR molecules are encompassed herein. In some cases, the antigen binding domain of the CAR is a scFv. In cases wherein an scFv is utilized in the extracellular domains, the variable heavy chain and the variable light chain for the scFv may be in any order in N-terminal to C-terminal direction. For example, the variable heavy chain may be on the N-terminal side of the variable light chain, or vice versa. The scFv and / or ligand that binds the antigen may or may not be codon optimized.B. CAR Antigen binding domains

[0040] Polypeptides of the present disclosure may comprise one or more antigen binding (receptor) domains. An “antigen binding domain” describes a region of a polypeptide capable of binding to an antigen under appropriate conditions. In some embodiments, an antigen binding domain is a single-chain variable fragment (scFv) based on one or more antibodies (e.g., CD20 antibodies). In some embodiments, an antigen binding domain comprise a variable heavy (VH)region and a variable light (VL) region, with the VH and VL regions being on the same polypeptide. In some embodiments, the antigen binding domain comprises a linker between the VH and VL regions. A linker may enable the antigen binding domain to form a desired structure for antigen binding.

[0041] The variable regions of the antigen-binding domains of the polypeptides of the disclosure can be modified by mutating amino acid residues within the VH and / or VL CDR 1, CDR 2 and / or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term “CDR” refers to a complementarity-determining region that is based on a part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B cells and T cells respectively, where these molecules bind to their specific antigen. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations may be introduced by site- directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions.

[0042] It is also contemplated that the antigen binding domain may be multi-specific or multivalent by multimerizing the antigen binding domain with VH and VL region pairs that bind either the same antigen (multi -valent) or a different antigen (multi-specific).

[0043] The binding affinity of the antigen binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10'5M, 10'6M, 10’7M, 10’8M, 10’9M, 10’10M, 10-nM, 10’12M, or 10’13M. In some embodiments, the KD of the antigen binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10’5M, 10’6M, 10’7M, 10’8M, 10’9M, 100M, 101M, 10’12M, or 10’13M (or any derivable range therein).

[0044] Binding affinity, KA, or KD can be determined by methods known in the art such as by surface plasmon resonance (SRP)-based biosensors, by kinetic exclusion assay (KinExA), by optical scanner for microarray detection based on polarization-modulated oblique-incidence reflectivity difference (OI-RD), or by ELISA.

[0045] In some embodiments, the polypeptide comprising the humanized binding region has equal, better, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 104, 106, 106, 108, 109, 110, 115, or 120% binding affinity and / or expression level in host cells, compared to a polypeptide comprising a non-humanized bindingC. CAR Transmembrane domains

[0046] In some aspects, the antigen-specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, a CAR includes a transmembrane domain fused to an extracellular domain of the CAR. In some embodiments, a transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some embodiments, a transmembrane domain is used that is not naturally associated with one of the domains of the CAR. In some instances, a transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In some embodiments, a transmembrane domain is derived either from a natural or from a synthetic source. In some embodiments, where the source is natural, a transmembrane domain is derived from any membrane-bound or transmembrane protein. In some embodiments, transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of; also “from”) the alpha, beta or zeta chain of the T- cell receptor, CD28, DAP12, DAP10, NKG2D, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, OX-40 (CD134), 4-1BB (CD137), CD154, ICOS / CD278, and so forth. In some embodiments, a transmembrane domain of the present disclosure is a transmembrane domain from CD8a.

[0047] Polypeptides of the present disclosure may comprise a transmembrane domain. In some embodiments, a transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0048] In some embodiments, the transmembrane domain is interposed between the extracellular spacer and the cytoplasmic region. In some embodiments, the transmembrane domain is interposed between the extracellular spacer and one or more costimulatory regions. In some embodiments, a linker is between the transmembrane domain and the one or more costimulatory regions.

[0049] In some embodiments, a transmembrane domain can have a length of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In some embodiments, a transmembrane domain is about 24 amino acids in length.

[0050] In some embodiments, any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some embodiments, a transmembrane domain is derived from CD30, CD28, CD8, CD4, CD3- zeta, OX-40 (CD 134), or CD7. In some embodiments, a transmembrane domain is derived the alpha, beta or zeta chain of the T- cell receptor, CD28, CD2, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8 (including CD8alpha), CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, OX-40 (CD134), 4-1BB (CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, PD-1, CTLA4, and DAP molecules. In some embodiments, a transmembrane domain is synthetic. In some aspects, a synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, a transmembrane domain comprises, consists essentially of, or consists of a sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the transmembrane domains listed above.D. CAR Cytoplasmic Regions (Intracellular Region)

[0051] After antigen recognition, receptors may cluster and a signal may be transmitted to the cell through the cytoplasmic (intracellular) region. In some embodiments, the costimulatory domains described herein are part of the cytoplasmic region. In some embodiments, the cytoplasmic region comprises an intracellular signaling domain. An intracellular signaling domain may comprise a primary signaling domain and one or more costimulatory domains. In embodiments, the cytoplasmic region comprises a polypeptide that transmits a Signal 1, a Signal 2, or both.

[0052] Cytoplasmic regions and / or costimulatory regions suitable for use in the CARs of the disclosure include any desired signaling domain that provides a distinct and detectable signal 1 or signal 2 (e.g., change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc. in response to activation by way of binding of theantigen to the antigen binding domain. In some embodiments, the cytoplasmic region includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described herein. In some embodiments, the cytoplasmic region includes DAP10 / CD28 type signaling chains.

[0053] Cytoplasmic regions suitable for use in the polypeptides of the disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. An ITAM motif is YX1X2(L / I), where XI and X2 are independently any amino acid. In some cases, the cytoplasmic region comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twice in an endodomain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids, e.g., (YXlX2(L / I))(X3)n(YXlX2(L / I)), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid.

[0054] In some embodiments, a suitable cytoplasmic region is an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable endodomain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3-zeta; and CD79A (antigen receptor complex-associated protein alpha chain).

