Compositions and systems for modulating function / abundance and delivery of polypeptide payloads - Patents.com

Abstract

Provided herein is an engineered regulatable polypeptide comprising a payload and a regulatory hub comprising at least two drug response domains (DRDs), the regulatory hub being operably linked to the payload, and the at least two DRDs being responsive to a ligand. Also provided is an engineered monomer comprising an oligomerization domain and a payload, a DRD, or both a payload and a DRD, the monomer being configured to assemble into an oligomer by the oligomerization domain, the oligomer comprising at least one payload operably linked to the regulatory hub. The abundance, availability and / or biological activity of the payload is regulated by the interaction between one or more DRDs and an effective amount of a ligand.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 262,646, filed October 18, 2021, and U.S. Provisional Patent Application No. 63 / 342,443, filed May 16, 2022, which are incorporated by reference in their entireties.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated by reference in its entirety. The Sequence Listing XML, created on October 18, 2022, is named 108407-1354052-Oct 18.xml and is 691,346 bytes in size. [Background technology]

[0003] Biological products (biologics) as defined by the US 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), cells from the same species of subject (homologous or allogeneic), or even cells from a different species (xenogeneic) can be administered to a subject as a biologic. In adoptive cell therapy ("ACT"), for example, T cells from a subject (or possibly a different source) are removed, genetically engineered to address the specific needs of the same or different subject, and then returned to the same or different subject. However, there are currently obstacles to the widespread adoption and success of biologics such as ACT. It can be difficult to maintain therapeutically effective levels of a biologic administered to a patient because the biologic may be expressed at toxic levels by the engineered cells or may be present or expressed for unacceptable sustained periods of time. Thus, there is a need for regulation of biologics to optimize therapeutic benefits and promote widespread adoption of biologic therapies. Summary of the Invention [Means for solving the problem]

[0004] This disclosure relates to compositions and systems capable of modulating payload function, as well as methods of making and using the compositions and systems. Payload function can be modulated, inter alia, by modifying and / or adjusting the abundance and / or activity of the payload. Described herein are compositions and systems capable of modifying and / or adjusting payload activity and / or abundance, as well as methods of making and using the compositions and systems.

[0005] The present disclosure provides compositions, termed "regulatory hubs," that include two or more drug-responsive domains ("DRDs"). A DRD is a polypeptide that can modulate the abundance, availability, and / or activity of a payload under appropriate conditions, for example, in response to a small molecule ligand. In some embodiments, the regulatory hub includes DRDs that are covalently linked in series. In such embodiments, the DRDs are linked, for example, directly or indirectly, via a linker or hinge. In certain embodiments, the regulatory hub includes a DRD associated with an oligomer (also referred to as a multimeric fusion composition or fusion composition) of at least two DRD oligomerization domain constructs, each of which includes a DRD linked directly or indirectly to an oligomerization domain configured to promote oligomerization of the DRD oligomerization domain construct. In some embodiments, where the oligomerization domain is a trimerizing domain, the multimeric fusion composition includes three DRD oligomerization domain constructs resulting in a regulatory hub with at least three DRDs and three trimerizing domains. In any of the embodiments of the coordination hub, the DRDs in the coordination hub may all be the same, all different, some the same, and some different.

[0006] The present disclosure also provides compositions, referred to as polypeptide monomers, that include an oligomerization domain configured to promote oligomerization of a polypeptide monomer, and at least one payload that has biological activity under appropriate conditions, or at least one drug-responsive domain (DRD) that is responsive to a ligand. In some embodiments, the regulatable polypeptide monomer includes an oligomerization domain configured to promote oligomerization of a polypeptide, at least one payload that has a biological activity level under appropriate conditions, and at least one drug-responsive domain. In some such embodiments, e.g., with the same monomer, multiple monomers oligomerize to form an oligomer that includes at least two payloads operably linked to a regulatory hub, which in turn includes at least two oligomerization domains and at least two DRDs. In other embodiments, e.g., with different monomers, multiple monomers oligomerize to form an oligomer that includes at least one payload operably linked to a regulatory hub, which in turn includes at least two oligomerization domains and at least two DRDs. In certain embodiments, the modulation of payload abundance and / or function in the resulting oligomer is improved compared to the same payload in a control construct. For example, in certain embodiments, the activity level of the payload in the oligomer has a range ranging from a basal activity level in the absence of a ligand to a maximal activity level in the presence of a saturating amount of ligand, and the basal activity level of the payload in the oligomer is lower than the basal activity level of the same payload in a control construct. In certain embodiments, the activity range of the payload in the oligomer is greater than the activity range of the same payload in a control construct. Thus, a better dose response is provided in the presence of a small molecule ligand specific for the DRD.

[0007] Also described herein is an engineered regulatable oligomer comprising at least two polypeptide monomers, each monomer comprising an oligomerization domain configured to promote oligomerization of the monomers. Also described herein is an engineered regulatable oligomer comprising at least two polypeptide monomers, each monomer comprising an oligomerization domain configured to promote oligomerization of the monomers, and at least one payload having biological activity, or at least one DRD responsive to a ligand, or at least one payload having biological activity and at least one DRD responsive to a ligand, with the proviso that the oligomer comprises at least one payload and at least two DRDs. In some embodiments, the oligomer comprises at least two payloads and at least two DRDs. In some embodiments, the at least two payloads and at least two DRDs are present in a 1:1 ratio.

[0008] In some embodiments, the engineered regulatable oligomer comprises at least two polypeptide monomers, each monomer comprising an oligomerization domain, at least one payload, and at least one DRD, such that the oligomer comprises at least two payloads operably linked to at least two DRDs. The oligomer can be formed by the same or different monomers, each of which can have one or more DRDs and / or one or more payloads. When a monomer has more than one DRD and / or more than one payload, the DRDs and payloads in the oligomer are the same if the oligomer is all formed from the same monomer and all DRDs and all payloads in the monomer are the same. Alternatively, when the oligomer is formed from different monomers with different payloads and DRDs, or when the oligomer is formed from the same monomer, the DRDs and payloads in the oligomer are different, but each monomer has different payloads and different DRDs. In certain embodiments, an oligomer may have multiple of the same payloads and multiple different DRDs, or multiple different payloads and multiple of the same DRD.

[0009] These engineered regulatable oligomers are optionally hetero-oligomers that contain either different DRDs, different payloads, or different DRDs and different payloads. In some hetero-oligomer embodiments, the oligomerization domain of one of the monomers is a hetero-oligomerization domain. In certain hetero-oligomer embodiments, the engineered regulatable oligomers contain at least two polypeptide monomers that constitute a first, second, and third polypeptide monomer, where the first and second polypeptide monomers are the same, each of the first and second polypeptide monomers contains an oligomerization domain and a DRD, and the third polypeptide monomer contains a hetero-oligomerization domain and a payload.

[0010] In embodiments of tunable engineered oligomers, the regulation of the function of the payload in the oligomer is improved compared to the same payload in a control construct. For example, in certain embodiments, the biological activity of the payload in the engineered tunable oligomer has a range that ranges from a basal activity level in the absence of a ligand to a maximum activity in the presence of a saturating amount of a ligand, and the basal activity level of the payload in the oligomer is lower than the basal activity level of the same payload in a control construct. In some embodiments, the range of biological activity of the payload in the oligomer is higher than the range of the same payload in a control construct.

[0011] Also provided is a composition, referred to as an engineered regulatable polypeptide, comprising at least one payload having a biological activity level and a regulatory hub comprising at least two drug responsive domains (DRDs), the regulatory hub being operably linked to the payload, and the at least two DRDs being responsive to a ligand. The biological activity of the payload is regulated by the interaction of at least two DRDs of the regulatory hub with an effective amount of a ligand under suitable conditions. In embodiments, the regulation of the function of at least one payload in the polypeptide is improved compared to the same payload in a control construct. For example, in some such embodiments, the activity of the at least one payload ranges from a basal activity level in the absence of a ligand to a maximum activity in the presence of a saturating amount of a ligand, and the basal activity of the at least one payload is lower than the basal activity of the same payload in the control construct. As another example, the range of activity level and dose response of the at least one payload in the engineered regulatable polypeptide is optionally greater than the range of activity level and dose response of the same payload in the control construct.

[0012] Provided herein are expressible nucleic acid constructs and vectors encoding one or more engineered regulatable polypeptide monomers, which upon expression can assemble into oligomers. Also provided herein are multiple nucleic acid constructs encoding the same or different engineered regulatable polypeptide monomers, which upon expression can assemble into oligomers, the oligomers comprising at least one payload operably linked to a regulatory hub comprising at least two DRDs. In an embodiment, the oligomer comprises at least two payloads operably linked to a regulatory hub comprising at least two DRDs, and the ratio of payload:DRD is 1:1. The present disclosure also provides nucleic acid constructs encoding engineered regulatable polypeptides.

[0013] Also provided is a cell containing one or more expressible nucleic acid constructs or vectors. Optionally, the cell is a human cell, e.g., an immune cell.

[0014] The present disclosure also provides a system for altering and / or modulating (hereinafter "modulating") the function of a payload, including a ligand and any of a reference polypeptide, oligomer, nucleic acid construct, vector, or cell.

