CA2-IL15 fusion protein for adjustable control

Abstract

The present disclosure provides drug-responsive domains derived from human carbonic anhydrase 2 that can modulate the protein stability of human interleukin-15 (IL15) payloads, as well as compositions and methods of use thereof.

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 898,520, filed on September 10, 2019. The entire content of the said application is incorporated herein by reference in its entirety. Reference to the Sequence Listing

[0002] This application includes a Sequence Listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on September 10, 2020, is named 268052_473951_SL.txt and has a size of 178,693 bytes.

Technical Field

[0003] The present disclosure relates to a drug - responsive domain (DRD) derived from human carbonic anhydrase 2 (CA2) that can modulate protein stability for at least one payload including human interleukin 15 (IL15), compositions, and methods of using the same. In the present disclosure, there are provided a CA2 biocircuit system, a CA2 effector module, a polypeptide of a stimulus - responsive element (SRE), polynucleotides encoding them, vectors and cells containing the polypeptide and / or polynucleotide, which are used for enhancing the reaction from immune cells.

Background Art

[0004] By using the DRD technology described herein in conjunction with methods of controlling cytokine function and / or expression, existing immunotherapy strategies can be significantly improved, expanding the area of protein therapeutics that can be safely and effectively incorporated into gene transfer and adoptive T cell transfer (ACT) therapies, including uses that have heretofore been considered unsuitable for therapeutic use. Improvements in natural killer cells (NK cells), tumor infiltrating lymphocytes (TIL), and T cell-based immunotherapies enhance the functionality of the therapy and are required to improve, for example, by improving the persistence and / or viability of engineered immune cells for use upon administration to a subject in various immunotherapies. CA2 DRDs that bind human IL15, modified cells, compositions, and methods that satisfy such needs are provided.

Summary of the Invention

[0005] The present disclosure provides a novel protein domain derived from human carbonic anhydrase 2 (CA2) that exhibits small molecule-dependent stability. Such a protein domain is referred to as a drug-responsive domain (DRD). In the absence of its binding (i.e., stabilizing) ligand, the DRD destabilizes, resulting in degradation of the payload (e.g., a protein of interest (POI)) operably linked to the DRD. On the other hand, in the presence of its binding ligand, the DRD and its operably linked payload are stabilized. The stability of the DRD and its operably linked payload is determined by the dose of the binding ligand.

[0006] In some embodiments, the present disclosure provides a stimulus-responsive element (SRE) that can include, in whole or in part, a drug-responsive domain (DRD) derived from human carbonic anhydrase 2 (CA2, having the amino acid sequence of SEQ ID NO: 1). In one embodiment, the DRD can be derived from the full-length CA2 polypeptide (SEQ ID NO: 1). In some embodiments, the DRD can be derived from a portion or region of human carbonic anhydrase. The portion or region of CA2 can be selected from amino acids 2-260 of CA2 (SEQ ID NO: 2).

[0007] In some embodiments, the SRE may include a DRD that contains one, two, three, four or more mutations in CA2 relative to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the SRE may include a DRD that contains one, two, three, four or more amino acid substitutions in CA2 relative to SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] In some embodiments, the SRE may include a DRD that contains one, two, three, four or more mutations in a part of CA2. In some embodiments, the SRE may include a DRD that contains one, two, three, four or more mutations in CA2 or a part thereof, and may further contain additional amino acids. In some embodiments, the SRE may include a DRD that contains one, two, three, four or more amino acid substitutions in a part of CA2. In some embodiments, the SRE may include a DRD that contains one, two, three, four or more amino acid substitutions in CA2 or a part thereof, and may further contain additional amino acids.

[0009] Also provided herein is an isolated polypeptide variant that contains at least one mutation relative to SEQ ID NO: 1. Non-limiting examples of CA2 mutations relative to SEQ ID NO: 1 include M1del and L156H. In another aspect, the DRD is a polypeptide that contains the amino acid sequence of SEQ ID NO: 4 or consists of the amino acid sequence of SEQ ID NO: 4, and the CA2 mutation includes Mdel1 (M1del) and L156H relative to SEQ ID NO: 1. In another aspect, the DRD is a polypeptide that contains the amino acid deletion M1del and the amino acid substitution L156H relative to SEQ ID NO: 1, and may further contain additional amino acids. In another aspect, the DRD is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4.

[0010] Also provided herein is a bio - circuit system comprising at least one effector module. The effector module of the bio - circuit may comprise a stimulus - responsive element (SRE), and the SRE may comprise a DRD derived from human carbonic anhydrase 2 (CA2; SEQ ID NO: 1) or a variant thereof, which comprises one, two, three, four or more mutations of CA2 with respect to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the effector module of the bio - circuit comprises an SRE comprising a DRD that comprises one, two, three, four or more amino acid substitutions in CA2 with respect to SEQ ID NO: 1 or SEQ ID NO: 2. The bio - circuit may comprise at least one payload that can be attached to, added to, or associated with the SRE. The payload may include, but is not limited to, (i) human IL15 comprising the amino acid sequence of SEQ ID NO: 8.

[0011] The SRE of the bio - circuit system may comprise one, two, three or more mutations of CA2 (SEQ ID NO: 1 or SEQ ID NO: 2), such as but not limited to Mdel1 and L156H. The SRE of the bio - circuit system may comprise one, two, three or more amino acid substitutions in CA2 (SEQ ID NO: 1 or SEQ ID NO: 2), such as but not limited to L156H.

[0012] In some embodiments, the SRE in the CA2 bio - circuit system may be CA2 having the mutations M1del and L156H, and the numbering is related to the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 4). In some embodiments, the SRE is a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or consisting of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the SRE is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4.

[0013] The bio - circuit system described herein may comprise an SRE that responds to one or more stimulants.

[0014] In some embodiments, the stimulant may be a small molecule, and the small molecule may be acetazolamide (ACZ).

[0015] In another aspect, the present disclosure provides an effector module comprising at least one payload. In some embodiments, the effector module comprises an SRE comprising a CA2 DRD operably linked to an IL15 payload. In some embodiments, the IL15 payload comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the IL15 payload may be partially encoded by a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the IL15 payload is the membrane-bound form of IL15. In some embodiments, the IL15 payload is the membrane-bound form of IL15 comprising a functional IL15 component or domain, a transmembrane domain, and an intracellular tail. In some embodiments, the IL15 payload is the membrane-bound form of IL15 comprising a functional IL15 component or domain, a transmembrane domain, an intracellular tail, and a leader sequence. In some embodiments, the present disclosure provides an SRE comprising a CA2 DRD operably linked to a membrane-bound IL15 polypeptide. In some embodiments, the present disclosure provides an SRE comprising a CA2 DRD operably linked to a membrane-bound IL15 polypeptide, and the membrane-bound IL15 polypeptide comprises, from N-terminus to C-terminus, a leader sequence, an IL15 polypeptide comprising the amino acid sequence of SEQ ID NO: 8, a peptide linker, a transmembrane domain, and an intracellular tail.

[0016] In another aspect, the present disclosure provides a method of creating a modified or genetically engineered cell, comprising the step of introducing into the cell a polynucleotide encoding an effector module. In some embodiments, the modified or engineered cell is an immune cell. In some embodiments, the immune cell is a T cell, a natural killer (NK) cell, or a tumor infiltrating lymphocyte (TIL). In some embodiments, the polynucleotide encodes a CA2 DRD operably linked to an IL15 payload. In some embodiments, the polynucleotide encodes a CA2 DRD operably linked to a membrane-bound IL15 payload. In some embodiments, the polynucleotide is introduced into the cell by a non-viral vector delivery method. In some embodiments, the polynucleotide is introduced into the cell by viral transduction. In some embodiments, the polynucleotide is introduced into the cell by lentiviral transduction. In some embodiments, the polynucleotide is introduced into the cell by lentiviral transduction of a T cell, an NK cell, or a TIL. In some embodiments, the present disclosure provides a method of creating a modified or genetically engineered T cell, NK cell, or TIL, comprising the step of introducing, by a viral vector such as a lentiviral vector, a polynucleotide encoding a CA2 DRD operably linked to a membrane-bound IL15 payload, into a T cell, an NK cell, or a TIL.

[0017] In another aspect, the present disclosure provides a method of treatment comprising: (a) administering to a subject having a disease or disorder a modified cell comprising a recombinant construct comprising an SRE coupled to a payload of the present disclosure, or a composition comprising a plurality of such modified cells; and (b) administering to the subject a therapeutically effective amount of an agent to which the SRE responds. In some embodiments of this aspect, the disease or disorder is cancer, a neoplasm, or a tumor. In some embodiments, the SRE is a CA2 DRD. In some embodiments, the payload is IL15 or membrane-bound IL15. In some embodiments, the modified cell comprises a CA2 SRE operably coupled to an IL15 payload. In some embodiments, the modified cell comprises a CA2 SRE operably coupled to a membrane-bound IL15 payload. In some embodiments, the agent is acetazolamide, celecoxib, baldecoib, rofecoxib, methazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. In some embodiments, the modified cell comprises a CA2 operably coupled to a membrane-bound IL15 payload, and the agent is acetazolamide. In some related aspects, the modified cell is an engineered or modified immune cell; for example, the CA2-IL15 biocircuit and system may be used with immune cells including T cells such as CD8+ T cells and CD4+ T cells, natural killer (NK) cells, NK T cells, cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer (LAK) cells, memory T cells, regulatory T cells (Tregs), helper T cells, cytokine-induced killer (CIK) cells, and any combination thereof. In other embodiments, the immune stimulatory cells for ACT may be generated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In some embodiments, autologous or allogeneic immune cells are used for ACT. In some embodiments, the immune cells are T cells, TILs, or NK cells.In some embodiments, the immune cells are NK cells derived from iPSCs, cord blood, or peripheral blood mononuclear cells, and the modified immune cells exhibit greater or longer proliferation and / or persistence in a subject than in a subject administered a reference cell composition lacking the SRE conjugated to the payload at the same or nearly the same dose.

[0018] In another aspect, the present disclosure provides a method for treating a malignancy in a subject, comprising: (a) administering to the subject a composition comprising a modified T cell, a modified NK cell, or a modified TIL, or a plurality of such modified cells, comprising a recombinant construct comprising an SRE coupled to a payload of the present disclosure; and (b) administering to the subject a therapeutically effective amount of a stimulant to which the SRE responds. In some embodiments, the tumor expresses a tumor-associated antigen. In some embodiments, the modified T cell or the modified NK cell further comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding domain specific for the tumor-associated antigen. In some embodiments, the modified T cell or the modified NK cell comprises a CAR comprising an antigen-binding domain specific for the tumor-associated antigen. In some embodiments, the modified T cell, the modified NK cell, or the modified TIL comprises an SRE that is CA2 DRD. In some embodiments, the modified T cell, the modified NK cell, or the modified TIL comprises a payload that is IL15 or membrane-bound IL15. In some embodiments, the modified T cell, the modified NK cell, or the modified TIL comprises a CA2 SRE operably linked to an IL15 payload. In some embodiments, the modified T cell, the modified NK cell, or the modified TIL comprises a CA2 SRE operably linked to a membrane-bound IL15 payload. In some embodiments, the stimulant is acetazolamide, celecoxib, baldecoib, rofecoxib, metazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. In some embodiments, the modified cell comprises a CA2 SRE operably linked to a membrane-bound IL15 payload and the stimulant is acetazolamide.

[0019] In another aspect, the present disclosure provides a polynucleotide and a vector encoding a biocircuit system, and a pharmaceutical composition comprising the biocircuit system and a pharmaceutically acceptable additive.

[0020] In another aspect, the present disclosure provides a recombinant protein encoded by a polynucleotide of the present disclosure. In some embodiments, the recombinant protein comprises an effector module comprising a CA2 DRD operably linked to an IL15 payload. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The foregoing and other objects, features, and advantages will become apparent from the following description of particular embodiments of the present disclosure as illustrated in the accompanying drawings. The drawings are not necessarily to scale, and emphasis is instead placed upon illustrating the principles of the various embodiments of the present disclosure.

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Mode for Carrying Out the Invention

[0022] Details of one or more embodiments of the present disclosure are set forth in the following accompanying description. Any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but the preferred materials and methods are described below. Other features, objects, and advantages of the present disclosure will become apparent from the description. In this description, the singular forms also include the plural forms unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present description will control.

[0023] Cancer immunotherapy aims to induce or restore the responsiveness of the immune system against cancer. Due to significant progress in immunotherapy research, various strategies have been developed that can be broadly classified into active immunotherapy and passive immunotherapy. Generally, these strategies can be utilized to directly kill cancer cells or to counteract the immunosuppressive tumor microenvironment. Active immunotherapy aims to induce an endogenous and persistent tumor antigen-specific immune response. The response can be further enhanced by non-specific stimulation of immune response modifiers such as cytokines. In contrast, passive immunotherapy involves approaches in which immune effector molecules such as tumor antigen-specific cytotoxic T cells or antibodies are administered to the host. This approach is short-lived and requires multiple treatments.

[0024] Efficient T cell activation requires three signals, namely T cell receptor (TCR) signaling (Signal 1), activation by co-stimulatory molecules (Signal 2), and immune-stimulatory cytokines (Signal 3). To date, the majority of CAR-based immunotherapies that have been designed and discussed have Signal 1 and Signal 2. However, Signal 3, which is typically provided by homeostatic cytokines, is not typically present in conventional CAR T cells and is not abundant in the tumor microenvironment either.

[0025] Therefore, it is necessary to engineer T cells (including, for example, CAR T cells) that can provide additional cytokine signaling to meet the need for Signal 3 for optimal T cell activation. The major cytokines involved in T cell activation that encompass Signal 3 cytokines belong to the γc class, such as IL-2, IL-7, IL-15, IL-21, and IL-9. These cytokines regulate the survival and proliferation of T cells, which ultimately play important roles in T cell persistence and efficacy. These cytokines are currently used in the ex vivo expansion of CAR T cells prior to combination or single therapy.

[0026] However, there may be a risk in supporting the extension of T cell lifespan by continuous exposure to IL15. This is because chronic high exposure to IL-15 may cause abnormal T cell proliferation or toxicity. In humans, dysregulated production of IL15, high serum levels, or abnormal IL15 signaling is associated with autoimmune diseases and may be involved in the development of large granular lymphocyte leukemia and cutaneous T cell lymphoma.

[0027] Natural killer (NK) cells are members of the natural lymphoid cell family and are characterized in humans by the expression of the phenotypic marker CD56 (neural cell adhesion molecule) in the absence of CD3 (T cell coreceptor). NK cells are powerful effector cells of the innate immune system that mediate cytotoxic attacks without the need for prior antigen priming and form the front line of defense against diseases including cancer malignancies and viral infections.

[0028] Several preclinical and clinical trials have demonstrated that adoptive transfer of NK cells is a promising treatment for cancers such as acute myeloid leukemia (Ruggeri et al., Science; 2002, 295:2097-2100; and Geller et al., Immunotherapy, 2011, 3:1445-1459). Adoptive transfer of NK cells expressing chimeric antigen receptors (CARs) such as DAP12-based activating CARs has been shown to promote tumor cell eradication (Topfer et al., J Immunol. 2015;194:3201-3212). NK cells engineered to express a CS-1-specific CAR have also been shown to promote cell lysis and interferon-γ (IFN-γ) production in multiple myeloma (Chu et al., Leukemia, 2014, 28(4):917-927).

[0029] NK cell activation is characterized by a series of receptors with activation and inhibitory functions. Important activating receptors in NK cells include CD94 / NKG2C and NKG2D (C-type lectin-like receptors), as well as natural cytotoxic receptors (NCR) NKp30, NKp44, and NKp46, which recognize ligands in tumor cells or virus-infected cells. NK cell inhibition is basically mediated by the α1 helix of HLA molecules through the interaction of polymorphic inhibitory killer cell immunoglobulin-like receptors (KIR) with their allogeneic human leukocyte antigen (HLA) ligands. The balance between signals generated from activating and inhibitory receptors mainly determines immediate cytotoxic activation.

[0030] NK cells may be isolated from peripheral blood mononuclear cells (PBMC) and cord blood, or may be derived from human embryonic stem (ES) cells and induced pluripotent stem cells (iPSC). NK cells can be further developed for adoptive immunotherapy. Strategies and protocols useful for the expansion of NK cells may include interleukin 2 (IL2) stimulation and the use of autologous feeder cells, or the use of genetically engineered allogeneic feeder cells. In some embodiments, NK cells can be selectively developed in combination with stimulatory ligands including IL15, IL21, IL2, 41BBL, IL12, IL18, MICA, 2B4, LFA-1, and BCM1 / SLAMF2 (e.g., U.S. Patent Publication No. US20150190471).

[0031] NK cell-based immunotherapy is rapidly evolving due to the ability of NK cells to directly lyse tumor targets, the emergence of antibodies and molecules that mediate NK cell-driven antibody-dependent cell-mediated cytotoxicity (ADCC), and the ability of NK cells to induce an inflammatory response. NK cells are being utilized in clinical trials using autologous and allogeneic NK cell infusion strategies alone or in combination with hematopoietic stem cell transplantation. In addition, there are signs of other modalities of NK cell therapy, such as the use of NK cell line products and NK cells transduced with chimeric antigen receptors (CARs). Others have shown that the in vivo persistence and proliferation of NK cells correlate with antitumor activity in patients with advanced AML. Among the preclinically evaluated strategies to address this issue, the utilization of cytokines that induce NK cell persistence and proliferation appears to be mainstream in current clinical trials. IL15 has a known physiological role in NK cell development and homeostasis without stimulating regulatory T cells, but experimental findings have shown that continuous treatment with IL-15 results in changes in functional NK cells consistent with exhaustion. For example, in at least one study, NK cells continuously treated with IL15 were experimentally shown to initially exhibit better proliferation and expansion during 9 days of continuous treatment with experimental IL15 but were more susceptible to the effects of cell death. In addition, cell cycle gene expression data have shown that NK cells continuously administered IL15 are enriched in the expression of cell cycle checkpoint genes and arrest genes, and on day 9 of culture, these cells have been shown to transition to a state of arrest due to cellular stress.

[0032] Tumor-infiltrating lymphocytes (TILs) consist of all lymphocyte cell populations that have invaded tumor tissue. The cellular components of tumors include TILs, NK cells, macrophages, dendritic cells, and myeloid lineage cells, suggesting a productive immune response. However, most immune cells present in the tumor microenvironment have some form of impaired normal function, as many of the immune cell populations are converted to phenotypes that further impair immune system responses. Tumors recruit regulatory T lymphocytes (Tregs), tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and cancer-associated fibroblasts (CAFs), which can help them evade immune recognition. Both Tregs and MDSCs exhibit immunosuppressive functions and limit responses by TILs and other cells. Depletion of CD4+ Tregs improves the clinical response of patients undergoing immune reconstitution treated with autologous TILs during TIL therapy. In mouse models, even a small number of Tregs can suppress adoptive cell therapy mediated by effective CD8+ T cells.

[0033] TILs are found in many solid tumors, including breast cancer and melanoma, and appear as important biomarkers in predicting the efficacy and outcome of treatment. In breast cancer, TILs mainly consist of cytotoxic (CD8+) and helper (CD4+) T cells, with low proportions of B cells and NK cells. Breast cancer patients with progressive tumors with high CD8+ T cell infiltrates or high TIL density have more favorable outcomes. In melanoma, TIL therapy is improved by including a lymphodepleting pretreatment therapy prior to cell injection. Investigations in human and mouse models of melanoma have suggested that lymphodepletion can suppress T cell proliferation in melanoma patients and serve to deplete negative regulatory cells, including regulatory T cells (Tregs) and peripheral myeloid-derived suppressor cells, which are beneficial for the proliferation of adoptively transferred T lymphocytes.

[0034] Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) is an individualized cancer treatment based on the infusion of autologous CD4+ and CD8+ T lymphocytes that have proliferated from tumors in the presence of interleukin-2 (IL-2) alone or in combination with IL-7, IL-15, and / or IL-21. TILs are a polyclonal population rich in lymphocytes that recognize tumor-specific antigens and include not only individual tumor neoantigens but also shared tumor-associated antigens. In a study at the National Cancer Institute (NCI) initiated in 1980, tumor regression was demonstrated in selected patients who received adoptive transfer of lymphokine-activated killer cells in combination with recombinant IL-2. Subsequent methods for large-scale expansion of human TILs, simplification and shortening of the TIL production process, and improvement of patient preconditioning and treatment protocols have resulted in increased patient response rates. However, the overall response rate of TIL therapy remains very low and improvement is needed.

[0035] The present disclosure provides systems, compositions, immunotherapeutic agents, and methods that avoid problems associated with continuously administered or expressed IL15 by providing regulatable control of IL15 gene expression and function for cancer immunotherapy. The invention also provides a biocircuit system, effector module, stimulus-responsive element (SRE), and IL15 payload, as well as polynucleotides encoding any of the foregoing. In one aspect, the systems, compositions, immunotherapeutic agents, and other components of the invention can be controlled by separately added stimulants that provide sufficient adaptability to control cancer immunotherapy.

[0036] The adjustable properties of the systems and compositions of the present invention have the potential to improve the efficacy and duration of effectiveness of immunotherapy. The ability to reversibly increase, decrease, or silence the biological activity of adoptively transferred cells using the compositions of the present invention enables the maximization of the potential of cell therapies that cannot use a "kill switch" that would terminate the therapy. Without being bound by any particular theory, the long-term engraftment of T cells can be achieved through transient intermittent exposure to IL15 in various cell populations, including NK cells, TIL, and T cells, used in various therapies, including cancer immunotherapy, without dysregulated proliferation or activation, and without phenotypic, functional, or chromosomal abnormalities.

[0037] The present invention provides a method for fine-tuning immunotherapy after administration to a patient. This subsequently improves the safety and effectiveness of immunotherapy and increases the population of subjects who benefit from immunotherapy. The effector modules described and disclosed in this disclosure form effector modules comprising mbIL15 by binding independently of, or integrally with, one or more stimulus-responsive elements (SREs) operably linked to IL15. The bio-circuits, SREs, and DRDs of the present disclosure can be used with immune cells and provide the desired signaling to enable adoptively transferred NK cells and T cells, including TIL, to extend their persistence and thereby provide persistent immune surveillance and therapeutic potential.

[0038] As used herein, a "biocircuit" or "biocircuit system" is defined as a circuit within a biological system or useful in a biological system that includes an agonist and at least one effector module that responds to the agonist, and the response to the agonist produces at least one signal or outcome within the biological system, between biological systems, as an indicator of the biological system, or in the biological system. A biological system is commonly understood to be any cell, tissue, organ, organ system, or living organism, regardless of whether it is an animal, plant, fungus, bacterium, or virus. A biocircuit may also be understood to be an artificial circuit that produces a signal or outcome in a cell-free environment, such as by a diagnostic, reporter system, device, assay, or kit, using an agonist or effector module taught by the present disclosure.

[0039] The biocircuits of the present disclosure include at least one effector module. As used herein, an "effector module" is a single or multi-component construct or complex that includes at least (a) one or more stimulus-responsive elements (SREs) and (b) one or more payloads (e.g., a protein of interest (POI)). In some embodiments, the effector module includes one SRE and one payload.

[0040] The effector module may be designed to include one or more payloads, one or more SREs, one or more cleavage sites, one or more signal sequences, and one or more additional features including the presence or absence of one or more linkers.

[0041] In one embodiment, the effector module includes at least one immunotherapeutic agent, such as IL15.

[0042] Effector modules include their SREs and payloads and may be nucleic acid-based, protein-based, or a combination thereof. They may be in the form of DNA, RNA, mRNA, protein, fusion protein, or any combination of the foregoing. In one embodiment, the effector module is a fusion protein. In one embodiment, the effector module is encoded by a nucleic acid such as DNA.

[0043] Effector modules include their SREs and payloads and may individually, collectively, or independently include peptides, polypeptides, or proteins. At the protein level, such payloads may be any natural or artificial peptide or polypeptide or fragment thereof. The natural peptide or polypeptide components of the payload may be from any known protein of any species.

[0044] Effector modules may be designed to operate in a group of one, two, three, four or more modules. When two or more effector modules are utilized in a biocircuit, it is known as the effector module system of that biocircuit.