[0055] In some embodiments, a suitable cytoplasmic region can comprise an ITAM motifcontaining portion of the full length DAP 12 amino acid sequence. In some embodiments, the cytoplasmic region is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcRgamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length FCER1G amino acid sequence.

[0056] In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3 -DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD35; CD3d antigen, delta polypeptide (TiT3 complex);, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 delta amino acid sequence. In some embodiments, the cytoplasmic region is derived from Tcell surface glycoprotein CD3 epsilon chain (also known as CD3e, CD3s; T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, 0KT3, AI504783, CD3, CD3-epsilon, T3e, etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 epsilon amino acid sequence. In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 gamma chain (also known as CD3G, CD3y, T cell receptor T3 gamma chain, CD3 -GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 gamma amino acid sequence. In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, CD3^, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 zeta amino acid sequence.

[0057] In particular embodiments of specific CAR molecules, a CAR may utilize CD28, DAP10, DAP12, 4-1BB, NKG2D, etc. or other costimulatory domains (which may be referred to herein as an intracytoplasmic domain). In some cases, CD3zeta is utilized without any costimulatory domains. In particular embodiments of specific CAR molecules, a CAR may utilize any suitable transmembrane domain, such as from CD30, DAP12, DAP10, 4-1BB, 2B4, 0X40, CD27, NKG2D, CD8, or CD28.

[0058] In some embodiments, the cytoplasmic region is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane- bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD79A amino acid sequence.

[0059] Non-limiting examples of suitable costimulatory regions, such as those included in the cytoplasmic region, include, but are not limited to, polypeptides from 4-1BB (CD 137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In some embodiments, a costimulatory region is derived from CD8, 4-1BB (CD137), CD27, CD28, CD30, OX-40 (CD134), CD3e, CD3 , CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, or CD154 In some embodiments, a costimulatory domain includes, but are not limited to one or more of CD28, CD27, OX-40 (CD 134), ICOS, HVEM, GITR, LIGHT, CD40L, DR3, CD30, SLAM, CD2, CD226 (DNAM-1), CD244, TMIGD2, BTNL3, NKG2D, DAP10, DAP12, 4-1BB (CD137), or a synthetic molecule. In some embodiments, in addition to a primary signal initiated by CD3^, an additionalsignal provided by a costimulatory receptor inserted in a CAR is important for full activation of immune cells and could help improve in vivo persistence and the therapeutic success of the cell therapy.

[0060] A costimulatory region may have a length of at least, at most, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids or any range derivable therein. In some embodiments, the costimulatory region is derived from the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA; etc.). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, 0X40, TXGP1L). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD27 (also known as S 152, T14, TNFRSF7, and Tp55). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5- 902P8.2, AITR, CD357, and GITR-D). In some embodiments, the costimulatory region derived from an intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2).

[0061] In some embodiments, the polypeptides described herein may further comprise a detection peptide. Suitable detection peptides include hemagglutinin, FLAG, c-myc, and the like. Other suitable detection peptides are known in the art.V. Reverse Fate Receptor (RFR) Polypeptides

[0062] Embodiment of the disclosure are a RFR that is used in an immune effector cell in combination with a AgRICA to target and kill disease cells. The RFR comprises at least an extracellular domain from a death receptor, a transmembrane domain, and an intracellular domain that transmits a cytokine signal (Signal 3) when the extracellular domain is activated. In embodiments, the RFR additionally can include one or more spacers, hinge domains, markers, intracellular domain, or a combination thereof.A. RFR Extracellular domain

[0063] In embodiments, the extracellular receptor domain of the RFR is an extracellular domain of a death receptor. In embodiments, the extracellular domain of a death receptor of the RFR is at least 90%, 95%, 98%, or 99% similar to wild-type sequence of an extracellular domain of a death receptor such as Fas (SEQ ID NO: 1), TNFR1 (SEQ ID NO: 2), DR3 (SEQ ID NO: 3), TRAIL-R1 (SEQ ID NO: 4), TRAIL-R2 (SEQ ID NO: 5), DR6 (SEQ ID NO: 6) and ED AR (SEQ ID NO: 7).B. RFR Transmembrane Domains

[0064] Polypeptides of the present disclosure may comprise a transmembrane domain. In some embodiments, a transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0065] In some embodiments, the transmembrane domain is interposed between the extracellular domain and the intracellular domain. In some embodiments, a spacer, hinge or linker is between the extracellular domain and the transmembrane domain.

[0066] In some embodiments, a transmembrane domain comprises, consists essentially of, or consists of a sequence derived from the transmembrane regions of the CD28, DAP12, DAP10, NKG2D, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD8a, CD9, CD 16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, OX-40 (CD134), 4-1BB (CD137), CD154, ICOS / CD278, Fas, TNFR1, DR3, TRAIL-R1, TRAIL-R2, DR6, or ED AR genes. In some embodiments, a transmembrane domain can have a length of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In some embodiments, a transmembrane domain is about 24 amino acids in length. In some embodiments, a transmembrane domain comprises, consists essentially of, or consists of a sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar to the transmembrane regions of the CD28, DAP 12, DAP 10, NKG2D, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD8a, CD9, CD 16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, OX-40 (CD134), 4-1BB (CD137), CD154, ICOS / CD278, or GITR / CD357, NKG2D, PD-1, CTLA4, DAP, Fas, TNFR1, DR3, TRAIL-R1, TRAIL-R2, DR6, and ED AR molecules. In some embodiments, a transmembrane domain is synthetic. In some aspects, a synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In someaspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.C. RFR Intracellular Domains

[0067] Embodiments of the disclosure comprise a RFR with a intracellular domain that transmits a cytokine signal (Signal 3) in the intracellular region of the RFR. In embodiments, the intracellular domain that transmits a cytokine signal is cMPL, MyD88, gpl30, gpl30(Sl)), TLR2, or TLR4. In specific embodiments, the intracellular polypeptide comprises at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence similarity to the sequences of the intracellular domain of cMPL (SEQ ID NO: 8), MyD88 (SEQ ID NO: 9), gpl30 (SEQ ID NO: 10), gpl30(Sl) (SEQ ID NO: 11), TLR2 (SEQ ID NO: 12), or TLR4 (SEQ ID NO: 13).VI. Additional Domains

[0068] In embodiments of the disclosure, a RFR, a AgRICA or both can comprise additional elements. In embodiments, the RFR, AgRICA, or both can comprise a space, a hinge, a marker or a combination thereof.