[0015] Disclosed herein is a method of modulating the function of a payload. In some embodiments, the modulating method comprises engineering a cell to express a payload operably linked to a regulatory hub comprising two or more DRDs. In such embodiments, the activity level of the payload in the absence of a ligand is reduced compared to a control cell, e.g., a cell engineered to express the payload independently of the DRDs. Optionally, the method comprises contacting a cell engineered to express a payload operably linked to a regulatory hub comprising two or more DRDs responsive to the ligand with an effective amount of a ligand such that the activity level of the payload is increased relative to a basal activity level. Optionally, the two or more DRDs are responsive to the same ligand, and the method alternatively comprises contacting the cell with a first selected dose of the ligand and a second selected dose of the same ligand, the first and second selected doses of the ligand resulting in a selected activity level of the payload, the first selected dose and the second selected dose of the ligand being close to zero, may be up to a saturating dose, may be the same or different. In embodiments in which the tuning hub comprises a first DRD responsive to a first ligand and a second DRD responsive to a second ligand, and further in which the first DRD is different from the second DRD and the first ligand is different from the second ligand, these embodiments optionally include contacting the hub with a selected amount of the first ligand and a selected amount of the second ligand, wherein the first ligand is different from the second ligand.

[0016] Disclosed herein is a method of controlling the dose or duration of administration of a payload to a subject, comprising administering to the subject one or more nucleic acid constructs, vectors, or cells described herein. The method optionally further comprises administering to the subject a selected amount of a ligand to deliver a selected activity of the payload to the subject, thereby controlling the activity level of the payload administered by the one or more nucleic acid constructs, vectors, or cells.

[0017] Also disclosed herein is a method for regulating the expression of target protein or peptide downstream of gene editing process. Such a method may include engineering cells to express engineered regulatable oligomers or polypeptides that contain payloads such as CAS9 protein or transcription factor proteins involved in the regulation of gene editors, and the payloads are operably linked to regulatory hubs. In such a method, the nucleic acid construct or vector described herein is administered to cells (in vivo, ex vivo, or in vitro), or a cell containing the nucleic acid or vector is administered to a subject to regulate the expression of target protein or peptide.

[0018] The specified embodiments are illustrative only and therefore non-limiting. The details of one or more non-limiting embodiments of the invention are set forth in the accompanying drawings and the description below. Other embodiments of the invention should be apparent to those skilled in the art after consideration of this disclosure. [Brief description of the drawings]