[0045] As used herein, a "stimulus-responsive element" (SRE) is a component of an effector module that links, attaches to, binds to, or associates with one or more payloads and, in some cases, causes the responsiveness of the effector module to one or more stimulants. As used herein, the "responsive" nature of an SRE to a stimulant may be characterized by covalent or non-covalent interactions, direct or indirect binding, or a structural or chemical reaction to the stimulant. Further, the reaction of any SRE to a stimulant may be a matter of degree or type. The reaction may be a partial reaction. The reaction may be a reversible reaction. The reaction may ultimately result in a control signal or output. Such an output signal may be from the relative nature to the stimulant and may produce, for example, a modifying effect of 1% to 100% or a factored increase or decrease such as 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more. In some embodiments, the SRE is a polypeptide that is operably bound to a polypeptide payload. In some embodiments, the SRE is a polypeptide fused to a polypeptide payload.

[0046] In some embodiments, the present disclosure provides methods for modulating protein expression, function, or level. In some aspects, modulating protein expression, function, or level refers to modulating expression, function, or level by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%, or 95-100%, etc., i.e., modulating expression, function, or level by at least about 20%.

[0047] A drug-responsive domain (DRD) is a small protein domain that can be added to a target protein of interest. In some embodiments, the DRD is operably linked to the target protein of interest. The DRD destabilizes the attached target protein in the absence of a DRD-binding ligand. However, when a specific small molecule ligand binds its intended DRD as a ligand-binding partner, the destabilization is overridden and protein function is restored. The conditional nature of DRD stability allows for a rapid and non-disruptive switch from a stable protein to an unstable degradation substrate. Furthermore, the dependence on the concentration of the ligand further provides tunable control of the degradation rate. The term drug-responsive domain (DRD) is interchangeable with the term destabilizing domain (DD).

[0048] In one embodiment, the SRE is a drug-responsive domain (DRD). In some embodiments, the CA2 drug-responsive domains described herein may be used as SREs in the bio-circuit systems of the present disclosure in connection with any of the IL15 payloads taught herein.

[0049] A region or part or domain of a wild-type protein (e.g., CA2) may be utilized as an SRE / DRD, in whole or in part. In one embodiment, the SRE is derived from the parent protein CA2 or a mutant CA2 protein. In various embodiments, the DRD comprises one, two, three, or four or more mutations compared to the parent CA2 protein, e.g., of human CA2, the amino acid sequence: MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY DPSLKPLSVS YDQATSLRIL NNGHAFNVEF DDSQDKAVLK GGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHL VHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVV DVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTP PLLECVTWIV LKEPISVSSE QVLKFRKLNF NGEGEPEELM VDNWRPAQPL KNRQIKASFK of SEQ ID NO: 1, or the amino acid sequence: SHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY DPSLKPLSVS YDQATSLRIL NNGHAFNVEF DDSQDKAVLK GGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHL VHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVV DVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTP PLLECVTWIV LKEPISVSSE QVLKFRKLNF NGEGEPEELM VDNWRPAQPL KNRQIKASFK of SEQ ID NO: 2.

[0050] Human CA2 having the amino acid sequence of SEQ ID NO: 1 is encoded by the polynucleotide having the nucleic acid sequence of SEQ ID NO: 3: atgtcccatcactgggggtacggcaaacacaacggacctgagcactggcataaggacttccccattgccaagggagagcgccagtcccctgttgacatcgacactcatacagccaagtatgacccttccctgaagcccctgtctgtttcctatgatcaagcaacttccctgaggatcctcaacaatggtcatgctttcaacgtggagtttgatgactctcaggacaaagcagtgctcaagggaggacccctggatggcacttacagattgattcagtttcactttcactggggttcacttgatggacaaggttcagagcatactgtggataaaaagaaatatgctgcagaacttcacttggttcactggaacaccaaatatggggattttgggaaagctgtgcagcaacctgatggactggccgttctaggtatttttttgaaggttggcagcgctaaaccgggccttcagaaagttgttgatgtgctggattccattaaaacaaagggcaagagtgctgacttcactaacttcgatcctcgtggcctccttcctgaatccctggattactggacctacccaggctcactgaccacccctcctcttctggaatgtgtgacctggattgtgctcaaggaacccatcagcgtcagcagcgagcaggtgttgaaattccgtaaacttaacttcaatggggagggtgaacccgaagaactgatggtggacaactggcgcccagctcagccactgaagaacaggcaaatcaaagcttccttcaaa It is encoded by a polynucleotide having the above sequence.

[0051] As used herein, the phrase "derived from" means that when it relates to an effector module, SRE, or payload, the effector module, SRE, or payload has its origin, at least in part, in the described parent molecule or sequence. For example, when designing an SRE, such an SRE may be derived from an epitope or region of a naturally occurring protein but is then modified by any of the methods taught herein and the SRE function is optimized.

[0052] In some embodiments, the DRD of the present disclosure may be derived from CA2 (SEQ ID NO: 1; Uniprot ID: P00918) which can be stabilized by a ligand such as a small molecule inhibitor of CA2. As used herein, the term "CA2 WT" refers to the human wild-type CA2 protein sequence defined as SEQ ID NO: 1 by GenBank Access NO. P00918. In some aspects, the DRD may be derived from CA2 of SEQ ID NO: 2.

[0053] In some embodiments, the DRD may be derived from CA2 having amino acids 2-260 of the wild-type human CA2 sequence of SEQ ID NO:1. This is referred to herein as the M1del mutation. The M1del mutation may also be referred to herein as an amino acid deletion. In some embodiments, the human DRD construct disclosed herein may not include the N-terminal methionine corresponding to the N-terminal methionine of SEQ ID NO:1. Regardless of the presence or absence of the N-terminal methionine in the disclosed CA2 DRD, the present disclosure identifies the position of the CA2 DRD in relation to the wild-type human CA2 of SEQ ID NO:1 (Uniprot ID: P00918), and the reference position 1 is the N-terminal methionine of SEQ ID NO:1. For example, a hypothetical CA2 DRD containing the G12A mutation refers to the CA2 DRD construct herein, and regardless of whether the CA2 DRD construct itself includes the N-terminal methionine corresponding to the N-terminal methionine of SEQ ID NO:1, glycine (G) is mutated to alanine (A) at a certain position in the CA2 DRD construct corresponding to the 12th amino acid of SEQ ID NO:1. The change from glycine (G) to alanine (A) in this example of a hypothetical CA2 DRD containing the G12A mutation is also referred to as an amino acid substitution.

[0054] In some embodiments, the DRD may be derived from human CA2 having amino acids 2-260 of the wild-type human CA2 sequence of SEQ ID NO:1. This may be referred to as the M1del mutation and has the amino acid sequence of SEQ ID NO:2. In some embodiments, the DRD of the present disclosure has an amino acid sequence as set forth in SEQ ID NO:4.

[0055] Table 1 provides the CA2 DRD. The positions of the mutant amino acids listed in Table 1 are related to the full-length CA2 of SEQ ID NO:1.

Table 1

[0056] In some embodiments, an exemplary DRD from CA2 that controls an operably linked IL15 payload comprises or consists of the amino acid sequence of SEQ ID NO:4 or is encoded by the nucleotide sequence of SEQ ID NO:5.

[0057] In some embodiments, a CA2 DRD useful for the controlled and regulatable expression of IL15 as described herein comprises one or more mutations associated with Uniprot ID:P00918 (SEQ ID NO:1) and may include, but is not limited to, (M1del, L156H) in relation to the amino acid sequence of SEQ ID NO:1. In some embodiments, a CA2 DRD useful for the controlled and regulatable expression of IL15 as described herein may include one mutation in relation to (SEQ ID NO:2)(L156H). In some embodiments, a CA2 DRD useful for the controlled and regulatable expression of IL15 as described herein may include one amino acid substitution in relation to (SEQ ID NO:2)(L156H). In some embodiments, the CA2 DRD comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the CA2 DRD consists of the amino acid sequence of SEQ ID NO:4.

[0058] The SREs described herein may include CA2 DRDs that contain one or two mutations, such as, but not limited to, M1del and L156H, relative to the CA2 WT of SEQ ID NO:1. In some embodiments, the CA2 DRD contains the L156H mutation relative to SEQ ID NO:1 and further contains one or more additional mutations. In some embodiments, the CA2 DRD contains the M1del and L156H mutations relative to SEQ ID NO:1 and further contains one or more additional mutations. In some embodiments, the CA2 DRD contains the M1del amino acid deletion and the L156H amino acid substitution relative to SEQ ID NO:1 and further contains one or more additional amino acid substitutions.

[0059] Also provided herein is a bio-circuit system comprising at least one effector module. The effector module of the bio-circuit may comprise a stimulus-responsive element (SRE) derived from CA2 (SEQ ID NO: 1 or SEQ ID NO: 2). In one embodiment, the SRE comprises, or consists of, the amino acid sequence of SEQ ID NO: 4. The bio-circuit may comprise at least one payload that can be attached to, added to, or associated with the SRE. The payload may comprise human IL15 comprising the amino acid sequence of SEQ ID NO: 8, and the payload may be encoded by a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 9.

[0060]

Table 2

[0061] In some embodiments, the present disclosure provides a method of modulating a protein, expression, function, or level by measuring a stabilization rate and a destabilization rate. As used herein, the stabilization rate may be defined as the ratio of the expression, function, or level of a target protein in response to a stimulant to the expression, function, or level of the target protein in the absence of a stimulant specific to the SRE. As used herein, the destabilization rate may be defined as the ratio of the expression, function, or level of a target protein in the absence of a stimulant specific to the effector module to the expression, function, or level of the target protein in the absence of a stimulant specific to the SRE and expressed structurally. As used herein, "structurally" refers to the expression, function, or level of a target protein that is not bound to the SRE and is thus expressed in both the presence and absence of a stimulant.

[0062] As used herein, "payload" or "target payload" or "payload of interest (POI)" is defined as any protein whose function changes. The payload may comprise any protein or fragment thereof.

[0063] In some embodiments, the payload of the present disclosure comprises IL15. It is technically understood that the nomenclature of a gene and / or protein regarding the same gene or protein may include or exclude punctuation such as the dash symbol "-" or symbols such as Greek letters. Whether or not these are included or excluded herein, the meaning is not intended to be changed as understood by those skilled in the art. For example, IL15, IL 15, and IL-15 refer to the same interleukin. In some embodiments, the payload of the present disclosure may be an IL15 interleukin cytokine that stimulates an immune response.

[0064] The payload of the present disclosure may include an amino acid sequence similar to the amino acid sequence of human IL15, such as UniProtKB-P40933 (IL15_HUMAN). In one embodiment, the IL15 payload comprises the amino acid sequence provided in Table 2 (SEQ ID NO: 8).

[0065] In some embodiments, the payload of the present disclosure may be utilized to improve the proliferation, survival, persistence, and efficacy of immune cells such as CD8+ TEM, natural killer (NK) cells, and tumor-infiltrating lymphocytes (TIL), as well as CAR T cells used in immunotherapy. In one aspect, the present disclosure provides bio-circuits and compositions that minimize the toxicity associated with cytokine therapy.

[0066] In some embodiments, the effector module may be a CA2 DRD-IL15 fusion polypeptide. In some embodiments, the IL15-containing construct of the present disclosure may be under the transcriptional regulation of a human cytomegalovirus (CMV) promoter, an elongation factor 1α (EF1α) promoter, an HIV LTR promoter, a 3-phosphoglycerate kinase (PGK) promoter, a Rous sarcoma virus long terminal repeat (RSV) promoter, a spleen focus-forming virus (SFFV) promoter, a synthetic MND promoter, a murine stem cell virus (MSCV) promoter, a synthetic RPBSA promoter, or a ubiquitin promoter.

[0067] A unique feature of IL15-mediated activation is the mechanism of trans-presentation, in which IL15 exists as a complex with the α subunit of the IL15 receptor (IL15Ra), which binds to and activates the membrane-bound IL15 beta / gamma receptor, either in the same cell or in different cells. In various embodiments, the payload of the present disclosure is membrane-bound IL15, and the amino acid sequence of the membrane-bound IL15 comprises the amino acid sequence of SEQ ID NO: 8.

[0068] The payload of the present disclosure may comprise a nucleic acid sequence as disclosed herein, but the payload may comprise additional or fewer nucleotides than those described. Such nucleic acid sequences may comprise more or fewer than about 1 nucleotide, more or fewer than about 2 nucleotides, more or fewer than about 3 nucleotides, more or fewer than about 4 nucleotides, more or fewer than about 5 nucleotides, more or fewer than about 6 nucleotides, more or fewer than about 7 nucleotides, more or fewer than about 8 nucleotides, more or fewer than about 9 nucleotides, more or fewer than about 10 nucleotides, or more than 10 nucleotides.

[0069] Effector modules, and bio-circuit components comprising the same, their SREs and payloads, may be nucleic acid-based. The term "nucleic acid" in a broad sense includes any compound and / or substance that is a polymer of nucleotides, for example, including linked nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribo configuration, α-LNA (a diastereomer of LNA) having an α-L-ribo configuration, 2'-amino-LNA having a 2'-amino functional group, and 2'-amino-α-LNA having a 2'-amino functional group), or hybrids thereof.

[0070] In some embodiments, the nucleic acid molecule is DNA. In some embodiments, the nucleic acid molecule is messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and can be translated to produce the polypeptide of interest in vitro, in vivo, in situ, or ex vivo. The polynucleotides of the present disclosure may be mRNA or any nucleic acid molecule, and may or may not be chemically modified.

[0071] In some embodiments, the polynucleotides of the present disclosure may encompass a 5’UTR sequence that plays a role in translation initiation. The 5’UTR sequence may include features such as the Kozak sequence, which is widely known to be involved in the process by which ribosomes initiate translation of a gene. The Kozak sequence has the consensus XCCR(A / G)CCAUG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), and X is any nucleotide. In one embodiment, the Kozak sequence is ACCGCC. By manipulating features typically found in genes highly expressed in the target cell or tissue, the stability of the polynucleotides of the present disclosure and protein production can be enhanced.

[0072] In one embodiment, the polynucleotides of the present disclosure may encode a variant polypeptide having a certain identity with a reference polypeptide sequence. As used herein, “reference polypeptide sequence” refers to the starting polypeptide sequence. The reference sequence may be a wild-type sequence or any sequence that is referred to in the design of another sequence.

[0073] As is well known in the art, the term "identity" refers to the relationship between two or more sequences as determined by comparing the sequences. Technically, identity also means the degree of sequence relatedness between sequences as determined by the number of matches between strings of two or more residues (amino acids or nucleic acids). Identity measures the percentage of exact matches between two or more sequences by means of a gap alignment (if any) handled by a particular mathematical model or computer program (i.e., an "algorithm"). The identity of related sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W. ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G. eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J. eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0074] In some embodiments, the variant array may have the same or similar activity as the reference array. Alternatively, the variant may have a changed activity (e.g., increased or decreased) relative to the reference array. Typically, a variant of a particular polynucleotide or polypeptide of the present disclosure will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity to that particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. Tools for such alignment include those of the BLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.).

[0075] The effector module of the present disclosure may further include a signal sequence that controls the distribution of the payload of interest, a cleavage mechanism and / or processing mechanism that facilitates cleavage of the payload from the effector module construct, a targeting signal and / or a penetration signal that can control the cellular localization of the effector module, a tag, and / or one or more linker sequences that link different components of the effector module.

[0076] In addition to the SRE and payload region, the effector module of the present disclosure may further include one or more additional features such as one or more signal sequences.

[0077] A signal sequence (also sometimes called a signal peptide, targeting signal, targeting peptide, localization sequence, transport peptide, leader sequence, or leader peptide) directs proteins (e.g., the effector modules of the present disclosure) to their designated cellular and / or extracellular locations. Protein signal sequences play a central role in the targeting and translocation of almost all secreted proteins and many integral membrane proteins.

[0078] A signal sequence is a short (5 - 30 amino acids in length) peptide present at the N-terminus of the majority of newly synthesized proteins that are targeted to specific locations. Signal sequences are recognized by the signal recognition particle (SRP) and can be cleaved using type I and type II signal peptidases. Signal sequences derived from human proteins can be incorporated as regulatory modules of effector modules and can direct effector modules to specific cellular and / or extracellular locations.

[0079] In some embodiments, the signal sequence may, but does not necessarily, be located at the N-terminus or C-terminus of the effector module and may, but does not necessarily, be cleaved from the desired effector module to produce a “mature” payload.

[0080] In some embodiments, the signal sequence used herein may exclude the methionine at position 1 of the amino acid sequence of the signal sequence. This may be referred to as the M1del mutation.

[0081] In addition to naturally occurring signal sequences, such as those from secreted proteins, signal sequences may be variants modified from known signal sequences of proteins.

[0082] In some cases, a signal sequence that directs the payload of interest to the surface membrane of the target cell may be used. Expression of the payload on the surface of the target cell may help to limit diffusion of the payload into the non-target in vivo environment, thereby potentially improving the safety profile of the payload. Additionally, membrane presentation of the payload may enable payload stabilization and recycling for a longer half-life, as well as physiological and qualitative signal transduction. The membrane sequence may be the endogenous signal sequence of the N-terminal component of the payload of interest. Optionally, it may be desirable to exchange this sequence for a different signal sequence. The signal sequence may be selected based on their compatibility with the secretory pathway of the cell type of interest such that the payload is present on the surface of the T cell. In some embodiments, the signal sequence may be an IgE signal sequence, a CD8a signal sequence (also referred to as the CD8a leader), or an IL15Ra signal sequence (also referred to as the IL15Ra leader), or an M1del CD8a signal sequence (also referred to as the M1del CD8 leader sequence).

[0083] In some embodiments, the effector module includes a cleavage mechanism and / or a processing mechanism. In some embodiments, the effector module of the present disclosure may include at least one protein cleavage signal / site. The protein cleavage signal / site may be located at the N-terminus, C-terminus, intermediate between the N-terminus and C-terminus, between the N-terminus and the midpoint, between the midpoint and the C-terminus, and any space between the N-terminus and C-terminus including but not limited to combinations thereof.

[0084] In some embodiments, the effector module includes a linker.

[0085] In some embodiments, the effector module of the present disclosure may further include a linker sequence. The linker region mainly functions as a spacer between two or more polypeptides within the effector module. As used herein, a "linker" or "spacer" refers to a molecule or group of molecules that connects two parts of a molecule, such as two molecules or two domains of a recombinant protein.

[0086] In some embodiments, a "linker" (L) or "linker domain" or "linker region" or "linker module" or "peptide linker" as used herein refers to an oligomer or polypeptide region of about 1 to 100 amino acids in length that covalently links any of the domains / regions of the effector module (also referred to as a peptide linker).

[0087] In some embodiments, an artificially designed peptide linker may consist of a polymer of flexible residues such as glycine (G) and serine (S) so that adjacent protein domains can move freely relative to each other. When it is desirable to ensure that two adjacent domains do not interfere with each other, a longer linker may be used. The selection of a particular linker sequence can be a concern if it affects the biological activity, stability, folding, targeting and / or pharmacokinetic properties of the fusion construct.

[0088] The linker sequence may be a natural linker derived from a multi-domain protein. A natural linker is a short peptide sequence that separates two different domains or motifs within a protein.

[0089] In one embodiment, the linker may be a BamHI site. By way of non-limiting example, the BamHI site has the amino acid sequence GS and / or the DNA sequence GGATCC.

[0090] The bio-circuits of the present disclosure are triggered by one or more stimulants. In some embodiments, the stimulant is a small molecule. In some embodiments, the small molecule is cell-permeable. In some embodiments, the small molecule is FDA-approved, safe, and orally administered.

[0091] In some embodiments, the ligand binds to carbonic anhydrase. In some embodiments, the ligand binds and inhibits carbonic anhydrase function and is referred to herein as a carbonic anhydrase inhibitor.

[0092] In some embodiments, the ligand is a small molecule that binds to carbonic anhydrase 2. In one embodiment, the small molecule is a CA2 inhibitor. In some embodiments, the ligand is a small molecule selected from acetazolamide, celecoxib, baldecozib, rofecoxib, methazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, and dichlorphenamide. In some embodiments, the ligand is a small molecule selected from acetazolamide, brinzolamide, dorzolamide hydrochloride, dichlorphenamide, chlorthalidone, methazolamide, topiramate, indapamide, ambroxol hydrochloride, glimepiride, tetracaine hydrochloride, and celecoxib. In some embodiments, the ligand is a small molecule selected from acetazolamide, brinzolamide, dorzolamide hydrochloride, dichlorphenamide, chlorthalidone, methazolamide, or topiramate. In some embodiments, the ligand is a CA2 inhibitor selected from acetazolamide, brinzolamide, dorzolamide hydrochloride, dichlorphenamide, or methazolamide. In some embodiments, the ligand is acetazolamide (ACZ).

[0093] In some embodiments, ligands and / or chimeric antigen receptors that do not affect the activity of immune cells are preferably selected in the absence of SRE.

[0094] In some embodiments, the compositions of the present disclosure include a promoter.

[0095] As used herein, a promoter is defined as a DNA sequence recognized by a cell's transcriptional apparatus that is necessary to initiate specific transcription of a polynucleotide sequence of the present disclosure. A vector can include a natural or non-natural promoter operably linked to a polynucleotide of the present disclosure. The selected promoter can be strong, weak, constitutive, inducible, tissue-specific, developmentally specific, and / or organism-specific. A strong constitutive promoter sequence can drive high-level expression of a polynucleotide sequence to which it is operably linked. Examples of strong constitutive promoters include, without limitation, the immediate early cytomegalovirus (CMV) promoter and Elongation Growth Factor-1 Alpha (EF-1α). Other constitutive promoters that can be used include, but are not limited to, simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, avian leukemia virus promoter, spleen focus-forming virus (SFFV) promoter, mouse stem cell virus (MSCV) promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and phosphoglycerate kinase (PGK) promoter, actin promoter, myosin promoter, hemoglobin promoter, ubiquitin C (Ubc) promoter, human U6 small nuclear protein promoter, and creatine kinase promoter, human gene promoters including, but not limited to. Synthetic promoters include the MND promoter and the RPBSA promoter. In some cases, inducible promoters such as, but not limited to, the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter may be used.

[0096] In some embodiments, an optimal promoter may be selected based on its ability to achieve minimal expression of the SRE and payload of the present disclosure in the absence of a ligand and detectable expression in the presence of a ligand.

[0097] Additional promoter elements, such as enhancers, may be used to control the frequency of transcription initiation. Such regions may be located 10 to 100 base pairs upstream or downstream of the start site. In some cases, two or more promoter elements may be used to activate transcription jointly or independently.

[0098] The bio - circuits of the present disclosure may include at least one effector module that can include at least one SRE derived from CA2 (referred to as "CA2 SRE") that is operably coupled to at least one target payload. These types of bio - circuits and effector modules are referred to as "CA2 bio - circuits" and "CA2 effector modules". Additionally, the CA2 effector module may include additional features including, but not limited to, signal sequences, linkers, spacers, tags, flags, cleavage sites, and IRES. Any of the exemplary SREs (e.g., DRD), target payloads, signal sequences, linkers, spacers, hinges, tags, flags, cleavage sites, and IRESs taught herein or known in the art may be combined to create the CA2 effector modules of the present disclosure.