[0069] A peptide spacer (e.g., a spacer), such as an extracellular spacer may link an antigen-binding domain to a transmembrane domain. In some embodiments, a peptide spacer is flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen binding. In some embodiments herein, a peptide spacer is a “hinge”, e.g., it is a flexible polypeptide connector region that connects one or more domains of a AgRICA and / or RFR to one or more other domains of a AgRICA or the RFR. As used herein, the term “hinge” refers to a flexible polypeptide connector region (also referred to herein as “hinge region”) providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides.

[0070] In some embodiments, an extracellular peptide spacer comprising a hinge can have a length of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids. In some embodiments, a peptide spacer is 12 amino acids in length.

[0071] The length of an extracellular spacer can have an effect on a AgRICA’ s signaling activity if present, a AgRICA’ s expression levels (transcription and / or translation), cytotoxicity and / or cancer cell killing efficacy, and / or a AgRICA immune effector cells’ expansion properties in response to antigen-stimulated AgRICA signaling. In some embodiments, a AgRICA’ s extracellular spacer sequence is dependent on the location of the target antigen. In someembodiments, where a target antigen is proximal to a cell membrane, a longer extracellular spacer is used. In some embodiments, where a target antigen is distal to a cell membrane, a shorter extracellular spacer is used. In some embodiments, where a more flexible AgRICA is desired, a longer extracellular spacer is used. In some embodiments, where a more rigid AgRICA is desired, a shorter extracellular spacer is used.

[0072] In some embodiments, a shorter spacer such as less than, or no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids is used. In some embodiments, a shorter spacer may have an advantage in AgRICA mediated signaling activity, a AgRICA’ s expression levels (transcription and / or translation), cytotoxicity and / or cancer cell killing efficacy, and / or a AgRICA cells’ expansion properties in response to antigen-stimulated AgRICA signaling.

[0073] The length of an extracellular spacer can have an effect on a RFR’s expression levels (transcription and / or translation), antigen binding, or a combination thereof. In some embodiments, a shorter spacer such as less than, or no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids is used.VII. Production of Polypeptides in Immune Effector Cells

[0074] It is contemplated that the engineered construct, such as a AgRICA or RFR, can be introduced into immune cells as naked DNA or RNA, a transposon, or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Patent No. 6,410,319. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid or viral expression vector in proper orientation for expression.

[0075] In some embodiments, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector) can be used to introduce the CAR construct into cells of any kind, including at least immune cells. Suitable vectors for use in accordance with the method of the present disclosure are non-replicating in the immune cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, MoMLV, MSCV, SV40, EBV, HSV, or BPV.

[0076] Certain embodiments of the present disclosure concern the use of nucleic acids, including nucleic acids encoding a cancer antigen-specific AgRICA polypeptide, including in some cases a CAR that has been humanized to reduce immunogenicity (hCAR), comprising at least one intracellular signaling domain, a transmembrane domain, and an extracellular domaincomprising one or more signaling motifs. In certain embodiments, the binding region can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and / or antigen binding fragments thereof. In another embodiment, that specificity is derived from a peptide (e.g., cytokine) that binds to a receptor.

[0077] It is contemplated that the CAR nucleic acids may or may not comprise at least parts of human genes used to enhance cellular immunotherapy for human patients. In a specific embodiment, the disclosure includes a full-length CAR cDNA or coding region. The antigen binding regions or domain can comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody (e.g., an antiCD 19 antibody). In some embodiments, the fragment can also be any number of different antigen binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is a cancer antigen-specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells. In some embodiments, an antigen binding region comprises a protein or polypeptide that acts as a ligand and / or receptor for another protein and / or polypeptide.

[0078] In some embodiments, an arrangement could be multimeric, such as a diabody or multimers. Multimers are most likely formed by cross pairing of the variable portion of the light and heavy chains into a diabody. The hinge portion of a construct can have multiple alternatives from being totally deleted, to having the first cysteine maintained, to a proline rather than a serine substitution, to being truncated up to the first cysteine. In some embodiments, an Fc portion can be deleted. In some embodiments, any protein that is stable and / or dimerizes can serve this purpose. In some embodiments, just one of the Fc domains, e.g., either the CH2 or CH3 domain from human immunoglobulin is utilized. In some embodiments, the hinge, CH2 and CH3 region of a human immunoglobulin that has been modified to improve dimerization can be utilized. In some embodiments, just the hinge portion of an immunoglobulin can be utilized.

[0079] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized e.g., via PCR), or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, as it is found that introns stabilize the mRNA. Also, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.VIII. Therapy

[0001] Certain aspects of the present embodiments can be used to prevent or treat a disease or disorder. In embodiments, the disease is a blood cancer such as leukemia, lymphoma, or myeloma. In a specific embodiment, the disease is acute myeloid leukemia (AML). In some embodiments, the disease is a solid tumor. In additional embodiments, the AgRICA immune effector cells targets pathogenic lymphocytes, for example treating autoimmune or alloimmune conditions.

[0002] Certain embodiments of the present disclosure concern obtaining and administering cells to a subject to target cancer cells. The cells may deliver antibody compositions encompassed herein but themselves may or may not be immune cells. In specific embodiments the cells are immune effector cells. Examples of cells include T cells (includingT cells or y5 T cells), Natural Killer (NK) cells, invariant NKT (iNKT) cells, B cells, macrophages, stem cells of any kind (including MSCs or induced pluripotent stem cells), or dendritic cells.