[0019] [Figure 1] Schematic representations of polypeptide constructs useful for understanding the scope of the present disclosure. A shows a control composition, a construct that produces a non-oligomerizing polypeptide. B shows an embodiment of a construct according to the present disclosure that includes a trimerizing oligomerization domain that produces monomers that self-assemble into a trimerized regulatable payload composition. C shows an engineered regulatable payload composition resulting from the association of monomers of B. D shows an illustrative example of an engineered regulatable payload composition having a regulatory hub with two DRDs operably connected to the payload according to certain embodiments of the present disclosure. The embodiment of D shows an embodiment of an intracellular polypeptide, since it lacks a transmembrane region. [Figure 2-1]Schematically illustrates various construct monomers, each having an oligomerization domain and a payload operably connected to a DRD. When expressed, the construct monomers self-assemble via the oligomerization domain to form a multimeric regulatable polypeptide (engineered regulatable polypeptide) according to an embodiment of the present disclosure. The degree of multimerization is generally determined by the selection of the oligomerization domain. Thus, for example, constructs having CD40L extracellular domain (ECD), collagen 18, Langerin, collectin 7, DAP12, and 4-1BB1 oligomerization domains tend to form trimerized regulatable payload compositions, while constructs having VASP oligomerization domains tend to form tetrameric regulatable payload compositions, and constructs having phospholamban oligomerization domains tend to form pentameric regulatable payload compositions. [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 3-1]Schematic diagrams of the polypeptide components encoded in the following constructs are shown: IL12-217 (SEQ ID NOs: 125 and 126), IL12-223 (SEQ ID NOs: 127 and 128), IL12-241-252 (SEQ ID NOs: 129-152), IL12-254-262 (SEQ ID NOs: 153-170), CD19-IL12-192 (SEQ ID NOs: 123 and 124), CD19-IL12-297-316 (SEQ ID NOs: 171-210), and CD19-IL12-319-332 (SEQ ID NOs: 211-238). The schematics of CD19-IL12-192 (SEQ ID NOs: 123 and 124), CD19-IL12-297-316, and CD19-IL12-319-332 do not show the CD19-CAR sequence located at the beginning of the construct, but is present separated by a P2A sequence and is included in the sequence listing for each construct. IL12-217 to IL12-262 contain mCherry separated by a P2A sequence in the construct, whereas IL12-192 and IL12-297 to IL12-332 contain CD19-CAR at the beginning of the construct. These additional domains are present and are included in the sequence listing for each construct. Certain constructs encode control compositions. Control compositions, referred to as "control constructs," are those that encode polypeptides and oligomers that lack an oligomerization domain and / or lack multiple DRDs, as defined in the definitions section herein (e.g., IL12-217 and IL12-223). Control compositions, referred to as "DRD controls," are those that lack a functional DRD. An example of a DRD control is a construct with a wild-type (wt) DRD, such as CA2(wt). Other constructs described herein are polypeptides with multiple DRDs and polypeptide monomers that can self-assemble into regulatable oligomers, according to embodiments of the present disclosure. Other constructs encode polypeptide monomers that can self-assemble into regulatable polypeptides, according to embodiments of the present disclosure. [Figure 3-2] Same as above [Figure 3-3] Same as above [Diagram 3-4] Same as above [Figure 4]A representative procedure for in vitro characterization and / or validation of CA2-regulatable membrane-bound IL12 ("mbIL12") expression in T cells is shown. On day 0, peripheral blood T cells were stimulated with Dynabeads (T-expander CD3 / CD28). On day 1, cells were transduced with lentiviral vectors. On day 6, cells were assessed for transduction efficiency using flow cytometry. As a readout for cell transduction, CD19-CAR expression was detected using CD19-Fc reagents followed by fluorescently labeled anti-human Fc antibodies. On day 8, normalized numbers of transduced (CAR+) cells were plated on 96-well plates and treated with either acetazolamide (ACZ) or an equal volume of DMSO vehicle control in the absence or presence of antigen restimulation. Stimulation conditions were either human Immunocult soluble CD3 / CD28 reagent (StemCell Technologies, Cat. No. 10971), K562 cells stably expressing the CAR antigen CD19 (Lozzio and Lozzio (1975) Blood 45:321-334) at an effector to target (E:T) ratio of 1:1, or parental control K562 cells. On day 9, mbIL12 expression on transduced T cells was analyzed. [Figure 5A] 1 provides flow cytometry graphs showing surface expression (abundance) of IL12 in control and inventive construct-transfected HEK cells, illustrating the modulation of IL12 function in HEK cells engineered according to embodiments of the present disclosure. The constructs include mCherry (not shown). [Figure 5B] 1 provides flow cytometry graphs showing surface expression (abundance) of IL12 in control and inventive construct-transfected HEK cells, illustrating the modulation of IL12 function in HEK cells engineered according to embodiments of the present disclosure. The constructs include mCherry (not shown). [Figure 5C]1 provides flow cytometry graphs showing surface expression (abundance) of IL12 in control and inventive construct-transfected HEK cells, illustrating the modulation of IL12 function in HEK cells engineered according to embodiments of the present disclosure. The constructs include mCherry (not shown). [Figure 6] 2 is a graph showing geometric mean fluorescence intensity ("MFI") indicating IL12 surface expression (abundance) in Jurkat cells transduced with control (217) and constructs of the invention (241, 243, 245, 247, 248, 254, 256, and 258, including mCherry), illustrating modulation of IL12 function in Jurkat cells according to embodiments of the present disclosure. [Figure 7] FIG. 7 is a graph showing levels of IL12p70 secreted from T cells transduced with control (192 and 223) and constructs of the invention (241, 245, 247, 256, and 260) consistent with the Jurkat cell results in FIG. 6, illustrating modulation of IL12 by embodiments of the present disclosure. Approximately equally transduced cells (15-25%) were plated in equal numbers. Constructs include mCherry (not shown) except for CD19 IL12-192. [Figure 8] Graph showing geometric MFI indicating IL12 abundance on the surface of CAR-T cells transduced with control (192) and constructs of the invention (241, 245, 247, 256, 260), demonstrating modulation of IL12 function and lower basal off-state levels compared to control constructs of the invention. Constructs include mCherry (not shown). [Figure 9] 9 provides a schematic diagram of polypeptide monomer constructs according to embodiments of the disclosure. CD19-IL12-302-306 (SEQ ID NOs: 181-189) and CD19-IL12-319-329 (SEQ ID NOs: 211-231). Although included within the test construct, the CD19-CAR sequence is not shown in FIG. 9. TM, transmembrane domain. Cyto, cytoplasmic tail. [Figure 10A]Representative MFI of membrane-bound IL12 expression in activated T cells transduced with inventive (CD19-IL12-302-306 and CD19-IL12-319-329) and control (CD19-IL12-192, empty vector (EV), untransduced (UT)) constructs is shown, demonstrating a lower basal off state of inventive engineered cells compared to control constructs. Results are shown for CD19-CAR T cells. [Figure 10B] Representative MFI of membrane-bound IL12 abundance (as determined by CD19-CAR positivity) in unactivated CAR T cells transduced with the same constructs shown in Figure 10A is shown, also demonstrating the lower basal off state of cells engineered with the present invention compared to the control construct. [Figure 11A] Graph of IL12 abundance excreted in 200 μl of media from 40,000 activated CD-19 CAR+ engineered T cells in FIG. 10A over a 20 hour period, showing that regulation was largely maintained and comparable to the results in FIG. 10A. [Figure 11B] Graph of IL12 abundance excreted in 200 μl of media from 40,000 CD-19 unactivated CAR+ in FIG. 10B over 20 hours, showing that regulation was largely maintained and comparable to the results in FIG. 10B. [Figure 12] A procedure is presented to test the abundance and activity of IL12 by measuring the phosphorylation of STAT4. [Figure 13A] 1 shows the MFI detected from pSTAT expression in the NK bystander assay described in Example 7, suggesting that modulation of the release portion of constructs according to embodiments of the present disclosure is functionally significant. [Figure 13B] A standard curve of pSTAT MFI values ​​relative to IL12 concentrations is shown. [Figure 14-1] 1A-1C are schematic diagrams illustrating additional embodiments of polypeptide monomer constructs, each comprising a payload, a DRD, and an oligomerization domain, that can self-assemble into oligomers in accordance with the present disclosure. [Figure 14-2]1A-1C are schematic diagrams illustrating additional embodiments of polypeptide monomer constructs, each comprising a payload, a DRD, and an oligomerization domain, that can self-assemble into oligomers in accordance with the present disclosure. [Figure 14-3] 1A-1C are schematic diagrams illustrating additional embodiments of polypeptide monomer constructs, each comprising a payload, a DRD, and an oligomerization domain, that can self-assemble into oligomers in accordance with the present disclosure. [Figure 15-1] FIG. 2 is a stylized diagram of a tunable payload composition (showing IL12 as an exemplary payload or a protein of interest (POI) as a generic payload, and showing CA2 as an exemplary DRD or DRD1-3 as generic DRDs) produced by cells engineered to express a nucleic acid construct according to an embodiment of the present disclosure. [Figure 15-2] FIG. 2 is a stylized diagram of a tunable payload composition (showing IL12 as an exemplary payload or a protein of interest (POI) as a generic payload, and showing CA2 as an exemplary DRD or DRD1-3 as generic DRDs) produced by cells engineered to express a nucleic acid construct according to an embodiment of the present disclosure. [Figure 16] 1 shows an exemplary stylized nucleic acid construct comprising a payload and a regulatory hub that includes more than one DRD. [Figure 17] 1 is an example of a tunable oligomer and a monomer construct that is assembled into the corresponding tunable oligomer, according to an embodiment of the present invention. [Figure 18] Schematically shows how to make the engineered regulatable constructs of the present disclosure by selecting a DRD / ligand pair, designing the polypeptide or oligomer for appropriate function, providing expression control, and providing effective delivery to target cells. SEQ ID NOs: 243 and 244 are disclosed in order of appearance, respectively. [Figure 19] 1 shows an exemplary process for designing engineered regulatable polypeptides, including membrane-bound cytokines, with sufficient dynamic range. [Figure 20]Schematic diagram of exemplary monomeric constructs for generating heterotrimers with a single payload and multiple DRDs, each construct containing DAP12 and NKG2C hetero-oligomerization domains. [Figure 21A] A histogram of cellular IL12 levels in the presence of acetazolamide (+) or DMSO (-) is shown. Cells were transduced with nucleic acids encoding the monomers shown in Figure 21B. [Figure 21B] Schematic showing assembly and oligomeric states using monomers (top panel) and an exemplary heterodimer (IL12-267 is a control construct with only one DRD) shown in Figure 20. IL12-266 and 268 represent engineered regulatable homodimers (IL12-266) and heterotrimers (IL12-268) with regulatory hubs containing multiple DRDs. [Figure 21C] Shown is the fold change in geometric MFI of IL12 following drug addition for each of IL12-266, 267, and 268. Transduced cells show a pattern of increasing fold regulation as they progress from monomer, dimer to trimer. [Figure 22] Graph showing surface IL23 expression on CD19 CAR+ cells plotted using Prism Software. [Figure 23] Graph showing IFNα expression on CD19 CAR+ cells plotted using Prism Software. [Figure 24] Graph showing IL2 expression on CD19 CAR+ cells plotted using Prism Software. [Diagram 25] FIG. 1 shows a schematic diagram illustrating the experimental design for determining IL18 expression. [Figure 26] FIG. 1 is a schematic diagram showing the experimental design for determining IFNγ levels. [Figure 27] FIG. 1 is a graph showing IL18 expression in unactivated T cells transformed with a non-regulated IL18 construct. [Figure 28]FIG. 1 is a graph showing IL18 expression in activated T cells transformed with a non-regulated IL18 construct. [Figure 29] Graph showing interferon gamma (IFNγ) expression in naive T cells transformed with unregulated IL18 constructs. [Diagram 30] FIG. 1 is a graph showing interferon gamma (IFNγ) expression in IL15-treated T cells transformed with a non-regulated IL18 construct. [Diagram 31] FIG. 1 is a graph showing IL18 expression in unactivated T cells transformed with regulated IL18 constructs. [Diagram 32] FIG. 1 is a graph showing IL18 expression in activated T cells transformed with regulated IL18 constructs. [Diagram 33] FIG. 13 is a graph showing IL18 expression on day 8 in unactivated T cells transformed with regulated IL18 constructs. [Diagram 34] FIG. 1 is a graph showing IL18 expression on day 10 in unactivated T cells transformed with regulated IL18 constructs. [Diagram 35] FIG. 1 is a graph showing IL18 expression on day 8 in activated T cells transformed with regulated IL18 constructs. [Diagram 36] FIG. 1 is a graph showing IFNγ-expressing T cells treated with 20 ng / mL IL15 and transformed with regulated IL18 constructs. [Figure 37] FIG. 1 is a graph showing IFNγ-expressing T cells treated with 20 ng / mL IL15 and transformed with regulated IL18 constructs. [Figure 38] FIG. 1 is a bar graph showing IL18 expression in T cells transformed with regulated IL18 constructs in the presence and absence of acetazolamide (ACZ). [Figure 39] 1 is a bar graph showing the geometric mean fluorescence intensity (GMFI) of T cells expressing membrane-bound IL18 in the presence and absence of ACZ. [Diagram 40]FIG. 1 is a bar graph showing IL18 expression in T cells transformed with regulated IL18 constructs in the presence and absence of ACZ. [Diagram 41] Figure 1 is a graph showing IL18 expression and excretion in untransformed (UT) cells or cells transformed with IL18-064 in the presence and absence of ACZ. The first three columns are unactivated cells and the last three columns are activated cells. Unactivated cells with IL18-064 treated with ACZ showed a 1.7-fold increase in excretion of IL18 with ACZ. Activated cells with IL18-064 treated with ACZ showed a 3.1-fold increase in IL18 expression and a 2.1-fold increase in IL18 excretion. [Diagram 42] Figure 1 is a graph showing IL18 expression and excretion in untransformed (UT) cells or cells transformed with IL18-066 in the presence and absence of ACZ. The first three columns are unactivated cells and the last three columns are activated cells. Unactivated cells with IL18-066 treated with ACZ showed a 2.0-fold increase in excretion of IL18 with ACZ. Activated cells with IL18-066 treated with ACZ showed a 3.1-fold increase in IL18 expression and a 2.6-fold increase in IL18 excretion. [Diagram 43] Figure 1 is a graph showing IL18 expression and excretion in non-transformed (UT) cells or cells transformed with IL18-069 in the presence and absence of ACZ. The first three rows are unactivated cells and the last three rows are activated cells. Activated cells with IL18-069 treated with ACZ showed a 1.9-fold increase in IL18 expression and a 1.3-fold increase in IL18 excretion. [Diagram 44] Figure 1 is a graph showing IL18 expression and secretion in non-transformed (UT) cells or cells transformed with IL18-070 in the presence and absence of ACZ. The first three rows are unactivated cells and the last three rows are activated cells. Activated cells with IL18-070 treated with ACZ showed a 1.8-fold increase in IL18 expression and a 1.1-fold increase in IL18 secretion. [Diagram 45]Figure 1 is a graph showing IL18 expression and secretion in non-transformed (UT) cells or cells transformed with IL18-071 in the presence and absence of ACZ. The first three rows are unactivated cells and the last three rows are activated cells. Activated cells with IL18-071 treated with ACZ showed a 1.8-fold increase in IL18 expression and a 1.2-fold increase in IL18 secretion. [Figure 46] FIG. 1 is a bar graph showing IL18 expression in T cells transformed with the IL18-048 construct and treated with 20 ng / mL IL15 in the presence and absence of ACZ. [Figure 47] FIG. 1 is a bar graph showing IL18 expression in T cells transformed with the IL18-064 construct and treated with 20 ng / mL IL15 in the presence and absence of ACZ. [Figure 48] FIG. 1 is a bar graph showing IL18 expression in T cells transformed with the IL18-066 construct and treated with 20 ng / mL IL15 in the presence and absence of ACZ. [Figure 49] FIG. 1 is a bar graph showing IL18 expression in T cells transformed with the IL18-069 construct and treated with 20 ng / mL IL15 in the presence and absence of ACZ. [Figure 50] FIG. 1 is a bar graph showing IL18 expression in T cells transformed with the IL18-070 construct and treated with 20 ng / mL IL15 in the presence and absence of ACZ. [Figure 51] FIG. 1 is a bar graph showing IL18 expression in T cells transformed with the IL18-071 construct and treated with 20 ng / mL IL15 in the presence and absence of ACZ. [Figure 52] 1 is a bar graph showing IL18 expression and shedding in T cells transformed with the IL18-064 construct in the presence and absence of ACZ. [Figure 53] 1 is a bar graph showing IL18 expression and shedding in T cells transformed with the IL18-066 construct in the presence and absence of ACZ. [Figure 54]FIG. 1 is a bar graph showing IL18 expression and shedding in T cells transformed with the IL18-048 construct in the presence and absence of ACZ. [Figure 55] FIG. 1 is a graph showing IL18 expression by Jurkat cells in the presence and absence of ACZ. [Figure 56] FIG. 1 is a graph showing surface recognition of IL12 on resting T cells. [Figure 57] FIG. 1 is a graph showing IL12 excretion in resting T cells transfected with control and experimental IL12 constructs. [Figure 58] 1 is a graph showing shed IL12 activity on NK cells. [Figure 59] FIG. 1 is a graph showing surface recognition of IL12 on CD3 / CD28 stimulated activated T cells. [Figure 60] FIG. 1 is a graph showing IL12 excretion in activated T cells transduced with control and experimental IL12 constructs. [Figure 61] 1 is a graph showing shed IL12 activity on NK cells. [Figure 62] FIG. 1 is a schematic showing the experimental design for in vivo studies of IL12 regulation. [Figure 63] Graphs A-C show that ACZ regulates mbIL12 on CD19-CART in vivo. [Figure 64] FIG. 13 is a graph showing that oligomerizing constructs excreted IL12 in plasma regulated by ACZ in a non-zero off state. [Figure 65] FIG. 1 is a schematic showing the scRaji solid tumor model study design. [Figure 66] Graph showing tumor growth in control groups CAR alone or empty vector (EV). [Figure 67] Graph showing tumor growth in mice given CAR-T cells with or without ACZ. [Figure 68]Graph showing that infusion of IL12-expressing CAR-T cells exhibits delayed but robust regulated anti-tumor activity, even at suboptimal doses of CAR-T cells. [Figure 69] A and B are graphs showing that regulatory hubs enable ACZ-induced control of IL12 in plasma. [Figure 70] (A and B) are graphs showing that the mbIL12 regulatory hub enables control of systemic IFNγ. Notably, by day 14, IFNγ levels return to baseline for all mbIL12 constructs except those turned on with ACZ. [Figure 71] A and B are graphs showing that the mbIL12 regulatory hub enables control of IFNy in tumors (7 days after ACT). [Figure 72] (A and B) are graphs showing that the mbIL12 regulatory hub enhances localization of IFNy production to tumors versus circulation. The higher IFNy ratio in tumors for regulated versus constitutive mbIL12 is likely due to the activation-dependence of IL12 regulation. [Figure 73] FIG. 1 is a graph showing that IL12 in the on state leads to increased activation of dendritic cells. [Figure 74] FIG. 1 is a graph showing that IL12 in the ON state leads to increased activation of MHCII monocytes. [Figure 75] 1 is a graph showing the percentage of tumor-associated macrophages decreased by IL12. [Figure 76] Graph showing the percentage of TAMs with a TAM phenotype decreased by IL12. [Figure 77] FIG. 1 is a graph showing that IL12 skews TAMs towards an M1-like phenotype. [Figure 78] Graph showing that PD-L1 is more upregulated with secreted IL12 than with mbIL12 across multiple cell types and tissues. [Figure 79]Flow cytometry readout showing IL12 expression in Jurkat cells transduced with IL12 constructs in the presence or absence of VDF 10 μM for 24 hours. [Figure 80] 1 is an image showing the percentage of Jurkat cells expressing IL12 in the presence or absence of VDF 10 μM over a 24 hour period. [Figure 81] 1 is a graph showing geometric MFI, which indicates IFNα abundance on the surface of tumor-infiltrating lymphocytes. [Figure 82] 1 is a graph showing regulation of IFNα by ACZ. [Figure 83] FIG. 1 is a schematic diagram showing T cell transduction experiments using constructs containing IL15 and IFNα. [Figure 84] 1 is a graph showing enrichment of mbIL15 on the surface of T cells over time. Patterned bars refer to +ACZ and solid bars refer to +Veh. The first set of bars (solid bars, then patterned bars) is for cells containing IFNα-003, the next set of bars is for cells containing IFNα-007, the third set of bars is for cells containing IFNα-015, the fourth set of bars is for cells containing IFNα-016, and the fifth and final set of bars is for cells containing IFNα-018. [Figure 85]Graphs showing that IFNα is regulated approximately 20-fold in the IFNα-016 and -018 constructs. Pattern bars refer to +ACZ and solid bars refer to +Veh. The first set of bars (solid bars, then pattern bars) is for cells containing IFNα-003, the next set of bars is for cells containing IFNα-007, the third set of bars is for cells containing IFNα-015, the fourth set of bars is for cells containing IFNα-016, the fifth set of bars is for cells containing IFNα-018, and the sixth and final set of bars is for control cells with empty vector. [Figure 86] FIG. 1 is a schematic diagram showing the Jurkat cell transduction experimental layout. [Figure 87] 1 is a bar graph showing the percentage of IL15 expressing cells following transduction with IL18 constructs. 300 μl of virus yielded 70%-90% transduced cells, 100 μl of virus yielded 40%-65% transduced cells, and 33 μl of virus yielded 7%-33% transduced cells. [Figure 88] FIG. 1 is a graph showing membrane-bound IL18 regulation observed with VEGFR-containing constructs. [Figure 89] 1 is a bar graph showing the 10-fold modulation of membrane IL18 (geometric MFI) observed in the IL18-080 construct. The bar graph refers to the left y-axis and indicates mbIL18 expression. The numbers at the bottom indicate the fold change of mbIL18. The circles refer to the right y-axis and indicate IL18 released in the medium. The numbers at the top indicate the fold change of excreted IL18. [Figure 90] The regulation of IL18 percentage on the cell membrane was more pronounced with the IL18-080 construct. The numbers indicate the fold change of mbIL18 percentage. IL18-081 and -082 show very high fold changes due to the very low values ​​of IL18 percentage in the off state. [Figure 91]1 is an image showing small-scale viral transduction in Jurkat cells for SOX2 constructs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure provides compositions and systems for regulatory control of a payload ("regulatory compositions and systems"). A regulatory composition according to the present disclosure comprises a regulatory hub, which comprises at least two drug responsive domains (DRDs) responsive to a ligand and at least one payload operably linked to the regulatory hub. The regulatory composition also comprises an engineered regulatable polypeptide monomer (or polypeptide monomer, or simply monomer), an engineered regulatable oligomer (or regulatable oligomer, or simply oligomer), and an engineered regulatable polypeptide (or regulated polypeptide, or simply polypeptide). The regulatable oligomer is formed from a regulatable monomer, and the regulatable oligomer and the regulatable polypeptide comprise at least one payload operably linked to the regulatory hub. A regulatory system according to the present disclosure comprises a regulatory composition and a stabilizing ligand.