[0099] In one embodiment, the CA2 effector module comprises a payload of interest. The payload of interest may be a wild-type sequence, a fragment of a wild-type sequence, and / or may contain one or more mutations. In one embodiment, the CA2 effector module produces a regulatory interleukin-15 (IL15). In some embodiments, the IL15 payload is at the N-terminus of the DRD. The CA2 effector module may comprise any of the IL15-related sequences of Table 3 or may be derived from any of the IL15-related sequences of Table 3. In some embodiments, at least one payload in the CA2 effector module is an IL15 (e.g., an IL15 payload) comprising an amino acid sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8, and the payload may be encoded by a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 9). In some embodiments, the payload is a membrane-bound form of IL15. In some embodiments, the payload is a membrane-bound form of IL15 comprising a transmembrane domain and an intracellular tail. In some embodiments, the payload is a membrane-bound form of IL15 comprising an IL15 polypeptide component comprising the amino acid sequence of SEQ ID NO: 8, a transmembrane domain, and an intracellular tail, the transmembrane domain is at the C-terminus of the IL15 polypeptide component, and the intracellular tail is at the C-terminus of the transmembrane domain. In some embodiments, the payload is a membrane-bound form of IL15 comprising a transmembrane domain, an intracellular tail, and one or more linkers. In some embodiments, the linker is a peptide domain that can be placed between the SRE or DRD and the payload or between different domains within the payload. In some embodiments, the linker is a peptide domain comprising glycine and serine amino acid residues. In some embodiments, the peptide linker comprising glycine and serine amino acid residues may be 2 to 36 amino acids in length.In one embodiment, at least one payload in the CA2 effector module is a membrane-bound form of IL15 that further includes a linker (GS)15, a B7.1 hinge, a B7.1 transmembrane domain, a B7.1 intracellular tail, and a linker (GS). The CA2 effector module may include not only the IL15 payload component but also payload components of transmembrane domains and / or cytoplasmic domains from another parent protein. In one embodiment, at least one payload in the CA2 effector module includes at least one mutation as compared to the wild-type sequence. In one embodiment, at least one payload in the CA2 effector module includes at least one amino acid substitution as compared to the wild-type sequence.

[0100]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

[0101] In various embodiments, the effector module produces a regulated membrane-bound interleukin-15 (IL15). In some embodiments, the effector module is IL15-293 or IL15-295 as described in Table 3. In various embodiments, the IL15 payload is expressed as a CA2 DRD fusion protein that includes a membrane-bound form of IL15.

[0102] The CA2 biocircuit and / or CA2 effector module of the present disclosure may be monocistronic or polycistronic, meaning that one (monocistronic) or two or more (polycistronic) messages (e.g., the payload of interest) are produced. If two messages are produced, the CA2 biocircuit or CA2 effector module is considered bicistronic. In one embodiment, at least one CA2 effector module of the present disclosure is monocistronic.

[0103] Various embodiments of the present disclosure provide nucleic acid molecules comprising one or more of the described polynucleotides. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a recombinant protein comprising a drug-responsive domain (DRD) operably linked to an IL15 payload, wherein the DRD is derived from human carbonic anhydrase II (CA2) and comprises one, two, three, four, or more mutations relative to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid molecule further comprises a second polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the CAR or TCR comprises an antigen-binding domain specific for an antigen of interest. In some embodiments, the CAR or TCR comprises an antigen-binding domain specific for an antigen of interest, e.g., the CAR comprises an antigen-binding domain specific for CD19.

[0104] The present teachings further include a pharmaceutical composition comprising one or more of the CA2 biocircuits, CA2 effector modules, or systems of the present disclosure, and optionally further comprising at least one pharmaceutically acceptable additive or inert ingredient.

[0105] As used herein, the term "pharmaceutical composition" refers to the preparation of one or more of the CA2 biocircuits or components described herein, or a pharmaceutically acceptable salt thereof, optionally containing other chemical components such as physiologically suitable carriers and additives.

[0106] The term "additive" or "inactive ingredient" refers to an inactive or inert substance that is added to a pharmaceutical composition to further facilitate the administration of the compound. Non-limiting examples of such inactive ingredients are disclosed herein.

[0107] In some embodiments, the composition is administered to a human, such as a human patient or subject. For the purposes of the present disclosure, the phrase "active ingredient" generally refers to any one or more CA2 bio-circuit components that are delivered as described herein.

[0108] The description of the pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, but it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, for example, a non-human animal, for example, a non-human mammal. Subjects to which the pharmaceutical composition is administered include non-human mammals including, but not limited to, farm animals such as cows, horses, chickens, and pigs, companion animals such as cats and dogs, or laboratory animals such as mice, rats, rabbits, dogs, and non-human primates.

[0109] The pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk as a single unit dose and / or as multiple single unit doses. As used herein, a "unit dose" is an individual quantity of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is usually equal to the dosage of the active ingredient administered to the subject and / or a convenient fraction of such dosage, for example, half or one-third of such dosage.

[0110] The relative amounts of the active ingredient, pharmaceutically acceptable additives or inactive ingredients, and / or any additional ingredients in the pharmaceutical compositions according to the present disclosure vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100%, for example, from 0.5% to 50%, from 1% to 30%, from 5% to 80%, at least 80% (w / w) of the active ingredient.

[0111] The therapeutic effect or remission of a disease can be evaluated, for example, by measuring the progression of the disease, remission of the disease, severity of symptoms, reduction of pain, quality of life, dosage of drug therapy required to maintain therapeutic effectiveness, level of disease markers, or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. Monitoring the effect of treatment or prevention by measuring any one of such parameters, or any combination of parameters, is well within the ability of one of ordinary skill in the art. With respect to administration of the compositions of the present disclosure, for example, “effective against” cancer means that administration in a clinically appropriate manner results in, among other things, improvement of symptoms, cure, reduction of disease burden, decrease in tumor mass or cell number, extension of lifespan, improvement of quality of life, or other effects that are recognized as positive by a physician ordinarily skilled in the treatment of a particular type of cancer, in at least a statistically significant proportion of patients.

[0112] The treatment or prevention effect becomes apparent when there is a statistically significant improvement in one or more parameters of the disease state, or when symptoms do not worsen or do not appear at the predicted location. As an example, a favorable change of at least 10%, and preferably at least 20%, 30%, 40%, 50% or more in a measurable parameter of the disease can suggest an effective treatment. The effectiveness of a given composition or formulation of the present disclosure can also be determined using an experimental animal model for a given disease as is well known in the art. When using an experimental animal model, the treatment effect is demonstrated when a statistically significant change is observed.

[0113] The compositions of the present disclosure may be formulated in any manner suitable for delivery. The formulations may be, but are not limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, and combinations thereof.

[0114] In some embodiments, a formulation or other preparation may include at least one additive that is an inert ingredient. As used herein, the term "inert ingredient" refers to one or more inert agents included in the preparation. In some embodiments, all or some of the inert ingredients that may be used in the formulations of the present disclosure may or may not be approved by the US Food and Drug Administration (FDA). The compositions of the present disclosure may be delivered to cells or a subject through one or more routes and modalities. A viral vector containing one or more of the CA2 biocircuits, CA2 effector modules, SREs, payloads, and other components described herein may be used to deliver them to cells and / or a subject. Other modalities may be used, such as mRNA, as a plasmid, and as a recombinant protein, etc.

[0115] A pharmaceutical composition, CA2 biocircuit, CA2 biocircuit component, CA2 effector module, comprising an SRE or payload of the present disclosure may be delivered to cells, tissues, organs, and / or a living body in a naked form. As used herein, the term "naked" refers to a pharmaceutical composition, CA2 biocircuit, CA2 biocircuit component, CA2 effector module, comprising an SRE or payload, that is delivered without including an agent or modification that promotes transfection or permeability. A naked pharmaceutical composition, CA2 biocircuit, CA2 biocircuit component, CA2 effector module, comprising an SRE or payload may be delivered to cells, tissues, organs, and / or a living body using routes of administration well known in the art and described herein. In some embodiments, naked delivery may include a preparation in a simple buffer such as saline or PBS.

[0116] In some embodiments, pharmaceutical compositions, CA2 biocircuits, CA2 biocircuit components, CA2 effector modules that include the SRE or payload of the present disclosure may be formulated using the methods described herein. Formulations may include modified and / or unmodified pharmaceutical compositions, CA2 biocircuits, CA2 biocircuit components, CA2 effector modules that include the SRE or payload. Formulations may further include, but are not limited to, cell permeant agents, pharmaceutically acceptable carriers, delivery agents, biodegradable or biocompatible polymers, solvents, and / or sustained release delivery depots. Formulations of the present disclosure may be delivered to cells using routes of administration well known in the art and described herein.

[0117] Pharmaceutical compositions, CA2 biocircuits, CA2 biocircuit components, CA2 effector modules that include the SRE or payload may be formulated for direct delivery to an organ or tissue by any of several methods in the art, including but not limited to using direct immersion or a water bath, catheters, gels, powders, ointments, creams, gels, lotions, and / or drops, or substrates such as fabrics or biodegradable materials coated with or impregnated with the composition.

[0118] In another aspect of the present disclosure, the CA2 biocircuits, CA2 effector modules, SREs (e.g., CA2 DRD), payloads of interest (e.g., IL15), and polynucleotides encoding the compositions of the present disclosure, and vectors comprising said polynucleotides may be introduced into cells. By way of non-limiting example, the cells may be effector immune cells.

[0119] In various embodiments, the present disclosure provides a cell comprising one or more nucleic acid molecules, one or more vectors, or one or more recombinant proteins of the present disclosure. In some embodiments, a method of modulating the expression, function, and / or level of an IL15 payload in a cell is provided, the method comprising administering to the cell an agent to which a DRD responds, the agent being administered in an amount sufficient to modulate the expression, function, and / or level of the IL15 payload. In some embodiments, the cell is isolated. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a mammalian cell. The mammalian cell may be a human cell. The human cell may be a T cell, a natural killer (NK) cell, or a tumor infiltrating lymphocyte (TIL). In some embodiments, the cell is a CD4+ or CD8+ T cell. In some embodiments, the human T cell or human NK cell further comprises a polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), the CAR or TCR comprising an antigen binding domain specific for an antigen of interest. In some embodiments, the CAR comprises an antigen binding domain specific for CD19.

[0120] In one aspect of the disclosure, the polynucleotides encoding the CA2 biocircuit, CA2 effector module, SRE (e.g., CA2 DRD), payload of interest (e.g., IL15), and compositions of the disclosure may be packaged into viral vectors or integrated into viral genomes that allow for transient or stable expression of the polynucleotides. Suitable viral vectors are retroviral vectors including lentiviral vectors and gammaretroviral vectors. To construct a retroviral vector, a polynucleotide molecule encoding the CA2 biocircuit, CA2 effector module, CA2 DRD, or payload of interest (e.g., an immunotherapeutic agent) is inserted into the viral genome in place of certain viral sequences to produce a replication-defective virus. The recombinant viral vector is then introduced into a packaging cell line that contains the gag, pol, and env genes but does not contain the LTR (for lentiviral vectors) and packaging components. The recombinant retroviral particles are secreted into the medium, subsequently collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are particularly suitable if they can affect both dividing and non-dividing cells.

[0121] The vector may be introduced into cells by physical means such as needles, electroporation, sonoporation, hydrooporation; chemical carriers such as inorganic particles (e.g., calcium phosphate, silica, gold), and / or non-viral methods by chemical means. In some embodiments, synthetic or natural biodegradable agents may be used for delivery such as cationic lipids, liquid nanoemulsions, nanoparticles, peptide-based vectors, or polymer-based vectors.

[0122] The CA2 biocircuit system, CA2 effector module, SRE and / or payload of the present disclosure may be delivered using one or more modalities. The present disclosure also provides vectors that package polynucleotides of the present disclosure encoding a CA2 biocircuit, a CA2 effector module, an SRE (e.g., CA2 DRD), and an IL15 payload of interest, and combinations thereof. The vectors of the present disclosure may be used to deliver the packaged polynucleotides to cells, local tissue sites, or subjects. These vectors may be of any type, including DNA vectors, RNA vectors, plasmids, viral vectors and particles. Viral vector technology is well known and is described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Viruses useful as vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes simplex virus vectors, retroviral vectors, oncolytic viruses, and the like. In some embodiments, viral vectors useful for introducing one or more nucleic acid molecules encoding the DRD and IL15 payloads exemplified herein into cells may be derived from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, or picornavirus.

[0123] Generally, a vector includes an origin of replication, a promoter sequence and convenient restriction endonuclease sites that function in at least one living organism, and one or more selectable markers, such as drug resistance genes.

[0124] In some embodiments, the recombinant expression vector may contain regulatory sequences, such as transcription and translation start and stop codons, specific to the type of host cell into which the vector is introduced.

[0125] In some embodiments, the vectors of the present disclosure may contain one or more payloads taught herein, and two or more payloads may be included in one CA2 effector module. In this case, two or more payloads are co-regulated by the same stimulant. In other embodiments, the vectors of the present disclosure may contain two or more CA2 effector modules, and each CA2 effector module contains a different payload. In this case, two or more CA2 effector modules and payloads are regulated by different stimulants, resulting in separate independent control of the two or more components. In other embodiments, the vectors of the present disclosure may contain one or more CA2 effector modules and one or more non-CA2 effector modules, and each CA2 effector module contains a different payload. In this case, the CA2 effector modules and payloads are regulated by different stimulants, resulting in separate independent control of the two or more components.

[0126] In some embodiments, the lentiviral vehicle / particle may be used as a delivery modality. Lentiviruses are a subgroup of the retrovirus family of viruses, named because reverse transcription of the viral RNA genome into DNA is required prior to integration into the host genome. Thus, the most important feature of lentiviral vehicles / particles is the integration of their genetic material into the genome of the target / host cell. Examples of lentiviruses include human immunodeficiency virus: HIV-1 and HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, Visna Maedi, and caprine arthritis encephalitis virus (CAEV).

[0127] Typically, lentiviral particles that make up a gene delivery vehicle are themselves unable to replicate (also referred to as "self-inactivating"). Lentiviruses can affect both dividing and non-dividing cells through an entry mechanism that crosses the intact host nuclear membrane (Naldini L et al., Curr. Opin. Biotechnol, 1998, 9:457-463). Recombinant lentiviral vehicles / particles are generated by complex attenuation of HIV pathogenic genes. For example, the genes Env, Vif, Vpr, Vpu, Nef, and Tat are deleted, and a biologically safe vector is created. Accordingly, for example, lentiviral vehicles derived from HIV-1 / HIV-2 can mediate efficient delivery, integration, and long-term expression of transgenes into non-dividing cells. As used herein, the term "recombinant" refers to vectors and other nucleic acids that contain both lentiviral sequences and non-lentiviral retroviral sequences.

[0128] Lentiviral particles may be generated in producer cells such as human HEK293T cells by co-expressing viral packaging elements and the vector genome itself. These elements are typically provided on three or four separate plasmids. The producer cells are co-transfected with plasmids encoding lentiviral components including the viral core (i.e., structural proteins) and enzymatic components, as well as envelope proteins (referred to as a packaging system), and a plasmid encoding a genome containing a foreign transgene, and transferred to target cells, the vehicle itself (also referred to as a transfer vector). Generally, the plasmid or vector is contained in a producer cell line. The plasmid / vector is introduced into the producer cell line by transfection, transduction, or infection. Methods of transfection, transduction, or infection are well known to those skilled in the art. As a non-limiting example, the packaging and transfer constructs are typically introduced into the producer cell line by calcium phosphate transfection, lipofection, or electroporation, along with a dominant selectable marker such as neo, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.

[0129] The producer cells produce recombinant viral particles containing a foreign gene, for example, the CA2 effector module of the present disclosure. The recombinant viral particles are recovered from the medium and titrated by standard methods used by those skilled in the art. The recombinant lentiviral vehicle can be used to affect target cells.

[0130] Cells that can be used to produce high-titer lentiviral particles include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol. Ther., 2005, 11:452-459), FreeStyle® 293 expression system (ThermoFisher, Waltham, MA), and other HEK293T-based production cell lines (e.g., Stewart et al., Hum Gene Ther. 2011, 22(3):357-369; Lee et al., Biotechnol Bioeng, 2012, 10996):1551-1560; Throm et al., Blood. 2009, 113(21):5104-5110), the content of each of which is hereby incorporated by reference in its entirety).

[0131] In some embodiments, the envelope protein may be a heterologous envelope protein from another virus, such as the G protein of vesicular stomatitis virus (VSV G) or the baculovirus gp64 envelope protein. The VSV-G glycoprotein is classified into species belonging to the genus Vesiculovirus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Aragua virus (VSAV), Vesicular stomatitis Indiana virus (VSIV), and Vesicular stomatitis New Jersey virus (VSNJV), and / or from among species temporarily classified into the genus Vesiculovirus such as Carp pox virus, BeAn157575 virus (BeAn157575), Boteke virus (BTKV), Cachiyuy virus (CQIV), Eel pox virus (EVA), Gray Lodge virus (GLOV), Jurona virus (JURY), Klamath virus (KLAV), Kwa tta virus (KWAV), La Joya virus (LJV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEBV), Perinet virus (PERV), Pike fry pox virus (PFRV), Porton virus (PORV), Rhadinovirus (RADIV), Carp spring viraemia virus (SVCV), Tupaia virus (TUPV), Ulcerative disease pox virus (UDRV), and Yug Bogdanova t virus (YBV).Other baculovirus env proteins can be derived from Autographa californica nucleopolyhedrovirus (AcMNPV), Anagrapha falcifera nucleopolyhedrovirus, Bombyx mori nucleopolyhedrovirus, Choristoneura fumiferana nucleopolyhedrovirus, Orgyia pseudotsugata single capsid nucleopolyhedrovirus, Epiphyas postvittana nucleopolyhedrovirus, American white moth nucleopolyhedrovirus, Hymenoptera nucleopolyhedrovirus, Dore virus, Sogo virus, Antheraea pemyi nucleopolyhedrovirus, or Batken virus.

[0132] Other elements provided to lentiviral particles can include a retroviral LTR (long terminal repeat) at either the 5' or 3' end, a retroviral export element, optionally, a lentiviral response element (RRE), a promoter or an active part thereof, and a locus control region (LCR) or an active part thereof. The CA2 effector module is bound to the vector.

[0133] Methods for generating recombinant lentiviral particles are discussed in the art, for example, in U.S. Pat. Nos. 8,846,385, 7,745,179, 7,629,153, 7,575,924, 7,179,903, and 6,808,905, the contents of each of which are hereby incorporated by reference in their entirety.

[0134] The lentiviral vector used may be selected from, but not limited to, pLVX, pLenti, pLenti6, pLJM1, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJM1-EGFP, pULTRA, pInducer20, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionII.

[0135] Lentiviral vectors, including recently approved products such as tisagenlecleucel (KYMRIAH®), are used to introduce transgenes into T cells (e.g., primary human T cells or Jurkat cells) for preclinical research and clinical applications. VSV-G pseudotyped third-generation lentiviral vectors provide high titers, high transduction efficiency, and safety, and have become the vector of choice for T cell manipulation. Without wishing to be bound by theory, T cell manipulation typically involves T cell activation with CD3 / CD28 antibodies, followed by lentiviral transduction, and further followed by cell proliferation that can last from 5 to 30 days (e.g., 9 to 14 days or 9 to 15 days). Generally, integration of the lentiviral transgene may take longer than 7 days to be sufficiently stable in T cells (e.g., primary human T cells or Jurkat cells).

[0136] In some embodiments, to determine transgene expression kinetics, lentiviruses carrying a transgene (e.g., IL15) can be transduced into CD3 / CD28-activated primary human T cells. The cells can be analyzed by methods described herein and / or well-known in the art with respect to viability, viral genome integration (e.g., using quantitative PCR), transcriptional level (e.g., using quantitative RT-PCR), and cell surface expression of the transgene. The cells can be analyzed prior to transduction and / or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or more after transduction. By way of non-limiting example, the cells can be analyzed at various time points between 3 and 14 days after transduction (e.g., 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, and / or 14 days). By way of non-limiting example, the cells can be analyzed 3 to 15 days after transduction. By way of non-limiting example, the cells can be analyzed 9 to 15 days after transduction.

[0137] In some embodiments, CD3 / CD28-activated primary human T cells can be reactivated by CD3 / CD28 beads after transduction. The cells can be reactivated 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 days or more after transduction. The cells can be analyzed by methods described herein and / or well-known in the art with respect to viability, viral genome integration (e.g., using quantitative PCR), transcription level (e.g., using quantitative RT-PCR), cell surface expression of the transgene, copy number, and / or mRNA level.

[0138] In some embodiments, the cell viability of activated primary human T cells transduced with a lentivirus carrying a transgene is greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%. As a non-limiting example, the cell viability is greater than 90%. As a non-limiting example, the cell viability is greater than 85%.

[0139] In some embodiments, the cell viability of Jurkat cells transduced with a lentivirus carrying a transgene is greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%. As a non-limiting example, the cell viability is greater than 90%. As a non-limiting example, the cell viability is greater than 85%.

[0140] In some embodiments, integration of the transgene into the genome of the cells can be at or above the saturation point. As a non-limiting example, the saturation point can be 3 copies per cell.

[0141] In some embodiments, the integration of the transgene into the genome can be high at the first time point evaluated and then decrease to a lower integration value until it stabilizes during the remaining culture. As a non-limiting example, the integration of the transgene into the genome can increase up to 20 copies per cell during the initial time point and then decrease to 2 copies per cell and remain stable throughout the remaining culture.

[0142] In some embodiments, the transduction of the T cell capabilities can be evaluated. T cells from at least one donor can be transduced with a lentivirus containing the transgene at a dose predicted to reach saturation levels (e.g., sufficient virus such that each cell should contain a copy, if a Poisson distribution is expected) and at a high lentivirus dose more than 5-fold saturation. The copies per cell, percentage of cells, and MFI (or concentration of the transgene in the medium) can be detected to determine whether all cells are expressing the transgene. As a non-limiting example, T cells from two different donors can be transduced with a lentivirus containing the transgene. The transduction can be at two doses, saturation and 5-fold saturation, and at 5 - 10 days post-transduction, it may be shown that all groups can reach or exceed the predicted saturation level of the integrated transgene and similar expression intensity throughout the group, although not all cells may be expressing the transgene. Even when provided from the same donor, not all T cells may have equal transduction susceptibility. The percentage of total cells expressing GFP (above the detection threshold) can vary by donor, lot, and / or virus dose.

[0143] In some embodiments, a certain percentage of cultured T cells (e.g., primary human T cells and / or Jurkat cells) may express the transgene. The percentage of cultured T cells that express the transgene may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more than 99%, but is not limited thereto. As a non-limiting example, the percentage may be more than 70%. As a non-limiting example, the percentage may be more than 75%. As a non-limiting example, the percentage may be more than 80%. As a non-limiting example, the percentage may be more than 85%. As a non-limiting example, the percentage may be more than 90%. As a non-limiting example, the percentage may be more than 95%.

[0144] In some embodiments, the mRNA level of the culture may decrease during the course of the study. The decrease may not be limited to a particular transgene, and the trend may be seen across multiple classes of the expressed protein. To increase the mRNA level, the cells may be reactivated after the mRNA level has decreased from the initial level. The cells may be reactivated 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, or more than 30 days after transduction. As a non-limiting example, to increase the mRNA level in the culture, the cells may be reactivated with CD3 / CD28 beads 13 days after transduction. As a non-limiting example, to increase the mRNA level in the culture, the cells may be reactivated with CD3 / CD28 beads 14 days after transduction. As a non-limiting example, to increase the mRNA level in the culture, the cells may be reactivated with CD3 / CD28 beads 15 days after transduction.

[0145] In some embodiments, surface expression of the culture may decline during the course of the study. For example, surface expression may decline between 3 to 13 days, 3 to 14 days, or 3 to 15 days after transduction. To increase surface expression, the cells may be reactivated after surface expression has decreased from its initial level. The cells may be reactivated 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more than 30 days after transduction. As a non-limiting example, to increase surface expression in the culture, the cells may be reactivated with CD3 / CD28 beads for 13 days after transduction. As a non-limiting example, to increase surface expression in the culture, the cells may be reactivated with CD3 / CD28 beads for 14 days after transduction. As a non-limiting example, to increase surface expression in the culture, the cells may be reactivated with CD3 / CD28 beads for 15 days after transduction.

[0146] In some embodiments, the transgene is IL15 (e.g., a membrane-bound IL15 payload when combined with other effector module components as described in Table 3). The cell viability may be greater than 90% in cells transduced with IL15. The cell viability may be greater than 85% in cells transduced with IL15. When the cells are primary T cells transduced with IL15, the number of viable cells may increase over an initial time point and then decrease. When the cells are Jurkat cells transduced with IL15, the number of viable cells may increase for at least 10 days. The copy number per cell for cells transduced with IL15 may be higher at an initial time point and decrease by 50% or more at a later time point. For primary human T cells transduced with IL15, the level of soluble IL15 in the medium may gradually decline over the course of the study, although there is a slight increase in the re-stimulated group. For Jurkat cells transduced with IL15, the level of soluble IL15 in the medium may decline in the first half of the culture with IL15 secretion decreasing and remain low throughout the second half of the culture time.