[0003] In some embodiments, the method further comprises administering a cancer therapy to the patient. The cancer therapy may be chosen based on the expression level measurements, alone or in combination with the clinical risk score calculated for the patient. In some embodiments, the cancer therapy comprises a local cancer therapy. In some embodiments, the cancer therapy excludes a systemic cancer therapy. In some embodiments, the cancer therapy excludes a local therapy. In some embodiments, the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy. In some embodiments, the cancer therapy comprises an immunotherapy, which may be an immune checkpoint therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.

[0004] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In some embodiments, the cancer is recurrent cancer. In some embodiments, the cancer is Stage I cancer. In some embodiments, the cancer is Stage II cancer. In some embodiments, the cancer is Stage III cancer. In some embodiments, the cancer is Stage IV cancer.

[0005] Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, ductal carcinoma, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0006] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig celltumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; B cell lymphoma; low grade / follicular nonHodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0007] Certain embodiments concern methods of treatment of leukemia. Leukemia is a cancer of the blood or bone marrow and is characterized by an abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). It is part of the broad group of diseases called hematological neoplasms. Leukemia is a broad term covering a spectrum of diseases. Leukemia is clinically and pathologically split into its acute and chronic forms.

[0008] In some embodiments, the present disclosure provides methods for immunotherapy comprising administering an effective amount of the compositions that comprise the AgRICA and RFR(s), of the present disclosure. In one embodiment, a medical disease or disorder is treated by administration of an AgRICA and RFR-expressing cell population that elicits an immune response. In certain embodiments of the present disclosure, cancer is treated by administration of an AgRICA and RFR immune cell population that elicits an immune response Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of an antigen-specific cell therapy. The present methods may be applied for the treatment of immune disorders, solid cancers, and hematologic cancers, as examples. Specifically, the cancer may be a B cell malignancy, such as diffuse large B-cell lymphoma, high-grade B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and chronic lymphocytic leukemia.

[0009] In some embodiments, the subject can be administered nonmyeloablative lymphodepleting chemotherapy prior to the cell therapy. The nonmyeloablative lymphodepleting chemotherapy can be any suitable such therapy, which can be administered by any suitable route. The nonmyeloablative lymphodepleting chemotherapy can comprise, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which can be metastatic. An exemplary route of administering cyclophosphamide and fludarabine is intravenously. Likewise, any suitable dose of cyclophosphamide and fludarabine can be administered. In particular aspects, around 60 mg / kg of cyclophosphamide is administered for two days after which around 25 mg / m2fludarabine is administered for five days.

[0010] Therapeutically effective amounts of immune cells can be administered by a number of routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrastemal, or intraarticular injection, or infusion.

[0011] Intratumoral injection, or injection into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors. Local, regional or systemic administration alsomay be appropriate. In some embodiments, for tumors of >4 cm, the volume to be administered will be about 4-10 ml (in particular 10 ml), while for tumors of <4 cm, a volume of about 1-3 ml will be used (in particular 3 ml). In some embodiments, multiple injections delivered as single doses can comprise about 0.1 to about 0.5 ml volumes.

[0012] In some embodiments, a cell population can be administered in treatment regimens consistent with the disease, for example a single or a few doses over one to several days to ameliorate a disease state or periodic doses over an extended time to inhibit disease progression and prevent disease recurrence. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient’s circumstances. The therapeutically effective amount of cells will be dependent on the subject being treated, the severity and type of the affliction, and the manner of administration. In some embodiments, doses that could be used in the treatment of human subjects range from at least 3.8><104, at least 3.8><105, at least 3.8x 106, at least 3.8x l07, at least 3.8xl08, at least 3.8 xlO9, or at least 3.8x l010cells / m2. In a certain embodiment, the dose used in the treatment of human subjects ranges from about 3.8 109to about 3.8xl010cells / m2. In additional embodiments, a therapeutically effective amount of cells can vary from about 5xl06cells per kg body weight to about 7.5x l08cells per kg body weight, such as about 2xl07cells to about 5xl08cells per kg body weight, or about 5xl07cells to about 2x 108cells per kg body weight. The exact amount of cells is readily determined by one of skill in the art based on the age, weight, sex, and physiological condition of the subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0013] In certain embodiments of the present disclosure, an effective amount of AgRICA and RFR-expressing immune cells are delivered to an individual in need thereof, such as an individual that has cancer. In some embodiments, cells can then enhance the individual’s immune system to attack cancer cells. In some cases, an individual is provided with one or more doses of immune cells (e.g., those described herein). In some embodiments, where the individual is provided with two or more doses of immune cells, the duration between the administrations should be sufficient to allow time for propagation in the individual, and in specific embodiments the duration between doses is 1, 2, 3, 4, 5, 6, 7, or more days.

[0014] In specific embodiments, the cells that have been engineered to express a AgRICA and a RFR are provided to an individual in a therapeutically effective amount (in a range from 103to 1010) that ameliorates at least one symptom related to cancer cells in the individual. A therapeutically effective amount may be from 103to IO10, 103to 109, 103to 108, 103to 107, 103to106, 103to IO5, 103to 104, 104to IO10, 104to 109, 104to IO8, 104to 107, 104to 106, 104to IO5, IO5to IO10, IO5to 109, IO5to 108, IO5to 107, IO5to 106, 106to IO10, 106to 109, 106to IO8, 106to 107, 107to IO10, 107to 109, 107to 108, 108to IO10, 108to 109, or 109to IO10cells. Thus, in particular embodiments an individual having a certain cancer is provided once or multiple times a therapeutically effective amount of cells expressing one or more AgRICA / RFRs.IX. Cellular Therapies

[0015] Certain embodiments relate to cells comprising AgRICA and RFR polypeptides or nucleic acids of the disclosure. In some embodiments the cell is an immune effecter cell. In certain T cell embodiments, as used herein, “T cell” includes all types of immune cells expressing CD3 including T-helper cells, invariant natural killer T (iNKT) cells, cytotoxic T cells, T-regulatory cells (Treg) gamma-delta T cells, natural-killer (NK) cells, and neutrophils. The T cell may refer to a CD4+ or CD8+ T cell.

[0016] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, Hut-78, Jurkat, HL- 60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0017] In some instances, the cell is not an immortalized cell line, but is instead a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell (e.g., peripheral blood stem cell) or progenitor cell obtained from an individual.