[0021] In some embodiments, the present disclosure is useful for enabling modulation of biologically active payloads. For example, the present disclosure describes improved drug responsive domain (DRD)-based modulation of payloads by improving control over the off-state of the payload (i.e., reducing the abundance or availability of the payload in the absence of a ligand) and / or optimizing the range of modulation, compared to DRD-modulating systems that include a single DRD.

[0022] Without meaning to be limited by theory, it is believed that DRDs are unstable polypeptides that degrade in the absence of a corresponding stabilizing ligand (also referred to as a paired ligand or ligand), but whose stability is rescued by binding to a stabilizing ligand. Because the binding of the ligand to the DRD is reversible, subsequent removal of the ligand causes the DRD to unfold, become unstable, and ultimately become tagged for degradation by the ubiquitin-proteasome system ("UPS"). Thus, when the DRD is operably linked to a payload, the entire construct (i.e., the DRD and payload) itself is believed to be destabilized and degraded by the UPS. However, in the presence of the paired ligand, the construct is stabilized and the payload remains available. Furthermore, it is believed that the conditional nature of DRD stability may allow for a rapid, non-perturbative switch from a stable polypeptide to an unstable UPS substrate, facilitating the regulation of the activity level of the payload and / or the tuning of the activity level of the payload. The underlying discovery that led to the improved regulatory control is that multiple DRDs associated together in a regulatory hub result in a payload with a lower off-state compared to the same payload operably linked to a single DRD. That is, in the absence of a ligand, increasing the density of DRDs (e.g., having multiple DRDs) will reduce the abundance or availability of payload compared to a payload that is operably linked to only a single DRD.

[0023] Payloads should be understood to include one or more polypeptides having one or more functions, such as one or more biological activities, that are desired to be modulated. Payloads include multiple classes of therapeutically important polypeptides (proteins and peptides), such as type I / II membrane proteins, cytokines, intracellular proteins, secreted proteins, CAS9 proteins, and transcription factor proteins.

[0024] Because the abundance and availability of a payload is related to the activity of the 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 expressly stated otherwise or meaningless in context. Furthermore, measurements of abundance or availability are used as proxies for and may be used herein to reflect activity levels. Thus, a change in abundance or availability of a payload in the presence of an effective amount of a ligand compared to the absence of the ligand optionally serves as a proxy for measuring a change in activity level.