[0147] In some embodiments, the lentivirus engineered cells described herein have genomic DNA integration that stabilizes after an initial decrease in copy number, with decreasing RNA and surface expression levels over time, and increasing RNA and surface expression after restimulation.

[0148] In some embodiments, lentivirus engineered cells may be evaluated using the following 14-day method in which the sample is collected 5-fold through culture. - On day 1, T cells (e.g., primary human T cells or Jurkat cells) may be thawed and CD3 / CD28 beads are added. On day 0, lentivirus for each condition is added (e.g., 4 mL of cells at 0.5e6 / mL), and there are control non-transduced cells. On day 1, the medium is made 2-fold to 8 mL, and on day 2, the medium is made 2-fold to 16 mL. On day 3, 4 mL is harvested, and on day 4, the medium is made 2-fold to 24 mL. After harvesting 4 mL on day 6, the medium is made 2-fold to 40 mL. The cells can be split on day 8 (e.g., 14 mL of 0.5e6 cells / mL), and then, 4 mL is harvested on day 6 and the medium is made 2-fold to 40 mL. After harvesting 4 mL on day 10, the medium may be made 2-fold to 20 mL. On day 13, after harvesting 4 mL, the medium is made 2-fold to 32 mL. The culture is split in half, and half of the culture is activated overnight (CD3 / CD28 activation beads 1:1) and stimulated. On day 14, 4 mL of each stimulated and non-stimulated cells are harvested and the culture is terminated. The transgene copy number per cell is assayed by harvesting the cells, extracting genomic DNA, and subsequently quantifying by standard curve qPCR against the endogenous genome and against the transgene sequence, and then converting the detected quantity to a ratio. The mean fluorescence intensity (MFI) is assayed by FLO in Attune with an appropriate staining method for each group. The percent expressed may also be assayed by FLO in Attune that quantifies the percent of cells that fluoresce above a threshold. Soluble payload can be quantified by harvesting the culture supernatant at each marked time point and performing a MesoScale Discovery plate assay (MSD) and standardizing against cell density.

[0149] In some embodiments, the CA2 effector module of the present disclosure may be designed as messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and can be translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo.

[0150] The present disclosure provides a method comprising administering to a subject in need thereof any one or more of the components of the CA2 bio-circuit system, which may be administered to the subject by any amount and by any route effective to prevent or treat a disease, disorder, and / or illness (e.g., a disease, disorder, and / or illness associated with insufficient working memory). The exact amount needed varies from subject to subject depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.

[0151] In some embodiments, the present disclosure provides a method of treating a disease or disorder responsive to regulated IL15 in a subject in need thereof, the method comprising: (a) administering to the subject a therapeutically effective amount of a nucleic acid molecule, vector, recombinant protein, cell, or pharmaceutical composition of the present disclosure; and administering to the subject a therapeutically effective amount of a stimulant, wherein the DRD responds to the stimulant and the expression of the IL15 payload is regulated in response to the stimulant. In some embodiments, the stimulant is selected from acetazolamide, celecoxib, baldecohib, rofecoxib, metazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. In some embodiments, the stimulant is acetazolamide.

[0152] In some embodiments, the present disclosure provides a method of treating a malignant tumor that expresses a tumor-associated antigen in a subject in need thereof, the method comprising: (a) administering to the subject a therapeutically effective amount of the human T cells or human NK cells of the present disclosure, further comprising a polynucleotide encoding a CAR or TCR, or a pharmaceutical composition thereof, wherein the CAR or TCR comprises an antigen-binding domain specific for the tumor-associated antigen; and (b) administering to the subject a therapeutically effective amount of a stimulant, wherein the CA2 DRD responds to the stimulant and the expression of the IL15 payload is regulated in response to the stimulant. In some embodiments, the stimulant is selected from acetazolamide, celecoxib, baldecoib, rofecoxib, methazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. In some embodiments, the stimulant administered to the subject is acetazolamide.

[0153] In some embodiments, the present disclosure provides a method of treating a malignant tumor in a subject in need thereof, the method comprising: (a) administering to the subject a therapeutically effective amount of the human TILs of the present disclosure; and (b) administering to the subject a therapeutically effective amount of a stimulant, wherein the CA2 DRD responds to the stimulant and the expression of the IL15 payload is regulated in response to the stimulant. In some embodiments, the stimulant is selected from acetazolamide, celecoxib, baldecoib, rofecoxib, methazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. In some embodiments, the stimulant administered to the subject is acetazolamide.

[0154] Compositions according to the present disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions of the present disclosure can be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dosage level for any particular patient will depend on the disorder being treated and the severity of that disorder; the activity of the particular compound being used; the particular composition being used; the patient's age, weight, general health, sex, and diet; the time of administration, the route of administration, and the excretion rate of the particular compound being used; the duration of treatment; drugs used in combination with or concurrently with the particular compound being used; and various factors including similar factors well known in the medical arts.

[0155] The compositions of the present disclosure may be used at various dosages to avoid T cell anergy, prevent cytokine release syndrome, and minimize toxicity associated with immunotherapy. For example, low dosages of the compositions of the present disclosure may be used to treat patients with high tumor burden early in the disease. On the other hand, patients with low tumor burden may be treated with high dosages and continuous dosages of the compositions of the present disclosure to ensure that a minimal tumor antigen load is recognized. In another example, the compositions of the present disclosure may be delivered pulsatilely to reduce tonic T cell signaling and enhance in vivo persistence. In some embodiments, toxicity may be minimized by first using a low dosage prior to administering the compositions of the present disclosure at a high dosage. Administration may be modified if serum markers such as ferritin, serum C-reactive protein, IL6, IFN-γ, and TNF-α are elevated.

[0156] Also provided herein is a method of administering a ligand according to the present disclosure to a subject in need thereof. The ligand may be administered to a subject or cell by any amount and by any route effective to modulate the CA2 biocircuit of the present disclosure. The exact amount needed will vary by subject depending on the subject's species, age, and general condition, the severity of the disease, the particular composition, its mode of administration, its mode of activity, etc. The subject may be a human, mammal, or animal. Compositions according to the present disclosure are typically formulated in unit dosage forms for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions of the present disclosure may be determined by the attending physician within the scope of sound medical judgment. In certain embodiments, the ligand according to the present disclosure is administered at a dosage level sufficient to deliver about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg, about 10 mg / kg to about 100 mg / kg, about 50 mg / kg to about 500 mg / kg, about 100 mg / kg to about 1000 mg / kg per day per subject's body weight, once or multiple times per day to obtain the desired effect. In some embodiments, the dosage level is 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 130 mg / kg, 140 mg / kg, 150 mg / kg, 160 mg / kg, 170 mg / kg, 180 mg / kg, 190 mg / kg or mg / kg per day per subject's body weight, once or multiple times per day to obtain the desired effect.

[0157] The present disclosure provides a method of delivering any of the ligands described herein to a cell or tissue, the method comprising contacting the cell or tissue with the ligand, and can be performed in vitro, ex vivo, or in vivo. In certain embodiments, the ligand according to the present disclosure may be administered to a cell at a dosage level sufficient to deliver from about 1 nM to about 10 nM, about 5 nM to about 50 nM, about 10 nM to about 100 nM, about 50 nM to about 500 nM, about 100 nM to about 1000 nM, about 1 μM to about 10 μM, about 5 μM to about 50 μM, about 10 μM to about 100 μM, about 25 μM to about 250 μM, about 50 μM to about 500 μM. In some embodiments, the ligand may be administered to a cell at a dosage selected from, but not limited to, 0.00064 μM, 0.0032 μM, 0.016 μM, 0.08 μM, 0.4 μM, 1 μM, 2 μM, 10 μM, 50 μM, 75 μM, 100 μM, 150 μM, 175 μM, 200 μM, 250 μM.

[0158] The desired dosage of the ligand of the present disclosure may be delivered only once, three times a day, twice a day, once a day, every other day, every three days, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered by multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). When multiple administrations are used, a divided dosing regimen as described herein may be employed. As used herein, "divided dosage" means dividing the "single unit dosage" or the total daily dosage into two or more administrations, e.g., administering the "single unit dosage" more than once. As used herein, "single unit dosage" is the dosage of any therapeutic agent administered in one administration / at one time / by a single route / a single contact, i.e., a single dosing event. The desired dosage of the ligand of the present disclosure may be administered as a "pulse dosage" or as a "continuous flow". As used herein, "pulse dosage" is a series of single unit dosages of any therapeutic agent administered at a set frequency over a period of time. As used herein, "continuous flow" is the dosage of a therapeutic agent administered continuously over a period of time at a single route / a single contact, i.e., a continuous dosing event. The total daily dosage, the amount given or prescribed in 24 hours, may be administered by any of these methods, or as a combination of these methods, or by any other method suitable for drug administration.

[0159] In some embodiments, the composition for immunotherapy may be administered ex vivo to cells and subsequently administered to a subject. Immune cells can be isolated and expanded ex vivo using various methods known in the art. For example, methods for isolating cytotoxic T cells are described in U.S. Pat. Nos. 6,805,861 and 6,531,451, the entire contents of each of which are incorporated herein by reference in their entirety. The isolation of NK cells is described in U.S. Pat. No. 7,435,596, the content of which is incorporated herein by reference in its entirety.

[0160] In some embodiments, depending on the nature of the cells, the cells may be introduced into a host organism, such as a mammal, in a variety of ways including injection, transfusion, infusion, topical instillation or transplantation. In some aspects, the cells described herein may be introduced into the site of a tumor. The number of cells used is determined by a number of circumstances, the purpose of the introduction, the lifespan of the cells, the protocol used, e.g., the number of administrations, the proliferative capacity of the cells, etc. The cells may be in a physiologically acceptable medium.

[0161] In some embodiments, the cells described herein may be administered to a subject having a disease or disorder in multiple administrations. The administration typically results in the improvement of one or more symptoms of cancer, and / or treats or prevents cancer or the disorder or its symptoms.

[0162] In some embodiments, the compositions for immunotherapy may be administered in vivo. In some embodiments, the CA2 biocircuit, CA2 effector molecule, SRE, payload (IL15) of the present disclosure, and the polypeptides of the present disclosure comprising the compositions may be delivered to a subject in vivo. The in vivo delivery of immunotherapeutic agents is well described technically. For example, the method of cytokine delivery is described in European Patent No. EP0930892A1, the content of which is incorporated herein by reference.

[0163] The pharmaceutical compositions, CA2 biocircuits, CA2 biocircuit components, CA2 effector modules comprising the SRE (e.g., CA2 DRD), payload (e.g., IL15), vectors, and cells of the present disclosure may be administered by any route and may result in a therapeutic effect.

[0164] Pharmaceutical compositions, CA2 biocircuits, CA2 biocircuit components, CA2 effector modules containing the SRE or payload of the present disclosure may be administered by any route and may result in a therapeutic effect. These include enteral (into the intestine), gastrointestinal, epidural (into the dura mater), oral (via the mouth), transdermal, peridural, intracerebral (into the cerebrum), intraventricular (into the ventricles), topical (application to the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into the vein), rapid intravenous injection, intravenous drip, intraarterial (into the artery), intramuscular (into the muscle), intracardiac (into the heart), intraosseous injection (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (injection or infusion into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavernosal injection (into the pathological cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extraamniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through the mucosa), transvaginal, insufflation (aspiration through the nose), sublingual, sublabial, enema, instillation (onto the conjunctiva), otic, auricular (into or through the ear), buccal (directed towards the cheek), conjunctival, cutaneous, dental (to one or more teeth), iontophoresis, endocervical, intracanalicular, intratracheal, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intraamniotic, intraarticular, intrahepatic, intratracheobronchial, intracapsular, intracartilaginous (inside the cartilage), intrasacral (inside the cauda equina), intracisternal (inside the cisterna magna cerebellomedularis), intracorneal (inside the cornea), dental intracornal, intracoronary (inside the coronary artery), intracavernosal (inside the corpus cavernosum of the peniswithin the dilatable space of the cavernosa), within the intervertebral disc (inside the intervertebral disc), within the duct (inside the glandular duct), within the duodenum (inside the duodenum), within the dura mater (inside or under the dura mater), within the epidermis (in the epidermis), within the esophagus (in the esophagus), within the stomach (inside the stomach), within the gingiva (inside the gingiva), within the ileum (intraileal) (inside the terminal part of the small intestine), within the lesion (inside the local lesion or directly introduced into the local lesion), within the lumen (inside the lumen of the tube), within the lymph (inside the lymph), within the medulla (inside the medullary cavity of the bone), within the meninges (inside the meninges), within the myocardium (inside the myocardium), within the eye (inside the eye), within the ovary (inside the ovary), within the pericardium (inside the pericardium), within the pleura (inside the pleura), within the prostate (inside the prostate), within the lung (inside the lung or its bronchi), within the nasal cavity (intrasinal) (inside the nose or the perinasal sinuses), within the spinal cord (inside the spinal column), within the synovial bursa of the joint (inside the synovial cavity of the joint), within the tendon (inside the tendon), within the testis (inside the testis), within the subarachnoid space (inside the cerebrospinal fluid at any level of the cerebrospinal axis), within the thoracic cavity (inside the chest), within the tube (inside the fine tube of the organ), within the tumor (inside the tumor), within the tympanic cavity (inside the middle ear (aurus media)), within the blood vessel (inside one or more blood vessels), within the ventricle (inside the ventricle), iontophoresis (using an electric current in which ions of a soluble salt move into the body's tissues), perfusion (immersing or flowing into an open wound or body cavity), larynx (directly into the larynx), nasogastric (through the nose into the inside of the stomach), occlusive dressing therapy (covered by a dressing material that closes the area following topical route administration), ocular region (to the external eye), oropharynx (directly to the mouth and pharynx), parenteral, percutaneous, perijoint, epidural (peridural), perineural, periodontal, rectal, respiratory (into the airways by oral or nasal inhalation for local or systemic effects), retrobulbar (behind the bridge or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, local, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), intratympanic (across or through the tympanic cavity), ureter (into the ureter), urethra (into the urethra), vagina, sacral block, diagnosis, nerve block, biliary perfusion, cardiac perfusion, photopheresis, or spinal cord, including but not limited to these.

[0165] In some embodiments, the pharmaceutical compositions, CA2 bio-circuits, CA2 bio-circuit components, CA2 effector modules comprising the SRE or payload of the present disclosure may be administered parenterally. Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage forms may include, for example, water and other solvents; solubilizing and emulsifying agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan; and inert diluents commonly used in the art such as mixtures thereof. In addition to the inert diluent, oral compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents. In some embodiments for parenteral administration, the composition is mixed with a solubilizing agent such as CREMOPHOR®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof. In other embodiments, surfactants such as hydroxypropylcellulose are included.

[0166] Injectable formulations, such as sterile intravenous administration formulations or aqueous or oily injection suspensions, may be formulated according to known techniques using suitable dispersants, wetting agents, and / or suspending agents. Sterile injectable formulations may be, for example, solutions in 1,3 - butanediol, sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents. Acceptable vehicles and solvents that may be used are water, Ringer's solution, U.S.P., and isotonic saline. Sterile fixed oils have heretofore been used as solvents or suspending agents. For this purpose, any sterile fixed oil containing synthetic mono - or diglycerides can be used. Fatty acids such as oleic acid can be used in the preparation of injection solutions.

[0167] Injectable formulations may be sterilized, for example, by filtration through a bacteria - retaining filter and / or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0168] The CA2 bio - circuits, CA2 effector modules, SREs, stimulants, compositions, or systems of the present disclosure that include one or more of a stimulant, CA2 bio - circuit, and CA2 effector module may be utilized in a wide variety of applications including, but not limited to, therapies, diagnosis and prognosis, bio - engineering, bioprocessing, bio - factories, research agents, metabolomics, gene expression, enzyme replacement, etc.

[0169] According to the present disclosure, the CA2 - IL15 bio - circuits and systems may be used in the development and implementation of cell therapies such as adoptive cell therapy. The CA2 - IL15 bio - circuits and systems may be used to effect CAR T - cell therapy, T - cell receptor (TCR) cell therapy, CAR NK - cell therapy, TCR NK - cell therapy, TIL therapy, any of which may be used in combination therapy with other treatment lines (e.g., radiation, cytokines).

[0170] In some embodiments, the CA2-IL15 biocircuit and system may be used to engineer immune cells including CD8 + T cells, CD4 + T cells, such as T cells, natural killer (NK) cells, NK T cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), lymphokine-activated killer (LAK) cells, memory T cells, regulatory T cells (Tregs), helper T cells, cytokine-induced killer (CIK) cells, and any combination thereof. In other embodiments, the CA2-IL15 biocircuit and system may be used to engineer immune-stimulatory cells generated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) that can be used for ACT. In some embodiments, the CA2-IL15 biocircuit and system may be used to engineer autologous or allogeneic immune cells that can be used for ACT. In some embodiments, the CA2-IL15 biocircuit and system may be used to engineer T cells, TILs, or NK cells. In some embodiments, the immune cells are NK cells derived from umbilical cord blood, iPSCs, or peripheral blood mononuclear cells.

[0171] In some embodiments, the cells engineered by CA2-IL15 used for ACT may be T cells that have also been engineered to express a chimeric antigen receptor (CAR) or a T cell receptor (TCR) containing an antigen-binding domain specific for an antigen in the target's tumor cells. In other embodiments, the cells engineered by CA2-IL15 used for ACT may be NK cells that have also been engineered to express a CAR or a TCR containing an antigen-binding domain specific for an antigen in the target's tumor cells. In some embodiments, the cells engineered by CA2-IL15 used for ACT may be a mixture of T cells and NK cells, either or both of which may express a CAR or a TCR.

[0172] When introduced into the phenotype of immune cells (e.g., T cells and NK cells), a chimeric antigen receptor (CAR) can redirect the immune cells against a target (e.g., a tumor cell) that expresses a molecule recognized by the extracellular targeting portion of the CAR. As used herein, the term "chimeric antigen receptor (CAR)" refers to a synthetic receptor that mimics the TCR on the surface of a T cell. Generally, a CAR consists of an extracellular targeting domain, a transmembrane domain / region, and an intracellular signaling / activation domain. In a standard CAR receptor, the extracellular targeting domain, transmembrane domain, and intracellular signaling / activation domain are linearly configured as a single fusion protein. The extracellular region includes a targeting domain / portion (e.g., scFv) that recognizes a specific tumor antigen (e.g., a tumor neoantigen) or other tumor cell surface molecule. The intracellular region may include an intracellular signaling domain (immunoreceptor tyrosine-based activation motif) of the TCR complex (e.g., the signaling region of CD3ζ), and / or one or more co-stimulatory signaling domains, such as those from CD28, 4-1BB (CD137), and OX-40 (CD134). When expressed by a T cell or NK cell, a CAR confers antigen specificity to the T cell or NK cell as determined by the extracellular targeting portion of the CAR.

[0173] In some embodiments, the extracellular target domain is linked to an intracellular signaling domain through a hinge (also called a spacer or a space region) and a transmembrane region. The hinge connects the extracellular target domain to the transmembrane domain that crosses the cell membrane and is connected to the intracellular signaling domain. The hinge may need to be modified to optimize the efficacy of the CAR-transformed cells on cancer cells depending on the size of the target protein to which the target moiety binds, as well as the size and affinity of the target domain itself. When the target moiety is recognized and binds to the target cell, the intracellular signaling domain induces an activation signal in the CAR T cell or CAR NK cell, which is further amplified by a "second signal" from one or more intracellular co-stimulatory domains. Once activated, the CAR T cell or CAR NK cell can destroy the target cell.

[0174] In some embodiments, the present disclosure provides an immune cell comprising a CA2-IL15 effector module and further comprising a chimeric antigen receptor (CAR). The CAR may be controlled by a DRD or may be constitutively expressed. In some embodiments, the CAR is constitutively expressed.

[0175] In some embodiments, the constitutively expressed or controlled CAR, and the CA2-IL15 effector module are encoded in different vectors. In some embodiments, a single vector includes both the CA2-IL15 effector module and the constitutively expressed or controlled CAR, forming a tandem construct. The CA2-IL15 effector module and the CAR may be separated from each other by an internal ribosome entry site (IRES); a ribosome skipping sequence 2A peptide selected from foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), porcine teschovirus-1 2A (P2A), or Thosea asigna virus 2A (T2A); or other ribosome skipping sequences or ribosome entry sequences, resulting in a bicistronic construct. In some embodiments, the 2A sequence is the P2A sequence. The IRES, 2A sequence, or other ribosome skipping sequences or ribosome entry sequences cause the expression of upstream and downstream sequences to be expressed as two independent polypeptides. In some embodiments, the single vector includes, in order, a sequence encoding the CAR, the P2A sequence, and a sequence encoding the CA2-IL15 effector module. The sequence encoding the CAR may be either 5' or 3' of the sequence encoding the CA2-IL15 effector module.

[0176] When introduced into immune cells (e.g., T cells and NK cells), the T cell receptor (TCR) can redirect the immune cells against a target (e.g., a tumor cell) that expresses a molecule recognized by the TCR. The TCR is a molecule that includes variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively), or an antigen-binding portion or fragment thereof that can specifically bind to a peptide bound to an MHC molecule. In some embodiments, the TCR is TCRαβ. Generally, the TCR is typically found on the surface of T cells that are responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0177] As used herein, the term TCR encompasses not only the antigen-binding portion or antigen-binding fragment thereof, but also the complete TCR. In some embodiments, the TCR is a full-length TCR that includes both an α chain and a β chain. In some embodiments, the TCR is an antigen-binding portion or fragment of the TCR, e.g., a portion of each of the α and β chains, that binds to a specific peptide bound in an MHC molecule. In some embodiments, the antigen-binding portion or fragment includes variable domains of the TCR, such as variable α (Vα) and variable β (Vβ) chains, that are sufficient to bind to a particular MHC-peptide complex.

[0178] The variable domains of the TCR include complementarity-determining regions (CDRs) that primarily contribute to MHC-peptide antigen recognition, binding, and specificity. The CDRs within the variable regions of the TCR chains are typically separated by framework regions (FRs) that are less variable than the CDRs. In some embodiments, one or more CDRs of the TCR form all or substantially all of the antigen-binding site of a given TCR molecule. In some embodiments, CDR3 is the major CDR responsible for antigen binding or specificity and interaction with the processed peptide portion of the peptide-MHC complex.

[0179] The α- and β-chains of the TCR may include constant domains, transmembrane domains, and short cytoplasmic tails. The cytoplasmic tail of the TCR firmly anchors proteins in the cell membrane and binds to invariant subunits of the CD3 complex, which is involved in the signaling ability of the TCR complex.

[0180] In some embodiments, the present disclosure provides CAR T cells or TCR T cells that are "armed" with a CA2-IL15 effector module and improve the efficacy and persistence of engineered cells.

[0181] In some embodiments, the present disclosure provides CAR NK cells or TCR NK cells that are "armed" with a CA2-IL15 effector module and improve the efficacy and persistence of engineered cells or prevent immune exhaustion and senescence.

[0182] In some embodiments, the present disclosure provides TILs that are engineered with a CA2-IL15 effector module and improve the efficacy and persistence of the engineered cells.

[0183] In some embodiments, the cells of the present disclosure may be autologous, allogeneic, syngeneic, or xenogeneic in the context of a particular individual subject. In some embodiments, the cells of the present disclosure may be mammalian cells, particularly human cells. The cells described herein may be primary cells or immortalized cell lines.

[0184] Cancer immunotherapy aims to induce or restore the responsiveness of the immune system to cancer. Adoptive cell therapy is a form of active immunotherapy that aims to induce an endogenous and persistent tumor antigen-specific immune response. The response can be enhanced by non-specific stimulation with immune response modifiers such as cytokines, but cytokine stimulation can cause toxicity or immune exhaustion.