[0018] The immune cells may be any kind of immune cells, including T cells (e.g., regulatory T cells, CD4+T cells, CD8+T cells, alpha beta T cells, gamma-delta T cells, or a mixture thereof), NK cells, invariant NKT cells, NKT cells, innate lymphoid cells, or a mixture thereof. The immune cells may be virus-specific, express a CAR, express a TCR, express a BiTE, orexpress a combination thereof. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells (DCs), mast cells, eosinophils, and / or basophils. Also provided herein are methods of producing and engineering the immune cells as well as methods of using and administering the cells for adoptive cell therapy, in which case the cells may be autologous or allogeneic. Thus, the immune cells may be used as immunotherapy, such as to target cancer cells. These immune cells may be used for therapy as a single cell type or as a combination of multiple immune cell types. In specific embodiments, the immune cells are CD3+, CD4+, CD8+, CD16+, or a mixture thereof.

[0019] The immune cells may be isolated from subjects, particularly human subjects. The immune cells can be obtained from a subject of interest, such as a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, or a subject who is undergoing therapy for a particular disease or condition. Immune cells can be collected from any location in which they reside in the subject including, but not limited to, blood, cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells may be used directly, or they can be stored for a period of time, such as by freezing.

[0020] The immune cells may be enriched / purified from any tissue where they reside including, but not limited to, blood (including blood collected by blood banks or cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained via biopsy procedures. Tissues / organs from which the immune cells are enriched, isolated, and / or purified may be isolated from both living and non-living subjects, wherein the non-living subjects are organ donors. In particular embodiments, the immune cells are isolated from blood, such as peripheral blood or cord blood. In some aspects, immune cells isolated from cord blood have enhanced immunomodulation capacity, such as measured by CD4- or CD8- positive T cell suppression. In specific aspects, the immune cells are isolated from pooled blood, particularly pooled cord blood, for enhanced immunomodulation capacity. The pooled blood may be from 2 or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0021] The population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity. Thus, the cells will be autologous to the subject in need of therapy. Alternatively, the population of immune cells can be obtained from a donor, such as a partially or fully histocompatibility matched donor or fully histocompatibility mismatched donor. The immune cell population can be harvested from the peripheral blood, cord blood, bone marrow, spleen, or any other organ / tissue in which immunecells reside in said subject or donor. The immune cells can be isolated from a pool of subjects and / or donors, such as from pooled cord blood.

[0022] When the population of immune cells is obtained from a donor distinct from the subject, the donor may be allogeneic, provided the cells obtained are subject-compatible in that they can be introduced into the subject. Allogeneic donor cells are may or may not be humanleukocyte-antigen (HLA)-compatible.B. T Cells

[0023] In some embodiments, the immune cells are T cells. Several basic approaches for the derivation, activation and expansion of functional anti-tumor effector cells have been described in the last two decades. These include: autologous cells, such as tumor-infiltrating lymphocytes (TILs); T cells activated ex-vivo using autologous DCs or PBMCs, lymphocytes, artificial antigen- presenting cells (APCs), soluble or plastic-coated antibodies or beads coated with T cell ligands and activating antibodies, or cells isolated by virtue of capturing target cell membrane; allogeneic cells naturally expressing anti-host tumor T cell receptor (TCR); and non-tumor-specific autologous or allogeneic donor-derived or iPSC-derived cells genetically reprogrammed or “redirected” to express tumor-reactive TCR, CAR or BiTE. These approaches have given rise to numerous protocols for T cell preparation and immunization which can be used in the methods described herein.

[0024] In some embodiments, the T cells are derived from the blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some aspects, the cells are human cells. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+cells, CD8+cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and / or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.

[0025] Among the sub-types and subpopulations of T cells (e.g., CD4+and / or CD8+T cells) are naive-like or naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and gamma / delta T cells. In certain embodiments, T cells are gamma / delta T cells.

[0026] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for a specific marker, such as surface markers, or that are negative for a specific marker. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (e.g., memory cells).

[0027] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+or CD8+selection step is used to separate CD4+helper and CD8+cytotoxic T cells. Such CD4+and CD8+populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0028] In some embodiments, CD8+T cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells or stem cell memory cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations.

[0029] In some embodiments, the T cells are autologous T cells. In this method, tumor samples are obtained from patients and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, e.g., mechanically (disaggregating the tumor using, e.g., a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Single-cell suspensions of tumor enzymatic digests are cultured in interleukin-2 (IL-2) or other growth factors.

[0030] The cultured T cells can be pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen-specific T-cells of at least about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 10 to about 14 days. More preferably, rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days.

[0031] Expansion can be accomplished by any of a number of methods as are known in the art. For example, T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. The non-specific T-cell receptor stimulus can include around 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, N.J.). Alternatively, T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as an human leukocyte antigen A2 (HLA-A2) binding peptide or peptides binding to other MHC class I or class II molecules, in the presence of a T-cell growth factor, such as 300 lU / ml IL-2 or IL-15, with IL-2 being preferred. The in vz / ro-induced T-cells are rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells or antigen-presenting cells expressing other HLA molecules. The in vz7 / v>-induced T-cells may also be expanded in the absence of antigen-presenting cells.C. NK Cells

[0032] In some embodiments, the immune cells are natural killer (NK) cells. NK cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells can be detected by specific surface markers, such as CD 16, CD56, and / or CD8 in humans. NK cells do not express T cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors.