[0025] Engineered Regulatable Polypeptides Provided herein is an engineered regulatable polypeptide having at least one payload and a regulatory hub comprising at least two drug responsive domains (DRDs), the regulatory hub being operably linked to the payload, and the at least two DRDs being responsive to a ligand. The at least two DRDs may be the same or different, and optionally, the at least two DRDs are responsive to the same ligand. For example, in certain embodiments, the regulatory hub comprises three DRDs, and the three DRDs are responsive to the same ligand. The at least one payload has a biological activity, and the biological activity of the payload is regulated by the interaction of two or more DRDs (e.g., two, three, four, or more, responsive to the same or different ligands) with an effective amount of a ligand. In the case of multiple different DRDs responsive to different ligands, reference to a "ligand" is understood to mean "multiple ligands." The activity level of at least one payload ranges from a basal activity level in the absence of a ligand to a maximum activity level in the presence of a saturating amount of the ligand, and the basal activity level of the payload in the regulatable polypeptide is lower than the basal activity level of the same payload in the control construct. A saturating amount of the ligand refers to an amount of any ligand equal to or greater than the amount that results in the maximum abundance and / or activity of the payload. As an example, the activity level of at least one payload ranges from a basal activity level in the absence of a ligand to a maximum activity level in the presence of a saturating amount of the ligand, and the activity level range of the payload in the regulatable polypeptide is higher than the activity level range of the same payload in the control construct. In some embodiments, the at least one payload is a multiple payload (e.g., two payloads or three payloads that are the same or different from each other). Thus, for example, in some embodiments, the at least one payload is a three payload, and the at least two DRDs are three DRDs, and the three payloads are the same, and the three DRDs are responsive to the same ligand.In certain embodiments, the at least one payload is a single payload and the at least two DRDs are three DRDs responsive to the same ligand. Figure ID shows an illustrative example of an engineered regulatable polypeptide having a regulatory hub with two DRDs operably connected to a payload.

[0026] Polypeptide Monomers and Engineered, Tunable Oligomers Provided herein are engineered polypeptide monomers and engineered regulatable oligomers of at least two engineered polypeptide monomers. The oligomers comprise at least one payload operably linked to a regulatory hub comprising two or more DRDs. The payload has a biological activity, and the biological activity of the payload is regulated by interaction of the two or more DRDs (e.g., two, three, four, or more, responsive to the same or different ligands) with an effective amount of a ligand.

[0027] In some embodiments, the monomer comprises at least one payload, an oligomerization domain (e.g., a dimerization domain, a trimerization domain, a tetramerization domain, a pentamerization domain, or a hexamerization domain) configured to promote oligomerization of the monomer, and at least one DRD responsive to a ligand, wherein the at least one payload is operably linked to the at least one DRD. In certain embodiments, the monomer comprises an oligomerization domain and at least one payload, or at least one DRD, or at least one payload and at least one DRD, provided that when at least two or more monomers oligomerize to form an oligomer, the resulting oligomer comprises at least one payload operably linked to a regulatory hub comprising at least two DRDs.

[0028] The oligomer according to the present disclosure is a multimeric association of at least two engineered regulatable polypeptide monomers. The monomers of the oligomer are associated through an oligomerization domain. The oligomer can be, for example, a dimer resulting from the association of monomers through a dimerization domain, or a trimer resulting from the association of monomers through a trimerization domain, or a tetramer resulting from the association of monomers through a tetramerization domain, or a pentamer resulting from the association of monomers through a pentamerization domain, or a hexamer resulting from the association of monomers through a hexamerization domain. In each case, the oligomer can be formed from the same or different monomers. Thus, the one or more payloads of each of the monomers are the same or different, and the DRD of each of the monomers are the same or different. Thus, by way of example, an oligomer may be an association of three monomers, each monomer comprising the same payload, oligomerization domain, and DRD, such that the oligomer comprises first, second, and third payloads that are the same, and a regulatory hub comprising first, second, and third DRDs that are responsive to the same ligand.

[0029] Thus, also provided herein is an engineered regulatable oligomer comprising at least two polypeptide monomers, each monomer comprising an oligomerization domain and at least one payload or one DRD, the oligomer comprising at least one payload operably linked to at least two DRDs, the at least one payload having biological activity, and the at least two DRDs being responsive to a ligand. Optionally, the oligomer is a hetero-oligomer, wherein at least two polypeptide monomers of the hetero-oligomer each comprise an oligomerization domain, and in some embodiments, the oligomerization domain of one of the polypeptide monomers comprises a hetero-oligomerization domain. Optionally, the DRDs in the hetero-oligomer are the same or different. Optionally, the at least two DRDs are responsive to the same ligand. As an example, an engineered tunable hetero-oligomer comprises a first, second and third polypeptide monomer, where the first and second polypeptide monomers are the same, each of the first and second polypeptide monomers comprises a DRD, and the third polypeptide monomer comprises a hetero-oligomerization domain and a payload. Figure 21B shows a heterotrimer comprising one payload and two or three DRDs, as well as a control construct having only one DRD. In certain hetero-oligomer embodiments, each monomer comprises the same oligomerization domain, but comprises a different payload and / or DRD. A hetero-oligomer construct may also comprise a first and second monomer, each comprising a DRD and a payload, as well as a third monomer comprising a DRD without a payload. However, an engineered tunable hetero-oligomer may have a payload:DRD ratio of 1:1.

[0030] The multiple payloads in an oligomer may be the same or different, but in embodiments, each of the payload(s) has biological activity under appropriate conditions. In some such embodiments, the activity level of each payload in the oligomer has a range ranging from a basal activity level in the absence of ligand to a maximum activity in the presence of a saturating amount of ligand, and the basal activity of each payload in the oligomer is lower than the basal activity of the same payload in a control construct. In some embodiments, the activity range of each payload in the oligomer is higher than the activity range of the same payload in a control construct. See, for example, Examples 3-5 below.

[0031] Also provided is an oligomer comprising at least two polypeptide monomers, each monomer comprising at least one payload, an oligomerization domain, and a DRD, the oligomer comprising at least two payloads operably linked to at least two DRDs, the at least two payloads having a level of biological activity, the DRD being responsive to a ligand, and in some embodiments, the at least two payloads and the at least two DRDs are present in a ratio of 1:1. In certain embodiments, the payload is present in the oligomer in a ratio of 1:2, 1:3, 2:3, 1:4, 3:4, 1:5, 2:5, 3:5, or 4:5 payload to DRD, or a ratio of 2:1, 3:1, 3:2, 4:1, 4:3, 5:1, 5:2, 5:3, or 5:4 payload to DRD, with the proviso that the oligomer always comprises at least two DRDs. In some embodiments, at least two DRDs may be the same or different, at least one payload may be multiple payloads, which may be the same or different, or multiple DRDs may be different but responsive to the same ligand. The oligomer may, for example, include a tuning hub including a first, second, and third DRD responsive to the same or different ligands, and a first, second, and third payload, which may be the same or different. In some embodiments, the first, second, and third DRDs are responsive to the same ligand, and the first, second, and third payloads are the same. The activity level of each payload in the oligomer has a range ranging from a basal activity level in the absence of ligand to a maximum activity in the presence of a saturating amount of ligand, and the basal activity of each payload in the oligomer is lower than the basal activity of the same payload in a control construct. Optionally, the activity level range of the payload in the oligomer is higher than the activity level range of the same payload in a control construct.

[0032] FIG. 1 shows exemplary polypeptide constructs useful for understanding the scope of the present disclosure provided. FIG. 1A shows a control construct having a payload, hinge region, and DRD. FIG. 1B shows a polypeptide monomer construct according to the present disclosure that includes an oligomerization domain that produces monomers that self-assemble into oligomers according to the present disclosure. FIG. 1C shows an engineered tunable oligomer resulting from the association of the monomers of FIG. 1B.

[0033] 2 shows a schematic diagram of various polypeptide monomer constructs, each having an oligomerization domain and a payload operably connected to a DRD. When expressed, the polypeptide monomers self-assemble via the oligomerization domain to form multimeric regulatable polypeptides (engineered regulatable payload compositions, also called oligomers when formed from monomers) according to embodiments of the present disclosure. The degree of multimerization is generally determined by the selection of the oligomerization domain. Thus, for example, constructs having CD40L ECD, collagen 18, lectin, DAP12, and 41BB1 oligomerization domains tend to form trimerized regulatable payload compositions, while constructs having VASP oligomerization domains tend to form tetrameric regulatable payload compositions, and constructs having phospholamban oligomerization domains tend to form pentameric regulatable payload compositions.

[0034] FIG. 3 shows a schematic diagram of the polypeptide components of constructs IL12-217 (SEQ ID NOs: 125 and 126), IL12-223 (SEQ ID NOs: 127 and 128), IL12-241-262 (SEQ ID NOs: 129-170), CD19-IL12-192 (SEQ ID NOs: 123 and 124), CD19-IL12-297-316 (SEQ ID NOs: 171-210), and CD19-IL12-319-332 (SEQ ID NOs: 211-238). Certain constructs are control constructs for the tunable compositions and lack an oligomerization domain resulting in a payload operably linked only to a single DRD (e.g., IL12-217, IL12-223, and CD19-IL12-192), others are control constructs for the DRD and include wild-type CA2 polypeptide in place of mutated CA2 (242, 246, 249, 252, 255, 257, 259, 262, 297), while other constructs (e.g., IL12-241) are polypeptide monomers that can self-assemble into tunable payload compositions according to embodiments of the present disclosure.

[0035] Monomers and components of regulatable polypeptides Exemplary components (building blocks) of engineered monomers that assemble to form regulatable engineered oligomers and exemplary components (building blocks) of engineered regulatable polypeptides described herein are referenced throughout this disclosure and provided below. As described herein, the monomers include at least an oligomerization domain and a payload, or a DRD, or a payload and a DRD. Also, as described herein, the oligomers and polypeptides include one or more payloads and a regulatory hub that includes at least two DRDs. The monomers, oligomers, and polypeptides optionally include one or more additional components, such as a linker, a hinge (e.g., excavable and non-excavable hinges), a tail (e.g., a cytoplasmic tail), and a transmembrane domain. As described below in the section on how to make, one of skill in the art can select from a variety of components using the guideposts provided below to achieve a desired result (e.g., the location of the oligomerization domain, payload, or DRD relative to the cell in which it is expressed, whether the payload is secreted from the cell or is membrane-linked, or the desired activity of the payload or payloads).

