Compositions and methods for tunable regulation of il15 and light

Abstract

Provided herein are nucleic acid molecules comprising a first nucleic acid encoding a polypeptide payload operably linked to a first drug responsive domain (DRD) and a second nucleic acid encoding a LIGHT polypeptide operably linked to a second DRD. The abundance, availability, and / or biological activity of the polypeptide payload and the LIGHT polypeptide is regulated by interaction of an effective amount of the first and second nucleic acid with an effective amount of at least one ligand. Vectors and cells comprising the nucleic molecules provided herein are provided, as are methods of making and using the nucleic molecules.

Description

[0001] COMPOSITIONS AND METHODS FOR TUNABLE REGULATION OF IL 15 AND LIGHT

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of priority to U.S. Provisional Patent Application No. 63 / 615,149, filed December 27, 2023, which is incorporated by reference herein in its entirety and for all purposes.

[0004] REFERENCE TO A “SEQUENCE LISTING”, A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK

[0005] This application contains a Sequence Listing submitted electronically and is hereby incorporated by reference in its entirety. The Sequence listing .xml file is entitled “108407- 1468685-055WO1”, is 127,000 bytes in size, and was created on December 23, 2024.

[0006] BACKGROUND

[0007] Biological products (biologies) as defined by the U.S. Food and Drug Administration include vaccines, blood and blood components, allergens, somatic cells, gene therapy, tissues, and recombinant therapeutic proteins. Cells from the same subject (autologous), from a subject of the same species (homologous or allogeneic), or even from a different species (heterologous) can be administered to a subject as a biologic. In adoptive cell therapy (ACT), for example, T cells originating from a subject (or sometimes from a different source) are removed, genetically engineered to address the specific needs of the same or different subject, and then transferred back into the same or different subject. There are currently obstacles to widespread adoption and success of biologies such as ACT. Maintaining therapeutically effective levels of a biologic administered to a patient can be difficult, as the biologic may be expressed by an engineered cell at toxic levels. For example, an engineered cell may express biologies for an unacceptably sustained duration. A need exists for regulation of the biologies for optimization of therapeutic benefits and to facilitate widespread adoption of biologic therapies.

[0008] BRIEF SUMMARY

[0009] Provided herein are nucleic acid molecules comprising a first nucleic acid encoding a polypeptide payload operably linked to a first drug responsive domain (DRD) and a second nucleic acid encoding a LIGHT polypeptide operably linked to a second DRD. The first and second nucleic acids can be under the control of the same or different promotors and, optionally, the nucleic acid molecule includes one or more cleavage sites (e.g., PA2) between the first and second nucleic acid. Each DRD within the nucleic acid molecule is responsive to a ligand. The biological activities of the polypeptide payload and the LIGHT polypeptide are regulatable by the interaction of the DRDs to an effective amount of a ligand. The first and second DRDs can be the same (i.e., responsive to the same ligand) or different (i.e., responsive to different ligands) and can be CA2, ecDHFR, hDHFR, FKBP, PDE5 ligand binding domain, PDE5, ER, or variants thereof. The ligands can be acetazolamide, methotrexate, trimethoprim, tacrolimus, celecoxib, topiramate, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide, and dichlorphenamide. The polypeptide payload can be a cytokine (e.g., a membrane bound IL15), a cytokine receptor, a TCR, a CAR, an immunomodulatory protein, or any combinations thereof.

[0010] Also provided herein are vectors and cells comprising one or more of the nucleic acid molecules. The cells are optionally human cells, including tumor infiltrating lymphocytes (TIL), T cells (e.g., CD4+ or CD8+ T cells)), or natural killer (NK) cells. Also provided are pharmaceutical compositions comprising the cells.

[0011] Also provided are methods of using the nucleic molecules to regulate control of payload and LIGHT activity in a cell in vivo or in vitro. The method includes administering to the cell at least one ligand to which the first DRD, the second DRD, or both are responsive. The amount of the at least one ligand is an amount sufficient to modulate the expression, function, and / or level of the polypeptide payload and LIGHT. Also provided are methods of producing a genetically engineered cell by introducing into the cell the nucleic acid molecules described herein.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows the frequency of LIGHT+ tumor infiltrating lymphocytes in tumor infiltrating lymphocytes from donors with either head and neck or lung cancer transduced with either IL15-293 (SEQ ID NO: 26) or LIGHT-005 (SEQ ID NO: 28).

[0014] FIG. 2 shows LIGHT expression on tumor infiltrating lymphocytes from donors with either head and neck or lung cancer transduced with either IL15-293 (SEQ ID NO: 26) or LIGHT-005 (SEQ ID NO: 28). FIG. 3 shows LIGHT activity on the herpes virus entry mediator (HVEM) receptor in tumor infiltrating lymphocytes from donors with head and neck cancer, lung cancer, sarcoma, or colorectal cancer transduced with either IL15-293 (SEQ ID NO: 26) or LIGHT-005 (SEQ ID NO: 28).

[0015] FIG. 4A shows LIGHT activity on the lymphotoxin receptor beta (LTpR) receptor in tumor infiltrating lymphocytes from donors with head and neck cancer or lung cancer transduced with either IL15-293 (SEQ ID NO: 26) or LIGHT-005 (SEQ ID NO: 28).

[0016] FIG. 4B shows a recombinant LTpR can block LIGHT activity in tumor infiltrating lymphocytes from donors with sarcoma transduced with either IL15-293 (SEQ ID NO: 26) or LIGHT-005 (SEQ ID NO: 28).

[0017] FIG. 5 shows expansion of tumor infiltrating lymphocytes from donors with head and neck or lung cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs in the presence of acetazolamide (ACZ).

[0018] FIG. 6 shows mbIL15 expression in tumor infiltrating lymphocytes from donors with head and neck and lung cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs in the presence or absence of ACZ in CD3 / CD28 activated tumor infiltrating lymphocytes.

[0019] FIG. 7 shows LIGHT expression in in tumor infiltrating lymphocytes from donors with head and neck and lung cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs in the presence or absence of ACZ in CD3 / CD28 activated tumor infiltrating lymphocytes.

[0020] FIG. 8 shows HVEM and LTBR receptor activation for tumor infiltrating lymphocytes from donors with head and neck cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs.

[0021] FIG. 9 shows HVEM receptor activation for tumor infiltrating lymphocytes from donors with head and neck cancer, lung cancer, and colorectal cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs.

[0022] FIG. 10A shows expansion of tumor infiltrating lymphocytes from donors with head and neck cancer (H&N) transduced with lentiviral co-regulated mbIL15 / LIGHT constructs in the presence of ACZ.

[0023] FIG. 10B shows expansion of tumor infiltrating lymphocytes from donors with colorectal cancer (CRC) transduced with lentiviral co-regulated mbIL15 / LIGHT constructs in the presence of ACZ. FIG. 11 shows mbIL15 and LIGHT expression in the presence or absence of ACZ in CD3 / CD28 activated tumor infiltrating lymphocytes from donors with head and neck cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs.

[0024] FIG. 12 shows HVEM and LTBR receptor activation for tumor infiltrating lymphocytes from donors with head and neck cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs.

[0025] FIG. 13 shows mbIL15 and LIGHT expression in the presence or absence of ACZ in CD3 / CD28 activated tumor infiltrating lymphocytes from donors with colorectal cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs.

[0026] FIG. 14 shows HVEM and LTBR receptor activation for tumor infiltrating lymphocytes from donors with colorectal cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs.

[0027] FIG. 15 shows mbIL15 and LIGHT expression in the presence or absence of ACZ and trimethoprim (TMP) in CD3 / CD28 activated T cells from donors with healthy donors transduced with lentiviral co-regulated mbIL15 / LIGHT constructs.

[0028] FIG. 16 shows cytotoxicity against patient derived tumor cells (PDc) in tumor infiltrating lymphocytes from donors with colorectal cancer or lung cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs. The right panel (dashed triangle) also shows the effect of a recombinant LTBR receptor.

[0029] FIG. 17A-B show cytotoxicity against patient derived tumor cells (PDc) in tumor infiltrating lymphocytes from a donor with sarcoma transduced with lentiviral co-regulated mbIL15 / LIGHT constructs. FIG. 17A shows co-localization of caspase (green) with PDc (red).

[0030] FIG. 18A-B show cytotoxicity against patient derived tumor cells (PDc) in tumor infiltrating lymphocytes from a donor with lung cancer transduced with lentiviral coregulated mbIL15 / LIGHT constructs. FIG. 18A shows co-localization of caspase (green) with PDc (red).

[0031] FIG. 19 shows cytotoxicity against patient derived tumor cells (PDc) in tumor infiltrating lymphocytes from a donor with head and neck cancer transduced with lentiviral co-regulated mbIL15 / LIGHT constructs is dependent on ACZ and could be blocked by recombinant LTBR.

[0032] FIG. 20A-B show cytotoxicity against patient derived tumor cells (PDc) in tumor infiltrating lymphocytes from a donor with head and neck cancer, in the presence and absence of ACZ, transduced with lentiviral co-regulated mbIL15 / LIGHT constructs. FIG. 20A shows co-localization of caspase (green) with PDc (red).

[0033] FIG. 21 shows mbIL15 expression in tumor infiltrating lymphocytes from donors with head and neck cancer transduced with retroviral co-regulated mbIL15 / LIGHT constructs in the presence of ACZ in both unactivated and CD3 / CD28 activated tumor infiltrating lymphocytes.

[0034] FIG. 22 shows mbIL15 expression in tumor infiltrating lymphocytes from donors with colorectal cancer with lung metastases transduced with retroviral co-regulated mbIL15 / LIGHT constructs in the presence of ACZ in both unactivated and CD3 / CD28 activated tumor infiltrating lymphocytes.

[0035] FIG. 23 shows LIGHT expression in tumor infiltrating lymphocytes from donors with head and neck cancer transduced with retroviral co-regulated mbIL15 / LIGHT constructs in the presence of ACZ in both unactivated and CD3 / CD28 activated tumor infiltrating lymphocytes.

[0036] FIG. 24 shows LIGHT expression in tumor infiltrating lymphocytes from donors with colorectal cancer with lung metastases transduced with retroviral co-regulated mbIL15 / LIGHT constructs in the presence of ACZ in both unactivated and CD3 / CD28 activated tumor infiltrating lymphocytes.

[0037] FIG. 25 shows the expansion of tumor infiltrating lymphocytes from donors with head and neck or colorectal cancer with lung metastases transduced with retroviral co-regulated mbIL15 / LIGHT constructs in the presence of ACZ.

[0038] FIG. 26A shows HVEM receptor activation for each of the retroviral co-regulated mbIL15 / LIGHT constructs in tumor infiltrating lymphocytes from donors with head and neck cancer.

[0039] FIG. 26B shows LTBR receptor activation for each of the retroviral co-regulated mbIL15 / LIGHT constructs in tumor infiltrating lymphocytes from donors with head and neck cancer.

[0040] FIG. 27A shows HVEM receptor activation for each of the retroviral co-regulated mbIL15 / LIGHT constructs in tumor infiltrating lymphocytes from donors with colorectal cancer with lung metastases.

[0041] FIG. 27B shows LTBR receptor activation for each of the retroviral co-regulated mbIL15 / LIGHT constructs in tumor infiltrating lymphocytes from donors with colorectal cancer with lung metastases. FIG. 28 shows cytotoxicity against a co-culture of patient derived cancer associated fibroblast (CAF) and patient derived tumor cells (PDc) in tumor infiltrating lymphocytes transduced with retroviral co-regulated mbIL15 / LIGHT constructs from donors with colorectal cancer with lung metastases or head and neck cancer.

[0042] FIG. 29 shows cytotoxicity against a co-culture of patient derived cancer associated fibroblasts (CAF) and patient derived tumor cells (PDc) in tumor infiltrating lymphocytes transduced with retroviral co-regulated mbIL15 / LIGHT constructs from donors with head and neck cancer is regulated by ACZ.

[0043] FIG. 30 shows selected construct components and sequences.

[0044] FIG. 31A are images showing staining of DAPI, CD3 and Granzyme B in a patient derived tumor cell (PDc) cancer associated fibroblast (CAF) spheroid model where C tumor infiltrating lymphocytes were transduced with a co-regulated LIGHT-IL15 construct or a regulated IL15 construct alone.

[0045] FIG. 3 IB are images showing granzyme B+ and granzyme B- tumor infiltrating lymphocytes in a patient derived tumor cell (PDc) cancer associated fibroblast (CAF) spheroid model. The tumor infiltrating lymphocytes were transduced with a co-regulated LIGHT-IL15 construct or a regulated IL 15 construct alone before seeding with the spheroids. FIG. 3 IB shows more Granzyme B+ tumor infiltrating lymphocytes infiltrated spheroids when transduced with a co-regulated LIGHT-IL15 construct.

[0046] FIG. 32A-B show tumor infiltrating lymphocyte infiltration depth in a patient derived tumor cell (PDc) spheroid model (tumor spheroid) (FIG. 32A) and in a PDc cancer associated fibroblast (CAF) spheroid model (tumor / CAF spheroid) (FIG. 32B) in granzyme B+ tumor infiltrating lymphocytes and granzyme B- tumor infiltrating lymphocytes.