[0033] In certain embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, tissues, or umbilical cord blood by methods well known in the art.D. NKT Cells

[0034] Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. Many of these cells recognize the non- polymorphic CD Id molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. They constitute only approximately 0.1% of all peripheral blood T cells. NKT cells are a subset of T cells that coexpress an up T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1. Invariant natural killer T (iNKT) cells express high levels of and are dependent on the transcriptional regulator promyelocytic leukemia zinc finger for their development. Currently, there are five major distinct iNKT cell subsets. These subset cells produce a different set of cytokines once activated. The subtypes iNKTl, iNKT2 and iNKT 17 mirror Th cell subsets in cytokine production. In addition, there are subtypes specialized in T follicular helper-like function and IL-10 dependent regulatory functions.E. Innate Lymphoid Cells

[0035] Innate lymphoid cells (ILCs) are a group of innate immune cells that are derived from common lymphoid progenitor (CLP) and belong to the lymphoid lineage. These cells are defined by absence of antigen specific B or T cell receptor because of the lack of recombination activating gene (RAG). ILCs do not express myeloid or dendritic cell markers. They play a role in protective immunity and the regulation of homeostasis and inflammation, so their dysregulation can lead to immune pathology such as allergy, bronchial asthma and autoimmune disease. ILCs can be divided based on the cytokines that they can produce, and the transcription factors that regulate their development and function.F. Cell Culture

[0036] In some embodiments, engineered cells may be cultured for at least between about 10 days and about 40 days, for at least between about 15 days and about 35 days, for at least between about 15 days and 21 days, such as for at least about 15, 16, 17, 18, 19 or 21 days. In some embodiments, the cells of the disclosure may be cultured for no longer than 60 days, or no longer than 50 days, or no longer than 45 days. The cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days. The cells may be cultured in the presence of a liquid culture medium. Typically, the medium may comprise a basal medium formulation as known in the art. Many basal media formulations can be used to culture cells herein, including but not limited to Eagle'sMinimum Essential Medium’ (MEM), Dulbecco Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essentia’ Modified Eagle's Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the cells cultured. In some embodiments, a culture medium formulation may be explants medium (CEM) which is composed of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 pg / ml streptomycin and 2 mmol / L L- glutamine. Other embodiments may employ further basal media formulations, such as chosen from the ones above.

[0037] Any medium capable of supporting cells in vitro may be used to culture the cells. Media formulations that can support the growth of cells include, but are not limited to, Dulbecco Modified Eagle's Medium (DMEM), alpha modified Minimal Essential Medium (oMEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum is added to the above medium in order to support the growth of cells. A defined medium, however, also can be used if the growth factors, cytokines, and hormones necessary for culturing cells are provided at appropriate concentrations in the medium. Media useful in the methods of the disclosure may comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells. The cells may be grown at temperatures between 27° C to 40° C, such as 31° C to 37° C, and may be in a humidified incubator. The carbon dioxide content may be maintained between 2% to 10% and the oxygen content may be maintained between 1% and 22%. The disclosure however, should in no way be construed to be limited to any one method of isolating and culturing cells. Rather, any method of isolating and culturing cells should be construed to be included in the present disclosure.

[0038] For use in the cell culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many media already contain amino acids, some amino acids may be supplemented later,e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with mammalian plasma or sera. Plasma or sera often contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated.

[0039] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. In particular embodiments, cells are cultured in a cell culture system comprising a cell culture medium, preferably in a culture vessel, in particular a cell culture medium supplemented with a substance suitable and determined for protecting the cells from in vitro aging and / or inducing in an unspecific or specific reprogramming.G. Cell Generation

[0040] Certain methods of the disclosure concern culturing the cells obtained from human tissue samples. In particular embodiments of the present disclosure, cells are plated onto a substrate that allows for adherence of cells thereto. This may be carried out, for example, by plating the cells in a culture plate that displays one or more substrate surfaces compatible with cell adhesion. When the one or more substrate surfaces contact the suspension of cells (e.g., suspension in a medium) introduced into the culture system, cell adhesion between the cells and the substrate surfaces may ensue. Accordingly, in certain embodiments cells are introduced into a culture system that features at least one substrate surface that is generally compatible with adherence of cells thereto, such that the plated cells can contact the said substrate surface, such embodiments encompass plating onto a substrate, which allows adherence of cells thereto.

[0041] Cells of the present disclosure may be identified and characterized by their expression of specific marker proteins, such as cell-surface markers. Detection and isolation ofthese cells can be achieved, for example, through flow cytometry, ELISA, and / or magnetic beads. Reverse-transcription polymerase chain reaction (RT-PCR) may be used to quantify cell-specific genes and / or to monitor changes in gene expression in response to differentiation. In certain embodiments, the marker proteins used to identify and characterize the cells are selected from the list consisting of CD3, CD4, CD8, CD5, CD7, CD45RA, CD45RO, CD45, and any combination thereof.X. Pharmaceutical Compositions

[0042] Also provided herein are pharmaceutical compositions and formulations comprising immune cell therapy and a pharmaceutically acceptable carrier. The cells of the disclosure can be cultured and infused in common media formulations, as known in the art. Examples of such are found below.

[0043] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral -active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.B. Combination Therapies

[0044] In certain embodiments, the compositions and methods of the present embodiments involve immune cell therapy used in combination with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy),chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, antibody-drug conjugates, siRNA therapy, vaccinations or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0045] In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side- effect limiting agents (e.g, agents intended to lessen the occurrence and / or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is therapy targeting PBK / AKT / mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and / or chemopreventative agent. In some embodiments the additional therapy is a monocolonal antibody therapy or antibody-based therapy. In some embodiments the additional therapy is an immunomodulator such as antibody against PD- 1, PDL-1, CTLA-4, or other checkpoint therapy or an immunoactivator such as IL2, IL7, ELI 5, IL-12, IL-18, or STING agonist. The additional therapy may be one or more of the chemotherapeutic agents known in the art.

[0046] A cell therapy may be administered before, during, after, or in various combinations relative to an additional cancer therapy, such as immune checkpoint therapy. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the immune cell therapy is provided to a patient separately from an additional therapeutic agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the antibody therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0047] Various combinations may be employed. For the example below an immune cell therapy is “A” and an anti-cancer therapy is “B”:A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / BB / B / B / A B / B / AZB A / A / B / B A / B / A / B A / B / B / A B / B / A / AB / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0048] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy.2. Chemotherapy

[0049] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5 -fluorouracil (5- FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, suchas fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti -adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above,3. Radiotherapy

[0050] Other factors that cause DNA damage and have been used extensively include what are commonly known as y-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation, and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.4. Immunotherapy

[0051] The skilled artisan will understand that immunotherapies may be used in combination or in conjunction with methods of the embodiments. In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells

[0052] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs and may be used in combination therapies. This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in “armed” MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index. Exemplary ADC drugs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T- DM1).