[0036] Adjustment Hub In general, a tuning hub is a polypeptide designed to facilitate modulation of a property (e.g., abundance or activity) of a target payload to which it is operably linked and whose basic building block is a DRD. A tuning hub according to the present disclosure comprises at least two DRDs, and in some embodiments, such a hub can be formed upon oligomerization of at least two polypeptide monomers as described herein. Thus, in some embodiments, at least two DRDs are linked together via a non-covalent bond formed by oligomerization, optionally by an oligomerization domain associated with each DRD, as shown in Figures 15A-E. In some embodiments, at least two DRDs are linked by a covalent bond, optionally via a linker, as shown in Figures 15F and 16.

[0037] DRD The DRD interacts with a ligand such that when the DRD is operably linked to a payload, it provides a ligand-dependent reversible modulation of the properties (e.g., activity) of the payload. Although referred to as a drug-responsive domain, the ligand to which the DRD is responsive does not have to be a drug. Suitable DRDs (and their paired ligands), which may be referred to as destabilization domains or ligand-binding domains, are also known in the art. See, for example, U.S. Patent Nos. 9,487,787 and 10,137,180, U.S. Publication Nos. 2019 / 0192691, 2020 / 0101142, 2020 / 0172879, and 2021 / 0069248, as well as U.S. Patent Application Nos. 17,251,635 and 17 / 288,373, and WO 2013 / 023666. O2018 / 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. 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:e1000376, and Feng et al. (2015) Elife 4:e10606, the contents of each of which are incorporated by reference in their entireties.

[0038] Specific examples of these and other exemplary DRDs suitable for use with a coordination hub according to this disclosure are provided elsewhere herein.

[0039] The DRD can be selected from, for example, FKBP (SEQ ID NO: 93), ecDHFR (SEQ ID NO: 18), hDHFR (SEQ ID NO: 37), ER (SEQ ID NO: 96), PDE5 full length (SEQ ID NO: 95), PDE5 ligand binding domain (SEQ ID NO: 94) and CA2 (SEQ ID NO: 1), or any portion of the above 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% identity to SEQ ID NO: 1, 18, 37, 93, 94, 95, or 96, or a DRD functional portion thereof. For example, one or more mutations (including truncations, substitutions, and deletions) in the amino acid sequences of FKBP, ecDHFR, hDHFR, ER, PDE5, and CA2 can be advantageous to further destabilize the DRD.

[0040] While numerous DRDs are described herein, one of skill in the art can identify additional DRDs suitable for use in the regulatory hubs and regulatable compositions of the present disclosure. By way of example, DRDs can be identified using library screening and structure-guided engineering to select optimal DRD variants that have sufficient instability in the absence of ligand and sufficient stability in the presence of ligand. Random mutagenesis screening can be used to generate variant libraries by transducing cells (e.g., Jurkat cells) with mutant DRD candidates. To generate enriched libraries, cells with the desired properties (low basal activity / expression and high dynamic range) are selected by testing polypeptide abundance over a range of ligand concentrations. Single cell clones are then generated and characterized to identify candidate DRDs.

[0041] The DRDs described herein are responsive to a paired ligand (also referred to as a "stabilizing ligand" or simply as a "ligand"). Optionally, the DRD is 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 a DRD and its paired ligand to meet the particular needs of the system. Table 1 provides examples of DRD / ligand pairs. [Table 1]

[0042] Optionally, the DRD of the present disclosure may be derived from carbonic anhydrase, a member of the superfamily of metalloenzymes. For example, human carbonic anhydrase (hCA2) may be adapted for use as a DRD. The DRD of the present disclosure may be derived from amino acids 1-260 of CA2 (Uniprot ID: P00918). Optionally, the DRD is derived from CA2 comprising amino acids 2-260 (e.g., amino acids 2-260) of the parent CA2 sequence. This is referred to herein as CA2 M1del mutation (CA2, SEQ ID NO: 5). Optionally, the DRD of the present disclosure comprises a region or the entirety of human carbonic anhydrase 2, further comprising one or more mutations to the full length sequence selected from M1del, L156H, and S56N. Optionally, the DRD is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, and 9.

[0043] Oligomerization Domain For purposes of this disclosure, an oligomerization domain is a sequence that promotes self-assembly from monomers into oligomers, for example, by non-covalent binding. Such domains may be found in nature and may be adapted for use in forming oligomers containing the payloads and regulatory hubs described herein. Examples of oligomerization domains include, but are not limited to, phospholamban, collectin, collagen, VASP, CD40L, TNFSF (e.g., TNFSF14, also referred to herein as LIGHT and 4-1BBL), Langerin, DAP12, and NKG2C. Optionally, the oligomerization domain is phospholamban transmembrane domain, collagen 18, avidin, streptavidin, CD40L extracellular domain (ECD), Langerin ECD, 4-1BBL ECD, VASP tetramer, DAP12, LIGHT extracellular domain (ECD), collectin 7, fungal lectin (5xzk), aspartate transcarbamoylase (1EKX), 4-OT trimer, RH3 designed coiled coil (1TGG), HIV-1 gp41, FIV dUTP pyrophosphate, SIV gp41, Ebola virus gp-2, HTLV-1 gp-21 domain, a foldon domain, a GCN4 domain, a T4 fibritin domain, yeast heat shock transcription factor, and human collagen VIII, collagen 18, collagen 15, and hnRNP. Certain oligomerization domains do not oligomerize by themselves, but only with a different oligomerization domain, such as NKG2C, such oligomerization domains are referred to herein as hetero-oligomerization domains.

[0044] Figures 15A-E provide non-limiting examples of embodiments of oligomerization domains described herein. Specifically, in the exemplary embodiment of Figures 15A and E, the ECD motif of CD40L (SEQ ID NO: 15) corresponds to the oligomerization domain. In Figure 15B, the trimerization motif of collagen 18 (SEQ ID NO: 19) corresponds to the oligomerization domain. In Figure 15C, the dimerization / trimerization motif of DAP12 (SEQ ID NO: 79) corresponds to the oligomerization domain. In Figure 15D, the trimerization motif of collectin 7 (SEQ ID NO: 23) corresponds to the oligomerization domain. Although Figure 15 shows the use of a trimerization motif as an oligomerization domain, any polypeptide motif that facilitates self-assembly or associates into a larger construct can be used as the basis for an oligomerization domain according to the present disclosure, including a dimerization motif or a motif that results in four or five or six or more polypeptide motifs assembled together. For example, the VASP motif represents an example of a suitable motif for use as an oligomerization domain that promotes self-assembly into a tetramer. As another example, the phospholamban motif represents an example of a suitable motif for use as an oligomerization domain that leads to self-assembly into a pentameric regulatory hub.

[0045] Thus, while the exemplary regulatory hubs shown in FIG. 16 all include three DRDs, regulatory hubs according to the present disclosure may include two or more, three or more, four or more, five or more, six or more, etc. DRDs. Furthermore, the DRDs forming the regulatory hubs may be the same or different. Thus, for example, in FIG. 15F, DRD1, DRD2, and DRD3 may each be the same DRD, or DRD1, DRD2, and DRD3 may each be different DRDs, or two of the DRDs (e.g., DRD1 and DRD2) may be the same DRD while DRD3 may be a different DRD. FIG. 17 shows a schematic construct (top) of a monomer having a payload, hinge, transmembrane region, cytoplasmic tail, oligomerization domain, and an oligomer resulting from self-assembly of the monomer. Any DRD may be used to form a regulatory hub, and specific non-limiting examples of suitable DRDs are referenced throughout this application, including the patents and applications referenced herein.

[0046] payload By way of example, the payload can be any polypeptide with a desired biological function. Such payloads can be modified polypeptides, such as glycosylated polypeptides or lipopeptides, that are modified intracellularly upon expression by constitutive enzymatic activity or by overexpression of a selected enzyme. 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 IL15, IL12, IL1β, IL2, IL7, IL18, IL21, IL23, IL36, TNFα, IFNγ, IFNα, IFNβ, including their membrane-bound forms 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 natural cytokines, or active portions thereof. The payload also includes transcription factors such as Foxp3, c-Myc, STAT5, and c-Jun, or their active parts, or their constitutively active forms. Other examples of suitable payloads include cytokine receptors, T cell receptors (TCRs), chimeric antigen receptors (CARs), immune-modulating proteins, or any active parts thereof, in addition to those already exemplified. The payload can also be a gene editing polypeptide or a transcription factor, in addition to those previously exemplified. The payload can also be a combination of polypeptides, or their active parts, with the desired combination of actions.

[0047] In some embodiments, the payload can be a therapeutic agent selected from a cancer therapeutic agent, a therapeutic agent for an autoimmune disease, an immunotherapeutic agent, an anti-inflammatory agent, an anti-pathogenic agent, a gene therapy agent, or a combination thereof. The immunotherapeutic agent can 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 mutant form of a cytokine (also called a mutein) with altered receptor binding properties, a cytokine receptor, a chemokine, a chemokine receptor, a metabolic factor, a clotting factor, an enzyme, a homing receptor, a kinase, a phosphatase, a dominant negative form of a phosphatase (such as SHP-1 or SHP-2), a dominant negative signaling molecule or receptor (e.g., a dominant negative Fas receptor), a dominant negative transcription factor, and a safety switch.