[0047] FIG. 33 depicts an experimental overview for assessing the effects of treating mice with an implanted colorectal cancer (CRC) patient-derived xenograft (PDx) with tumor infiltrating lymphocytes transduced with either regulated IL 15 or co-regulated IL 15 and LIGHT .

[0048] FIG. 34 shows the frequency of CD45-aSMA+ cells in a colorectal cancer (CRC) patient-derived xenograft (PDx) implanted into a murine model.

[0049] FIG. 35 shows tumor volume over time in mice administered tumor infiltrating lymphocytes transduced with a co-regulated LIGHT-IL15 construct compared to mice administered tumor infiltrating lymphocytes transduced a regulated IL15 construct alone. ACT is adoptive cell therapy. FIG. 36A shows tumor volume over time in mice administered tumor infiltrating lymphocytes transduced with a co-regulated LIGHT-IL15 construct compared to mice administered tumor infiltrating lymphocytes transduced a regulated IL15 construct alone. ACT is adoptive cell therapy.

[0050] FIG. 36B shows the frequency of CD3+ cells over time in mice administered tumor infiltrating lymphocytes transduced with a co-regulated LIGHT-IL15 construct compared to mice administered tumor infiltrating lymphocytes transduced a regulated IL15 construct alone. ACT is adoptive cell therapy.

[0051] FIG. 37 shows soluble (shed) LIGHT in plasma (left) and in tumor (right) in mice treated with tumor infiltrating lymphocytes from donors with colorectal cancer transduced with co-regulated mbIL15 / LIGHT constructs in the presence of ACZ (for the duration of the experiment or 14 days on and 3 days off) or vehicle.

[0052] FIG. 38 shows LIGHT expression in tumor infiltrating lymphocytes from donors with head and neck cancer or lung cancer untransduced or transduced with regulated mbIL15 or co-regulated mbIL15 / LIGHT constructs after ACZ withdrawal (0, 2, 4, 5, 8, and 24 hours after withdrawal).

[0053] FIG. 39 shows the fold increase over Jurkat-HVEM cells of LIGHT in tumor infiltrating lymphocytes transduced with regulated mbIL15 or co-regulated mbIL15 / LIGHT constructs in the presence of ACZ and / or varying concentrations of decoy receptor 3 (DcR3).

[0054] DETAILED DESCRIPTION

[0055] The present disclosure provides compositions and systems for regulatory control of both a LIGHT polypeptide and a polypeptide payload. Regulation compositions according to this disclosure include nucleic acid molecules, wherein a nucleic acid molecule comprises a first nucleic acid encoding a polypeptide payload operably linked to a first drug responsive domain (DRD) and a second nucleic acid encoding a LIGHT polypeptide operably linked to a second DRD. Optionally, the first DRD provides translational control of payload activity that is temporally linked to ligand administration. Optionally, the second DRD provides translational control of LIGHT activity that is temporally linked to ligand administration. Regulation systems according to this disclosure comprise regulation compositions with at least one stabilizing ligand to which the first or second DRD or both the first and second DRDs are responsive.

[0056] Adoptive cellular therapies (ACT) have encountered challenges in the treatment of solid tumors due in part to the immunosuppressive tumor microenvironment (TME). Tumor infiltrating lymphocytes engineered to express mbIL15 regulatable using the cytoDRiVE® platform has been developed, allowing for tumor infiltrating lymphocyte expansion, persistence, and anti-tumor efficacy under control of the FDA-approved small molecule ligand, acetazolamide (ACZ), eliminating the need for co-administration of IL2. LIGHT, a tumor necrosis factor family member, interacts with lymphotoxin beta receptor (LTpR) and herpes virus entry mediator (HVEM) found on various TME cell types, including stromal cells such as cancer associated fibroblasts (CAF). In preclinical studies, LIGHT expression within a tumor has been linked to the formation of tertiary lymphoid structures and vascular normalization (Ramachandran Cancer Cell 2023), both associated with better clinical outcomes (Sautes-Fridman Nat Rev Cancer 2019). As shown herein, engineering tumor infiltrating lymphocytes with both regulatable mbIL15 and LIGHT expression enhance their efficacy by modifying the TME. Tumor infiltrating lymphocytes from colorectal (CRC) and head and neck squamous cell carcinoma (HNSCC) were transduced with retroviral vectors to express regulatable mbIL15 and LIGHT. ACZ-induced surface expression of mbIL15 and LIGHT in expanded tumor infiltrating lymphocytes was examined using flow cytometry. Functional signaling of LIGHT was assessed through co-culture with Jurkat-HVEM-NF-KB reporter cells and LTPR+ HUVEC cells. In vitro, engineered tumor infiltrating lymphocytes were tested in stromal-rich tumor models (CRC and HNSCC) by co-culturing with autologous patient-derived tumor / CAF hybrid spheroids. In vivo, antigen independent tumor infiltrating lymphocytes persistence was assessed in NSG mice without exogenous IL2. Syngeneic studies were performed to assess the efficacy of adoptively transferred mbIL15 and LIGHT -engineered Pmel cells (CD8+ T cells transgenic for a gplOO specific T cell receptor) in a subcutaneous cold tumor model (B16-F10).

[0057] As described herein, engineered tumor infiltrating lymphocytes were successfully expanded without exogenous IL2. ACZ-dependent mbIL15 and LIGHT expression were confirmed, validating co-regulation and functionality in vitro. Tumor infiltrating lymphocytes engineered with mbIL15 and LIGHT displayed significantly increased cytotoxicity against autologous tumor / CAF spheroids compared to tumor infiltrating lymphocytes expressing mbIL15 alone (p<0.005) in CRC and HNSCC tumor / CAF hybrid models. Tumor infiltrating lymphocytes with mbIL15 and LIGHT expanded in vivo and persisted for >42 days without exogenous IL2 support. Moreover, Pmel cells engineered with mbIL15 and LIGHT demonstrated durable anti -turn or efficacy in B16-F10 tumor-bearing mice, which was greater than Pmel cells engineered with mbIL15 alone (p< 0.01). These preclinical results show that tumor infiltrating lymphocytes engineered with regulatable mbIL15 and LIGHT using the cytoDRiVE platform can address the high unmet clinical need in cold tumors with suppressive TMEs, which are currently not amenable to ACT.

[0058] LIGHT, also called tumor necrosis factor superfamily member 14, is a protein important in T cell activation. LIGHT binds to two receptors: lymphotoxin beta receptor (LTpR) and Herpes virus entry mediator (HVEM). Expression of LTBR and HVEM has been shown in several types of tumors. HVEM binding mediates T cell activation, proliferation, and survival, and LTpR binding leads to tumor cell apoptosis. However, overexpression of LIGHT induces systemic inflammation linked to autoimmunity. Therefore, regulating LIGHT transcription with a DRD as described herein controls the development of negative LIGHT - related effects.

[0059] DRDs are unstable polypeptides that degrade in the absence of their corresponding stabilizing ligand (also referred to as the paired ligand or ligand), but whose stability is rescued by binding to the stabilizing ligand. Because binding of the ligand to the DRD is reversible, later removal of the ligand results in the DRD unfolding, becoming unstable, and ultimately being tagged for degradation by the ubiquitin-proteasome system (“UPS”). Accordingly, it is believed that when a DRD is operably linked to a payload or polypeptide, for example LIGHT, the entire construct (i.e., DRD plus payload or DRD plus LIGHT polypeptide) is itself rendered unstable and is degraded by the UPS. However, in the presence of the paired ligand, the construct is stabilized, and the payload or LIGHT polypeptide remains available for use. In this way, the DRD operably linked to the payload or LIGHT polypeptide provides a temporal regulation of translation (concurrent with ligand delivery).

[0060] Further, the conditional nature of DRD stability allows a rapid and non-perturbing switch from stable polypeptide to unstable UPS substrate, facilitating regulation of a payload or LIGHT polypeptide’s activity level, and / or modulation of a payload or LIGHT polypeptide’s activity level. Multiple DRDs associated together in a polypeptide monomer result in a payload or other polypeptide having a lower off-state as compared to the same payload or LIGHT polypeptide operably linked to a single DRD. That is, in the absence of ligand, increasing the density of DRDs (for example with multiple DRDs) is thought to reduce the abundance or availability of a payload or LIGHT polypeptide as compared to a payload or LIGHT polypeptide that is operably linked to only a single DRD.

[0061] Payloads should be understood to include one or more polypeptides having one or more functions, such as one or more biological activities, desired to be regulated. Reference to a biological activity is understood to mean a desired activity under appropriate conditions even if not so stated. Payloads include multiple classes of therapeutically important polypeptides (proteins and peptides) such as Type I / II membrane proteins, cytokines, immunomodulatory proteins, intracellular proteins, secreted proteins, CAS9 proteins, and transcription factor proteins.

[0062] Because the abundance and availability of a payload are related to the activity of a payload, for purposes of this disclosure, the terms abundance, availability, activity, and the phrase abundance and / or activity (and similarly level of abundance, level of availability, level of activity, and level of abundance and / or activity) are used interchangeably throughout this disclosure and are generally referred to as activity, unless explicitly stated otherwise or nonsensical in context. Further, measurements of abundance or availability are used as a proxy for activity level and may be used herein to reflect the activity level. Consequently, changes in the abundance or availability of a payload in the presence of an effective amount of ligand as compared to in the absence of ligand optionally serves as a proxy for measuring changes in activity level.

[0063] Nucleic Acid Molecules

[0064] Provided herein is a nucleic acid molecule comprising a first nucleic acid sequence encoding a polypeptide payload operably linked to a first drug responsive domain (DRD) and a second nucleic acid sequence encoding a LIGHT polypeptide operably linked to a second DRD. The first and second DRDs are each responsive to a ligand. Optionally, the first and second DRD are responsive to the same ligand. In other cases, the first and second DRD are responsive to different ligands. The at least one polypeptide payload and LIGHT polypeptide have a biological activity, and the biological activity is regulated by the interaction of the first and second DRDs with an effective amount of at least one ligand. Exemplary constructs are provided in FIG. 30.

[0065] As used throughout, the term nucleic acid or nucleotide refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or doublestranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. A person of skill in the art would recognize that a particular nucleic acid sequence can be modified to encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences while retaining the function of the reference sequence, in this case the sequence encoding myocardin. Any of the nucleic acid sequences described herein can be codon-optimized.

[0066] As used throughout, the terms polypeptide, peptide, and protein are used interchangeably herein to refer to a polymer of amino acid residues. The terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0067] As used herein, operably linked generally means that two moieties are directly or indirectly linked such that one moiety influences the other moiety. In the context of the DRD being operably linked to a sequence encoding a polypeptide payload or LIGHT, operable linkage means that the DRD is linked to the payload or LIGHT polypeptide directly or indirectly so as to alter a measurable characteristic of the payload or LIGHT polypeptide in the presence of the DRD’s paired ligand. For example, the DRD bound to the ligand can alter the level of activity of the payload or LIGHT polypeptide as compared to the level of activity in the absence of the paired ligand or can alter the level of translation of the sequence encoding the polypeptide payload or LIGHT polypeptide as compared to the level of translation in the absence of the paired ligand. Optionally, the measured level of amount and / or activity of the payload or LIGHT polypeptide increases in the presence of an effective amount of ligand as compared to the measured level of expression or activity in the absence of ligand. An effective amount of ligand means the amount of ligand needed to see an increase in the measure of the amount or activity of the payload or LIGHT polypeptide. Optionally, the effective amount is not so great as to produce unacceptable toxicity or off- target effects. Optionally, the measurable characteristic is a therapeutic outcome, e.g., an amount of the payload or LIGHT polypeptide in a sample, or a biological activity level of the payload or LIGHT polypeptide (for which measuring the amount of payload or LIGHT polypeptide can serve as a proxy for activity).

[0068] Nucleic acids encoding polypeptide payloads

[0069] The nucleic acid molecule provided herein comprises a first nucleic acid encoding a polypeptide payload. The term payload refers to the polypeptide whose abundance, activity, availability, expression, function, or other characteristic is desired to be regulated by a DRD. By way of example, the payload can be any polypeptide having a desired biological function. Optionally, the payload can be an active portion or variant of a polypeptide with a desired biological function, so long as the variant retains the desired biological function. Such payloads can be modified polypeptides such as glycosylated polypeptides or lipopeptides, which upon expression are modified in a cell by constitutive enzymatic activity or by overexpression of selected enzymes.

[0070] Payloads include multiple classes of therapeutically important polypeptides or any active portion thereof. For example, payloads include Type I / II membrane proteins such as CD40L, 4-1BBL, and CAR or active portions thereof. Payloads also include cytokines such as IL 15 (SEQ ID NO: 19, encoded by the nucleic acid sequence of SEQ ID NO: 20), IL 12, ILip, IL2, IL7, IL 18, IL21, IL23, IL36, TNFa, IFNy, IFNa, IFNP, etc., including membrane- tethered forms thereof or active portions thereof. Payloads also include intracellular polypeptides such as dnSHP2, T7, RNA polymerase, Cas9, or active portions thereof. Payloads include secreted proteins such as VEGF-trap, and native cytokines, or active portions thereof. And payloads further include transcription factors such as Foxp3, c-Myc, STAT5, and c-Jun, or active portions thereof, or constitutively active versions thereof. Other examples of suitable payloads include cytokine receptors, T-cell receptors (TCR), chimeric antigen receptors (CAR), immunomodulatory proteins in addition to those already exemplified, or any active portion thereof. The payload can also be a gene editing polypeptide or transcription factor in addition to those previously exemplified. The payload can also be a combination of polypeptides having a desired combination of actions, or active portions thereof.