[0053] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, z.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, erb b2 and pl55. An alternative aspect of immunotherapy is to combine anti cancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.

[0054] Examples of immunotherapies include immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds); cytokine therapy, e.g., interferons a, P, and y, IL-1, GM-CSF, and TNF; gene therapy, e.g., TNF, IL-1, IL-2, and p53; and monoclonal antibodies, e.g., anti-CD20, anti -ganglioside GM2, and anti-pl85. It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.

[0055] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either turn up a signal e.g., co-stimulatory molecules) or turn down a signal. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD 152), indol eamine 2,3 -dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0056] The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies. Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used. As the skilled person will know, alternative and / or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and / or equivalent names are interchangeable in the context of the present disclosure. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.

[0057] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0058] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti- PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesincomprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e. ., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP -224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody that may be used. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an exemplary anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP -224, also known as B7-DCIg, is aPDL2-Fc fusion soluble receptor.

[0059] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD 152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number LI 5006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0060] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0061] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acidsequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).5. Surgery

[0062] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).

[0063] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.6. Other Agents

[0064] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti- hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin.XI. Articles of Manufacture or Kits

[0065] An article of manufacture or a kit is provided comprising polynucleotides, polypeptides, immune cells, antibodies, reagents, buffers, or a combination thereof is also providedherein. The article of manufacture or kit can further comprise a package insert comprising instructions for using the immune cells to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer. Any of the antigen-specific immune cells described herein may be included in the article of manufacture or kits. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti -neoplastic agent). Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.XII. Sequences used in Certain EmbodimentsTable 1: Sequences of the disclosure.

[0066] As used herein, a “protein” or “polypeptide” refers a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, wild-type versions of a protein or polypeptide are employed, however, in many embodiments of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity.

[0067] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solidphase peptide synthesis (SPPS) or other in vitro methods. In particular embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refersto a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0068] In certain embodiments the size of a protein or polypeptide (wild-type or modified) may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or 525 amino acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.

[0069] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids or nucleotides, or any range derivable therein, of any one of the SEQ ID NOs of Table 1, respectively.

[0070] It is contemplated that in certain embodiments of compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0071] Variant Polypeptides

[0072] The following is a discussion of changing the amino acid subunits of a protein to create an equivalent, or even improved, second-generation variant polypeptide or peptide. For example, certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.

[0073] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0074] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to the sequences in Table 1. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. Avariant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.

[0075] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0076] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.

[0077] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0078] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesisrather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0079] Alternatively, substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.

[0080] One skilled in the art can determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan will also be able to identify amino acid residues and portions of the molecules that are conserved among similar proteins or polypeptides. In further embodiments, areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or without adversely affecting the protein or polypeptide structure.

[0081] In making such changes, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain embodiments, the substitution of amino acids whose hydropathy indices are within ±2 is included. In some aspects of the present disclosure, those thatare within ±1 are included, and in other aspects of the present disclosure, those within ±0.5 are included.

[0082] It also is understood in the art that the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554, 101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (—0.4); proline (-0.5+1); alanine (_0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other embodiments, those which are within ±1 are included, and in still other embodiments, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0083] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0084] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acidresidue. These variants can then be screened using standard assays for binding and / or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy.org / proteomics / protein_structure.

[0085] In some embodiments of the disclosure, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and / or (5) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).XIII. Examples

[0086] The following examples are included to demonstrate particular embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1Screening of an exemplary RFR candidate

[0087] The effect of exemplary RFRs were evaluated with CAR-transduced V62 T cells. Here, we utilized an anti-CD19 CAR construct containing 4-1BB and the CD3z endodomain (19BBz). The CAR construct allows V52 T cells to recognize and mediate cytotoxicity against CD19-expressing tumor cells. Hence, in combination with the RFR, these effector cells are expected to be able to resist apoptosis and proliferate as a result of cytokine signaling, allowing these V52 T cells to mediate superior tumor control. We designed several RFR candidates toevaluate the feasibility of this design (AFas, Fas-cMPL, Fas-MyD88, Fas-gpl30, and Fas- gpl30(Sl)) (Fig. 3A). A Fas transmembrane domain was used in these constructs. Since the natural Fas homo-trimer is activated upon engagement of FasL, the immunostimulatory domains chosen here form natural homo-dimers, which can be utilized to transfer signal 3 in lieu of the natural Fas death signal.

[0088] We first transduced RFR candidates into 19BBz-V52 T cells and evaluated their expression. As shown in Fig. 3B, RFRs can be co-expressed with 19BBz as detected by the NGFR reporter gene included to detect the RFRs. Next, to investigate whether RFRs function as dominant-negative Fas receptors, engineered V52 T cells were incubated with soluble FasL and evaluated in their apoptosis status via Annexin V and 7-AAD staining. While soluble FasL induced apoptosis in non-transduced (NT) and 19BBz-V82 T cells, the expression of RFRs was able to protect 19BBz-V52 T cells from Fas-induced apoptosis (Fig. 3C-D).