[0048] In some embodiments, the payload of the present disclosure may be a cytokine, as well as fragments, variants, analogs, and derivatives thereof, including, but not limited to, interleukins, tumor necrosis factors (TNFs), interferons (IFNs), TGFβ, and chemokines. Interleukins may be selected from IL15, IL12, IL1β, IL2, IL7, IL18, IL21, IL23, IL36, and variants thereof, including membrane-bound, secreted, fusion polypeptides, or cytokine mutants (such as muteins) with altered receptor binding properties, and bicistronic forms of interleukins, and combinations thereof.

[0049] For example, IL12 may contain both p35 and p40 subunits encoded by a single nucleic acid that produces a single chain polypeptide. The single chain polypeptide may be generated by placing the p35 subunit at the N-terminus or C-terminus of the single chain polypeptide. Similarly, the p40 subunit may be at the N-terminus or C-terminus of the single chain polypeptide. As another example, the payload may be a bicistronic IL12 that contains p40 and p35 subunits.

[0050] In some embodiments, the payload can be an active portion or variant of a polypeptide having a desired biological function, so long as the payload retains the desired biological function. By way of example, the payload can be the p40 subunit of IL12 or the p35 subunit of IL12, or a variant of IL12 that promotes NK cell survival, regulates NK cell and T cell activation and proliferation, and / or supports NK cell development from hematopoietic stem cells.

[0051] In some embodiments, the payload of the present disclosure may be a chimeric antigen receptor (CAR) that includes an extracellular targeting domain (e.g., an scFv that recognizes a specific tumor antigen or other tumor cell surface molecule), a transmembrane domain / region, and an intracellular signaling / activation domain (e.g., the signaling region of CD3ζ, and / or one or more costimulatory signaling domains, such as the signaling domains from CD28, 4-1BB (CD137), and OX-40 (CD134)).

[0052] In some embodiments, a payload can be selected that reduces the immune response in a subject. For example, the payload can be a neutralizing antibody against tumor necrosis factor (TNF)-α or an anti-cytokine such as an interleukin. In some embodiments, the payloads of the present disclosure target B cell depletion, such as neutralizing antibodies against CD20, CD22, CD28, CTLA-4, and B lymphocyte stimulator (BLyS).

[0053] In some embodiments, the payload may also be a contractile protein (e.g., actin and myosin), an enzyme (e.g., lactase and pepsin), a hormone (e.g., insulin, oxytocin, and somatotropin), a structural protein (e.g., keratin, collagen, and elastin), a storage protein (e.g., ovalbumin and ferritin), a transport protein (e.g., hemoglobin), a membrane-associated protein (e.g., class I, II, or III transmembrane proteins, receptors, transporters, etc.).

[0054] In some embodiments, the payload of the present disclosure can be one or more components of a gene editing system. In such embodiments, the oligomer or engineered regulatable polypeptide regulates the activity of the gene editing system, thereby regulating the expression of a downstream target protein. As used herein, a target protein refers to a protein selected for gene editing, including, for example, a protein with a genetic mutation that causes a deleterious effect in a subject or cell. For example, the payload of the present disclosure can be a Cas protein (CRISPR-associated protein), including Cas9 and Cas12. The Cas protein can be altered or otherwise modified. For example, the Cas protein can be deadCas9. 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 nucleases, TALENs (transcription activator-like effector-based nucleases), or meganucleases) and / or recombinases, such as Cre recombinase.

[0055] Payloads useful in the present disclosure also include polypeptides involved in nucleic acid synthesis and replication, such as DNA and RNA polymerases, transcription factors, primases, helicases, RNases, ligases, topoisomerases, endonucleases, IRES, and telomerases.

[0056] Linker Linker sequences (linkers) are known in the art and described in the references cited herein. Linkers include, for example, GS linkers, GSG linkers, and GGSG (SEQ ID NO: 239) linkers. These linkers are one or more repeats of a subunit. Thus, a GS linker is a repeat of GS nlinker (SEQ ID NO: 240), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater. Similarly, the GSG linker is n linker (SEQ ID NO:241), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater. n linker (SEQ ID NO:242), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater.

[0057] Hinge and transmembrane domains A hinge sequence is a short sequence of amino acids that promotes flexibility between the connected components. The hinge sequence can be any suitable sequence derived from or obtained from any suitable molecule. The hinge sequence can be derived from all or part of the immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence between the CH1 and CH2 domains of the immunoglobulin (e.g., IgG4 Fc hinge), or the extracellular region of type 1 membrane proteins, such as CD8α CD4, CD28, and CD7, which can be wild-type sequences or derivatives thereof. Some hinge regions include an immunoglobulin CH3 domain or both the CH3 and CH2 domains. In some embodiments, the hinge is derived from a transmembrane domain.

[0058] Transmembrane domains useful in the engineered regulatable polypeptide constructs of the present disclosure can include, for example, an MHC1 transmembrane domain, a CD8α transmembrane domain, a B7-1 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.

[0059] Intracellular / cytoplasmic or transmembrane tails Optionally, the monomer or polypeptide construct provided herein comprises an intracellular / cytoplasmic or transmembrane tail.Optionally, the intracellular / cytoplasmic or transmembrane tail is CD8, CD40L, LIGHT, NKG2C, or B7.1 intracellular tail.The absence of a transmembrane region in the construct can be designed, for example, for secreted payloads or payloads with intracellular or nuclear activity.

[0060] tag Optionally, the monomer or polypeptide described herein comprises a tag. Such a tag allows for the isolation or detection of the monomer or polypeptide, or the isolation or detection of the payload. Such a tag optionally comprises a fluorescent protein (e.g., green fluorescent protein), His tag, HA tag, Myc tag, FLAG tag, mCherry, CD20, CD34, nerve growth factor receptor (NGFR), truncated NGFR (tNGRR), epidermal growth factor (EGFR), or truncated EGFR (tEGFR).

[0061] Ligand The ligand may be any agent that binds to the DRD of the regulatory hub of an engineered regulatable polypeptide or oligomer described herein, an effective amount of which results in a measurable change in the alteration of the properties (e.g., abundance, availability, activity) of the payload operably linked to the DRD. In some embodiments, the ligand may be a synthetic molecule. In some embodiments, the stabilized ligand of the present disclosure may be a small molecule compound. The stabilized ligand is optionally a small molecule therapeutic that has been previously approved by a regulatory agency, such as the US Food and Drug Administration (FDA). Examples of stabilized ligands and their corresponding DRDs suitable for use in the regulatory hubs described herein are described in Table 1, in the patents and applications referenced above in the section illustrating suitable DRDs, 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, U.S. Patent No. 6,313,233, filed May 29, 2018, U.S. Patent No. 5,137,180 ... 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. Patent Application No. 16 / 558,224, filed September 2, 2019, the entire disclosures of which are incorporated herein by reference in their entireties.

[0062] Nucleic acids, vectors, and cells Provided herein are expressible nucleic acid constructs that encode one or more engineered regulatable polypeptide monomers or regulatable polypeptides described herein. In embodiments, the nucleic acid constructs encoding the monomers include an oligomerization domain and at least one DRD and / or at least one payload, and optionally include additional components such as hinges, linkers, transmembrane domains, tags, and intracellular / cytoplasmic and transmembrane tails as described herein. In certain embodiments, a single nucleic acid construct includes nucleic acid sequences that encode all components of the monomers described herein. For example, the nucleic acid construct encodes at least the payload, the DRD, and the oligomerization domain. When the monomers are expressed multiple times, the monomers can assemble into oligomers, such as, for example, dimers, trimers, tetramers, pentamers, or hexamers, depending on and via the oligomerization domain. Optionally, a single nucleic acid construct encodes multiple copies of the same or different monomers, which assemble into oligomers upon expression. Optionally, certain nucleic acid constructs encode an oligomerization domain and at least one payload, and certain nucleic acid constructs encode an oligomerization domain and at least one DRD, which when expressed, assemble into an oligomer comprising at least one payload and at least two DRDs.

[0063] In embodiments, the nucleic acid construct encoding a regulatable polypeptide comprises at least one payload and at least one regulatory hub comprising at least two DRDs. The nucleic acid construct encoding a regulatable polypeptide may comprise additional components such as hinges, linkers, transmembrane domains, tags, and intracellular / cytoplasmic and transmembrane tails as described herein.

[0064] The nucleic acid constructs encoding the monomers and regulatable polypeptides also optionally encode additional components, such as a signal sequence and a cleavage site containing an excretion domain. The constructs optionally further comprise promoter sequences and other regulatory elements, such as enhancers, translational control elements (e.g., IRES), and elements that control half-life.

[0065] Further provided are multiple nucleic acid constructs, each construct encoding one or more monomers or polypeptides described herein. Such multiple constructs, upon expression, optionally provide monomers that oligomerize. The multiple constructs may be the same or different. Thus, the multiple nucleic acids may encode the same polypeptide or monomer, or may encode polypeptides or monomers that include different DRDs, payloads, and / or additional components.

[0066] Also provided herein is a vector for expressing one or more of the nucleic acids. Such vectors can be selected from viral and non-viral vectors, plasmids, cosmids, 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 includes a nucleic acid sequence encoding a transposase and / or a nuclease. Examples of non-viral vectors include physical vectors, such as electroporation, and chemical vectors, such as lipid nanoparticles.

[0067] Cells are provided that contain one or more nucleic acid constructs or vectors described herein. The cells provide an expression system or therapeutic target for the monomers, oligomers, or polypeptides described herein. Suitable cells include immune cells or stem cells. Optionally, the immune cells are primary human T cells, such as T cells derived from human peripheral blood mononuclear cells (PBMCs), PBMCs collected after stimulation with G-CSF, bone marrow, or umbilical cord blood. In embodiments, the immune cells are tumor infiltrating lymphocytes (TILs), for example, collected from tumors. The immune effector cells may also be NK cells, αβ T cells, iNKT cells, γδ T cells, macrophages, B cells, dendritic cells, bone marrow-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.