[0071] Optionally, payloads are therapeutic agents chosen from a cancer therapeutic agent, a therapeutic agent for an autoimmune disease, an immunotherapeutic agent, an antiinflammatory agent, an anti-pathogenic agent, a gene therapy agent, or combinations thereof. The immunotherapeutic agent may be an antibody or fragments and variants thereof, a TCR, a CAR, a chimeric switch receptor, an antagonist of a co-inhibitory molecule, an agonist of a co-stimulatory molecule, a cytokine, a mutated version of a cytokine possessing altered receptor binding properties (also called a mutein), a cytokine receptor, a chemokine, a chemokine receptor, a metabolic factor, a coagulation factor, an enzyme, a homing receptor, a kinase, a phosphatase, a dominant negative version of a phosphatase (such as SHP-1 or SHP- 2), a dominant negative signaling molecule or receptor (such as a dominant negative Fas Receptor), a dominant negative transcription factor, and a safety switch.

[0072] Optionally, payloads of the present disclosure may be cytokines or fragments, variants, analogs, and derivatives thereof, including but not limited to interleukins, tumor necrosis factors (TNFs), interferons (IFNs), TGFP, and chemokines. The interleukins may be chosen from IL15 (SEQ ID NO: 19, encoded by the nucleic acid sequence of SEQ ID NO: 20), IL 12, ILip, IL2, IL7, IL 18, IL21, IL23, IL36, and variants thereof including membranebound, secreted, fusion polypeptide, or cytokine mutants with altered receptor binding properties (such as muteins), and bicistronic forms of the interleukins, and combinations thereof.

[0073] Optionally, payloads of the present disclosure may be chimeric antigen receptors (CARs) comprising an extracellular targeting domain (e.g., a scFv that recognizes a specific tumor antigen or other tumor cell-surface molecules), a transmembrane domain / region, and an intracellular signaling / activation domain (e.g., the signal region of CD3^, and / or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX- 40 (CD 134)).

[0074] Optionally, payloads can be selected that reduce immune responses in a subject. For example, the payload can be an anti-cytokine, such as neutralizing antibodies to tumor necrosis factor (TNF)-a or an interleukin. Optionally, payloads of the present disclosure target B-cell depletion, such as neutralizing antibodies to CD20, CD22, CD28, CTLA-4, and B-lymphocyte stimulator (BlyS).

[0075] Optionally, payloads can also be contractile proteins (e.g., actin and myosin), enzymes (e.g., lactase and pepsin), hormones (e.g., insulin, oxytocin, and somatotropin), structural proteins (e.g., keratin, collagen, and elastin), storage proteins (e.g., ovalbumin and ferritin), transport proteins (e.g., hemoglobin), membrane-bound proteins (e.g., class I, II, or III transmembrane proteins; receptors, transporters, and the like).

[0076] Optionally, payloads of the present disclosure may be one or more components of a gene editing system. In such examples, the oligomer or engineered, regulatable polypeptide regulates activity of the gene editing system and consequently expression of a downstream target protein. A target protein, as used herein, refers to a protein selected for gene editing, including, for example, a protein having a genetic mutation that results in a deleterious effect in a subject or in a cell. For example, payloads of the present disclosure may be a Cas protein (CRISPR-associated protein), including Cas9 and Casl2. The Cas protein may be altered or otherwise modified. For example, the Cas protein may be a dead Cas9. Optionally, the Cas9 protein is an enzymatically active Cas9 protein, a Cas9 protein wild-type protein, a Cas9 protein nickase or a nuclease null or nuclease deficient Cas9 protein. Such payloads optionally include nucleases (e.g., Zinc finger nuclease, TALEN (Transcription activator-like effector-based nucleases), or meganucleases) and / or recombinases, such as a Cre recombinase. Payloads useful in the present disclosure also include polypeptides involved in nucleic acid synthesis and replication, for example, DNA and RNA polymerases, transcription factors, primases, helicases, RNases, ligases, topoisomerases, endonucleases, IRES, and telomerases.

[0077] Optionally, the payload is IL15. It is understood in the art that certain gene and / or protein nomenclature for the same gene or protein may be inclusive or exclusive of punctuation such as a dash or other symbol. Whether these are included or excluded herein, the meaning is not meant to be changed as would be understood by one of skill in the art. For example, IL15, IL 15 and IL-15 refer to the same interleukin. In some embodiments, payloads of the present disclosure may be an IL15 interleukin cytokine that stimulate certain immune responses.

[0078] Payloads of the present disclosure may comprise amino acid sequences similar to the amino acid sequence of human IL15, for example, UniProtKB - P40933 (IL15 HUMAN). Optionally, the IL15 payload comprises the amino acid sequence of SEQ ID NO: 19. Optionally, the IL 15 payload comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20 as determined by sequence alignment programs and parameters known to those skilled in the art. Such tools for 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.)

[0079] Optionally, the IL 15 payload of the present disclosure 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: 19, a transmembrane domain and an intracellular tail, wherein the transmembrane domain is C-terminal to the IL15 polypeptide component, and the intracellular tail is C-terminal to the transmembrane domain. In some embodiments, the payload is a membrane-bound form of IL 15 comprising a transmembrane domain, intracellular tail and one or more linkers.

[0080] Nucleic acids encoding LIGHT polypeptides

[0081] The nucleic acid molecule provided herein further comprises a second nucleic acid encoding a LIGHT polypeptide. Optionally, the LIGHT polypeptide comprises the amino acid sequence of SEQ ID NO: 23. Optionally, the LIGHT polypeptide comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 24 as determined by sequence alignment programs and parameters known to those skilled in the art.

[0082] DRDs

[0083] In the nucleic acid molecule provided herein, the first and second nucleic acid sequences are each operably linked to a DRD. DRD is understood to mean a domain responsive to a ligand. DRDs interact with a ligand such that, when the DRD is operatively linked to a payload or LIGHT polypeptide, it confers ligand-dependent reversible regulation of a characteristic of the payload or LIGHT polypeptide (for example, activity). Although referred to as drug responsive domains, the ligand to which a DRD is responsive need not be a drug. Suitable DRDs (and their paired ligands), which may be referred to as destabilizing domains or ligand binding domains, are also known in the art. See, e.g., U.S. Pat. Nos. 9,487,787 and 10,137,180, U.S. Publication Nos.: 2019 / 0192691; 2020 / 0101142; 2020 / 0172879; 2021 / 0069248, and U.S. Pat. App. Nos.: 17,251,635; and 17 / 288,373, and W02018 / 161000; WO2018 / 231759; WO2019 / 241315; US8, 173,792; US8,530,636; WO2018 / 237323; WO2017 / 181119; US2017 / 0114346; US2019 / 0300864; WO2017 / 156238; Miyazaki et al., J Am Chem Soc, 134:3942 (2012); Banaszynski et al. (2006) Cell 126:995- 1004; Stankunas, K. et al. (2003) Mol. Cell 12: 1615-1624; Banaszynski et al. (2008) Nat. Med. 14: 1123-1127; Iwamoto et al. (2010) Chem. Biol. 17:981-988; Armstrong et al. (2007) Nat. Methods 4: 1007-1009; Madeira da Silva et al. (2009) Proc. Natl. Acad. Sci. USA 106:7583-7588; Pruett-Miller et al. (2009) PLoS Genet. 5:el000376; and Feng et al. (2015) Elife 4:el0606, the contents of each of which are hereby incorporated by reference in their entirety.

[0084] The DRDs, by way of example, can be chosen from CA2 (SEQ ID NO: 1), FKBP (SEQ ID NO: 11), ecDHFR (SEQ ID NO: 7), hDHFR (SEQ ID NO: 9), ER (SEQ ID NO: 17), PDE5 full length (SEQ ID NO: 15), PDE5 ligand binding domain (SEQ ID NO: 13). Optionally the DRD is a modified CA2 such as CA2 with an Mldel mutation (SEQ ID NO: 5) or L156H mutation in CA2 (SEQ ID NO: 3, encoded by the nucleic acid sequence of SEQ ID NO: 4) or a portion of any of the foregoing that maintains DRD function or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17 or the DRD functional portion thereof. One or more mutations (including truncations, substitutions, and deletions) in the amino acid sequence of CA2, FKBP, ecDHFR, hDHFR, ER, and PDE5, for example, can be advantageous to further destabilize the DRD.

[0085] Numerous DRD are described herein, but one of skill in the art can identify additional DRDs suitable for use in regulatable compositions according to this disclosure. By way of example, DRDs can be identified using library screening and structure-guided engineering to select the optimal DRD variant with sufficient instability in the absence of the ligand and sufficient stability in the presence of the ligand. A variant library can be generated using random mutagenesis screening by transducing cells (e.g., Jurkat cells) with mutant DRD candidates. To produce an enriched library, cells with the desired characteristics (low basal activity / expression and high dynamic range of activity / expression) are selected by testing polypeptide abundance across a range of concentrations of ligand. Single cell clones are then produced and characterized to identify candidate DRDs.

[0086] The DRDs described herein are responsive to a paired ligand (also referred to as a stabilizing ligand or simply as a ligand. Optionally, the DRDs are responsive to a paired ligand that is a small molecule drug, such as an FDA-approved small molecule. However, one of skill in the art can select the DRD and its paired ligand to meet the specific needs of the system. Examples of DRD / ligand pairs are shown in Table 1.

[0087] Table 1. Listing of DRD and exemplary ligands

[0088] Optionally, a DRD of the present disclosure may be a modified carbonic anhydrase, which is a member of a superfamily of metalloenzymes. For example, human carbonic anhydrase (hCA2) can be adapted for use as a DRD. A DRD of the present disclosure may be derived from amino acids 1-260 of CA2 (Uniprot ID: P00918). Optionally, DRDs are derived from CA2 comprising amino acids 2-260 of the parent CA2 sequence (e.g., amino acids 2- 260). This is referred to herein as a CA2 Mldel mutation (SEQ ID NO: 5). Optionally, a DRD of the present disclosure comprises a region of or the whole human carbonic anhydrase 2 and further comprises one or more mutations relative to the full-length sequence selected from Mldel, L156H, and S56N. Optionally, the DRD is selected from the group consisting of SEQ ID NOs: 1, 3, or 5.

[0089] Promoters

[0090] In some cases, the first and second nucleic acids contained in the nucleic acid molecules of the disclosure may be placed under the transcriptional control one or more promoters. As used herein, promoter refers to a DNA sequence recognized by transcription machinery of the cell, required to initiate specific transcription of the first and second nucleic acid sequences of the present disclosure. The promoters selected may be strong, weak, constitutive, inducible, tissue specific, development stage-specific, and / or organism specific. Examples of promoters which may be used herein include, but are not limited to, human cytomegalovirus (CMV) promoter, an Elongation Factor la (EFla) promoter, HIV LTR promoter, 3 -phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus long terminal repeat (RSV) promoter, spleen focus forming virus (SFFV) promoter, synthetic MND promoter, murine stem cell virus (MSCV) promoter, synthetic RPBSA promoter or a ubiquitin promoter.

[0091] Optionally, the first nucleic acid is under the control of a different promoter than the second nucleic acid. In other cases, the first nucleic acid is under the control of the same promoter as the second nucleic acid. The optimal promoter may be selected by one skilled in the art based on its ability to achieve minimal expression of first and second nucleic acids of the disclosure in the absence of the ligand and detectable expression in the presence of the ligand.

[0092] Transmembrane domains, linkers, and cleavage sites

[0093] Transmembrane domains, useful in the nucleic acid molecules of the present disclosure can include, for example, a MHC1 transmembrane domain, a CD8a transmembrane domain, a B7-l transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, or a human IgG4 Fc region. Optionally, the intracellular tail is a B7.1 intracellular tail.

[0094] As used herein, a linker refers to peptide domains that may be placed between or between different domains within the payload. Linkers include, for example, GS linkers, GSG linkers, and GGSG linkers. These linkers are repeats of the subunit one or more times. Thus, a GS linker is a GSn linker where n is a numerical number being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Similarly, a GSG linker is a GSGn linker wherein n is a numerical number being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. A GGSG linker is a GGSGn linker where n is a numerical number being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0095] Optionally, the nucleic acid molecules provided herein contain one or more cleavage sites between the first and second nucleic acid sequences of the molecule. As used herein, a cleavage site refers to an amino acid sequence that induces ribosomal skipping during translation, resulting in cellular expression of multiple, discrete polypeptide sequences. Optionally, the one or more cleavage sites is a 2A peptide chosen from the group of porcine teschovirus-1 (P2A), foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), and Thosea asigna virus (T2A). Optionally, the one or more cleavage sites is a P2A domain (SEQ ID NO: 21) encoded by the nucleic acid sequence of SEQ ID NO: 22 or a nucleic acid sequence having at least 85, 90, 95, or 99% identity to SEQ ID NO: 22.