[0185] Despite significant progress, the effectiveness of current immunotherapy strategies is limited by associated toxicities. These are often related to the narrow therapeutic window associated with immunotherapy, arising from the need to push therapeutic doses to the brink of potentially lethal toxicity to obtain clinically meaningful treatment efficacy. Additionally, the dose expands in vivo because adoptively transferred immune cells often unexpectedly continue to proliferate rapidly in the patient's body.

[0186] The major risk associated with immunotherapy is off-target but extra-tumoral side effects resulting from T cell activation in response to normal tissue expression of tumor-associated antigens (TAAs).

[0187] Immunotherapy can also cause on-target, on-tumor toxicity that appears when tumor cells die in response to immunotherapy. Adverse effects include tumor lysis syndrome, cytokine release syndrome, and related macrophage activation syndrome. Importantly, these adverse effects can occur during tumor destruction, so even if on-tumor immunotherapy is successful, toxicity can result. Therefore, an approach to manage immunotherapy controllably is highly desirable as it has the potential to reduce toxicity and maximize effectiveness.

[0188] The present disclosure provides systems, compositions, immunotherapeutic agents, and methods for cancer immunotherapy. These compositions provide regulatable control of gene expression and function in immunotherapy. The present disclosure also provides CA2 biocircuits, CA2 effector modules, stimulus-responsive elements (SREs), and payloads, as well as polynucleotides encoding any of the foregoing. In one aspect, the systems, compositions, immunotherapeutic agents, and other components of the present disclosure can be controlled by separately added stimulants that provide significant adaptability in controlling cancer immunotherapy. Further, the systems, compositions, and methods of the present disclosure may be combined with therapeutic agents such as chemotherapeutic drugs, small molecules, gene therapy, and antibodies.

[0189] The adjustable properties of the systems and compositions of the present disclosure have the potential to increase the efficacy and duration of the effectiveness of immunotherapy. By reversibly silencing the biological activity of adoptively transferred cells using the compositions of the present disclosure, the potential of cell therapy can be maximized without causing irreversible killing or ending the therapy.

[0190] The present disclosure provides a method for fine-tuning immunotherapy after administration to a patient. This subsequently improves the safety and effectiveness of immunotherapy and increases the population of subjects who benefit from immunotherapy.

[0191] In one embodiment, the CA2 biocircuit, CA2 effector module, SRE, and components that regulate expression levels and the activity of any agent may be used in immunotherapy. As a non-limiting example, the immunotherapy agent used in the constructs of the present disclosure is IL15, which induces an immune response in cells and subjects.

[0192] In some embodiments, the composition for inducing an immune response may include a CA2 effector module. In some embodiments, the CA2 effector module may include a stimulus-responsive element (SRE) operably linked to human IL15 comprising the amino acid sequence of SEQ ID NO: 8.

[0193] In some embodiments, the CA2 biocircuit, CA2 effector module, and compositions of the present disclosure are related to the post-translational regulation of the anti-tumor immune response of the protein (payload) function of immunotherapy agents.

[0194] In some embodiments, cells that are genetically engineered to express at least one CA2 biocircuit, a CA2 effector module, an SRE (e.g., CA2 DRD), and / or a payload (immunotherapeutic agent) of interest may be used in adoptive cell therapy (ACT). As used herein, adoptive cell transfer refers to the administration of immune cells (from autologous, allogeneic, or genetically engineered hosts) that have direct anti-cancer activity. ACT has demonstrated efficacy in clinical applications against malignant and infectious diseases.

[0195] According to the present disclosure, CA2 biocircuits and systems may be used in the development and implementation of cell therapies such as adoptive cell therapy. CA2 biocircuits, CA2 effector modules, and their SREs and payloads may be used in cell therapy and may be provided by immune cell therapy alone or in combination with other treatment lines (e.g., radiation, cytokines).

[0196] Provided herein are methods for use in adoptive cell therapy. In one embodiment, the method comprises preconditioning a subject in need thereof, removing a portion of the subject's T cells, engineering the subject's T cells with a CA2 effector module of the present disclosure, and administering the engineered T cells that express the CA2 effector module to the subject, wherein the engineered cells successfully engraft in the subject's body.

[0197] In another embodiment, the method comprises preconditioning a subject in need thereof and administering to the subject allogeneic engineered T cells that express a CA2 effector module, wherein the engineered cells successfully engraft in the subject's body.

[0198] In some embodiments, the method comprises removing a malignant tumor from a subject, isolating TILs from the tumor, engineering the TILs with the CA2 effector module of the present disclosure, and administering the engineered TILs to the subject, wherein the TILs successfully infiltrate remaining tumors or metastases in the subject's body.

[0199] In some embodiments, the SRE, CA2 biocircuit, and compositions of the present disclosure may be used to minimize preconditioning regimens associated with adoptive cell therapy. As used herein, "preconditioning" refers to any treatment regimen administered to a subject to improve the outcome of adoptive cell therapy. Preconditioning strategies include, but are not limited to, total body irradiation and / or lymphodepleting chemotherapy. Adoptive cell therapy clinical trials without preconditioning have not been able to demonstrate clinical benefit, indicating its importance in ACT. However, preconditioning is associated with significant toxicity and limits the cohort of subjects suitable for ACT. In some cases, immune cells for ACT may be engineered to express cytokines such as IL15 using the SRE of the present disclosure, thereby reducing the need for preconditioning.

[0200] In some embodiments, NK cells engineered to express the compositions of the present disclosure may be used for ACT. NK cell activation induces perforin / granzyme-dependent apoptosis in target cells. NK cell activation also induces secretion of cytokines such as IFNγ, TNF-α, and GM-CSF. These cytokines enhance phagocytic functions of macrophages and their antibacterial activities, and increase adaptive immune responses using upregulation of antigen presentation by antigen-presenting cells such as dendritic cells (DCs).

[0201] Immune cells can be isolated and expanded ex vivo using a variety of methods well known in the art. For example, methods for isolating and expanding cytotoxic T cells are described in U.S. Pat. Nos. 6,805,861 and 6,531,451; U.S. Patent Publication No. US20160348072A1 and International Patent Publication No. WO2016168595A1, the contents of each of which are incorporated herein by reference in their entirety. The isolation and expansion of NK cells are described in U.S. Patent Publication No. US20150152387A1, U.S. Pat. No. 7,435,596; and Oyer, J.L. (2016). Cytotherapy. 18(5):653-63, the contents of each of which are incorporated herein by reference in their entirety. In particular, human primary NK cells can be expanded in the presence of feeder cells, such as a bone marrow cell line that has been genetically recombined to express membrane-bound IL15 and 4-1BBL.

[0202] In some embodiments, the activation and expansion of T cells for ACT is accomplished by antigen stimulation of a chimeric antigen receptor (CAR) transiently expressed on the cell surface. Such activation methods are taught in International Patent No. WO2017015427, the content of which is incorporated herein by reference in its entirety.

[0203] In some embodiments, immune cells may be activated by an antigen conjugated to an antigen-presenting cell (APC). In some embodiments, the APC may be a dendritic cell, macrophage, or B cell, either antigen-specific or non-specific. The APCs may be autologous or syngeneic in their organs. In some embodiments, the APC may be an artificial antigen-presenting cell (aAPC), such as a cell-based aAPC or a cell-free aAPC. The cell-based aAPC may be selected from either genetically recombinant allogeneic cells, such as human erythroleukemia cells, or heterologous cells, such as mouse fibroblasts and Drosophila cells. Alternatively, the APC may be cell-free, and the antigen or co-stimulatory domain is presented on a synthetic surface, such as latex beads, polystyrene beads, lipid vesicles, or exosomes.

[0204] In some embodiments, adoptive cell therapy is performed by autologous transplantation, where the cells are derived from a subject in need of treatment and are administered to the same subject following isolation and processing. In other examples, ACT may involve allogeneic transplantation, where the cells are isolated and / or prepared from a donor subject other than the recipient subject who will ultimately receive the cell therapy. The donor and recipient subjects may be genetically identical, similar, or may express the same HLA class or subtype.

[0205] After genetic modification using the SRE, CA2 biocircuit, and compositions of the present disclosure, the cells are administered to a subject in need thereof. Methods of administering cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, for methods of adoptive T cell therapy, see, e.g., U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338, each of which is incorporated herein by reference in its entirety.

[0206] In some embodiments, the immune cells engineered with CA2-IL15 for ACT may be further modified to express one or more immunotherapeutic agents that promote immune cell activation, infiltration, proliferation, survival, and anti-tumor function. The immunotherapeutic agent may be a chimeric antigen receptor (CAR) or a T cell receptor (TCR) specific for a target molecule in a tumor cell; a second cytokine or cytokine receptor; a chimeric switch receptor that converts an inhibitory signal to a stimulatory signal; a homing receptor that directs adoptively transferred cells to a target site such as a tumor tissue; an agent that optimizes the metabolism of immune cells; or a safety switch gene (e.g., a suicide gene) that kills activated T cells when serious events are observed after adoptive cell transfer or when the transferred immune cells are no longer needed.

[0207] In some embodiments, the immune cells used for adoptive cell transfer are genetically engineered with the overall goal of further improving their ability to kill tumors in cancer patients, and their persistence, cytotoxicity, tumor targeting ability, and ability to be directed to the site of disease in vivo can be improved. One example is to introduce the CA2 effector module of the present disclosure containing IL15 into immune cells to promote the proliferation and survival of immune cells. Transduction of IL15 into cells allows immune cells to proliferate without the addition of exogenous cytokines, and cytokine-expressing NK cells can increase tumor cell cytotoxicity.

[0208] In some embodiments, the CA2 biocircuit, SRE, or CA2 effector module may be utilized to prevent T cell exhaustion. As used herein, "T cell exhaustion" refers to the gradual and progressive loss of T cell function resulting from chronic T cell activation. T cell exhaustion is a major factor limiting the effectiveness of anti-viral and anti-tumor immunotherapies. In exhausted T cells, the apoptosis rate of multiple inhibitory receptors is high, surface expression is high, and at the same time, the proliferative and cytokine-producing abilities are low. T cell activation leading to exhaustion can occur either in the presence or absence of antigen.

[0209] In some embodiments, the CA2 biocircuits and their components may be used to prevent T cell exhaustion in the context of chimeric antigen receptor-T cell therapy (CAR-T). In this regard, exhaustion may in some cases result from oligomerization of the scFv of the CAR on the cell surface, which may lead to constitutive activation of the intracellular domain of the CAR. As a non-limiting example, the CARs of the present disclosure may include scFvs that cannot oligomerize. As another non-limiting example, a CAR that is rapidly internalized and re-expressed following antigen exposure may be selected to prevent chronic scFv oligomerization on the cell surface. In one embodiment, the framework region of the scFv may be modified to prevent constitutive CAR signaling.

[0210] The adjustable CA2 biocircuits of the present disclosure may be used to control surface expression of the CAR on the T cell surface and prevent chronic T cell activation. The CARs of the present disclosure may be engineered to minimize exhaustion. As a non-limiting example, the 4-1-BB signaling domain may be incorporated into the CAR design, along with membrane-bound IL15 expression controlled by the CA2 biocircuit, SRE, or CA2 effector module exemplified in Table 3 of the present disclosure, to improve T cell exhaustion.

[0211] In some embodiments, the adjustable nature of the CA2-IL15 biocircuit of the present disclosure may be utilized to reverse human T cell exhaustion observed with tonic CAR signaling. Using the compositions of the present disclosure to reversibly silence the biological activity of adoptively transferred cells may be utilized to reverse tonic signaling, which may then reactivate the T cells. Reversal of exhaustion may be measured by downregulation of multiple inhibitory receptors associated with exhaustion.

[0212] In some embodiments, the compositions of the present disclosure may be used to alter the TIL (tumor-infiltrating lymphocyte) population in a subject. In one embodiment, any of the payloads described herein may be used to alter the ratio of CD4-positive cells to the CD8-positive population. In some embodiments, TILs may be sorted ex vivo and engineered to express any of the cytokines described herein. The payloads of the present disclosure may be used to expand the CD4 and / or CD8 populations of TILs and enhance the TIL-mediated immune response.

[0213] Provided herein is a method of reducing tumor volume or burden in a subject in need thereof, the method comprising introducing into the subject's body a composition of the present disclosure.

[0214] The present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of effector immune cells in which the genes are recombined to express at least one CA2 effector module of the present disclosure.

[0215] A variety of cancers may be treated with the pharmaceutical compositions, CA2 biocircuits, CA2 biocircuit components, CA2 effector modules of the present disclosure that include the SRE and IL15 payloads. As used herein, the term "cancer" refers to any of a variety of malignant neoplasms characterized by the growth of undifferentiated cells that tend to invade surrounding tissues and metastasize to new body sites, and also refers to the pathological conditions characterized by the growth of such malignant neoplasms. Cancers include tumors or hematological malignancies and may include, but are not limited to, all types of lymphoma / leukemia, carcinomas, and sarcomas, such as cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovary, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, coccyx, testis, thyroid, and uterus.

[0216] The types of tumors that can be treated with the compositions of the present disclosure include, but are not limited to, papilloma / carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma / adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, hemangioma, osteoma, chondroma, glioma, lymphoma / leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, basal cell carcinoma, and sinonasal undifferentiated carcinoma.

[0217] The types of tumors that can be treated with the compositions of the present disclosure include, but are not limited to, soft tissue sarcomas such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, fibrous histiocytoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, pericytic tumor, hemangiosarcoma, Kaposi sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin tumor, Ewing sarcoma (primitive neuroectodermal tumor), malignant angioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.

[0218] As non-limiting examples, treatable carcinomas include acute granulocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenoma, adrenal cancer, adrenocortical cancer, anal cancer, anaplastic astrocytoma, angiosarcoma, appendiceal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, non-invasive ductal breast cancer, endometrial cancer, epithelioma, epitheloid sarcoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumor, general, germ cell tumor, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma, Hodgkin disease, Hodgkin's lymphoma, hypopharyngeal cancer, invasive ductal breast cancer, invasive lobular breast cancer, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, invasive / infiltrating breast cancer, islet cell cancer, jaw cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, leukemia, lip cancer, liposarcoma, liver cancer, non-invasive lobular breast cancer, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary cancer, medulloblastoma, melanoma, meningioma, Merkel cell cancer, mesenchymal chondrosarcoma, mesenchymal, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous cell carcinoma of the neck, mixed glioma, mouth cancer, mucinous cancer, mucosal melanoma, multiple myeloma, nasal cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oat cell cancer, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cancer, oral cavitycancer), pharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, primary peritoneal cancer of the ovary, ovarian sex cord stromal tumor, Paget's disease, pancreatic cancer, papillary cancer, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nervous system cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal gland tumor, pineoblastoma, pituitary cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal column cancer, spinal cord cancer, spinal tumor, squamous cell cancer, stomach cancer, synovial sarcoma, T cell lymphoma), testicular cancer, throat cancer, thymoma / thymus cancer, thyroid cancer, tongue cancer, tonsil cancer, transitional cell cancer, transitional cell cancer, transitional cell cancer, triple negative breast cancer, tubal cancer, tubular cancer, ureteral cancer, ureteral cancer, urethral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0219] In some embodiments, the pharmaceutical compositions, CA2 biocircuits, CA2 biocircuit components, CA2 effector modules comprising the SRE or payload of the present disclosure may be used to modulate or alter or harness the immune system to target one or more cancers. This approach may be considered in combination with other such biological approaches, such as immunomodulatory therapies, such as the administration of interferons, interleukins, colony stimulating factors, other monoclonal antibodies, vaccines, gene therapies, and non-specific immunosuppressive drugs, and is also envisioned as an anti-cancer treatment in combination with the pharmaceutical compositions, CA2 biocircuits, CA2 biocircuit components, CA2 effector modules comprising the SRE or payload of the present disclosure.

[0220] Cancer immunotherapy refers to a variety of treatment regimens designed to induce the patient's own immune system to fight cancer. In some embodiments, pharmaceutical compositions, CA2 biocircuits, CA2 biocircuit components, CA2 effector modules comprising the SRE or payload of the present disclosure are designed as immuno - oncology therapies.

[0221] There are several types of cellular immunotherapies, including natural killer (NK) cells, tumor - infiltrating lymphocyte (TIL) therapy, and genetically engineered T cells with chimeric antigen receptor (CAR) or recombinant T cell receptor (TCR) technology.

[0222] In one embodiment, the CAR T cells or TCR T cells of the present disclosure may be "armed" T cells transformed by the CA2 - IL15 effector module to improve efficacy and persistence.

[0223] In one embodiment, patients may be stratified according to antigenic peptides presented by their immune cells, used as parameters, and a suitable patient cohort that may therapeutically benefit from the compositions of the present disclosure may be determined.

[0224] In some embodiments, the cells of the present disclosure may be autologous, allogeneic, syngeneic, or xenogeneic in the context of a particular individual subject.

[0225] In some embodiments, the cells of the present disclosure may be mammalian cells, particularly human cells. The cells of the present disclosure may be primary cells or immortalized cell lines.

[0226] The engineered immune cells can be completed by transducing a cell composition with one or more polynucleotides encoding the CA2 biocircuit, the CA2 effector module, the SRE, and the IL15 payload polypeptides, or a vector comprising said polynucleotides. The vector may be a viral vector such as a lentiviral vector or a gammaretroviral vector. In some embodiments, the immune cells of the present disclosure express at least one immunotherapeutic agent of the present disclosure whose gene is recombined and regulatable using a stimulus. Definitions

[0227] Throughout the specification, features or functions of the compositions of the present disclosure are disclosed in groups or ranges. In particular, the present disclosure is intended to include each and every individual sub-combination of the members of such groups and ranges. The following is a non-limiting list of term definitions.

[0228] Activity: As used herein, the term "activity" refers to the state in which something is happening or being done. The compositions of the present disclosure may have activity, and this activity may be accompanied by one or more biological events. In some embodiments, the biological event may include a cell signaling event. In some embodiments, the biological event may include a cell signaling event associated with a protein interaction with one or more corresponding proteins, receptors, small molecules, or biocircuit components described herein.

[0229] Adoptive cell therapy (ACT): As used herein, the terms "adoptive cell therapy" or "adoptive cell transfer" refer to cell therapies involving the transfer of cells to a patient, where the cells may be derived from the patient or another individual and are manipulated (altered) prior to being transferred back into the patient's body. The therapeutic cells may be of immune system origin, such as effector immune cells: CD4+ T cells; CD8+ T cells, natural killer cells (NK cells); and B cells and tumor-infiltrating lymphocytes (TILs) derived from resected tumors. The most commonly transferred cells are autologous anti-tumor T cells after ex vivo expansion or manipulation. For example, autologous peripheral blood lymphocytes can be genetically engineered to express a T cell receptor (TCR) or chimeric antigen receptor (CAR) to recognize specific tumor antigens.

[0230] Agent (drug): As used herein, the term "agent (drug)" refers to a biological, pharmaceutical, or chemical compound. Non-limiting examples include single or complex organic or inorganic molecules, peptides, proteins, oligonucleotides, antibodies, antibody derivatives, antibody fragments, receptors, and soluble factors.

[0231] Antigen: As used herein, the term "antigen" is defined as a molecule that, when introduced into a subject or produced by a subject, such as a tumor antigen resulting from cancer growth itself, elicits an immune response. This immune response may involve either, or both, antibody production or activation of specific immunologically competent cells such as cytotoxic T lymphocytes and T helper cells. Antigens can be derived from living organisms, protein / antigen subunits, whole or lysed dead or inactivated cells. In the context of the present disclosure, the terms "target antigen" or "desired antigen" refer to those proteins and / or other biomolecules provided herein that immunospecifically bind to or interact with the antibodies of the present disclosure and / or the fragments, variants, variants, and / or modified forms thereof described herein. In some embodiments, the target antigen may comprise any of the polypeptides or payloads or proteins described herein, or fragments or portions thereof.

[0232] Linked to: As used herein, the terms "linked to", "joined", "bound", "attached", and "connected" when used with respect to two or more moieties mean that the moieties are physically bound or connected to each other, either directly or via one or more additional moieties that function as a linker, to form a sufficiently stable structure such that the moieties remain physically bound in the state in which the structure is used, e.g., under physiological conditions. "Binding" need not be strictly by direct covalent chemical bonds. Sufficiently stable ionic or hydrogen bonds or hybridization-based connectivity may be proposed such that the "bound" entities remain physically bound.

[0233] Self: As used herein, the term "self" is intended to refer to any substance derived from an individual that is later reintroduced into the same individual.

[0234] Cancer: As used herein, the term "cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and proliferation result in the formation of malignant tumors that invade adjacent tissues and ultimately metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0235] Costimulatory molecule: As used herein, in the context of immune T cell activation, refers to a group of immune cell surface receptors / ligands that bind between T cells and APCs and generate stimulatory signals in T cells that bind to stimulatory signals in T cells resulting from the recognition of the antigen / MHC complex (pMHC) in APCs by the T cell receptor (TCR).

[0236] Cytokine: As used herein, the term "cytokine" refers to a family of small soluble factors with multifaceted functions that are produced by many cell types that can affect and control the functions of the immune system.

[0237] Delivery: As used herein, the term "delivery" refers to the act or method of delivering a compound, substance, entity, moiety, cargo, or payload. A "delivery agent" refers to any agent that facilitates the in vivo delivery of one or more substances (including, but not limited to, the compounds and / or compositions of the present disclosure) to cells, a subject, or other biological system cells to some extent.

[0238] Destabilized: As used herein, the terms "destable", "destabilize", or "destabilized region" mean a region or molecule that is less stable than the starting, reference, wild-type, or native form of the same region or molecule.

[0239] Engineered: As used herein, embodiments of the present disclosure are "engineered" when designed to have features or properties that are different, either structurally or chemically, from the starting point, wild-type, or native molecule.

[0240] Formulation: As used herein, "formulation" includes at least the compounds and / or compositions of the present disclosure, as well as delivery agents.

[0241] Fragment: As used herein, "fragment" refers to a part of a molecule that is smaller than the whole molecule. For example, a fragment of a protein may include a polypeptide obtained by digesting the full-length protein. In some embodiments, a fragment of an antibody includes a portion of the antibody.

[0242] Functional: As used herein, a "functional" biomolecule is a biological entity that has a structure and exhibits the properties and / or activities by which it is characterized.

[0243] Immune cell: As used herein, the term "immune cell" refers to any cell of the immune system that has its origin in hematopoietic stem cells in the bone marrow and gives rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (which give rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD8-double negative T cells, Tγδ cells, Tαβ cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells may be referred to as "antigen-presenting cells" or "APCs," and are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with the T cell receptor on the surface of the T cell.

[0244] Immunotherapy: As used herein, the term "immunotherapy" refers to a type of treatment of a disease by inducing or restoring the responsiveness of the immune system to the disease.

[0245] Immunotherapy drug: As used herein, the term "immunotherapy drug" refers to the treatment of a disease by inducing or restoring the reactivity of the immune system to the disease by a living body, medicine, or chemical compound.

[0246] in vitro: As used herein, the term "in vitro" refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than inside a living organism (e.g., an animal, a plant, or a microorganism).

[0247] in vivo: As used herein, the term "in vivo" refers to an event that occurs inside a living organism (e.g., an animal, a plant, or a microorganism or a cell or its tissue).

[0248] Linker: As used herein, a linker refers to a moiety that connects two or more domains, moieties, or entities. In one embodiment, the linker may comprise 10 or more atoms. In a further embodiment, the linker may comprise a group of atoms, e.g., 10 to 1,000 atoms, and may include atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. In some embodiments, the linker may comprise one or more nucleic acids comprising one or more nucleotides. In some embodiments, the linker may comprise an amino acid, a peptide, a polypeptide, or a protein. In some embodiments, the moieties linked by the linker may include, but are not limited to, atoms, chemical groups, nucleosides, nucleotides, nucleobases, sugars, nucleic acids, amino acids, peptides, polypeptides, proteins, protein complexes, payloads (e.g., therapeutic agents), or markers (including, but not limited to, chemical, fluorescent, radioactive, or bioluminescent markers). The linker can be used for any beneficial purpose, such as, but not limited to, administering a payload as described herein or forming multimers or conjugates. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomer units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetrapropylene glycol), and dextran polymers. Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, disulfide bonds (-S-S-) or azo bonds (-N=N-) that can be cleaved using a reducing agent or photolysis.Non-limiting examples of selectively cleavable linkages include, for example, not only ester linkages that can be cleaved by acidic or basic hydrolysis, but also, for example, the use of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and / or amide linkages that can be cleaved by photolysis.