[0089] To evaluate the benefit of downstream signaling of RFR, we performed coculture experiments where engineered V52 T cells were cocultured with the CD19+NALM6 cell line in the absence of exogenous cytokines. We monitored both T cell and target cell expansion over 9 days by flow cytometry. 19BBz-V62 T cells exhibited short-term cytotoxicity compared with NT V82 T cells against NALM6, and co-expression of AFas enhanced their anti-tumor effect with minimum proliferation of V82 T cells. Amongst the RFR candidates, co-expression of Fas-MyD88 on 19BBz-V82 T cells showed superior tumor killing with the highest level of V82 T cell proliferation (Fig. 3E-F), indicating that signaling through the RFR can provide a proliferative advantage to 19BBz-V82 T cells in the absence of exogenous cytokines.Example 2Fas-MyD88 RFR enhanced in vivo functionality of CAR V62 T cells

[0090] The impact of RFRs on CAR-V62 T cells was further evaluated in an in vivo xenograft murine model of B cell leukemia. For this, we used V82 T cells modified to express Aka-luciferase (AkaLuc), 19BBz, with or without the Fas-MyD88 RFR. NALM6 cells (0.5 x 106) expressing click beetle green luciferase (CBG) were intravenously infused into NSG mice, followed by engineered V32 T cells (4 x 106) 5 days later (Fig. 4A). Both T cell and tumor cell proliferation were monitored by IVIS imaging, with mouse tail vein bleeding at specific timepoints.

[0091] 19BBz-V82 T cells and AFas + 19BBz V82 T cells delayed tumor growth compared to control NT V32 T cells(Fig. 4B), but showed limited persistence (Fig. 4C). While enrichment of the NGFR(+) / FLAG(+) population of human T cells in mouse blood was observed for Fas-MyD88 + 19BBz V82 T cells, a more variable response was observed for AFas + 19BBz V62 T cells (Fig. 4D). In contrast, treatment with Fas-MyD88 + 19BBz-V62 T cells showed improved tumor control with enhanced in vivo T cell expansion (Fig. 4B-C), resulting in a prolonged overall survival of mice (Fig. 4E).Example 3Fas-MyD88 RFR provided functional enhancement of iPSC-derived T-cells

[0092] Separately, we found that Fas-MyD88 improved the cytotoxicity of induced pluripotent stem cell (iPSCs)-derived T cells (iT cells hereafter). iPSCs were genetically modified to express CD19.CAR and differentiated to iT cells. We further modified CD19.CAR iT cells to express Fas-MyD88 RFR and evaluated its effect on CD19.CAR iT cells. In coculture experiments against CD19(+) NALM6 target cells, CD19.CAR iT cells co-expressing Fas-MyD88 showed improved killing over CD19.CAR iT cells (Fig. 5), indicating that the ability of RFRs to enhance T cells activity is not only limited to V62 T cells.Example 4Applications of the RFR construct

[0093] In the example, we used CAR-V82 T cells to investigate the utility of the RFR. In examples we 1) a single construct was used to provide both anti-apoptosis and signal 3 immunostimulatory signaling, 2) the signal 3 immunostimulatory signaling was transmitted under the control of the Fas extracellular domain, and 3) signaling was constrained to only cells that express the RFR, preventing potential toxicity driven by the endogenous immune system

[0094] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.U.S. Patent No. 11,725,210Oda SK, Anderson KG, Ravikumar P, Bonson P, Garcia NM, Jenkins CM, Zhuang S, Daman AW, Chiu EY, Bates BM, Greenberg PD. A Fas-4-lBB fusion protein converts a death to a pro-survival signal and enhances T cell therapy. J Exp Med. 2020 Dec 7;217(12):e20191166. doi: 10.1084 / jem.20191166. PMID: 32860705; PMCID: PMC7953733.U.S. Patent Application No: 20230133554CA3174812

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a first polypeptide comprising an Antigen Receptor for Immune Cell Activation (AgRICA) and a second polypeptide comprising an extracellular domain of a death receptor, a transmembrane domain, and an intracellular domain that transmits cytokine signaling.

2. A composition comprising a first polynucleotide that encodes a first polypeptide comprising an AgRICA and a second polynucleotide that encodes a second polypeptide comprising an extracellular domain of a death receptor, a transmembrane domain, and an intracellular domain that transmits cytokine signaling, optionally wherein the first polynucleotide and the second polynucleotide is comprised on one or more vectors.

3. The composition of claim 1-2, wherein the first polynucleotide and the second polynucleotide are attached.

4. The composition of any one of claims 1-3, wherein the AgRICA comprises a extracellular receptor domain, a transmembrane domain, and interacelluar Signal 1 domain.

5. The composition of any one of claims 1-3, wherein the AgRICA comprises a extracellular receptor domain, a transmembrane domain, and interacelluar Signal 2 domain.

6. The composition of any one of claims 1-3, wherein the AgRICA is a BiTE, a CAR, a TCR, or a combination thereof.

7. The composition of any one of claims 165, wherein the intracellular domain that transmits cytokine signaling is all or a functional portion of cMPL, myD88, gpl30, gpl30(51), TLR2, TLR4, or a combination thereof.

8. The composition of claim 7, wherein the intracellular domain that transmits cytokine signaling domain is any one or more of a sequence that is at least 95% similar to SEQ ID NOs: 8- 13.

9. The composition of any one of claims 1-8, wherein the extracellular domain of a death receptor is all of or a functional portion of Fas, TNFR1, DR3, TRAIL-R1, TRAIL-R2, DR6, or ED AR.

10. The composition of claim 9, wherein the extracellular domain of a death receptor comprises any one or more of a sequence at least 95% similar to SEQ ID NOs. 1-7.

11. A cell comprising the composition of any one of claims 1-10.

12. The cell of claim 11, wherein the cell does not natively express Jakl or Jak3.

13. The cell of claim 11, wherein the cell is a T cell.

14. The cell of claim 11, wherein the cell is gamma-delta T cell, NK cell, IPS cell, or a mixture thereof.

15. A plurality of a cell of any one of claims 11-14.

16. The plurality of claim 15, wherein the plurality is frozen.

17. A method of treating a cancer in an individual comprising administering to the individual a therapeutically effective amount of a plurality of cells of any one of claims 15-16, the composition of any one of claims 1-10, or a combination thereof.

18. The method of claim 17, wherein the cancer is a hematological cancer.

19. The method of claim 17, wherein the cancer is a solid tumor.

20. The method of any one of claims 17-19, further comprising administering at least a second therapeutic agent to the subject.

21. The method of claim 20, wherein the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiotherapy, drug therapy, targeted therapy, hormone therapy, biotherapy, or a combination thereof.

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