[0096] Ligands

[0097] As used herein, ligand refers to any agent that binds to a first DRD, a second DRD, or both the first and second DRD of the nucleic acid molecules described herein. An effective amount of such a ligand results in a measurable change in a characteristic (e.g., abundance, availability, or activity) of a payload or LIGHT polypeptide operably linked to the DRD. The terms ligand, paired ligand, and stabilizing ligand are used interchangeably and mean the same thing when used in reference to a DRD. Optionally, ligands may be synthetic molecules. Optionally, stabilizing ligands of the present disclosure may be small molecule compounds. Stabilizing ligands are optionally small molecule therapeutic drugs previously approved by a regulatory agency, such as the U.S. Food and Drug Administration (FDA). Examples of stabilizing ligands and their corresponding DRDs suitable for use described herein are shown in Table 1 and in U.S. Patent No. 9,487,787 filed March 33, 2012, U.S. Patent No. 10,137,180 filed September 6, 2013, PCT Application No. PCT / US2018 / 037005, filed June 12, 2018, PCT Application No. PCT / US2019 / 036654 filed June 12, 2019, PCT Application No. PCT / US2019 / 057698 filed October 23, 2019, PCT Application No. PCT / US2020 / 021596 filed March 6, 2020, and U.S. Application No. 16 / 558,224 filed September 2, 2019, the disclosures of which are all are incorporated herein by reference in their entireties.

[0098] Vectors and Cells

[0099] Also provided herein are vectors for expressing one or more of the nucleic acid molecules. Such a vector can be chosen from viral vectors and non-viral vectors, plasmids, cosmids, transposons, and artificial chromosomes. By way of example, the vector can be a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno- associated viral vector. The vector optionally comprises nucleic acid sequences that encode transposases and / or nucleases. Non-viral vector examples include physical vectors such as electroporation and chemical vectors such as lipid nanoparticles.

[0100] Cells containing one or more nucleic acid molecules or vectors as described herein are provided. The cell provides an expression system or a therapeutic target for the monomers, oligomers, or polypeptides described herein. Suitable cells include somatic cells, such as immune cells, epithelial cells, stem cells, or germline cells. Optionally, the immune cells are primary human T cells, such as T cells derived from human peripheral blood mononuclear cells (PBMC), PBMC collected after stimulation with G-CSF, bone marrow, or umbilical cord blood. Optionally, the immune cells are tumor infiltrating lymphocytes, for example collected from a tumor. The immune effector cells may also be NK cells, aP T cells, iNKT cells, yS T cells, macrophages, B cells, dendritic cells, myeloid derived progenitor cells, eosinophils, basophils, neutrophils, or Tregs. Optionally, the stem cells are hematopoietic stem cells, human embryonic stem cells, or iPSCs. The cells provided herein are optionally mammalian cells, or, more specifically, human cells.

[0101] Pharmaceutical compositions

[0102] Provided herein is a pharmaceutical composition suitable for use in ACT comprising the cells described herein and a pharmaceutically acceptable carrier. Optionally, the cell is a human tumor infiltrating lymphocyte, human T cell, or NK cell. Optionally, the cells are allogeneic, for example expanded tumor infiltrating lymphocytes. The population of allogeneic cells in the pharmaceutical composition is optionally a mixed population of cells comprising a subpopulation of modified cells and unmodified cells (e.g., tumor infiltrating lymphocytes engineered to express mbIL15 and LIGHT together with untransduced or unengineered tumor infiltrating lymphocytes).

[0103] The term carrier means a compound, composition, substance, or structure that, when in combination with a compound or cells, aids, or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or cells for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the cells in the pharmaceutical composition and to minimize any adverse side effects in the subject. Such pharmaceutically acceptable carriers include sterile biocompatible pharmaceutical carriers, including, but not limited to, saline, buffered saline, artificial cerebral spinal fluid, dextrose, and water. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the cells in the pharmaceutical composition without causing unacceptable biological effects or interacting in a deleterious manner.

[0104] Optionally the pharmaceutical composition further comprises a cryoprotectant (cryopreservative). Such a cryoprotectant serves to prevent unacceptable cell lysis or damage should the cells be frozen for future use. Cryoprotectants are known in the art. Such cryoprotectants can be selected from among glycerol, ethylene glycol, propylene glycol, or dimethylsulfoxide (DMSO).

[0105] The pharmaceutical compositions described herein optionally further comprise one or more pharmaceutically acceptable excipients ((e.g., human serum albumin or polymeric materials (e.g., PEG)).

[0106] The compositions of the present disclosure can be formulated in any manner suitable for delivery. The cells can be administered in nanoparticles, poly (lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids, or combinations thereof.

[0107] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human mammals. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, agricultural animals, such as cattle, horses, chickens, and pigs; domestic animals, such as cats, dogs; or research animals such as mice, rats, rabbits, dogs, and non-human primates.

[0108] Methods of Use

[0109] Disclosed herein are methods of regulating the biological activity levels of both a polypeptide payload and LIGHT. Optionally, the method of regulation is a method of modifying the activities of both the polypeptide payload and LIGHT comprising administering to a cell containing the nucleic acid molecules described herein a selected dose of at least one ligand to which the first DRD, the second DRD, or both are responsive. Optionally, the activities of both the polypeptide payload and LIGHT (e.g., corresponding to the abundance and / or availability of the payload and LIGHT) is reduced as compared to the activities of the polypeptide payload or LIGHT in a control cell, for example, a cell not containing one or more of the nucleic acid molecules described herein or not in the presence of the ligand.

[0110] As used herein administering is understood to mean providing an agent (such as a ligand) to a target (such as a DRD) such that the agent and target may come into contact with one another. Administration can be in vitro, ex vivo, or in vivo, whereby the agent is added to cell culture medium or administered to the patient. As a non-limiting example, administering also includes providing the ligand to a cell, wherein the DRD is located intracellularly, such that the ligand reaches the cytoplasm or the nucleus or other cellular organelles. Similarly, administration can be in vivo, for example by administering a ligand to a subject such that the ligand reaches a cell or the DRD contained on the surface or in the interior of the cell. In each case, the agent needs to reach a minimum intracellular concentration to exert its stabilizing effect on the target. Optionally both the polypeptide payload and LIGHT have biological activity levels ranging from a basal activity level in the absence of ligand to a maximum activity in the presence of a saturating amount of ligand. Basal level as used herein can be zero, near zero, or any amount in the absence of exogenous ligand. Basal activity may occur because of endogenous levels of the same or different ligand or may occur because of a resting level of payload production in the absence of exogenous ligand. Optionally, the method is a method of modulating the activities of a payload and LIGHT comprising administering to a cell comprising the nucleic acid molecule a selected dose of at least one ligand such that the activities of both the polypeptide payload and LIGHT are increased relative to the basal activity levels. Optionally, the method comprises administering to the cell a selected amount of ligand, wherein the selected amount of ligand results in selected activity levels of both the polypeptide payload and LIGHT. In certain examples, the method comprises alternatively administering to the cell varying selected amounts of ligand, to achieve varying selected activity levels ranging from the basal level to the maximum level.

[0111] Methods of Producing Genetically Engineered Cells

[0112] Also disclosed herein are methods of producing a genetically engineered cell such as a tumor infiltrating lymphocyte, T cell, or NK cell, comprising introducing into the tumor infiltrating lymphocyte, T cell, or NK cell the nucleic acid molecule described herein.

[0113] As used herein, introducing in the context of introducing a nucleic acid molecule described herein, refers to the translocation of the nucleic acid molecule from outside a cell to inside the cell. In some cases, introducing refers to translocation of the nucleic acid molecule from outside the cell to inside the nucleus of the cell. Various methods of such translocation are contemplated, including but not limited to, electroporation, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, and the like.

[0114] Cells for engineering can be isolated from any biological sample, including for example, blood (e.g., umbilical cord blood or peripheral blood), bone marrow, embryonic or fetal tissue (e.g., human embryonic stem cells), or tumors. Cells can be modified cells, such as a stem cell modified to be pluripotent (e.g., an induced pluripotent stem cell (iPSC)) or CAR T cells, prior to transduction with the described nucleic acids and vectors.

[0115] By way of example, isolated T cells or tumor infiltrating lymphocytes can be isolated from a biological sample, transduced, and, optionally, expanded in culture. Such expansion can be performed, for example, in the presence of feeder cells, recombinant antigen, or an antibody that stimulates cell expansion. Optionally expansion is performed in the presence of IL2. However, for T cells or tumor infiltrating lymphocytes engineered to express mbIL15, contacting the transduced cell with IL2 is not required for expansion.

[0116] The cell to which the nucleic acid molecule is delivered is selected based, at least in part, on the ability of the cell to allow expression of the first and second nucleic acids described herein and to allow payload and LIGHT activities in a sufficient dynamic range. Optionally, the cell expresses little or no payload or LIGHT in the absence of the provided nucleic acid molecule. In certain examples, one of skill in the art would select a cell in need of an increase in payload or LIGHT activity or abundance in a cell that expresses the payload or LIGHT. In certain examples, one of skill in the art would select a cell in need of gene editing. Optionally, the cell is selected as an effector cell, for example, an immune effector cell. In certain cases, the cells are selected to provide a payload to a subject in need of the payload.

[0117] Methods of Treatment

[0118] Also disclosed are methods of delivering a payload to a subject, for example a therapeutically effective payload to a subject in need thereof, whereby a nucleic acid construct or a vector as described herein is administered to the subject. The method results in expression, for example in target cells of the subject, of the payload and LIGHT. The methods may further comprise controlling the dose or duration of administration of a payload to a subject. For example, the method optionally further comprises administering to the subject a selected amount of one or more paired ligands to deliver a selected activity of the payload and LIGHT to the subject. The one or more ligands can be delivered to achieve continuous or intermittent payload activity in the subject. Continuous payload and LIGHT activity may be a substantially consistent level of activity, or the level of activity may be modulated. Intermittent activity, between the off-state and on-state includes modulating activity between the off-state and a substantially consistent on-state, or between the off-state and varying on-state activity levels. A higher dose or longer duration of administration of the one or more ligands is administered when more activity of the payload and / or LIGHT is desired, and reduction or elimination of the one or more ligand doses is chosen when less activity is desired. The dose and duration of administration of the one or more ligands and the resulting activity of the payload and LIGHT may be selected to avoid unacceptable or undesired side effects or toxicity in the subject. Dosages of ligand and schedules for administering the dosages of ligand may be determined empirically by one skilled in the art based on the amount of resulting payload or LIGHT, the activity of the payload or LIGHT , or based on one or more signs of the effect of the payload or LIGHT activity. The ranges for administration of the one or more ligands range from any amount above zero to a saturating dose and the resulting payload or LIGHT activity ranges from a basal level to a maximal level, optionally with a sufficient dynamic range that allows for the desired dose-response to the ligand and concomitant activity range for the payload (e.g., for a given ligand and payload, the range of difference in off-state and maximum payload activity would result from at least a 10 fold range of ligand). This sufficient dynamic range allows for fine tuning and a dose response curve that is not unacceptably steep. In certain embodiments, the dosage or frequency of administration of ligand and resulting abundance and activity of payload is chosen to avoid, mitigate against, or limit unacceptable or undesired adverse side effects and will vary with the age, condition, and / or sex of the subject, and type of condition being treated, the extent of the condition, or, and whether other therapeutic agents are included in the treatment regimen. Guidance can be found in the literature for appropriate dosages for given classes of ligands.

[0119] Notably, when the payload and LIGHT are operably linked to the same DRDs, one of skill in the art would select and modulate a dose of ligand that provides the desired abundance or activity or both. When the payload and LIGHT are operably linked to different DRDs, one of skill in the art would select and modulate a dose of each ligand to provide the desired effect of the combination of payload and LIGHT.

[0120] By way of further example, for a subject with cancer, a nucleic acid construct, vector, or cell according to this disclosure is provided to a subject, wherein the nucleic acid construct, vector, or cell encodes a payload and LIGHT operably linked to one or more DRDs and wherein the payload targets a tumor cell or an immune cell that in turn targets the tumor cell. By way of example, the payload can be an immune checkpoint inhibitor, a cytokine, CAR, or TCR. Additional Definitions

[0121] As used in this specification and the appended claims, the singular forms a, an, and the include plural reference unless the context clearly dictates otherwise.

[0122] The use of any and all examples or exemplary language (e.g., for example or such as) provided herein, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0123] The terms may, may be, can, and can be, and related terms are intended to convey that the subject matter involved is optional (that is, the subject matter is present in some examples and is not present in other examples), not a reference to a capability of the subject matter or to a probability, unless the context clearly indicates otherwise.

[0124] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0125] The terms about and approximate, when used to refer to a measurable value such as an amount, concentration, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, weight, position, length and the like, is meant to account for variations due to experimental error, which could encompass variations of ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount, concentration, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, weight, position, length and the like. All measurements or numbers are implicitly understood to be modified by the word about, even if the measurement or number is not explicitly modified by the word about. In instances in which the terms about and approximate are used in connection with the location or position of regions within a reference polypeptide, these terms encompass variations of ± up to 20 amino acid residues, ± up to 15 amino acid residues, ± up to 10 amino acid residues, ± up to 5 amino acid residues, ± up to 4 amino acid residues, ± up to 3 amino acid residues, ± up to 2 amino acid residues, or even ± 1 amino acid residue.