[0249] Modified: As used herein, the term “modified” refers to a change in the state or structure of a molecule or entity as compared to a parent or reference molecule or entity. A molecule may be modified in many ways, including chemically, structurally, and functionally. In some embodiments, the compounds and / or compositions of the present disclosure are modified by the introduction of non-natural amino acids.

[0250] Mutation: As used herein, the term “mutation” refers to a change and / or alteration. In some embodiments, a mutation may be a change and / or alteration to a protein (including peptides and polypeptides) and / or a nucleic acid (including polynucleic acids). In some embodiments, a mutation includes a change and / or alteration to a protein and / or nucleic acid sequence. Such changes and / or alterations may include the addition, substitution, and / or deletion of one or more amino acids (in the case of proteins and / or peptides) and / or nucleotides (in the case of nucleic acids and / or polynucleic acids, e.g., polynucleotides). In some embodiments, a mutation includes the addition and / or substitution of amino acids and / or nucleotides, such addition and / or substitution may include one or more amino acid and / or nucleotide residues, and may include modified amino acids and / or nucleotides. A construct, molecule, or sequence resulting from a mutation, change, or alteration may be referred to herein as a variant.

[0251] Neoantigen: As used herein, the term "neoantigen" refers to a tumor antigen that is present in tumor cells but not in normal cells and does not induce deletion of their allogeneic antigen-specific T cells in the thymus (i.e., central tolerance). These tumor neoantigens may provide a "foreign" signal similar to that of a pathogen and induce an effective immune response necessary for cancer immunotherapy. A neoantigen may be limited to a particular tumor. A neoantigen is a peptide / protein having a missense mutation (missense neoantigen) or a new peptide having a long, completely novel stretch of amino acids from a novel open reading frame (neoORF). NeoORFs can be generated in some tumors by out-of-frame insertions or deletions (due to defects in DNA mismatch repair that cause microsatellite instability), gene fusions, read-through mutations at stop codons, or translation of inappropriately spliced RNA (e.g., Saeterdal et al., Proc Natl Acad Sci USA, 2001, 98:13255-13260).

[0252] Off-target: As used herein, "off-target" refers to any unintended effect on any one or more targets, genes, cellular transcripts, cells, and / or tissues.

[0253] Operably linked: As used herein, the phrase "operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties, etc.

[0254] Payload or Payload of Interest (POI): The terms "payload" and "payload of interest (POI)" are used interchangeably when used in this specification. Payload of interest (POI) refers to any protein or compound whose function is altered. In the context of the present disclosure, the POI is a component in the immune system, including both the innate and adaptive immune systems. The payload of interest may be a protein, a fusion construct encoding a fusion protein, or a non-coding gene, or variants and fragments thereof. When the payload of interest is amino acid-based, it may be referred to as the protein of interest.

[0255] Pharmaceutically acceptable additives: The term "pharmaceutically acceptable additives" as used herein refers to any component other than the active agent (e.g., as described herein) that is present in a pharmaceutical composition and has substantially non-toxic and non-inflammatory properties in a subject. In some embodiments, the pharmaceutically acceptable additive is a vehicle capable of suspending and / or dissolving the active agent. Additives may include, for example, antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, excipients (diluents), film formers or coatings, flavors, fragrances, flow enhancers, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary additives include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0256] Pharmaceutically acceptable salts: The pharmaceutically acceptable salts of the compounds described herein are in the form of the disclosed compounds, where the acid or base moiety is in its salt form (e.g., as produced by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., and non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts from non-toxic inorganic or organic acids. In some embodiments, the pharmaceutically acceptable salts are prepared from the parent compound containing a basic or acidic moiety by conventional chemical methods.Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water, an organic solvent, or a mixture of the two; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Remington’s Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is hereby incorporated by reference in its entirety. Pharmaceutically acceptable solvates: The term “pharmaceutically acceptable solvate” as used herein refers to the crystalline form of a compound, where molecules of a suitable solvent are incorporated into the crystal lattice. For example, the solvate may be prepared by crystallization, recrystallization, or precipitation from a solution containing an organic solvent, water, or a mixture thereof. Examples of suitable solvents include ethanol, water (e.g., mono-, di-, and trihydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N’-dimethylformamide (DMF), N,N’-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is called a “hydrate”. In some embodiments, the solvent incorporated into the solvate is of a type or level that is physiologically tolerable to the living organism to which the solvate is administered (e.g., in a unit dosage form of a pharmaceutical composition).

[0257] Stable: As used herein, "stable" refers to a compound or entity that is robust enough to survive separation from a reaction mixture to a useful purity and is preferably capable of being formulated into an effective therapeutic agent.

[0258] Stabilized: As used herein, the terms "stabilize", "stabilized", and "stabilized region" mean to stabilize or cause to be stabilized. In some embodiments, stability is measured relative to an absolute value. In some embodiments, stability is measured relative to a secondary situation or state, or relative to a reference compound or entity.

[0259] Standard CAR: As used herein, the term "standard CAR" refers to the standard design of a chimeric antigen receptor. The components of a CAR fusion protein, which includes an extracellular scFv fragment, a transmembrane domain, and one or more intracellular domains, are linearly configured as a single fusion protein.

[0260] Subject: As used herein, the terms "subject" or "patient" refer to any living organism to which a composition according to the disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0261] T cells: T cells are immune cells that produce T cell receptors (TCRs). T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA; and decreased expression of CD45RO compared to TCM), memory T cells (TM) (experienced antigen and long-lived), and effector cells (experienced antigen, cytotoxic). TM can be further classified into subsets of central memory T cells (TCM; increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95; and decreased expression of CD54RA compared to naive T cells) and effector memory T cells (TEM; decreased expression of CD62L, CCR7, CD28, CD45RA; and increased expression of CD127 compared to naive T cells or TCM). Effector T cells (TE) refer to antigen-experienced CD8+ cytotoxic T lymphocytes with decreased expression of CD62L, CCR7, CD28, and positive for granzyme and perforin compared to TCM. Other exemplary T cells include regulatory T cells such as Tr1, Th3, CD8+CD28-, and Qa-1 restricted T cells, as well as CD4+CD25+ (Foxp3+) regulatory T cells and Treg17 cells.

[0262] T cell receptor: The T cell receptor (TCR) refers to a member of the immunoglobulin superfamily that has a variable antigen-binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail, and can specifically bind to an antigen peptide bound to an MHC receptor. TCRs can be found on the surface of cells or in a soluble form and usually contain a heterodimer with α and β chains (also known as TCRα and TCRβ, respectively), or γ and δ chains (also known as TCRγ and TCRδ, respectively). The extracellular portion of a TCR chain (e.g., an α-chain, β-chain) contains two immunoglobulin domains, a variable domain at the N-terminus (e.g., an α-chain variable domain or Vα, β-chain variable domain or Vβ), and one constant domain adjacent to the cell membrane (e.g., an α-chain constant domain or Cα and β-chain constant domain or Cβ). Similar to immunoglobulins, the variable domain contains complementarity-determining regions (CDRs) separated by framework regions (FRs). TCRs are usually associated with the CD3 complex to form a TCR complex. As used herein, the term "TCR complex" refers to the complex formed by the association of TCR with CD3. For example, a TCR complex can consist of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of CD3ζ chains, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex can consist of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of CD3ζ chains, a TCRγ chain, and a TCRδ chain. "Components of the TCR complex" as used herein refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0263] Amount effective for treatment: As used herein, the term "amount effective for treatment" means an amount of an agent (e.g., nucleic acid, drug, therapeutic, diagnostic, prophylactic, etc.) delivered that is sufficient to treat, ameliorate, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or affliction in a subject suffering from or susceptible to such infection, disease, disorder, and / or affliction. In some embodiments, the amount effective for treatment is provided as a single dose. In some embodiments, the amount effective for treatment is administered in a dosage regimen that includes multiple doses. One of ordinary skill in the art will understand that in some embodiments, a unit dosage form may be considered to contain a particular agent or entity in an amount effective for treatment if it contains an amount effective when administered as part of such a dosage regimen.

[0264] Treat or treating: As used herein, the terms "treat" or "treating" refer to an approach for obtaining a beneficial or desired result, preferably including a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of cancerous cells or other abnormal cells (or destroying cancerous cells or other abnormal cells), reducing the metastasis of cancerous cells found in cancer, reducing the size of a tumor, reducing the symptoms resulting from a disease, increasing the quality of life of a person suffering from a disease, reducing the dosage of other pharmaceutical therapies necessary to treat a disease, delaying the progression of a disease, and / or extending the lifespan of an individual.

[0265] Modulate: As used herein, the term "modulate" means to prepare, balance, or adapt one thing in response to a stimulus or towards a particular result. In one non-limiting example, the SREs and / or DRDs of the present disclosure modulate the function or structure of a composition to which they are added, attached, or bound in response to a particular stimulus and / or environment. Equivalents and Ranges

[0266] One of ordinary skill in the art will recognize, and be able to ascertain, many equivalents to the specific embodiments of the present disclosure described herein with only routine experimentation. The scope of the present disclosure is not intended to be limited to the above description, but rather is set forth in the appended claims.

[0267] In the claims, articles such as "a," "an," and "the" may mean one or more than one unless contrary to indicated or clear from the context. A claim or description that includes "or" between one or more members of a group is considered to be satisfied if, unless contrary to indicated or clear from the context, one, more than one, or all of the group members are present in, used in, or relevant to a given product or process. The present disclosure includes embodiments where only one member of the group is present in, used in, or relevant to a given product or process. The present disclosure includes embodiments where two or more, or all of the group members are present in, used in, or relevant to a given product or process.

[0268] Note also that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps, but does not require them. When the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0269] When ranges are given, endpoints are included. Further, unless otherwise indicated or clear from the context and understanding of one of ordinary skill in the art, values expressed as ranges are to be understood as including any and all subranges between the lower and upper limits of the range, down to the unit of one tenth of the lower limit of the range, and including any specific value or subrange within the range described in different embodiments of the present disclosure, unless the context clearly dictates otherwise.

[0270] In addition, it is understood that any particular embodiment of the present disclosure that falls within the scope of the prior art may be expressly excluded from any one or more of the claims. Since such embodiments are considered to be known to those skilled in the art, they may be excluded even if the exclusion is not expressly described herein. Any particular embodiment of the compositions of the present disclosure (e.g., any antibacterial, therapeutic or active ingredient; any production method; any method of use, etc.) may be excluded from any one or more of the claims for any reason, regardless of whether it relates to the existence of the prior art.

[0271] It is understood that the words used are words of description and not of limitation, and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.

[0272] Although the present disclosure has been described in some detail and somewhat particularly with respect to several described embodiments, it is not intended that any such matter or embodiment or any particular embodiment be limiting, and in view of the prior art, the appended claims should be construed so as to provide the broadest interpretation of such claims so as to effectively encompass the intended scope of the present disclosure. The present disclosure is further illustrated by the following non-limiting examples.

Example

[0273] Figure 1 shows a representative procedure for the in vitro characterization and / or verification of ACZ-controlled mbIL15 expression in T cells. As shown in Figure 1, for example, T cells may be transduced with an mbIL15 construct according to the procedure described in Example 1. After transduction, for example, according to the procedure described in Example 2, the T cells may be treated under control conditions or with ACZ and assayed for IL15 expression and / or antigen-independent cell proliferation in vitro.

[0274] Figure 2 shows a representative procedure for in vivo characterization and / or verification of ACZ-controlled mbIL15 expression in T cells. As shown in Figure 2, for example, according to the procedure described in Example 1, T cells may be transduced with an mbIL15 construct. After transduction, the T cells are injected into a mouse subject (e.g., NSG mouse) according to the procedure described in Example 3, for example, and specimens of mice treated with a vehicle or ACZ may be assayed for IL15 expression and / or antigen-independent cell proliferation. Example 1. T cell transduction with an acetazolamide (ACZ)-controlled mbIL15 construct

[0275] This example shows a method that can be used to prepare an ACZ-controlled mbIL15 construct and a method that can be used for transduction of T cells with an ACZ-controlled mbIL15 construct. IL15 construct assembly

[0276] OT-IL15-292, OT-IL15-293, OT-IL15-294, and OT-IL15-295 were each constructed in a pELNS vector (a third-generation self-inactivating lentiviral expression vector) using standard molecular biology techniques. Gene fragments (Gblocks) encoding codon-optimized IL15, a GS linker, a B7-1 hinge, a transmembrane domain, and a cytoplasmic tail were purchased from Integrated DNA Technologies, Inc. (IDT, Coralville, Iowa). The gene fragments were inserted into the pELNS vector and placed under the control of the EF1a promoter using Gibson assembly (NEBuilder Hifi). The assembled plasmids were transformed into Escherichia coli (NEB stable) for amplification and sequence confirmation prior to proceeding with virus production.

[0277] Table 4 presents the nucleic acid and amino acid sequences for the components of the constitutive IL15 constructs (OT-IL15-292 and OT-IL15-294) and the ACZ-controlled IL15 constructs (OT-IL15-293 and OT-IL15-295) disclosed herein. The amino acids that are bolded and underlined in Table 4 indicate the differences in the B7-1 cytoplasmic tail between the constructs OT-IL15-292 / OT-IL15-293 and OT-IL15-294 / OT-IL15-295. The constructs OT-IL15-293 and OT-IL15-295 contain destabilizing domains classified as CA2 (M1del, L156H) in Table 4.

[0278]

Table 4-1

Table 4-2

Table 4-3

[0279] Table 5 presents the nucleic acid and amino acid sequences for the constitutive IL15 (IL15-292 and IL15-294) and the ACZ-controlled IL15 (IL15-293 and IL15-295 constructs.

[0280]

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

[0281] HEK293T cells were seeded on collagen-coated tissue culture plates until 70% confluent. Cells were transfected in Opti-MEM medium using Lipofectamine 3000 transfection reagent with constitutive (IL15-292 or IL15-294) or control (IL15-293 or IL15-295) IL15 constructs, and a pELNS transfer vector carrying packaging plasmids (pRSV.REV, pMDLg / p.RRE, and pMD2.G). The medium was replaced with serum-free medium 6 - 8 hours after transfection. Supernatants containing virus were harvested 24 hours after transfection, fresh medium was added, and the supernatants were harvested again 48 hours after transfection. Virus supernatants were filtered to remove debris and concentrated by ultracentrifugation on a 20% sucrose density gradient. Viruses were resuspended, aliquoted, and stored in a -80°C freezer.

[0282] The nucleotide sequences of the pELNS transfer vectors OT-IL15-292, OT-IL15-293, OT-IL15-294, and OT-IL15-295 are SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively.

[0283] As used herein, the lentiviruses used to transduce cells are referred to by their construct names (e.g., IL15-292, IL15-293, IL15-294, IL15-295, CD19-IL15-057, CD19-IL15-058, or CD19-063) or their transplantation vector names (e.g., OT-IL15-292, OT-IL15-293, OT-IL15-294, OT-IL15-295, OT-CD19-IL15-057, OT-CD19-IL15-058, or OT-CD19-063). T cell stock

[0284] T cells were isolated from Leukopak collected from healthy human donors. After PBMC isolation by Ficoll density gradient, T cells were isolated using a negative selection kit (StemCell Technologies) according to the manufacturer's protocol. T cells were resuspended in cell freezing medium (Bambanker), aliquoted, and stored in liquid nitrogen. Lentiviral transduction of T cells

[0285] T cells were thawed, the cells were washed and counted. T cells were mixed with CD3 / CD28 beads (Invitrogen cat#11141D) at a bead-to-T cell ratio of 3:1. 5×10 5 cells / well were added to a 24-well plate in 500 μL of medium. The cells were activated for 24 hours. The next day, the lentivirus was thawed and added to each well in different volumes. After 24 hours, 500 μL of fresh medium was added to the wells and the cells were grown by adding equal volumes of fresh medium every 2 - 3 days, maintaining a cell density of 0.5 - 1×10 6 / mL. The cells were analyzed by flow cytometry and expression was confirmed on day 5 or 6. The cells were grown for 9 - 10 days. Example 2. In vitro analysis of mbIL15 expression controlled by ACZ and T cell proliferation controlled by ACZ

[0286] This example shows in vitro verification of (i) ACZ-controlled mbIL15 expression in T cells and (ii) ACZ-controlled proliferation of T cells expressing ACZ-controlled mbIL15.

[0287] Human primary T cells capable of constitutively expressing mbIL15 or ACZ-controlled mbIL15 were prepared according to the method described in Example 1 above. See Figure 1.

[0288] After transduction and proliferation of T cells, CD3 / CD28 beads were removed using a magnet, the cells were washed twice, resuspended in fresh medium, and counted. The cells were placed in a 12-well plate at 1×10 6 in 2 mL. Non-transduced cells in one well were cultured in the presence of 2 ng / mL of IL15 as a control. T cells expressing the control construct were cultured in the absence or presence of acetazolamide (ACZ, 30, 10, 3, 1, 0.3 μM). Cell numbers were monitored by flow cytometry every 3 - 4 days, and the cells were cultured for 10 - 12 days. At each time point, 100 μL of cells were collected from the well and analyzed by flow cytometry. The cells were split as needed. Cell culture was maintained in a 12-well plate by taking a portion and adding medium to a new plate. The volume of each well was recorded before and after splitting, and the final volume for cell number assessment was calculated. IL15 or ACZ was replenished at the final concentration in the fresh medium added during each split. See Figure 1.

[0289] The number of T cells was determined by flow cytometry. The number of cells transduced with the empty vector (EV) decreased to background levels within 3 - 5 days in the absence of IL15, and the cells proliferated 11-fold in the presence of 2 ng / mL exogenous IL15 (Figure 3A). T cells expressing constitutive IL15-292 and IL15-294 proliferated 18 - 21-fold respectively in 10 days (Figure 3A). In T cells expressing IL15-293, the maximum proliferation was 9.5 - 11-fold at 30, 10, 3 μM (Figure 3B). At the lowest concentration tested (0.3 μM), the cells proliferated 3.3-fold. These cells survived longer (0.8-fold) compared to EV cells without drug treatment (0.8-fold). In T cells expressing IL15-295, the maximum proliferation was 8.2 - 10-fold at 30, 10, 3 μM, and the cells proliferated 4.9-fold at the lowest concentration tested (0.3 μM) (Figure 3C). Without drug treatment, these cells survived longer (0.9-fold) compared to EV.

[0290] The effect of different concentrations of ACZ on IL15 expression was examined. T cells were started at 100 μM, treated with ACZ for 24 hours, and diluted 3-fold between 9 points. %IL15+ T cell (Figure 4A) and IL15 mean fluorescence intensity (MFI) (Figure 4B) analyses showed similar dose curves for both OT-IL15-293 and OT-IL15-295. Expression increased 4 - 5-fold between the highest and lowest concentrations of ACZ (both %IL15+ T cells and IL15 MFI). EC 50 values were 0.29 μM and 0.22 μM EC50 based on %IL15+ T cells and 0.65 μM and 0.44 μM based on MFI of IL15 for OT-IL15-293 and OT-IL15-295 respectively. Example 3. In vivo analysis of ACZ-controlled mbIL15 expression and ACZ-controlled T cell proliferation

[0291] This example demonstrates (i) ACZ-controlled mbIL15 expression in T cells, and (ii) in vivo verification of ACZ-controlled proliferation of T cells expressing ACZ-controlled mbIL15. NK cell proliferation

[0292] A portion of PBMCs isolated from Leukopak was used to expand NK cells using a negative selection kit (StemCell Technologies) according to the manufacturer's protocol. Cells were cultured for 7 - 14 days at a 1:1 ratio with feeder K562 cells expressing 4-1BB-L and membrane-bound IL21, and recombinant IL2 (100 U / mL). Proliferation was monitored by cell counting and purity was evaluated by flow cytometry. In vivo analysis

[0293] Human primary T cells capable of constitutively expressing mbIL15 or ACZ-controlled mbIL15 were prepared according to the method described in Example 1 above. After transduction and proliferation of T cells, CD3 / CD28 beads were removed using a magnet, the cells were washed twice, resuspended in fresh medium, and counted. T cells were mixed with expanded NK cells and each animal received 5×10 6 T cells and 2×10 6 expanded NK cells. The cells were injected into NSG mice by intravenous injection. Animals injected with T cells expressing a control construct were administered 200 mg / kg of ACZ or vehicle daily by PO injection. Every 3 - 4 days, 50 μL of blood was analyzed by flow cytometry for the presence of T cells and NK cells using antibodies against mouse and human CD45, CD3, and CD56. IL15 expression on day 25 was analyzed using an IL15Ra-Fc and an anti-human IgG antibody conjugated with a fluorescent dye. See Figure 2.

[0294] The proliferation of T cells expressing constitutive and controlled IL15 constructs, and their impact on bystander NK cells, were evaluated in vivo in NSG mice for 25 days (Figures 5A - 5B). The number of cells transduced with the empty vector (EV) decreased slowly over time. T cells expressing constitutive IL15 - 292 and IL15 - 294 proliferated up to 13 - fold compared to the frequency on day 3. In mice injected with T cells expressing the control construct, the frequency of cells decreased in the presence of vehicle treatment. In the group treated daily with ACZ, the cells proliferated up to 7 - 8 - fold compared to the frequency on day 3. Bystander NK cells survived and proliferated in the presence of T cells expressing constitutive IL15 constructs or T cells expressing controlled IL15 constructs treated daily with ACZ.

[0295] IL15 expression in T cells in vivo on day 25 was analyzed by flow cytometry (Figure 5C). T cells transduced with the constitutive constructs IL15 - 292 and IL15 - 294 expressed IL15 at high levels (82% and 61%). IL15 expression was <1.5% in the EV group as well as in vehicle - treated IL15 - 293 and IL15 - 295. IL15 expression levels were 11% and 9% in T cells transduced with IL15 - 293 and IL15 - 295 in the group treated with ACZ. Example 4: In vivo analysis of efficacy and proliferation in CART cells expressing constitutive and controlled mbIL15

[0296] This example shows that control mbIL15 conjugated with ACZ administration enhances the anti - tumor efficacy and proliferation of CD19 CART cells in the presence of CD19 - positive tumors. Generation of tandem CD19 CAR and mbIL15 constructs and lentiviral stocks

[0297] Lentiviral vector constructs and lentiviral stocks co-expressing CD19 CAR and mbIL15 were basically generated as described in Example 1. The CD19 CAR sequence (AA sequence: SEQ ID NO: 38; NA sequence: SEQ ID NO: 39) consists of a CD8a leader sequence (aa1-21 in Uniprot ID P01732), the FMC63 (anti-CD19) single-chain variable fragment (scFv), a hinge and transmembrane domain derived from CD8 (aa138-206 in Uniprot ID P01732), a co-stimulatory domain derived from 4-1BB (aa214-255 in Uniprot ID Q07011), and a CD3zeta signaling domain (aa52-164 in Uniprot ID P20963).