[0126] The details of one or more embodiments of the present disclosure are set forth in the description and accompanying drawings. It is to be understood that other embodiments may be utilized and structural or process changes made without departing from the scope of the disclosure. In other words, illustrative embodiments and aspects are described below. But it will be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions may be made to achieve the developer’s specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it will be appreciated that such development effort might be complex and time-consuming but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

[0127] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.

[0128] The examples below are intended to further illustrate certain aspects of the methods and compositions described herein and are not intended to limit the scope of the claims.

[0129] Examples

[0130] Example 1. Testing lentiviral vector co-regulated LIGHT and mbIL15 constructs

[0131] The present example demonstrates nucleic acid molecules provided herein, as shown in Table 2 below, enable co-regulation of LIGHT and a polypeptide payload, for example mbIL15.

[0132] Table 2. Lentiviral constructs used in Example 1.

[0133] Regulating LIGHT expression with lentiviral constructs

[0134] In a first set of experiments, LIGHT was regulated by adding a CA2 domain to the wildtype sequence of LIGHT and maintaining a constitutive mbIL15 in a lentiviral construct (LIGHT-005 (SEQ ID NO: 28). This construct confirmed LIGHT expression required two signals: T cell activation and DRD stabilization with a ligand, for example, ACZ (FIGS. 1-2). Tumor infiltrating lymphocytes were isolated from different donors with either head and neck cancer or lung cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol (see WO 2022 / 159939) and transduced with lentiviral vectors containing SEQ ID NOS: 26 and 28. The tumor infiltrating lymphocytes were either left resting or activated with CD3 / CD28 in the presence or absence of 25 pM ACZ for 24 hours. LIGHT expression was assessed using flow cytometry. The frequency of LIGHT+ CD3 cells was calculated, as well as the geometric mean of fluorescence intensity (gMFI).

[0135] As shown in FIGS. 1-2, LIGHT expression required both an activated T cell and a ligand to stabilize the DRD and prevent degradation of LIGHT, evinced by LIGHT-005 (SEQ ID NO: 27), which contained a regulatable DRD coupled to LIGHT and a constitutive mbIL15. In contrast, the construct designated IL-293 (SEQ ID NO: 26), which expressed a regulatable DRD coupled to mbIL15 showed significantly less LIGHT expression even when both signals were present.

[0136] Regulated LIGHT expression by tumor infiltrating lymphocytes transduced with either IL-293 (SEQ ID NO: 26) or LIGHT-005 (SEQ ID NO: 28) activated T cells through HVEM receptor binding (FIG. 3). Tumor infiltrating lymphocytes were isolated from different donors with either head and neck cancer, lung cancer, sarcoma, or colorectal cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26 or 28. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ for 48 hours and then co-cultured with Jurkat-HVEM T cells for 3 hours at a 10:1 ratio of tumor infiltrating lymphocytes: Jurkat-HVEM. Luciferase was used as a reporter gene to assess HVEM receptor binding and T cell activation. Luciferase expression was assessed using a luminometer and the fold increase in luciferase expression in the co-culture compared to in Jurkat-HVEM cells was calculated. In all of the donor indication groups HVEM-mediated activation was highest in ACZ-treated tumor infiltrating lymphocytes transduced with LIGHT-005, which contained a regulatable DRD (FIG. 3).

[0137] Regulated LIGHT expression by tumor infiltrating lymphocytes transduced with either IL-293 (SEQ ID NO: 26) or LIGHT-005 (SEQ ID NO: 28) promoted monocyte chemotactic protein- 1 (MCP-1) release via LTpR receptor binding in human umbilical vein endothelial cells (HUVECs) (FIG. 4A). Tumor infiltrating lymphocytes were isolated from different donors with either head and neck cancer or lung cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26 and 28. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ for 48 hours and then co-cultured with LTpR- expressing HUVECs for 24 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: HUVECs. LTpR activation induces MCP-1 cytokine expression (see, for example, C.W. Shuptrine et al.. European Journal of Cancer 187: 154 (2023)). MCP-1 expression, analyzed by Meso Scale Discovery technology (Meso Scale Diagnostics, LLC, Rockville, Maryland), was used to assess LTpR activation. In all of the donor indication groups, MCP-1 release, and thus LTpR activation, was highest in ACZ-treated tumor infiltrating lymphocytes transduced with LIGHT-005, which contained a regulatable DRD (FIG. 4A).

[0138] Further, pre-incubation of tumor infiltrating lymphocytes with a recombinant LTpR significantly blocked LIGHT activity (FIG. 4B). Tumor infiltrating lymphocytes were isolated from different donors with sarcoma. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26 and 28. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ for 48 hours and then co-cultured recombinant LTpR for 30 minutes at 37°C to block LIGHT protein. Tumor infiltrating lymphocytes were then co-cultured with LTpR- expressing HUVECs for 24 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: HUVECs. LTpR activation induces MCP-1 cytokine expression (see, for example, C.W. Shuptrine et al., European Journal of Cancer 187: 154 (2023)). MCP-1 expression, analyzed by Meso Scale Discovery technology (Meso Scale Diagnostics, LLC, Rockville, Maryland), was used to assess LTpR activation from LIGHT binding. In each group, pre-incubation with recombinant LTpR significantly blocked LIGHT activity, decreasing LTpR-mediated MCP-1 expression (FIG. 4B).

[0139] Comparing mbIL 15 and LIGHT expression between LIGHT-010, 012, 013, and 018

[0140] In this series of experiments, various arrangements of a co-regulated lentiviral mbIL15 / LIGHT construct were tested. In LIGHT-010 (SEQ ID NO: 30), the same CA2 DRD was added to both mbIL15 and LIGHT, and mbIL15 was encoded before the P2A sequence with LIGHT after the P2A sequence. In the lentiviral construct LIGHT-012 (SEQ ID NO: 32), the CA2 DRD controlling mbIL15 was altered and LIGHT was encoded before the P2A sequence, with mbIL15 after the P2A sequence. In the lentiviral construct LIGHT-013 (SEQ ID NO: 34), two different promoters were used. LIGHT expression was controlled by EFla, and mbIL15 expression was controlled by the MND promoter. Finally, in the construct designated LIGHT-018 (SEQ ID NO: 36), the position of LIGHT and mbIL15 from LIGHT- 012 was switched. mbIL15 with an altered CA2 DRD was expressed first and LIGHT expressed after the P2A sequence.

[0141] In each of the co-regulated LIGHT and mbIL15 constructs, mbIL15 expression expanded significantly in the presence of ACZ. (FIG. 5). Tumor infiltrating lymphocytes were isolated from different donors with either head and neck cancer or lung cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26, 28, 30, 32, 34, and 36. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ. On days 4 and 18, tumor infiltrating lymphocytes were counted and mbIL15 frequency was assessed using flow cytometry. Fold expansion was calculated based on the total number of mbIL15+ cells at day 18 divided by the total number of mbIL15+ cells at day 4. In each group, Tumor infiltrating lymphocytes transduced with the co-regulated mbIL15 / LIGHT constructs efficiently expanded at least 400-fold in the presence of ACZ (FIG. 5).

[0142] To assess IL15 and LIGHT expression, tumor infiltrating lymphocytes were isolated from different donors with either head and neck cancer or lung cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26, 28, 30, 32, 34, and 36. The tumor infiltrating lymphocytes were either left resting or activated (FIGS. 6-7) with CD3 / CD28 in the presence or absence of 25 pM ACZ for 24 hours. mbIL15 and LIGHT expression were assessed using flow cytometry and the frequencies of mbIL15+ and LIGHT+ cells in total CD3+ cells were calculated.

[0143] As shown in FIG. 6, all of the co-regulated mbIL15 / LIGHT lentiviral constructs treated with ACZ show similar IL15 expression except LIGHT-013 (SEQ ID NO: 34). LIGHT-013 lost IL 15 expression in activated tumor infiltrating lymphocytes (FIG. 6). The construct designated LIGHT-018 (SEQ ID NO: 36) showed the highest mbIL15 expression (FIG. 6). Likewise, all of the co-regulated mbIL15 / LIGHT lentiviral constructs showed LIGHT expression and regulation (FIG. 7). The construct designated LIGHT-010 (SEQ ID NO: 30) showed the lowest LIGHT expression in activated tumor infiltrating lymphocytes treated with ACZ (FIG. 7).

[0144] In each of the co-regulated mbIL15 / LIGHT lentiviral constructs, LIGHT activated HVEM and LTpR receptors (FIG. 8). Tumor infiltrating lymphocytes were isolated from different donors with head and neck cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26, 28, 30, 32, 34, and 36. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ for 48 hours. To assess HVEM activity, tumor infiltrating lymphocytes were co-cultured with Jurkat-HVEM T cells for 3 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: Jurkat-HVEM. Luciferase was used as a reporter gene to assess HVEM receptor binding and T cell activation. Luciferase expression was assessed using a luminometer, and the fold increase in luciferase expression in the co-culture compared to in Jurkat-HVEM cells was calculated. To assess LTpR activity, following ACZ treatment, tumor infiltrating lymphocytes were then co-cultured with LTpR-expressing HUVECs for 24 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: HUVECs. MCP-1 expression, analyzed by Meso Scale Discovery technology (Meso Scale Diagnostics, LLC, Rockville, Maryland), was used to assess LTpR activation from LIGHT binding. As shown in FIG. 8, each of the co-regulated mbIL15 / LIGHT lentiviral constructs showed functional regulation of LIGHT both on the HVEM and LTpR pathways. LIGHT-005 showed lower LIGHT activity on the LTpR pathway.

[0145] Further, HVEM pathway activation was consistent across tumor infiltrating lymphocyte donor indication (FIG. 9). Tumor infiltrating lymphocytes were isolated from different donors with head and neck cancer, lung cancer, and colorectal cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26, 28, and 36. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ for 48 hours. To assess HVEM activity, tumor infiltrating lymphocytes were co-cultured with Jurkat-HVEM T cells for 3 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: Jurkat-HVEM. Luciferase was used as a reporter gene to assess HVEM receptor binding and T cell activation. Luciferase expression was assessed using a luminometer and the fold increase in luciferase expression in the coculture compared to in Jurkat-HVEM cells was calculated. In each donor indication, the coregulated mbIL15 / LIGHT lentiviral constructs showed functional regulation of LIGHT on the HVEM pathway (FIG. 9).

[0146] Comparing mbIL15 and LIGHT expression between LIGHT-022 and 023

[0147] In this series of experiments, a series of modifications were made to previously tested co-regulated mbIL15 / LIGHT lentiviral constructs. In the construct designated LIGHT-022 (SEQ ID NO: 38), the same construct arrangement from LIGHT-012 (SEQ ID NO: 32) was fully codon optimized. In the construct designated LIGHT-023 (SEQ ID NO: 40), the same construct arrangement from the construct designated LIGHT-018 (SEQ ID NO: 36) was fully codon optimized. In constructs LIGHT-027 and -028 (SEQ ID NOS: 46 and 48, respectively), a GCCACC (SEQ ID NO: 49) Kozak sequence was introduced before the ATG start codon, and in LIGHT-028, a CD34 leading sequence, and mbIL15 were encoded as a pre-protein sequence.

[0148] Each of the co-regulated mbIL15 / LIGHT lentiviral constructs tested showed efficient expansion of mbIL15 in the presence of ACZ (FIG. 10A and FIG. 10B). Tumor infiltrating lymphocytes were isolated from different donors with either head and neck cancer or colorectal cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 25, 27, 35, 37, and 39. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ. On days 4 and 18, tumor infiltrating lymphocytes were counted and mbIL15 frequency was assessed using flow cytometry. Fold expansion was calculated based on the total number of mbIL15+ cells at day 18 divided by the total number of mbIL15+ cells at day 4. In each group, tumor infiltrating lymphocytes transduced with the co-regulated mbIL15 / LIGHT lentiviral constructs efficiently expanded mbIL15 expression at least 400-fold in the presence of ACZ (FIG. lOA and FIG. 10B).

[0149] To assess both mbIL15 and LIGHT expression, tumor infiltrating lymphocytes were isolated from different donors with head and neck cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26, 28, 36, 38, and 40. The tumor infiltrating lymphocytes were either left resting or activated (FIG. 11) with CD3 / CD28 in the presence or absence of 25 pM ACZ for 24 hours. mbIL15 and LIGHT expression were assessed using flow cytometry and the frequency of mbIL15+ and LIGHT+ cells in total CD3+ cells was calculated. Each of the tested co-regulated mbIL15 / LIGHT lentiviral constructs treated with ACZ showed similar mbIL15 and LIGHT expression, except for LIGHT-022, which showed lower mbIL15 expression in cells without ACZ treatment, but higher LIGHT expression in cells treated with ACZ (FIG. 11).

[0150] LIGHT-018, LIGHT-022, and LIGHT-023 showed similar LIGHT functionality on both the HVEM- and LTpR-mediated pathways (FIG. 12). Tumor infiltrating lymphocytes were isolated from different donors with head and neck cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26, 28, 36, 38, and 40. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ for 48 hours. To assess HVEM activity, Tumor infiltrating lymphocytes were co-cultured with Jurkat-HVEM T cells for 3 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: Jurkat-HVEM in the presence or absence of 25 pM ACZ. Luciferase was used as a reporter gene to assess HVEM receptor binding and T cell activation. Luciferase expression was assessed using a luminometer and the fold increase in luciferase expression in the co-culture compared to in Jurkat-HVEM cells was calculated. To assess LTpR activity, following ACZ treatment, tumor infiltrating lymphocytes were then co-cultured with LTpR-expressing HUVECs for 24 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: HUVECs in the presence of absence of 25 pM ACZ. MCP-1 expression, analyzed by Meso Scale Discovery technology (Meso Scale Diagnostics, LLC, Rockville, Maryland), was used to assess LTpR activation from LIGHT binding. As shown in FIG. 12, each of the co-regulated mbIL15 / LIGHT constructs tested showed functional regulation of LIGHT both on the HVEM and LTpR pathways. In tumor infiltrating lymphocytes from donors with colorectal cancer, each of the coregulated mbIL15 / LIGHT constructs LIGHT-018, LIGHT-022, and LIGHT-023 showed similar IL 15 and LIGHT expression when treated with ACZ (FIG. 13). Tumor infiltrating lymphocytes were isolated from different donors with colorectal cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26, 28, 36, 38, and 40. The tumor infiltrating lymphocytes were either left resting or activated (FIG. 13) with CD3 / CD28 in the presence or absence of 25 pM ACZ for 24 hours. mbIL15 and LIGHT expression were assessed using flow cytometry and the frequency of mbIL15+ and LIGHT+ cells in total CD3+ cells was calculated. Each of the tested co-regulated mbIL15 / LIGHT constructs treated with ACZ showed similar mbIL15 and LIGHT expression, except for LIGHT-022, which showed lower mbIL15 expression in cells without ACZ treatment, but the highest LIGHT expression in cells treated with ACZ (FIG. 13).

[0151] In the same tumor infiltrating lymphocytes from donors with colorectal cancer as in FIG. 13, co-regulated mbIL15 / LIGHT constructs LIGHT-018, LIGHT-022, and LIGHT-023 showed similar LIGHT functionality on both the HVEM- and LTBR-mediated pathways (FIG. 14). Tumor infiltrating lymphocytes were isolated from different donors with colorectal cancer. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with lentiviral vectors containing SEQ ID NOS: 26, 28, 36, 38, and 40. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ for 48 hours. To assess HVEM activity, Tumor infiltrating lymphocytes were co-cultured with Jurkat-HVEM T cells for 3 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: Jurkat-HVEM in the presence or absence of 25 pM ACZ. Luciferase was used as a reporter gene to assess HVEM receptor binding and T cell activation. Luciferase expression was assessed using a luminometer and the fold increase in luciferase expression in the co-culture compared to in Jurkat-HVEM cells was calculated. To assess LTpR activity, following ACZ treatment, tumor infiltrating lymphocytes were then co-cultured with LTpR-expressing HUVECs for 24 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: HUVECs in the presence of absence of 25 pM ACZ. MCP-1 expression, analyzed by Meso Scale Discovery technology (Meso Scale Diagnostics, LLC, Rockville, Maryland), was used to assess LTpR activation from LIGHT binding. As shown in FIG. 14, each of the co-regulated mbIL15 / LIGHT lentiviral constructs tested showed functional regulation of LIGHT on the HVEM pathway; however, LIGHT-023 showed low functionality on the LTpR pathway. Assessing different DRDs in LIGHT-024 and 025

[0152] In this set of experiments, mbIL15 and LIGHT were coupled to different DRDs. In the polypeptides encoded by LIGHT-024 and 025 (see amino acid sequences of SEQ ID NOs: 41 and 43, respectively), LIGHT is regulated by a CA2 DRD and mbIL15 is regulated by a hDHFR DRD. In the construct designated LIGHT-024 (SEQ ID NO: 42), LIGHT is encoded before the P2A sequence and mbIL15 encoded after the P2A sequence, whereas in the construct designated LIGHT-025 (SEQ ID NO: 44), mbIL15 is encoded before the P2A sequence and LIGHT is encoded after the P2A sequence.

[0153] To assess co-regulation of mbIL15 and LIGHT in T cells, activated T cells were transduced with either constructs designated LIGHT-024 (SEQ ID NO: 42) or LIGHT-025 (SEQ ID NO: 44). T cells were then left resting in DMSO or treated with ACZ (50pM) and TMP (lOpM) for 24 hours. Expression of LIGHT and IL15 was assessed using flow cytometry. As shown in FIG. 15, both LIGHT-024 and 025 showed good regulation in T cells treated with ACZ and TMP. LIGHT-024 showed the highest LIGHT expression and comparable levels of mbIL15 to LIGHT-025 (FIG. 15).

[0154] Testing cytotoxicity with tumor infdtrating lymphocytes transduced with co-regulated lentiviral constructs

[0155] Next, the cytotoxicity against autologous patient-derived tumor cells (PDc) by tumor infiltrating lymphocytes transduced with a lentiviral regulated-LIGHT-constitutive-mbIL15 construct (LIGHT-005; SEQ ID NO: 28) was assessed. See FIG. 16.

[0156] Autologous PDc cells and cancer associated fibroblasts (CAF) cells were pre-stained with CellTracker Deep Red dye (Invitrogen; Waltham, MA) and plated at 1 :1 ratio into Aggrewell 400 (Stemcell Technologies; Vancouver, Canada) with DMEM / F12+10% fetal bovine serum (FBS) supplemented with 10 pM Y-27632 Rho-Kinase inhibitor (Stemcell Technologies). The PDc and CAF formed spheroids overnight. The tumor infiltrating lymphocytes were thawed and rested for 48 hours in tumor infiltrating lymphocytes media before transduction with LIGHT-005 and co-culture with the tumor / CAF spheroids. To set up co-culture, tumor infiltrating lymphocytes were counted and enriched for viable cells using dead cell removal kit (Miltenyi Biotec; Gaithersburg, MD). For LTBR block conditions, regulated-LIGHT-constitutive-mbIL15 TIL were pre-incubated with recombinant human LTBR (Aero Biosystems; Newark, USA) for 1 hour before addition to the spheroids. Tumor / CAF spheroids were harvested and plated into ultra-low attachment 96 well plate (Revvity; Waltham, MA) at 20 spheroids per well. Viable tumor infiltrating lymphocytes were seeded with the spheroids at 250,000 tumor infiltrating lymphocytes per well. The Incucyte Caspase 3 / 7 green dye (Sartorius; Gottingen, Germany)) was then loaded into the co-culture system to label apoptotic cells. The co-culture plate was then loaded onto the Incucyte S3 for imaging. Cytotoxicity was calculated by dividing detected Caspase 3 / 7 signal inside the spheroids against the spheroid total area.

[0157] As shown in FIG. 16, tumor infiltrating lymphocytes from donors with colorectal cancer and lung cancer transduced with LIGHT-005 (SEQ ID NO: 28) showed ACZ- dependent cytotoxicity against PDc. A recombinant LTBR receptor blocked LIGHT functionality and cytotoxicity in tumor infiltrating lymphocytes transduced with lentiviral coregulated mbIL15 / LIGHT constructs (FIG. 16, right, dashed triangle).

[0158] FIGS. 17A-B show tumor infiltrating lymphocytes transduced with a regulated- LIGHT-constitutive-mbIL15 construct (LIGHT-005; SEQ ID NO: 28) showed ACZ- dependent cytotoxicity towards autologous tumor spheroids from a sarcoma donor. Autologous PDc cells and cancer associated fibroblasts (CAF) cells were pre-stained with CellTracker Deep Red dye (Invitrogen; Waltham, MA) and plated at 1 : 1 ratio into Aggrewell 400 (Stemcell Technologies; Vancouver, Canada) with DMEM / F12+10% fetal bovine serum (FBS) supplemented with 10 pM Y-27632 (Stemcell Technologies). The PDc and CAF formed spheroids overnight. The tumor infiltrating lymphocytes were thawed and rested for 48 hours in tumor infiltrating lymphocytes media before transduction with LIGHT-005 (SEQ ID NO: 28) and co-culture with the tumor / CAF spheroids. To set up co-culture, tumor infiltrating lymphocytes were counted and enriched for viable cells using dead cell removal kit (Miltenyi Biotec; Gaithersburg, MD). Tumor / CAF spheroids were harvested and plated into ultra-low attachment 96 well plate (Revvity; Waltham, MA) at 20 spheroids per well. Viable tumor infiltrating lymphocytes were seeded with the spheroids at 250,000 tumor infiltrating lymphocytes per well. The Incucyte Caspase 3 / 7 green dye (Sartorius; Gottingen, Germany)) was then loaded into the co-culture system to label apoptotic cells. The co-culture plate was then loaded onto the Incucyte S3 for imaging. Cytotoxicity was calculated by dividing detected Caspase 3 / 7 signal inside the spheroids against the spheroid total area. As shown in FIG. 17 A, tumor infiltrating lymphocytes transduced with the regulated-LIGHT- constitutive-mbIL15 construct showed the highest cytotoxicity, measured by caspase 3 / 7 fluorescence, in the presence of ACZ. FIG. 17B shows regulation of LIGHT with IL 15 in tumor infiltrating lymphocytes displays ACZ-dependent cytotoxicity towards autologous tumor spheroids from a sarcoma donor. FIGS. 18A-B show tumor infiltrating lymphocytes transduced with a regulated- LIGHT-constitutive-mbIL15 construct (LIGHT-005; SEQ ID NO: 28) showed ACZ- dependent cytotoxicity towards autologous tumor / CAF hybrid spheroids from a lung cancer donor. Autologous PDc cells and cancer associated fibroblasts (CAF) cells were pre-stained with CellTracker Deep Red dye (Invitrogen) and plated at 1 : 1 ratio into Aggrewell 400 (Stemcell Technologies) with DMEM / F12+10% fetal bovine serum (FBS) supplemented with 10 pM Y-27632 (Stemcell Technologies). The PDc and CAF formed spheroids overnight. The tumor infiltrating lymphocytes were thawed and rested for 48 hours in tumor infiltrating lymphocytes media before transduction with LIGHT-005 (SEQ ID NO: 28) and co-culture with the tumor / CAF spheroids. To set up co-culture, tumor infiltrating lymphocytes were counted and enriched for viable cells using dead cell removal kit (Miltenyi Biotec). Tumor / CAF spheroids were harvested and plated into ultra-low attachment 96 well plate (Revvity) at 20 spheroids per well. Viable tumor infiltrating lymphocytes were seeded with the spheroids at 250,000 tumor infiltrating lymphocytes per well. The Incucyte Caspase 3 / 7 green dye (Sartorius) was then loaded into the co-culture system to label apoptotic cells. The co-culture plate was then loaded onto the Incucyte S3 for imaging. Cytotoxicity was calculated by dividing detected Caspase 3 / 7 signal inside the spheroids against the spheroid total area. As shown in FIG. 18 A, tumor infiltrating lymphocytes transduced with the regulated-LIGHT-constitutive-mbIL15 construct showed the highest cytotoxicity, measured by caspase 3 / 7 fluorescence, in the presence of ACZ. FIG. 18B shows regulation of LIGHT with IL 15 in tumor infiltrating lymphocytes displays ACZ-dependent cytotoxicity autologous tumor / cancer-associated fibroblast (CAF) hybrid spheroids from a lung donor.

[0159] To assess whether tumor infiltrating lymphocytes cytotoxicity could be blocked by recombinant LTpR-Fc, tumor infiltrating lymphocytes from donors with head and neck cancer were isolated and transduced with either a regulated mb IL 15 construct, a constitutive mbIL15-regulated LIGHT construct, or a regulated mbIL15-regulated LIGHT construct. Results are shown in Fig. 19.

[0160] Autologous PDc cells and cancer associated fibroblasts (CAF) cells were pre-stained with CellTracker Deep Red dye (Invitrogen; Waltham, MA) and plated at 1 :1 ratio into Aggrewell 400 (Stemcell Technologies; Vancouver, Canada) with DMEM / F12+10% fetal bovine serum (FBS) supplemented with 10 pM Y-27632 Rho-Kinase inhibitor (Stemcell Technologies). The PDc and CAF formed spheroids overnight. The tumor infiltrating lymphocytes were thawed and rested for 48 hours in tumor infiltrating lymphocytes media before transduction and co-culture with the tumor / CAF spheroids. To set up co-culture, tumor infiltrating lymphocytes were counted and enriched for viable cells using dead cell removal kit (Miltenyi Biotec; Gaithersburg, MD). For LTBR block conditions, regulated-LIGHT- constitutive-mbIL15 TIL were pre-incubated with recombinant human LTBR (Aero Biosystems; Newark, USA) for 1 hour before addition to the spheroids. Tumor / CAF spheroids were harvested and plated into ultra-low attachment 96 well plate (Revvity; Waltham, MA) at 20 spheroids per well. Viable tumor infiltrating lymphocytes were seeded with the spheroids at 250,000 tumor infiltrating lymphocytes per well. The Incucyte Caspase 3 / 7 green dye (Sartorius; Gottingen, Germany)) was then loaded into the co-culture system to label apoptotic cells. The co-culture plate was then loaded onto the Incucyte S3 for imaging. Cytotoxicity was calculated by dividing detected Caspase 3 / 7 signal inside the spheroids against the spheroid total area.