[0298] The bicistronic transgene expression cassette (from 5' to 3' as described) included a regulatory or constitutive mbIL15, a P2A sequence (AA sequence: SEQ ID NO: 40; NA sequence: SEQ ID NO: 41), and an anti-CD19 CAR downstream of P2A (see Figure 6). For the regulatory construct (CD19-IL15-058; AA sequence: SEQ ID NO: 42; NA sequence: SEQ ID NO: 43), an IL15-293 construct containing mbIL15 operably linked to CA2 (L156H) DRD was used (AA sequence: SEQ ID NO: 24, NA sequence: SEQ ID NO: 25). For the constitutive construct (CD19-IL15-057; AA sequence: SEQ ID NO: 45; NA sequence: SEQ ID NO: 46), a construct containing mbIL15 operably linked to the CA2 wild-type sequence was used (see Figure 6). The nucleotide sequence of lentivirus OT-CD19-IL15-058 is SEQ ID NO: 44, and the nucleotide sequence of lentivirus OT-CD19-IL15-057 is SEQ ID NO: 45. Expression of the mbIL15-CAR construct in peripheral blood T cells

[0299] Peripheral blood T cells were activated, transduced, and expanded until day 10, and then frozen in cell freezing medium basically as described in Example 1 and used in an in vivo human Nalm6-Luc xenograft tumor model. mbIL15 and CAR expression were analyzed by flow cytometry using a recombinant protein containing an anti-IL15 antibody and the extracellular domain of human CD19 fused to the human IgG1 Fc domain (CD19-Fc), respectively. Cells transfected with only CD19 CAR or non-transfected cells were used as controls. 72% of the cells transfected with the control CD19 CAR construct were CAR+mbIL15- (Figure 7A). For cells transfected with a lentiviral vector expressing constitutive mbIL15 and CAR (CD19-IL15-057), 26% of the cells were CAR+, and 13% were double positive for CAR+mbIL15+. For cells transfected with a vector expressing control mbIL15 and CAR (CD19-IL15-058), 25% were CAR+mbIL15- in the absence of ACZ, and 9.7% were CAR+mbIL15+ double positive after 24-hour exposure to 10 μM ACZ. These results confirmed the expression of both CAR and mbIL15 after transduction of T cells with a lentiviral vector expressing CD19 CAR in combination with constitutive or control mbIL15. Evaluation of mbIL15-CART cells in the Nalm6-Luc xenograft model

[0300] To evaluate the effect of mbIL15 on the anti-tumor activity of CART cells, T cells transfected with a lentiviral vector expressing CD19 CAR with or without constitutive or control mbIL15 were injected into mice after transplantation of CD19+Nalm6-Luc tumors. CD19+Nalm6 cells expressing luciferase (Nalm6-Luc) were injected via the intravenous route (1×10 6 / mouse), and injected into NSG mice. Tumor growth was measured once or twice a week by bioluminescence imaging measurement (total flux unit of photons / second (p / s)) after intraperitoneal injection of D-luciferin. On day 6, when the average tumor size reached approximately 10 6 total p / s, the animals were randomly withdrawn into new cages (N = 8 per group). CD19 CART cells (engineered with or without constitutive or regulated mbIL15) were thawed for injection into mice with tumors. CAR expression in T cells was determined after thawing and 24-hour restimulation with anti-CD3 / CD28 beads. CART cells across different groups were normalized based on %CAR+ cells. Each mouse received 0.3×10 6 CAR+ cells, and the total number of T cells in the injected product was adjusted to 7×10 6 T cells by the addition of T cells transduced with EV.

[0301] Group 1 received T cells engineered with EV as a negative control, Group 2 received control CART cells without mbIL15 (CD19 - 063; AA sequence: SEQ ID NO: 48; NA sequence: SEQ ID NO: 49; vector sequence: SEQ ID NO: 50), and Group 3 received CART cells that constitutively express mbIL5 (CD19 - IL15 - 057). Groups 4 and 5 received CART cells that co - expressed control mbIL15 (CD19 - IL15 - 058). One group was administered ACZ at 200 mg / kg PO daily, while the other group was treated daily with vehicle until the end of the study (~50 days). To monitor T - cell proliferation, animals were added to each group (n = 4 for blood and n = 4 for bone marrow). Blood (50 μL) was taken from the submandibular vein on days 7, 14, and 21, and bone marrow (from the femur) was collected on day 14 after T - cell injection. Red blood cells were lysed and stained with fluorescent - dye - conjugated antibodies against human CD45, CD3, and mouse CD45, and the cells were analyzed by flow cytometry. Tumor growth was measured until 55 days after tumor implantation, and the evaluation items included not only the effects on animal health such as hind - limb paralysis and weight loss, but also 10 10 total flux units were included.

[0302] Tumor growth in individual mice of each group is shown in Fig. 7B and group averages are shown in Fig. 7C. Rapid tumor growth was observed in all animals of group 2 treated with T cells transduced with EV, and tumor growth was delayed until day 25 with treatment by control CART cells that did not achieve complete remission. Tumor growth rates in mice treated with CART cells expressing control mbIL15 and vehicle treatment were similar to the control CART group. In contrast, tumors regressed to background levels in 5 out of 8 and 6 out of 8 animals, respectively, in the groups treated with CART cells expressing constitutive mbIL15 and CART cells expressing control mbIL15 plus ACZ. The number of T cells decreased over time in the blood of mice treated with control T cells, control CART, and CART expressing control mbIL15 by vehicle treatment (<0.2% on day 21, Fig. 7D) and was low in the bone marrow (<1% on day 14, Fig. 7E). In contrast, in mice treated with CART expressing control mbIL15 by constitutive mbIL15 or ACZ, the number of T cells increased over time in the blood (>1% on day 21, Fig. 7D) and was high in the bone marrow (20% for constitutive, 10% for control plus ACZ, Fig. 7E). These results demonstrate that control mbIL15 conjugated with ACZ treatment enhanced the CART anti-tumor response and promoted the expansion of CAR-engineered T cells after tumor clearance compared to T cells expressing only the CAR after injection of a sub-optimal CART cell dose. Example 5: Isolation of TILs from patient tumor specimens

[0303] Tumor specimens of the head and neck were obtained from the Cooperative Human Tissue Network. The tumor specimens were cut into 1-3 mm fragments in Hank's balanced salt solution (HBSS) buffer, and the fragments were placed in 24-well plates at 1 fragment / well in 2 mL of medium (RPMI-1640 supplemented with 1X penicillin / streptomycin, 1 mM sodium pyruvate, 1X HEPES, 50 μM 2-mercaptoethanol (Invitrogen), and 10% heat-inactivated human AB serum (Valley Bio)) containing 6000 IU / mL of IL2. Half of the medium was replaced with fresh medium containing IL2 on day 5 from the start, and the cells were split into multiple wells to become confluent for 3 weeks. This culture process is called the pre-rapid expansion protocol (REP). TILs of other tumor types have been isolated using basically the same process.

[0304] To determine the change in the frequency of T cells before and after pre-REP culture, a part of the tumor fragment was digested with collagenase and deoxyribonuclease I prior to pre-REP culture to generate a single-cell suspension, which was compared with the cells obtained after pre-REP culture. The frequency of T cells was analyzed by flow cytometry using fluorescent dye-conjugated anti-CD45 and anti-CD3 antibodies. As shown in Figure 8A, almost half (44.29±21.67%) of the cells in the pre-culture tumor cell suspension were CD45+, and only ~39.85±23.69% of these were CD3+ T cells. Three weeks after culture (pre-REP) in the presence of IL2, more than half of the cells were CD45+ (90.35±7.28%), indicating an increase in hematopoietic cells, and CD3+ (80.64±15.19%), indicating an increase in T cells.

[0305] TILs of many other human tumor types, including melanoma and malignancies of the breast, lung, kidney, endometrium, liver, pancreas, and ovary, have been isolated in the same way. Example 6. In vitro analysis of controlled mbIL15 expression by ACZ in TIL BaEV pseudotyped lentivirus production

[0306] HEK293T cells were seeded on collagen-coated tissue culture plates until 70% confluent. Cells were transfected in Opti-MEM medium using Lipofectamine 3000 transfection reagent with a constitutive (IL15-292) or control (IL15-293) IL15 construct, and a pELNS transfer vector carrying packaging plasmids (pRSV.REV, pMDLg / pRRE, and OT-BaEVg-002 (SEQ ID NO: 51)). The medium was replaced with serum-free medium 6 - 8 hours after transfection. The supernatant containing the virus was collected 24 hours after transfection, fresh medium was added, and the supernatant was collected again 48 hours after transfection. The virus supernatant was filtered to remove debris and concentrated by low-speed ultracentrifugation. The virus was resuspended, aliquoted, and stored in a -80°C freezer. Transduction of TILs with lentivirus

[0307] A 96-well non-coated tissue culture plate was incubated with 35 μg / mL of RetroNectin (Takara Bio) in PBS at 37 °C for 2 hours or at 4 °C overnight. The RetroNectin was removed and the plate was washed with PBS. BaEV pseudotyped lentivirus and TIL cell medium prepared as described above were added to each well at a total volume of 50 μL / well, and the plate was centrifuged at low speed at 32 °C for 2 hours. TIL generated from head and neck tumor specimens prepared as described in Example 5 were manipulated in pre-REP culture 3 weeks later. TIL were activated with anti-CD3 / CD28 beads at a bead-to-T cell ratio of 3:1 in a 24-well plate for 24 hours. The activated TIL were placed on virus-coated plates, centrifuged at 800 g for 2 hours, and cultured in medium at 37 °C for 4 days. Cells from one well were treated similarly without adding virus and used as a negative control (“non-transduced”). Cells transduced with the control mbIL15 construct were treated with either 10 μM of ACZ or DMSO for 24 hours. Expression of control mbIL15 in TIL in response to ACZ

[0308] mbIL15 expression was determined by flow cytometry using two staining reagents: a fluorescent dye-conjugated anti-IL15 antibody and a recombinant protein containing the extracellular domain of IL15Ra fused to the human IgG1 Fc domain (IL15Ra-Fc). The frequency of mbIL15+ cells was determined based on co-staining with anti-IL15 and IL15Ra-Fc (identified as the IL15+IL15Ra-Fc+ double-positive population). As shown in Figure 8B, 45.5% of the TIL expressed constitutive mbIL15 (IL15-292). In the presence of DMSO, control mbIL15 (IL15-293) expression was 17.1% with a low MFI. In contrast, in the presence of ACZ, control mbIL15 expression increased to 42.5% with a high MFI. These data indicate that ACZ induces CA2 DRD-controlled mbIL15 in transduced TIL. Example 7: In vivo analysis of TILs expressing constitutive and controllable mbIL15

[0309] To evaluate the effect of controllable mbIL15 on the anti-tumor activity of tumor-infiltrating lymphocytes (TILs), a human patient-derived xenograft (hPDX) model is used. TILs are isolated from patient tumor specimens, such as head and neck tumor specimens as described in Example 6. TILs are transduced with either a BaEV pseudotyped lentiviral vector containing either the IL15-292 construct or the IL15-293 construct, or a BaEV pseudotyped lentiviral empty vector (EV) as described in Example 6, and subsequently optionally frozen. Patient-derived xenografts (hPDXs) engrafted with TILs from tumor specimens are made by subcutaneous implantation into the right flank of NSG mice. Tumor growth is measured once or twice a week using calipers. At the start of the study, tumors are measured and the mice are randomly withdrawn and placed in new cages in cohorts based on similar mean tumor volumes across all groups (N = 8 per group). Engineered and engrafted TILs are thawed if necessary, stimulated with PMA, and subsequently injected into mice bearing tumors. Each mouse receives an equal number of engineered TILs.

[0310] Group 1 receives non-transduced TIL supplemented with recombinant human IL2 (hIL2) as a benchmark control, and Group 2 receives TIL transduced with constitutive mbIL15 (IL15-292). Groups 3 and 4 receive TIL transduced with control mbIL15 (IL15-293). Group 3 is administered 200 mg / kg of ACZ PO daily, and Group 4 is treated daily with vehicle until the end of the study. To monitor TIL persistence, animals are added to each group (n = 4 for blood). Blood (50 μL) is taken from the submandibular vein on the pre-determined day. Red blood cells are lysed and stained with fluorescent dye-conjugated antibodies against human CD45, CD3, and mouse CD45, and the cells are analyzed by flow cytometry. Tumor growth is measured up to approximately 90 days, and the evaluation items include not only the effects on the health of the animals, such as tumor necrosis and weight loss, but also the maximum caliper measurement.

[0311] Tumor growth is continued in individual mice of each group, and group averages are collected. In the animals of Group 1 treated with hIL2 in addition to non-transduced TIL, a delay in tumor growth is expected. Since little mbIL15 is expressed by TIL, no tumor growth inhibition is observed in Group 4. In contrast, in Groups 2 and 3, both groups have TIL that express mbIL15, which enhances their persistent and correlated anti-tumor activity, and thus the tumors will regress substantially, in some cases to baseline. Example 8: Isolation of NK Cells from Umbilical Cord Blood

[0312] Cryopreserved mononuclear cell fraction cord blood units were obtained from BioBridge Global. Cord blood was diluted 1:1 with phosphate buffered saline (PBS) and centrifuged on a cushion of Ficoll-Paque+ (Sigma Cat.No.GE17-1440-02). The buffy coat containing mononuclear cells (MNC) was collected, and the MNC were washed and counted. NK cells were isolated from the MNC using the EasySep Human NK Cell Isolation Kit (Stemcell Technologies Cat.No.17955). NK cells were counted and their purity was checked by FACS using CD56, CD16, CD3, and viability staining. Example 9: In vitro analysis of ACZ-regulated mbIL15 expression in NK cells NK cell proliferation

[0313] One day prior to NK cell isolation as described in Example 8, feeder cells (K562 cells expressing 4-1BBL and mbIL-21) were thawed in complete NK cell medium (RPMI with Glutamax (ThermoFisher), 10% heat-inactivated fetal bovine serum (Gibco), 1X penicillin / streptomycin, 1 mM sodium pyruvate, 1X HEPES, 50 μM 2-mercaptoethanol). On the day of NK cell isolation, 10×10 6 of the feeder cells were treated with mitomycin C to inhibit their proliferation, washed to remove excess drug. NK cells were added to the feeder cells at an effector-to-target ratio of 1:2 in NK cell medium with 200 U / mL of recombinant human IL2 (rhIL2; PeproTech). The NK cell culture was increased by replenishing the cell culture with NK cell medium and rhIL2 every two days and analyzed by FACS for NK cell proliferation. Lentiviral transduction of NK cells

[0314] The 96-well non-coated tissue culture plates were cultured with 35 μg / mL of RetroNectin (Takara Bio) in PBS at 37 °C for 2 hours or at 4 °C overnight. RetroNectin was removed and the plates were washed with PBS. BaEV pseudotyped lentivirus prepared as described in Example 6, and NK cell medium were added to each well at a total volume of 50 μL / well, and the plates were centrifuged at low speed at 32 °C for 2 hours. 1 × 10 5 NK cells in 100 μL of NK cell transduction medium (NK cell medium with 1 mg / mL of Synperonic F 108 (Sigma-Aldrich) and 200 U / mL of rhIL2) were added to each well and the cells were grown in NK cell medium for 4 days. Control of mbIL15 construct by ACZ

[0315] NK cells from three different donors were isolated, grown, and transduced with BaEV pseudotyped lentivirus containing either the IL15-292 construct or the IL15-293 construct. The titer of the IL15-292 lentivirus was 2.38 × 10 8 TU / mL while the titer of the IL15-293 lentivirus was 6.51 × 10 8 TU / mL (as measured by Jurkat qPCR titer), and 4 μL of lentivirus and 46 μL of NK cell medium were added to each well. After the cells were grown for 4 days, 10 μM of ACZ or vehicle (DMSO) was added and the cells were cultured overnight. Expression of mbIL15 was analyzed the next day (5 days after transduction) using FACS.

[0316] mbIL15 expression was determined by flow cytometry using two staining reagents: IL15Ra-Fc and anti-CD56 antibody. The frequency of mbIL15+ cells was determined in relation to the number of NK cells as determined by anti-CD56 antibody. As shown in Figure 9, more than 40% of NK cells derived from umbilical cord blood of two out of three donors expressed mbIL15, and approximately 70% of NK cells derived from umbilical cord blood of one out of three donors expressed mbIL15. In the presence of DMSO, constitutive mbIL15 expression was 10% or less in NK cells regardless of the donor. In contrast, in the presence of ACZ, constitutive mbIL15 expression increased by more than 40% in NK cells regardless of the donor. These data indicate that ACZ induces mbIL15 controlled by CA2 DRD in transduced NKs. Example 10: In vivo analysis of NK cells expressing constitutive and regulated mbIL15

[0317] To evaluate the effect of regulated mbIL15 on the anti-tumor activity of NK cells, an HL-60 animal model of acute myeloid leukemia is used. Umbilical cord blood NK cells are transduced with a BaEV pseudotyped lentiviral vector containing either the IL15-292 construct or the IL15-293 construct as described in Example 9, or a BaEV pseudotyped lentiviral empty vector (EV), and optionally frozen after transduction. HL-60 cells expressing luciferase (HL-60-luc) are intravenously injected into NSG mice (1×10 6 / mouse), and tumor growth is measured once or twice a week by bioluminescence imaging measurement (total flux units of photons / second (p / s) after intraperitoneal injection of D-luciferin). On day 6, when the average tumor size reaches approximately 10 6 total p / s, animals are randomly withdrawn into new cages (N = 8 per group). Engineered NK cells are thawed if necessary and injected into mice bearing HL-60 tumors. Each mouse receives an equal number of mbIL15+ cells, and the total number of NK cells in the injected product is adjusted by the addition of EV-transduced NK cells.

[0318] Group 1 receives NK cells engineered with EV as a negative control, and Group 2 receives NK cells transduced with constitutive mbIL5 (construct IL15-292). Groups 3 and 4 receive NK cells transduced with control mbIL15 (construct IL15-293). Group 3 is administered 200 mg / kg of ACZ PO daily, and Group 4 is treated daily with vehicle until the end of the study. To monitor NK proliferation, animals are added to each group (n = 4 for blood analysis). Blood (50 μL) is taken from the submandibular vein on days 7, 14, and 21. Red blood cells are lysed and stained with fluorescent dye-conjugated antibodies against human CD45, CD3, and mouse CD45, and the cells are analyzed by flow cytometry. Tumor growth is measured up to approximately 30 days, and the evaluation items include not only the effects on the health of the animals, such as hind limb paralysis and weight loss, but also the maximum total flux units (10 10 ) are included. Tumor growth is measured in individual mice in each group, and group averages are collected. Rapid tumor growth is expected in all animals in Group 1 injected with NK cells engineered with EV. The tumor growth rate in the mice of Group 4 will be similar to the control EV group because little mbIL15 is expressed. In contrast, in Groups 2 and 3, the mice in both groups express mbIL15 in NK cells, and these cells show higher proliferation and persistence compared to the NK cells in Groups 1 and 4, so the tumors will substantially regress. This example will demonstrate that ACZ can induce in vivo expression of mbIL15 in transduced NK cells and result in a higher NK anti-tumor response compared to transduced and vehicle-treated NK cells that express little mbIL15.

[0319] In the above detailed description, the present invention has been described with reference to specific embodiments. However, it will be recognized that various modifications and changes can be made without departing from the scope of the present invention as described in the appended claims.

[0320] The following items are examples of various embodiments of the present disclosure.

[0321] Item 1. A nucleic acid molecule comprising a polynucleotide encoding a recombinant protein comprising a drug-responsive domain (DRD) operably linked to an IL15 payload, wherein the DRD is derived from human carbonic anhydrase II (CA2) and comprises one, two, three, four or more mutations relative to SEQ ID NO: 1 or SEQ ID NO: 2.

[0322] Item 2. The nucleic acid molecule of Item 1, wherein the DRD comprises one, two, three or four amino acid additions, substitutions, and / or deletions relative to SEQ ID NO: 1 or SEQ ID NO: 2.

[0323] Item 3. The nucleic acid molecule of Item 2, wherein the DRD comprises the amino acid sequence of SEQ ID NO: 4.

[0324] Item 4. The nucleic acid molecule of Item 3, wherein the DRD consists of the amino acid sequence of SEQ ID NO: 4.

[0325] Item 5. The nucleic acid molecule of any one of Items 1 to 4, wherein the IL15 payload comprises the amino acid sequence of SEQ ID NO: 8.

[0326] Item 6. The nucleic acid molecule of any one of Items 1 to 5, wherein the IL15 payload is at the N-terminus of the DRD.

[0327] Item 7. The nucleic acid molecule of Item 6, wherein the IL15 payload is a membrane-bound IL15 polypeptide.

[0328] Item 8. The nucleic acid molecule of Item 7, wherein the membrane-bound IL15 polypeptide comprises an IL15 polypeptide component comprising the amino acid sequence of SEQ ID NO: 8, a transmembrane domain, and an intracellular tail, the transmembrane domain is at the C-terminus of the IL15 polypeptide component, and the intracellular tail is at the C-terminus of the transmembrane domain.

[0329] Item 9. The nucleic acid molecule of item 8, wherein the membrane-bound IL15 polypeptide further comprises a linker between the IL15 polypeptide component and the transmembrane domain.

[0330] Item 10. The nucleic acid molecule of any one of items 1 to 7, wherein the IL15 payload further comprises one or more components selected from the group consisting of: (a) a leader sequence; (b) a GS linker; (c) a hinge domain; (d) a transmembrane domain; and (e) an intracellular tail.

[0331] Item 11. The nucleic acid molecule of any one of items 1 to 7, wherein the IL15 payload further comprises: (a) a leader sequence; (b) a GS linker; (c) a hinge domain; (d) a transmembrane domain; and (e) an intracellular tail.

[0332] Item 12. The nucleic acid molecule of item 11, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 24.

[0333] Item 13. The nucleic acid molecule of item 12, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 25.

[0334] Item 14. The nucleic acid molecule of item 11, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 28.

[0335] Item 15. The nucleic acid molecule of item 14, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 29.

[0336] Item 16. A nucleic acid molecule further comprising a second polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the CAR or TCR comprises an antigen-binding domain specific for an antigen of interest, and the nucleic acid molecule is any one of items 1 to 15.

[0337] Item 17. The nucleic acid molecule of item 16, wherein the second polynucleotide encodes a CAR comprising an antigen-binding domain specific for an antigen of interest.

[0338] Item 18. The CAR is the nucleic acid molecule of Item 17, comprising an antigen-binding domain specific for CD19.

[0339] Item 19. A vector comprising any one of the nucleic acid molecules of Items 1 to 18.

[0340] Item 20. The vector of Item 19, which is a plasmid or viral vector.

[0341] Item 21. The vector of Item 20, which is a viral vector derived from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, or picornavirus.

[0342] Item 22. The vector of Item 21, wherein the viral vector is selected from lentiviral vector, adenoviral vector, AAV vector, herpes simplex virus vector, retroviral vector, or oncolytic virus vector.

[0343] Item 23. The vector of Item 22, wherein the viral vector is selected from lentiviral vector or gammaretroviral vector.

[0344] Item 24. A recombinant protein encoded by any one of the nucleic acid molecules of Items 1 to 15.

[0345] Item 25. A cell comprising any one of the nucleic acid molecules of Items 1 to 18, any one of the vectors of Items 18 to 23, or the recombinant protein of Item 24.

[0346] Item 26. The cell of Item 25, which is a bacterial cell.

[0347] Item 27. The cell of Item 25, which is a mammalian cell.

[0348] Item 28. The cell of Item 27, wherein the mammalian cell is a human cell.

[0349] Item 29. The cell of Item 28, wherein the human cell is a T cell, a natural killer (NK) cell, or a tumor infiltrating lymphocyte (TIL).

[0350] Item 30. The cell of Item 29, which is a CD4+ or CD8+ T cell.

[0351] Item 31. The cell of Item 29, which is isolated.

[0352] Item 32. The cell of Item 29, wherein the human cell is a T cell or an NK cell, and the human T cell or human NK cell further comprises a second polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the CAR or TCR comprises an antigen-binding domain specific for an antigen of a subject.

[0353] Item 33. The cell of Item 32, wherein the second polynucleotide encodes a CAR comprising an antigen-binding domain specific for an antigen of a subject.

[0354] Item 34. The cell of Item 33, wherein the CAR comprises an antigen-binding domain specific for CD19.

[0355] Item 35. A pharmaceutical composition comprising the cell of any one of Items 25 to 34 and a pharmaceutically acceptable carrier.

[0356] Item 36. The pharmaceutical composition of Item 35, wherein the cell is a human T cell, a human NK cell, or a human TIL.

[0357] Item 37. A method for regulating the expression, function, and / or level of IL15 in the cell of any one of Items 25 to 34, the method comprising administering to the cell a stimulant to which DRD responds, wherein the stimulant is administered in an amount sufficient to regulate the expression, function, and / or level of IL15.