[0161] As shown in FIG. 19, tumor infiltrating lymphocytes from donors with head and neck cancer transduced with a lentiviral co-regulated mbIL15-LIGHT construct showed ACZ- dependent cytotoxicity against PDc. A recombinant LTBR receptor blocked LIGHT functionality and cytotoxicity in tumor infiltrating lymphocytes transduced with lentiviral coregulated mbIL15 / LIGHT constructs (dashed triangle).

[0162] Tumor infiltrating lymphocytes transduced with a regulated-LIGHT-constitutive- mbIL15 construct showed ACZ-dependent cytotoxicity towards autologous tumor / CAF hybrid spheroids from a head and neck cancer donor (FIGs. 20A-B). Autologous PDc cells and cancer associated fibroblasts (CAF) cells were pre-stained with CellTracker Deep Red dye (Invitrogen) and plated at 1 : 1 ratio into Aggrewell 400 (Stemcell Technologies) with DMEM / F12+10% fetal bovine serum (FBS) supplemented with 10 pM Y-27632 (Stemcell Technologies). The PDc and CAF formed spheroids overnight. The tumor infiltrating lymphocytes were thawed and rested for 48 hours in tumor infiltrating lymphocytes media before the co-culture with the tumor / CAF spheroids. To set up co-culture, tumor infiltrating lymphocytes were counted and enriched for viable cells using dead cell removal kit (Miltenyi Biotec). Tumor / CAF spheroids were harvested and plated into ultra-low attachment 96 well plate (Revvity) at 20 spheroids per well. Viable tumor infiltrating lymphocytes were seeded with the spheroids at 250,000 tumor infiltrating lymphocytes per well. The Incucyte Caspase 3 / 7 green dye (Sartorius) was then loaded into the co-culture system to label apoptotic cells. The co-culture plate was then loaded onto the Incucyte S3 for imaging. Cytotoxicity was calculated by dividing detected Caspase 3 / 7 signal inside the spheroids against the spheroid total area. As shown in FIG. 20A, tumor infiltrating lymphocytes transduced with the regulated-LIGHT-constitutive-mbIL15 construct showed the highest cytotoxicity, measured by caspase 3 / 7 fluorescence, in the presence of ACZ. FIG. 20B shows regulation of LIGHT with IL 15 in tumor infiltrating lymphocytes displays ACZ-dependent cytotoxicity towards autologous tumor / CAF hybrid spheroids from a head and neck donor.

[0163] Example 2, Testing retroviral vector co-regulated LIGHT and mbIL15 constructs

[0164] The present example demonstrates nucleic acid molecules provided herein, as shown in Table 3 below, enable co-regulation of LIGHT and a polypeptide payload, for example mbIL15.

[0165] In this set of experiments, retroviral vectors were used to deliver the co-regulated mbIL15 / LIGHT nucleic acid construct to tumor infiltrating lymphocytes. The constructs tested in this example varied in both promoters used and the placement of each LIGHT and mbIL15 encoding moiety. IL15-293 (SEQ ID NO: 26) was used as a control, without LIGHT and with mbIL15 placed under the control of a MPSV promoter. In LIGHT-037, 038, and 039, LIGHT was encoded before the P2A sequence with mbIL15 after. In LIGHT-037, LIGHT and mbIL15 encoding regions were placed under the control of the MPSV promoter, whereas LIGHT-038 used the MND promoter and LIGHT-039 used the EFS promoter.

[0166] Conversely, in constructs designated LIGHT-041, 042, and 043 , mbIL15 was encoded before the P2A sequence with LIGHT after. In the construct designated LIGHT-041, mbIL15 and LIGHT were placed under the control of the MPSV promoter, whereas the construct designated LIGHT-042 used the MND promoter and the construct labeled LIGHT- 043 used the EFS promoter.

[0167] Table 3. Retroviral constructs used in Example 2.

[0168] Testing mbIL15 expression in retroviral constructs mbIL15 expression in unactivated and CD3 / CD28 activated tumor infiltrating lymphocytes from donors with head and neck cancer and colorectal cancer with lung metastases was tested. Tumor infiltrating lymphocytes were isolated from donors then expanded using a rapid expansion protocol and transduced with retroviral vectors containing SEQ ID NOS: 50, 52, 54, 56, 59, and 60. The tumor infiltrating lymphocytes were either left resting or activated with CD3 / CD28 in the presence or absence of 25 pM ACZ for 24 hours. mbIL15 expression was assessed using flow cytometry and the gMFI was calculated. In each donor, the tested retroviral constructs showed good regulated mbIL15 expression. LIGHT- 039, which was under the control of the EFS promoter, underperformed in inactive cells, but overperformed in CD3 / CD28 activated cells relative to IL15-239 in both donor indications. See FIGs. 21-22.

[0169] Testing LIGHT expression in retroviral constructs

[0170] LIGHT expression in unactivated and CD3 / CD28 activated tumor infiltrating lymphocytes from donors with head and neck cancer and colorectal cancer with lung metastases was assessed. Tumor infiltrating lymphocytes were isolated from donors then expanded using a rapid expansion protocol and transduced with retroviral vectors containing SEQ ID NOS: 50, 52, 54, 56, 58, and 60. The tumor infiltrating lymphocytes were either left resting or activated with CD3 / CD28 in the presence or absence of 25 pM ACZ for 24 hours. LIGHT expression was assessed using flow cytometry and the gMFI was calculated. In each donor, the tested retroviral constructs showed good regulated LIGHT expression. LIGHT expression was stronger in the constructs with LIGHT positioned before mbIL15 (LIGHT- 037, 038, and 039 (SEQ ID NOS: 50, 52, and 54, respectively) for both types of donors. FIGS. 23-24 Tumor infiltrating lymphocytes expansion in retroviral constructs

[0171] As shown in FIG. 25, each of the co-regulated mbIL15 / LIGHT constructs tested showed efficient expansion of mbIL15 in the presence of ACZ. Tumor infiltrating lymphocytes were isolated from different donors with head and neck cancer or with colorectal cancer with lung metastases. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with retroviral vectors containing SEQ ID NOS: 26, 50, 52, 54, 56, 58, and 60. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ for 24 hours. On days 4 and 18, tumor infiltrating lymphocytes were counted and mbIL15 frequency was assessed using flow cytometry. Fold expansion was calculated based on the total number of mbIL15+ cells at day 18 divided by the total number of mbIL15+ cells at day 4. In each group, tumor infiltrating lymphocytes transduced with the co-regulated mbIL15 / LIGHT retroviral constructs efficiently expanded over 1000-fold in the presence of ACZ (FIG. 25).

[0172] LIGHT activity on LTBR and HVEM pathways in retroviral constructs

[0173] Most of the co-regulated mbIL15 / LIGHT constructs tested in this example showed LIGHT functionality on both the HVEM- and LTBR-mediated pathways (FIGS. 26-27). Tumor infiltrating lymphocytes were isolated from different donors with head and neck cancer or with colorectal cancer with lung metastases. Tumor infiltrating lymphocytes were then expanded using a rapid expansion protocol and transduced with retroviral vectors containing SEQ ID NOS: 26, 50, 52, 54, 56, 58, and 60. The tumor infiltrating lymphocytes were cultured with or without 25 pM ACZ for 48 hours. To assess HVEM activity, tumor infiltrating lymphocytes were co-cultured with Jurkat-HVEM T cells for 3 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: Jurkat-HVEM in the presence or absence of 25 pM ACZ. Luciferase was used as a reporter gene to assess HVEM receptor binding and T cell activation. Luciferase expression was assessed using a luminometer and the fold increase in luciferase expression in the co-culture compared to in Jurkat-HVEM cells was calculated (FIG. 26A). To assess LTpR activity, following ACZ treatment, tumor infiltrating lymphocytes were then co-cultured with LTpR-expressing HUVECs for 24 hours at a 10: 1 ratio of tumor infiltrating lymphocytes: HUVECs in the presence of absence of 25 pM ACZ. MCP-1 expression, analyzed by Meso Scale Discovery technology (Meso Scale Diagnostics, LLC, Rockville, Maryland), was used to assess LTpR activation from LIGHT binding (FIG. 26B). In tumor infiltrating lymphocytes from donors with head and neck cancer, constructs driven by the EFS promoter, e.g., the constructs designated LIGHT-039 (SEQ ID NO: 54) and LIGHT-043 (SEQ ID NO: 60), however, failed to stimulate the LTpR pathway more than the control construct IL15-293 (SEQ ID NO: 26) (FIG. 26B). As shown in FIG. 26A, other constructs showed higher LIGHT functionality on the HVEM pathway than the control IL 15- 293. Assessing LTpR-mediated activation, constructs LIGHT-039 and 043, however, failed to stimulate MCP-1 expression more than the control IL 15-293 construct (FIG. 26B).

[0174] Likewise, in tumor infiltrating lymphocytes from donors with colorectal cancer with lung metastases, constructs driven by the EFS promoter, e.g., LIGHT-039 (SEQ ID NO: 54) and LIGHT-043 (SEQ ID NO: 60), failed to stimulate the LTpR pathway more than the control construct IL15-293 (SEQ ID NO: 26) (FIG. 27B). As shown in FIG. 27A, each of the test constructs showed higher LIGHT functionality on the HVEM pathway than the control IL15-293. Assessing LTpR-mediated activation, constructs LIGHT-039 and 043, however, failed to stimulate MCP-1 expression more than the control IL15-293 construct (FIG. 27B).

[0175] Cytotoxicity against PDc in retroviral constructs

[0176] Next, the cytotoxicity against autologous PDc by tumor infiltrating lymphocytes transduced with retroviral constructs IL15-293, LIGHT-037, and LIGHT-041 (SEQ ID NOS: 26, 50, and 56, respectively) was assessed. Tumor infiltrating lymphocytes transduced with the tested constructs showed enhanced cytotoxicity towards autologous tumor spheroids from donors with colorectal cancer with lung metastases and head and neck cancer (FIG. 28); moreover, the cytotoxicity was ACZ-dependent (FIG. 29).

[0177] Autologous PDc cells and CAF cells were pre-stained with CellTracker Deep Red dye (Invitrogen) and plated at 1 : 1 ratio into Aggrewell 400 (Stemcell Technologies) with DMEM / F12+10% FBS supplemented with 10 pM Y-27632 (Stemcell Technologies). The PDc and CAF formed spheroids overnight. The tumor infiltrating lymphocytes were thawed and rested for 48 hours in tumor infiltrating lymphocytes media before the co-culture with the tumor / CAF spheroids. To set up co-culture, tumor infiltrating lymphocytes were counted and enriched for viable cells using dead cell removal kit (Miltenyi Biotec). Tumor / CAF spheroids were harvested and plated into ultra-low attachment 96 well plate (Revvity) at 20 spheroids per well. Viable tumor infiltrating lymphocytes were seeded with the spheroids at 250,000 tumor infiltrating lymphocytes per well. The Incucyte Caspase 3 / 7 green dye (Sartorius) was then loaded into the co-culture system to label apoptotic cells. The co-culture plate was then loaded onto the Incucyte S3 for imaging. Cytotoxicity was calculated by dividing detected Caspase 3 / 7 signal inside the spheroids against the spheroid total area. The final tumor infiltrating lymphocytes:tumor:CAF:PDc ratio was 1 :38: 1 : 1.

[0178] As shown in FIG. 28, tumor infiltrating lymphocytes transduced with the tested retroviral constructs showed the highest cytotoxicity, measured by caspase 3 / 7 fluorescence, in the presence of ACZ. FIG. 29 shows tumor infiltrating lymphocytes cytoxocity against PDc / CAF hybrid spheroids was ACZ dependent.

[0179] Example 3. Testing LIGHT-IL15 tumor infiltrating lymphocytes in a PDc / CAF spheroid model

[0180] Next, the infiltration of tumor infiltrating lymphocytes transduced with a regulated LIGHT-IL15 construct into spheroids of autologous patient-derived tumor cells (PDc) and cancer-associated fibroblasts (CAF) was assessed. Tumor infiltrating lymphocytes were collected from a donor with head and neck cancer and were transduced with either a CA2- regulated IL15 construct (IL15-293; SEQ ID NO: 26) or a co-regulated IL15 and LIGHT construct (LIGHT-037; SEQ ID NO: 50). The tumor infiltrating lymphocytes were seeded with tumor only or 1 : 1 mixed tumor / CAF spheroids and co-cultured for 24 hours. The spheroids were fixed and tissue cleared to render the tissue transparent for further imaging. The spheroids were then stained for DAPI / CD3 (T cells) and Granzyme B and imaged on a confocal microscope. Tumor infiltrating lymphocyte infiltration and depth, as well as granzyme B expression were analyzed and quantified using Imaris software.