[0358] Item 38. The method of item 37, wherein the stimulant is selected from acetazolamide, celecoxib, baldecohib, rofecoxib, metazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide.

[0359] Item 39. The method of item 38, wherein the stimulant is acetazolamide.

[0360] Item 40. The method of item 37, wherein the cell is a human T cell or a human NK cell, and the human T cell or the human NK cell further comprises a second polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the CAR or TCR comprises an antigen-binding domain specific for an antigen of the subject.

[0361] Item 41. The method of item 40, wherein the second polynucleotide encodes a CAR comprising an antigen-binding domain specific for an antigen of the subject.

[0362] Item 42. The method of item 41, wherein the CAR comprises an antigen-binding domain specific for CD19.

[0363] Item 43. A method of treating a disease or disorder responsive to control IL15 in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules of items 1-18, any one of the vectors of items 19-23, the recombinant protein of item 24, any one of the cells of items 25-34, or any one of the pharmaceutical compositions of items 35-36; and (b) administering to the subject a therapeutically effective amount of a stimulant, wherein the DRD responds to the stimulant and the expression of the IL15 payload is regulated in response to the stimulant.

[0364] Item 44. The method of item 43, wherein the stimulant is selected from acetazolamide, celecoxib, baldecohib, rofecoxib, metazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide.

[0365] Item 45. The method of item 44, wherein the stimulant is acetazolamide.

[0366] Item 46. The method according to any one of items 43 - 45, wherein the disease or disorder is cancer.

[0367] Item 47. A method for treating a malignant tumor that expresses a tumor - associated antigen in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of any one of the human T cells or human NK cells of items 32 - 34, or a pharmaceutical composition thereof, wherein the CAR or TCR comprises an antigen - binding domain specific for a tumor - associated antigen; and (b) administering to the subject a therapeutically effective amount of a stimulant, wherein the DRD responds to the stimulant and the expression of the IL15 payload is regulated in response to the stimulant.

[0368] Item 48. The method of item 47, wherein the subject is administered a therapeutically effective amount of human T cells comprising a CAR, or a pharmaceutical composition thereof.

[0369] Item 49. The method according to any one of items 47 or 48, wherein the stimulant is selected from acetazolamide, celecoxib, baldecoib, rofecoxib, metazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide.

[0370] Item 50. The method of item 49, wherein the stimulant is acetazolamide.

[0371] Item 51. A method for producing genetically engineered T cells, natural killer (NK) cells, or tumor - infiltrating lymphocytes (TIL), comprising introducing into the T cells, NK cells, or TIL a polynucleotide encoding a protein comprising a drug - responsive domain (DRD) operably linked to an IL15 payload, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 24 or 28.

[0372] Item 52. The method of Item 51, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 25 or 29.

[0373] Item 53. The method of Item 52, wherein the polynucleotide is introduced into T cells, NK cells, or TILs by lentiviral transduction.

[0374] Item 54. The method of Item 52, wherein the polynucleotide is introduced into T cells, NK cells, or TILs by a non-viral vector delivery method.

[0375] Item 55. A modified cell comprising a recombinant protein, wherein the recombinant protein comprises: (i) an effector module comprising a stimulus-responsive element (SRE) comprising a drug-responsive domain (DRD), wherein the DRD is derived from a mutant protein having one or more amino acid mutations in the amino acid sequence of a parent protein or human carbonic anhydrase 2 (CA2) (SEQ ID NO: 1) and comprises the amino acid sequence of SEQ ID NO: 4; and (ii) recombinant IL15 bound to the SRE.

[0376] Item 56. The cell of Item 55, wherein the recombinant IL15 comprises the amino acid sequence of SEQ ID NO: 8.

[0377] Item 57. The cell of Item 55 or 56, wherein the recombinant IL15 can be expressed on the cell surface.

[0378] Item 58. The cell of Item 55 or 56, wherein the recombinant IL15 is membrane-bound IL15 (mbIL15).

[0379] Item 59. The cell of any one of Items 55 to 58, wherein the recombinant protein comprises all or part of SEQ ID NO: 16.

[0380] Item 60. The cell of any one of Items 55 to 59, wherein the recombinant protein comprises all or part of SEQ ID NO: 18.

[0381] Item 61. The cell of item 55, wherein the recombinant protein further comprises one or more components selected from the group consisting of (a) a leader sequence; (b) a GS linker; (c) a hinge domain; (d) a transmembrane domain; and (e) a cytoplasmic tail domain.

[0382] Item 62. The cell of item 55, wherein the recombinant protein comprises the amino acid sequence of SEQ ID NO: 24.

[0383] Item 63. The cell of item 55, wherein the recombinant protein comprises the amino acid sequence of SEQ ID NO: 28.

[0384] Item 64. The cell of any one of items 55 to 63, wherein the recombinant IL15 is further bound to at least one of (a) a leader sequence; (b) a signal peptide; (c) a linker; (d) a spacer; (e) a cleavage site; (f) a tag; (g) a co-stimulatory domain; (h) a fluorescent protein; and (i) a hinge.

[0385] Item 65. The cell of any one of items 55 to 64, wherein the SRE responds to or interacts with acetazolamide (ACZ).

[0386] Item 66. The cell of item 55, wherein the cell is a T cell, a natural killer cell (NK cell), or a tumor infiltrating lymphocyte (TIL).

[0387] Item 67. A polynucleotide, optionally a nucleic acid molecule comprising a first expression cassette, encoding a first recombinant protein comprising a stimulus-responsive element (SRE) that binds to an IL15 polypeptide, wherein the SRE comprises a DRD, and the DRD comprises the amino acid sequence of SEQ ID NO: 4.

[0388] Item 68. The nucleic acid molecule of item 67, wherein IL15 is under the control of the SRE.

[0389] Item 69. The polynucleotide is the nucleic acid molecule of Item 67, further encoding (a) a leader sequence; (b) a GS linker; (c) a hinge domain; (d) a transmembrane domain; and (e) a cytoplasmic tail domain.

[0390] Item 70. The polynucleotide is the nucleic acid molecule of Item 67, comprising the nucleic acid sequence of SEQ ID NO: 25.

[0391] Item 71. The polynucleotide is the nucleic acid molecule of Item 67, comprising the nucleic acid sequence of SEQ ID NO: 29.

[0392] Item 72. An isolated nucleic acid molecule of any one of Items 67 - 71.

[0393] Item 73. A recombinant protein encoded by the nucleic acid molecule of any one of Items 67 - 72.

[0394] Item 74. The recombinant protein of Item 73, comprising the amino acid sequence of SEQ ID NO: 24 or 28.

[0395] Item 75. A vector comprising the nucleic acid molecule of any one of Items 67 - 72.

[0396] Item 76. The vector of Item 75, which is a plasmid or a lentiviral vector.

[0397] Item 77. The vector of Item 76, lacking integrase.

[0398] Item 78. A T cell, NK cell, or TIL comprising the nucleic acid molecule of any one of Items 67 - 72, the recombinant protein of Items 73 - 74, or the vector of any one of Items 75 - 77.

[0399] Item 79. The T cell of Item 78, which is a CD4+ or CD8+ T cell.

[0400] Item 80. The T cell of Item 79 or Item 42, which is a human T cell.

[0401] Item 81. A T cell according to any one of Items 78 to 80, which is isolated.

[0402] Item 82. A pharmaceutical composition comprising any one cell of Items 55 to 66 or 78 to 81, a T cell, an NK cell, or a TIL, and a pharmaceutically acceptable carrier.

[0403] Item 83. A method for producing a genetically engineered T cell, NK cell, or TIL, the method comprising introducing into the T cell, NK cell, or TIL a first polynucleotide encoding a stimulatory response element comprising a CA2 DRD bound to an IL15 polypeptide payload, wherein the first polynucleotide has the nucleotide sequence of SEQ ID NO: 25 or 29.

[0404] Item 84. The method of Item 83, wherein the first polynucleotide is introduced into the T cell, NK cell, or TIL by lentiviral viral transfection of the T cell, NK cell, or TIL.

[0405] Although the present disclosure has been described in some detail and somewhat specifically with respect to several described embodiments, it is not intended that any such matter or embodiment or any specific embodiment be limiting, and in view of the prior art, the broadest interpretation of the scope of such claims can be provided, and accordingly, the appended claims should be construed with reference to the scope intended to be effectively encompassed by the present disclosure.

[0406] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the definition including this specification shall prevail. In addition, the section headings, materials, methods, and examples are for illustration only and are not intended to be limiting. The present invention provides, for example, the following items. (Item 1) A nucleic acid molecule comprising a polynucleotide encoding a recombinant protein comprising a drug-responsive domain (DRD) operably linked to an IL15 payload, wherein the DRD is derived from human carbonic anhydrase II (CA2) and comprises one, two, three, four or more mutations relative to SEQ ID NO: 1 or SEQ ID NO: 2. (Item 2) The nucleic acid molecule of Item 1, wherein the DRD comprises one, two, three or four amino acid additions, substitutions, and / or deletions relative to SEQ ID NO: 1 or SEQ ID NO: 2. (Item 3) The nucleic acid molecule of Item 2, wherein the DRD comprises the amino acid sequence of SEQ ID NO: 4. (Item 4) The nucleic acid molecule of Item 3, wherein the DRD consists of the amino acid sequence of SEQ ID NO: 4. (Item 5) The nucleic acid molecule of any one of Items 1 to 4, wherein the IL15 payload comprises the amino acid sequence of SEQ ID NO: 8. (Item 6) The nucleic acid molecule of any one of Items 1 to 5, wherein the IL15 payload is the N-terminus of the DRD. (Item 7) The nucleic acid molecule of Item 6, wherein the IL15 payload is a membrane-bound IL15 polypeptide. (Item 8) The membrane-bound IL15 polypeptide comprises an IL15 polypeptide component comprising the amino acid sequence of SEQ ID NO: 8, a transmembrane domain, and an intracellular tail, wherein the transmembrane domain is the C-terminus of the IL15 polypeptide component and the intracellular tail is the C-terminus of the transmembrane domain. (Item 9) The nucleic acid molecule of Item 8, wherein the membrane-bound IL15 polypeptide further comprises a linker between the IL15 polypeptide component and the transmembrane domain. (Item 10) The nucleic acid molecule of any one of Items 1 to 7, wherein the IL15 payload further comprises one or more components selected from the group consisting of: (a) a leader sequence; (b) a GS linker; (c) a hinge domain; (d) a transmembrane domain; and (e) an intracellular tail. (Item 11) The nucleic acid molecule of any one of Items 1 to 7, wherein the IL15 payload further comprises: (a) a leader sequence; (b) a GS linker; (c) a hinge domain; (d) a transmembrane domain; and (e) an intracellular tail. (Item 12) The polynucleotide is the nucleic acid molecule of item 11 that encodes the amino acid sequence of SEQ ID NO: 24. (Item 13) The polynucleotide is the nucleic acid molecule of item 12 that contains the nucleic acid sequence of SEQ ID NO: 25. (Item 14) The polynucleotide is the nucleic acid molecule of item 11 that encodes the amino acid sequence of SEQ ID NO: 28. (Item 15) The polynucleotide is the nucleic acid molecule of item 14 that contains the nucleic acid sequence of SEQ ID NO: 29. (Item 16) A nucleic acid molecule further comprising a second polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the CAR or TCR contains an antigen-binding domain specific for an antigen of interest, the nucleic acid molecule of any one of items 1 to 15. (Item 17) The second polynucleotide is the nucleic acid molecule of item 16 that encodes a CAR containing an antigen-binding domain specific for an antigen of interest. (Item 18) The CAR is the nucleic acid molecule of item 17 that contains an antigen-binding domain specific for CD19. (Item 19) A vector containing the nucleic acid molecule of any one of items 1 to 18. (Item 20) The vector of item 19, which is a plasmid or a viral vector. (Item 21) The viral vector of item 20, which is a viral vector derived from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, or picornavirus. (Item 22) The viral vector of item 21, which is selected from a lentiviral vector, an adenoviral vector, an AAV vector, a herpes simplex virus vector, a retroviral vector, or an oncolytic virus vector. (Item 23) The viral vector of item 22, which is selected from a lentiviral vector or a gammaretroviral vector. (Item 24) A recombinant protein encoded by the nucleic acid molecule of any one of items 1 to 15. (Item 25) A cell containing the nucleic acid molecule of any one of items 1 to 18, the vector of any one of items 18 to 23, or the recombinant protein of item 24. (Item 26) The cell of item 25, which is a bacterial cell. (Item 27) The cell of item 25, which is a mammalian cell. (Item 28) The mammalian cell of item 27, which is a human cell. (Item 29) The human cell of item 28 is a T cell, a natural killer (NK) cell, or a tumor infiltrating lymphocyte (TIL). (Item 30) The cell of item 29 is a CD4+ or CD8+ T cell. (Item 31) The cell of item 29 is isolated. (Item 32) The human cell is a T cell or an NK cell, and the human T cell or the human NK cell further comprises a second polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the CAR or TCR comprises an antigen binding domain specific for an antigen of interest. The cell of item 29. (Item 33) The second polynucleotide of item 32 encodes a CAR comprising an antigen binding domain specific for an antigen of interest. (Item 34) The CAR of item 33 comprises an antigen binding domain specific for CD19. (Item 35) A pharmaceutical composition comprising the cell of any one of items 25 to 34 and a pharmaceutically acceptable carrier. (Item 36) The cell of item 35 is a human T cell, a human NK cell, or a human TIL. (Item 37) A method for regulating the expression, function, and / or level of IL15 in the cell of any one of items 25 to 34, which comprises administering to the cell a stimulant to which the DRD responds, and the stimulant is administered in an amount sufficient to regulate the expression, function, and / or level of IL15. A method comprising the step. (Item 38) The stimulant of item 37 is selected from acetazolamide, celecoxib, baldecozib, rofecoxib, metazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. (Item 39) The stimulant of item 38 is acetazolamide. (Item 40) The cell is a human T cell or a human NK cell, and the human T cell or the human NK cell further comprises a second polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the CAR or TCR comprises an antigen binding domain specific for an antigen of interest. The method of item 37. (Item 41) The second polynucleotide of item 40 encodes a CAR comprising an antigen binding domain specific for an antigen of interest. (Item 42) The CAR of item 41 comprises an antigen binding domain specific for CD19. (Item 43) A method for treating a disease or disorder responsive to control IL15 in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of a nucleic acid molecule of any one of items 1 to 18, a vector of any one of items 19 to 23, a recombinant protein of item 24, a cell of any one of items 25 to 34, or a pharmaceutical composition of any one of items 35 to 36; (b) administering to the subject a therapeutically effective amount of a stimulant, wherein the DRD responds to the stimulant and the expression of the IL15 payload is regulated in response to the stimulant. A method comprising the steps of. (Item 44) The method of item 43, wherein the stimulant is selected from acetazolamide, celecoxib, baldecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. (Item 45) The method of item 44, wherein the stimulant is acetazolamide. (Item 46) The method of any one of items 43 to 45, wherein the disease or disorder is cancer. (Item 47) A method for treating a malignant tumor that expresses a tumor-associated antigen in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of a human T cell or human NK cell of any one of items 32 to 34, or a pharmaceutical composition thereof, wherein the CAR or TCR comprises an antigen-binding domain specific for the tumor-associated antigen; (b) administering to the subject a therapeutically effective amount of a stimulant, wherein the DRD responds to the stimulant and the expression of the IL15 payload is regulated in response to the stimulant. A method comprising the steps of. (Item 48) The method of item 47, wherein the subject is administered a therapeutically effective amount of the human T cell comprising a CAR, or a pharmaceutical composition thereof. composition thereof. (Item 49) The method of any one of items 47 or 48, wherein the stimulant is selected from acetazolamide, celecoxib, baldecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. (Item 50) The method of item 49, wherein the stimulant is acetazolamide. (Item 51) A method for producing genetically engineered T cells, natural killer (NK) cells, or tumor-infiltrating lymphocytes (TILs), the method comprising introducing into the T cells, NK cells, or TILs a polynucleotide encoding a protein comprising a drug-responsive domain (DRD) operably linked to an IL15 payload, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 24 or 28. (Item 52) The method of Item 51, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 25 or 29. (Item 53) The method of Item 52, wherein the polynucleotide is introduced into the T cells, NK cells, or TILs by lentiviral transduction. (Item 54) The method of Item 52, wherein the polynucleotide is introduced into the T cells, NK cells, or TILs by a non-viral vector delivery method.

Claims

1. A nucleic acid molecule comprising a polynucleotide encoding a recombinant protein comprising a drug-responsive domain (DRD) operably linked to an IL15 payload, wherein said DRD is derived from human carbonic anhydrase II (CA2) and comprises the amino acid sequence of SEQ ID NO: 4, and wherein said IL15 payload further comprises an intracellular tail derived from B7-1 comprising the amino acid sequence of SEQ ID NO:

18.

2. The nucleic acid molecule of claim 1, wherein said DRD consists of the amino acid sequence of SEQ ID NO:

4.

3. The nucleic acid molecule of claim 1 or 2, wherein said IL15 payload comprises the amino acid sequence of SEQ ID NO:

8.

4. The nucleic acid molecule of any one of claims 1 to 3, wherein said IL15 payload is at the N-terminus of said DRD.

5. The nucleic acid molecule of claim 4, wherein said IL15 payload is a membrane-bound IL15 polypeptide.

6. The nucleic acid molecule of claim 5, wherein said membrane-bound IL15 polypeptide comprises an IL15 polypeptide component comprising the amino acid sequence of SEQ ID NO: 8, a transmembrane domain, and a B7-1 intracellular tail comprising the amino acid sequence of SEQ ID NO: 18, wherein said transmembrane domain is at the C-terminus of said IL15 polypeptide component, and wherein said intracellular tail is at the C-terminus of said transmembrane domain.

7. The nucleic acid molecule of claim 6, wherein said membrane-bound IL15 polypeptide further comprises a linker between said IL15 polypeptide component and said transmembrane domain.

8. The nucleic acid molecule of any one of claims 1 to 5, wherein said IL15 payload further comprises one or more components selected from the group consisting of: (a) a leader sequence; (b) a GS linker; (c) a hinge domain; (d) a transmembrane domain; and (e) Intracellular tail. **Claim 9** The nucleic acid molecule according to any one of claims 1 to 5, wherein the IL15 payload further comprises: (a) A leader sequence; (b) A GS linker; (c) A hinge domain; (d) A transmembrane domain; and (e) An intracellular tail. **Claim 10** The nucleic acid molecule according to claim 9, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO:

28. **Claim 11** The nucleic acid molecule according to claim 10, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:

29. **Claim 12** A nucleic acid molecule further comprising a second polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the CAR or TCR comprises an antigen-binding domain specific for an antigen of interest, according to any one of claims 1 to 11. **Claim 13** The nucleic acid molecule according to claim 12, wherein the second polynucleotide encodes a CAR comprising an antigen-binding domain specific for an antigen of interest. **Claim 14** The nucleic acid molecule according to claim 13, wherein the CAR comprises an antigen-binding domain specific for CD19. **Claim 15** A vector comprising the nucleic acid molecule according to any one of claims 1 to 14. **Claim 16** The vector according to claim 15, which is a plasmid or a viral vector. **Claim 17** The vector according to claim 16, which is a viral vector derived from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, or picornavirus. **Claim 18** The virus vector is the vector according to claim 17, which is selected from a lentivirus vector, an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, or an oncolytic virus vector.

19. The virus vector is the vector according to claim 18, which is selected from a lentivirus vector or a gammaretrovirus vector.

20. A recombinant protein encoded by the nucleic acid molecule according to any one of claims 1 to 11.

21. A cell comprising the nucleic acid molecule according to any one of claims 1 to 14, the vector according to any one of claims 15 to 19, or the recombinant protein according to claim 20.

22. The cell according to claim 21, which is a bacterial cell.

23. The cell according to claim 21, which is a mammalian cell.

24. The cell according to claim 23, wherein the mammalian cell is a human cell.

25. The cell according to claim 24, wherein the human cell is a T cell, a natural killer (NK) cell, or a tumor-infiltrating lymphocyte (TIL).

26. The cell according to claim 25, which is a CD4+ or CD8+ T cell.

27. The cell according to claim 25, which is isolated.

28. The cell according to claim 25, wherein the human cell is a T cell or an NK cell, and the human T cell or the human NK cell further comprises a second polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the CAR or TCR comprises an antigen-binding domain specific for an antigen of interest.

29. The cell according to claim 28, wherein the second polynucleotide encodes a chimeric antigen receptor (CAR) comprising an antigen-binding domain specific for a target antigen.

30. The cell according to claim 29, wherein the CAR comprises an antigen-binding domain specific for CD19.

31. A pharmaceutical composition comprising the cell according to any one of claims 21 to 30 and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition according to claim 31, wherein the cell is a human T cell, a human NK cell, or a human TIL.

33. An in vitro method for regulating the expression, function, and / or level of IL15 in the cell according to any one of claims 21 to 30, the method comprising administering to the cell a stimulant to which the DRD responds, wherein the stimulant is administered in an amount sufficient to regulate the expression, function, and / or level of IL15.

34. The method according to claim 33, wherein the stimulant is selected from acetazolamide, celecoxib, baldecoxib, rofecoxib, metazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide.

35. The method according to claim 34, wherein the stimulant is acetazolamide.

36. The method according to claim 33, wherein the cell is a human T cell or a human NK cell, and the human T cell or the human NK cell further comprises a second polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the CAR or TCR comprises an antigen-binding domain specific for a target antigen.

37. The method according to claim 36, wherein the second polynucleotide encodes a CAR comprising an antigen-binding domain specific for a target antigen.

38. The CAR is the method of claim 37, comprising an antigen-binding domain specific for CD19. **Claim 39** A composition for use in the treatment of a disease or disorder responsive to control of IL15, comprising: (a) a therapeutically effective amount of a nucleic acid molecule of any one of claims 1 to 14, a vector of any one of claims 15 to 19, a recombinant protein of claim 20, a cell of any one of claims 21 to 30, or a pharmaceutical composition of any one of claims 31 to 32; and (b) a therapeutically effective amount of a stimulant, wherein the DRD responds to the stimulant and the expression of the IL15 payload is regulated in response to the stimulant. A composition comprising the above. **Claim 40** The composition of claim 39, wherein the stimulant is selected from acetazolamide, celecoxib, baldecozib, rofecoxib, metazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide. **Claim 41** The composition of claim 40, wherein the stimulant is acetazolamide. **Claim 42** The composition of any one of claims 39 to 41, wherein the disease or disorder is cancer. **Claim 43** A composition for use in the treatment of a malignant tumor that expresses a tumor-associated antigen, comprising: (a) a therapeutically effective amount of a human T cell or human NK cell of any one of claims 28 to 30, wherein the CAR or TCR comprises an antigen-binding domain specific for the tumor-associated antigen; and (b) a therapeutically effective amount of a stimulant, wherein the DRD responds to the stimulant and the expression of the IL15 payload is regulated in response to the stimulant. A composition comprising the above. **Claim 44** The composition according to claim 43, wherein the composition comprises a therapeutically effective amount of the human T cells comprising the CAR.

45. The composition according to any one of claims 43 or 44, wherein the stimulant is selected from acetazolamide, celecoxib, baldecozib, rofecoxib, metazolamide, dorzolamide, brinzolamide, dichlorphenamide, ethoxzolamide, zonisamide, dansylamide, or dichlorphenamide.

46. The composition according to claim 45, wherein the stimulant is acetazolamide.

47. An in vitro method for producing genetically engineered T cells, natural killer (NK) cells, or tumor infiltrating lymphocytes (TIL), the method comprising introducing into the T cells, NK cells, or TIL a polynucleotide encoding a protein comprising a drug-responsive domain (DRD) operably linked to an IL15 payload, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 24 or 28.

48. The method according to claim 47, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 25 or 29.

49. The method according to claim 48, wherein the polynucleotide is introduced into the T cells, NK cells, or TIL by lentiviral transduction.

50. The method according to claim 48, wherein the polynucleotide is introduced into the T cells, NK cells, or TIL by a non-viral vector delivery method.

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