[0181] Tumor infiltrating lymphocytes transduced with a regulated LIGHT-IL15 construct showed enhanced infiltration into PDc / CAF spheroids compared to tumor infiltrating lymphocytes transduced a regulated IL15 construct alone (FIG. 3 IB). Tumor infiltrating lymphocytes transduced with a regulated LIGHT -IL15 construct also showed greater granzyme B release than tumor infiltrating lymphocytes transduced with a regulated IL 15 construct alone, suggesting enhanced cytotoxicity (FIG. 31A-31B). Moreover, infiltration depth was greater in tumor infiltrating lymphocytes transduced with a regulated LIGHT-IL15 construct than that of tumor infiltrating lymphocytes transduced with a regulated IL 15 construct alone (FIG. 32).

[0182] Thus, as shown in FIGs. 31-32, tumor infiltrating lymphocytes transduced with a regulated LIGHT-IL15 construct showed increased infiltration, cytotoxicity, and infiltration depth in a PDc / CAF spheroid model compared to tumor infiltrating lymphocytes transduced with regulated IL 15 alone. Example 4: Assessing LIGHT in a PDx CRC model

[0183] Tumor infiltrating lymphocytes expressing co-regulated mbIL15 and LIGHT show improved anti-tumor activity in a PDx model of CRC

[0184] Tumor infiltrating lymphocytes transduced with regulated IL 15 and LIGHT were tested in a patient-derived xenograft (PDx) model of colorectal cancer (CRC). An experimental overview is depicted in FIG. 33. Briefly, a CRC lung metastasis was excised from a patient and approximately 100 mg tumor fragment was implanted subcutaneously within 24 hours of surgery (Passage 0 [P0]). One fragment was homogenized, and analyzed by flow cytometry with anti-human CD45, anti-human EpCAM, and anti-human aSMA to characterize the tumor microenvironment (FIG. 34). Two NOD scid gamma (NSG) female mice were implanted with similarly sized fragments that were allowed to grow for approximately 150 days. Serial passaging was performed until there was enough viable tumor tissue available to support a large-scale implantation for an efficacy study. Animals (n=8 per treatment group) were randomized on Day 32 following the large-scale PDX tumor implant. Tumor infiltrating lymphocytes were transduced with either a CA2 -regulated IL15 construct (IL15-293; SEQ ID NO: 26) or a co-regulated IL15 and LIGHT construct (LIGHT-037; SEQ ID NO: 50) and infused intravenously the following day. Both treatment groups were dosed ACZ daily via oral gavage at 200 mg / kg from day of tumor infiltrating lymphocytes infusion (Day 0) until termination of the study.

[0185] As shown in FIG. 34, PDx cells showed a high CAF content, signified by CD45- aSMA+ cells. Tumor volume decreased in mice administered tumor infiltrating lymphocytes transduced with a regulated LIGHT-IL15 construct compared to mice administered tumor infiltrating lymphocytes transduced a regulated IL 15 construct alone (FIG. 35). As shown in FIG. 36A, tumor volume decreased in mice administered tumor infiltrating lymphocytes transduced with a regulated LIGHT-IL15 construct and further administered ACZ compared to mice which did not receive ACZ. Further, tumor infiltrating lymphocytes frequency and persistence, signified by CD3+ cells, was increased in mice administered tumor infiltrating lymphocytes transduced with a regulated LIGHT -IL15 construct and further administered ACZ compared to mice which did not receive ACZ (FIG. 36B).

[0186] Thus, tumor infiltrating lymphocytes transduced with a regulated LIGHT-IL15 construct showed cytotoxicity against a CAF-rich CRC PDx model compared to tumor infiltrating lymphocytes transduced with regulated IL 15 alone (FIGs. 34-35). Further, LIGHT-IL15 construct was regulated by ACZ and tumor infiltrating lymphocytes containing the LIGHT-IL15 construct were increased in frequency and persistency in the presence of ACZ (FIG. 36A-B).

[0187] Tumor infiltrating lymphocytes transduced with a regulated IL15-LIGHT construct demonstrates significant regulated LIGHT production in a CRC PDx model

[0188] LIGHT regulation and production of tumor infiltrating lymphocytes transduced with a regulated IL15-LIGHT construct was assessed in a CRC PDx model. A CRC lung metastasis was excised from a patient and approximately 100 mg tumor fragment was implanted subcutaneously within 24 hours of surgery (passage 0 [P0]). NOD scid gamma (NSG) mice were implanted with similarly sized fragments that were allowed to grow for approximately 28 days. Serial passaging was performed until there was enough viable tumor tissue available to support a large-scale implantation for an efficacy study. Animals were randomized and tumor infiltrating lymphocytes transduced with either a CA2-regulated IL15 construct (IL15- 293; SEQ ID NO: 26) or a co-regulated IL 15 and LIGHT construct (LIGHT-041; SEQ ID NO: 56) and infused intravenously the next day. Groups were dosed ACZ (treatment group) or vehicle (control group) daily via oral gavage at 200 mg / kg from day of tumor infiltrating lymphocytes infusion (day 0). For the treatment group, two dosing regimens of ACZ were administered: one group received ACZ until each day until termination of the study (day 17) and one group received ACZ until day 14 of the study and did not receive ACZ for the last three days (days 15-17) of the study. Whole blood collected weekly and human tumor infiltrating lymphocytes were identified by flow cytometry as human CD3+ (hCD3+) cells.

[0189] The CRC lung metastasis implanted into mice and tumor infiltrating lymphocytes and ACZ were administered as described above. Tumors and plasma samples were collected from a group of mice at day 17 post-tumor infiltrating lymphocytes infusion to determine LIGHT regulation. Tumors were homogenized, centrifuged and supernatants collected for MSD analysis to detect human LIGHT. Data was normalized to total protein concentration as determined by using bicinchoninic acid (BCA) protein assay.

[0190] As shown in FIG. 37, LIGHT production is regulatable — soluble (shed) LIGHT increases in plasma (FIG. 37, left) and tumor (FIG. 37, right) in the presence of ACZ versus control vehicle ligand. Example 5: Assessing LIGHT off-state upon ACZ withdrawal

[0191] LIGHT production upon ACZ withdrawal was assessed. Tumor infiltrating lymphocytes isolated from donors having head and neck cancer and donors having lung cancer were expanded using a rapid expansion protocol (see, e.g., WO 2022 / 159939). Tumor infiltrating lymphocytes were transduced with retroviral vectors containing SEQ ID NOS: 26 and 51. After thawing, tumor infiltrating lymphocytes were activated with CD3 / CD28 in presence or absence of 25uM of Acetazolamide (ACZ). The samples cultured in presence of ACZ were washed out from ACZ 2, 4, 8 and 24 hours prior to harvesting. Tumor infiltrating lymphocytes were then assessed for LIGHT expression using flow cytometry.

[0192] As shown in FIG. 38, in both head and neck cancer and lung cancer models, tumor infiltrating lymphocytes expressing regulated LIGHT (LIGHT-037; SEQ ID NO: 50) had a fast decrease in production upon ACZ withdrawal compared to unengineered tumor infiltrating lymphocytes and tumor infiltrating lymphocytes expressing regulated IL15 (IL15- 293; SEQ ID NO: 26).

[0193] Example 6: Assessing LIGHT in response to DcR3

[0194] LIGHT functionality in tumor infiltrating lymphocytes expressing regulated LIGHT - IL15 was assessed in response to decoy receptor 3 (DcR3). DcR3 expression is low or absent under normal conditions but highly expressed in malignant cells. Secreted DcR3 was assessed in two sarcoma spheroid samples 24 after start of culture using an ELISA assay. DcR3 concentration was 1.45 ng / mL in one sample and 0.27 ng / mL in the other sample. Tumor infiltrating lymphocytes isolated from sarcoma donors were expanded using a rapid expansion protocol (see, e.g., WO 2022 / 159939) and cryopreserved. After thawing, tumor infiltrating lymphocytes were cultured with or without ACZ for 48 hours and then cocultured with Jurkat-herpes virus entry mediator (HVEM) for 3 hours at a 10: 1 ratio tumor infiltrating lymphocytes: Jurkat-HVEM in presence or absence of 25 uM ACZ and varying concentrations of recombinant DcR3 (1, 10, and 1000 ng / mL). Tumor infiltrating lymphocytes were transduced with either a CA2-regulated IL15 construct (IL15-293; SEQ ID NO: 26) or a co-regulated IL15 and LIGHT construct (LIGHT-037; SEQ ID NO: 50).

[0195] LIGHT functionality of LIGHT expressed by tumor infiltrating lymphocytes transduced with a regulated LIGHT-IL15 construct was not affected by a concentration of DcR3 similar to that produced by tumor cells in a sarcoma model (FIG. 39). As shown in 5 FIG. 39, 1 and 10 ng / mL DcR3 did not affect functionality of LIGHT expressed by tumor infiltrating lymphocytes transduced with a regulated LIGHT-IL 15 construct.

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid molecule comprising(a) a first nucleic acid encoding a polypeptide payload operably linked to a first drug responsive domain (DRD); and(b) a second nucleic acid encoding a LIGHT polypeptide operably linked to a second DRD, wherein the polypeptide payload and LIGHT each have a biological activity and wherein the first and second DRDs are each responsive to a ligand.

2. The nucleic acid molecule of claim 1, wherein the LIGHT polypeptide payload comprises SEQ ID NO: 24.

3. The nucleic acid molecule of claim 1 or 2, wherein the biological activities of the polypeptide payload and LIGHT are regulated by the interaction of the first and second DRDs with an effective amount of at least one ligand.

4. The nucleic acid molecule of claim 3, wherein the first DRD, the second DRD, or both the first and second DRDs are CA2 or a variant thereof.

5. The nucleic acid molecule of claim 4, wherein either the first DRD or second DRD comprises one, two, three or four mutations relative to SEQ ID NO: 2 or SEQ ID NO: 6.

6. The nucleic acid molecule of any one of claims 1-3, wherein the first DRD is selected from the group consisting of CA2, ecDHFR , hDHFR, FKBP, PDE5 ligand binding domain, PDE5, and ER, and variants thereof, wherein the variants of CA2, ecDHFR, hDHFR, FKBP, PDE5 ligand binding domain, PDE5, and ER.

7. The nucleic acid molecule of any one of claims 1-3, wherein the second DRD is selected from the group consisting of CA2, ecDHFR, hDHFR, FKBP, PDE5 ligand binding domain, PDE5, and ER, and variants thereof.

8. The nucleic acid molecule of any one of claims 1-7, wherein the first and second DRDs are responsive to the same ligand.

9. The nucleic acid molecule of any one of claims 1-7, wherein the first and second DRDs are responsive to different ligands.

10. The nucleic acid molecule of claim 1-9, wherein the first and second ligands are selected from the group consisting of acetazolamide, methotrexate, trimethoprim, tacrolimus, celecoxib, topiramate, valdecoxib, rofecoxib, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide, and di chlorphenamide.

11. The nucleic acid molecule of claim 10, wherein the ligand is acetazolamide.

12. The nucleic acid molecule of any one of claims 1-11, wherein the polypeptide payload is selected from the group consisting of a cytokine, a cytokine receptor, a TCR, a CAR, an immunomodulatory protein, and combinations thereof.

13. The nucleic acid molecule of claim 12, wherein the polypeptide payload is a cytokine and wherein the cytokine is a membrane-bound IL15 polypeptide.

14. The nucleic acid molecule of claim 13, wherein the membrane-bound IL15 polypeptide comprises an amino acid sequence of SEQ ID NO: 19, a transmembrane domain and an intracellular tail, wherein the transmembrane domain is C-terminal to SEQ ID NO: 19 and the intracellular tail is C-terminal to the transmembrane domain.

15. The nucleic acid molecule of claim 14, wherein the membrane-bound IL 15 polypeptide further comprises a linker between the IL 15 polypeptide component and the transmembrane domain.

16. The nucleic acid molecule of any one of claims 1-15, wherein the first and second nucleic acids are under the control of the same promoter.

17. The nucleic acid molecule of any one of claims 1-15, wherein the first and second nucleic acids are under the control of different promoters.

18. The nucleic acid molecule of any one of claims 1-17, wherein the nucleic acid molecule comprises one or more cleavage sites between the first and second nucleic acids.

19. The nucleic acid molecule of claim 18, wherein the one or more cleavage sites comprise a P2A domain.

20. A vector comprising one or more nucleic acid molecules of any one of claims 1-19.

21. A cell comprising one of more nucleic acid molecules of any one of claims 1-19.

22. The cell of claim 21, wherein the cell is a human cell.

23. The cell of claim 22, wherein the human cell is a tumor infiltrating lymphocyte (TIL),T cell, or natural killer (NK) cell.

24. The cell of claim 23 wherein the cell is a CD4+ or CD8+ T cell.

25. A method of regulating a biological activity level of both a polypeptide payload andLIGHT in the cell of any one of claim 21-24, comprising administering to the cell at least one ligand to which the first DRD, the second DRD, or both are responsive, wherein the at least one ligand is administered in an amount sufficient to modulate the expression, function, and / or level of the polypeptide payload and LIGHT.

26. A method of producing a genetically engineered tumor infiltrating lymphocyte (TIL), T cell, or natural killer (NK) cell, comprising introducing into the TIL, T cell, or NK cell the nucleic acid molecule of any of claims 1-19.

27. A pharmaceutical composition comprising the cell of any one of claims 20-24 and a pharmaceutically acceptable carrier.

28. The pharmaceutical composition of claim 27, wherein the cell is a human TIL, human T cell, or human NK cell.

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