[0068] How to make it The present disclosure provides methods of making nucleic acid constructs and vectors encoding the monomers and polypeptides of the present disclosure, methods of making the monomers and polypeptides of the present disclosure by expression of the nucleic acid constructs, and methods of making cells containing the nucleic acid constructs or vectors described herein.

[0069] FIG. 18 provides an embodiment of a process for designing nucleic acid constructs and vectors encoding monomers or polypeptides of the invention and then delivering the constructs or vectors for expression in cells. As shown, a DRD / ligand pair is selected for modulation of the desired payload. Optionally, a DRD is selected that has a ligand pair that is an FDA approved small molecule drug. In some embodiments, the DRD / FDA approved small molecule drug combination is selected so that it is clinically tractable (e.g., the ligand can modulate the abundance of the payload or modulate the activity of the payload within the approved FDA dose range of the ligand). The nucleic acid or vector is then designed to encode the building blocks / components of the polypeptides or monomers described herein, which in the case of polypeptides include at least the payload and two of the selected DRDs, and in the case of monomers include at least an oligomerization domain and at least one of the selected DRDs, or at least one payload, or at least one payload and at least one of the selected DRDs. The nucleic acid or vector may be further designed to encode additional building blocks / components such as transmembrane domains, linkers, hinges, and tags. Using this disclosure, one skilled in the art can select appropriate components and order them in the construct to achieve the desired result. For example, the order of the building blocks affects whether the DRD regulates the payload at the N-terminus or C-terminus.

[0070] As another example, the linker and the length of the linker may affect the "constitutive" activity level (i.e., basal activity in the absence of ligand), and in embodiments, a particular linker and length are selected to maximize the on-state (e.g., maximum activity level) while maintaining a low basal activity level and ligand (e.g., drug) responsiveness. As yet another example, a particular hinge may allow conformational changes, thereby affecting ligand responsiveness, and thus is selected to provide sufficient dynamic range to obtain the desired range of payload abundance and biological activity (i.e., acceptable payload activity range corresponding to ligand fluctuations from zero or minimum to maximum saturation). As an example, as shown in FIG. 19, if the goal is to produce a membrane-bound cytokine such as IL15 with sufficient dynamic range, the polypeptide optionally includes from the N-terminus the payload (IL15), the linker, the hinge, the transmembrane region, the tail, and the DRD.

[0071] In some embodiments, the nucleic acid sequences encoding the polypeptides or monomers may be engineered to include one or more additional components such that, when expressed intracellularly, the payload is localized to the interior of the cell membrane (optionally linked to the membrane or released to the cytoplasm), and in certain embodiments, to the exterior of the cell membrane (optionally linked to the membrane or released extracellularly (i.e., released or cleaved from the membrane). Similarly, the nucleic acid constructs are optionally configured to encode polypeptide monomers that oligomerize by association of intracellular, transmembrane, or extracellular oligomerization domains. Thus, all or a portion of the regulatory hub or oligomerization domains can be engineered to be intracellular, in the cell membrane, or on the surface of the cell (e.g., linked to the cell surface). See FIG. 15.

[0072] In some embodiments, the nucleic acid sequence encoding the polypeptide monomer construct or regulatable payload composition may be engineered to include one or more of these additional components such that, when expressed in a cell, the regulatable payload composition is engineered with a signal sequence to transport part or all of the engineered regulatable polypeptide construct into the secretory pathway.

[0073] After designing the construct for appropriate function, including localization, appropriate components such as promoters, enhancers, multicistronic expression, translational control, and half-life control elements are selected to achieve the desired control of payload abundance or activity. In addition, nucleic acid constructs are designed for cistronic or multicistronic expression necessary for the desired expression of the various engineered components.

[0074] In addition, the vehicle / vector is selected and designed to deliver the nucleic acid construct into the desired cell. For example, the vehicle can be selected from the above, including viral vectors (such as lentiviral vectors, retroviral vectors, and adeno-associated vectors), plasmids, cosmids, and artificial chromosomes. Such vectors can be designed to encode transposases, nucleases, and elements that control translation (e.g., IRES). The choice of vector can also affect the selection of various building block components. For example, a vector that requires a smaller construct may require the use of a smaller DRD.

[0075] Cells for engineering can be isolated from any biological sample, including, for example, blood (e.g., umbilical cord blood or peripheral blood), bone marrow, fetal tissue (human embryonic stem cells), or tumors. The cells can be modified cells, such as stem cells that have been modified to be pluripotent (e.g., induced pluripotent stem cells (iPSCs)) or CAR T cells prior to transduction with the described nucleic acids and vectors.

[0076] By way of example, isolated T cells or tumor infiltrating lymphocytes (TILs) can be isolated from a biological sample, transduced, and optionally expanded in culture, for example by contacting the transduced cells with IL2, feeder cells, recombinant antigens, or antibodies that stimulate cell proliferation.

[0077] The cells to which the nucleic acid is delivered are selected at least in part based on the cell's ability to allow expression of the polypeptide or monomer disclosed herein and to allow payload activity in a sufficient dynamic range. Optionally, the cell expresses little or no payload in the absence of the nucleic acid or vector provided. In certain embodiments, the skilled artisan will select cells that require increased payload activity or abundance in cells that express the payload. In certain embodiments, the skilled artisan will select cells that require gene editing. In certain embodiments, the cells are selected as effector cells, for example, immune effector cells.

[0078] Those skilled in the art who apply knowledge from the present disclosure can construct various monomers, polypeptides, and oligomers, as well as the nucleic acids and vectors encoding them, beyond those explicitly exemplified herein, within the scope of the present disclosure. For example, as described, the regulatory hub, payload, DRD, oligomerization domain, and additional components such as linkers, tails, transmembrane domains, signal sequences, hinges, etc. can be selected to create various polypeptides, oligomers, and nucleic acids encoding them, taking into consideration factors such as the desired therapeutic outcome, whether the payload is membrane-bound or secreted, whether the payload acts intracellularly or extracellularly, and whether the oligomerization domain should be located intracellularly, extracellularly, or transmembrane. Thus, the exemplary embodiments are non-limiting and serve to provide guidance for those skilled in the art to implement other embodiments within the scope of the present disclosure.

[0079] How to use Disclosed herein are methods of modulating payload function, e.g., modulating the abundance, availability and / or activity ("activity") of a payload. In some embodiments, the modulating method is a method of altering the activity of a payload, comprising engineering a cell to express a payload operably linked to a regulatory hub comprising two or more DRDs responsive to a ligand. In some such embodiments, the activity of the payload (e.g., corresponding to the abundance and / or availability of the payload) is decreased compared to the activity of the payload in a control cell, e.g., a cell engineered to express the payload independent of the DRDs.

[0080] Optionally, the payload has a biological activity level ranging from a basal activity level in the absence of ligand to a maximal activity in the presence of a saturating amount of ligand, and the method is a method of modulating the activity of the payload comprising contacting a cell engineered to express the payload operably linked to a regulatory hub with an effective amount of the ligand such that the activity of the payload is increased relative to the basal activity level. In some embodiments, the method comprises contacting the cell with a selected amount of the ligand, the selected amount of the ligand resulting in a selected activity level of the payload. In certain embodiments, the method alternatively comprises contacting the cell with different selected amounts of the ligand to achieve different selected activity levels ranging from basal to maximal levels.

[0081] The contacting step can be performed in vitro, ex vivo, or in vitro. The contacting step is optionally performed to achieve a continuous selected activity of the payload (i.e., to achieve a continuous on-state of the payload) or to achieve an intermittent activity of the payload (i.e., to provide a pulsatile delivery of the payload between an on-state and an off-state). By "off-state" is meant that the payload activity is at a basal activity level. By "on-state" is meant that the selected activity level is greater than the off-state in the presence of an effective amount of the ligand. The continuous activity of the payload can be achieved by contacting the DRD with an effective amount of the ligand continuously, or by providing one or more steps of contacting the DRD with the ligand, where the one or more subsequent contacting steps are performed before the activity level of the payload from the previous contacting step reaches the basal activity level. Each contacting step can be varied with respect to the amount of the ligand, such that when more ligand is used, more payload activity is provided, and when less ligand is used, less payload activity is provided. Thus, the amount of ligand can be varied in subsequent contacting steps to up- or down-tune the amount and / or activity of the payload over time. Each contacting step can also be varied in frequency to achieve a desired pattern of activity levels. Because the tuning hubs described herein provide a lower off-state and optionally a wider dynamic range of activity for a payload compared to the same payload in a control construct, the activity of the payload can be modulated from a lower off-state and, optionally, varied over a larger range compared to the payload in the control construct.

[0082] Also disclosed are methods of delivering a payload to a subject, e.g., a therapeutically effective payload, to a subject in need thereof, whereby a nucleic acid construct or vector described herein is administered to the subject. The method results in expression of one or more polypeptides or monomers, e.g., in target cells of the subject, which monomers self-assemble to form oligomers described herein. The method may further include controlling the dose or duration of administration of the payload to the subject. For example, the method optionally further includes administering to the subject a selected amount of a paired ligand to deliver a selected activity of the payload to the subject. The ligand can be delivered to achieve continuous or intermittent payload activity in the subject. The continuous payload activity may be a substantially consistent activity level, or the activity level may be modulated. Intermittent activity between an off state and an on state includes modulating activity between an off state and a substantially consistent on state, or between an off state and a range of on state activity levels. A higher dose or longer duration of administration of the ligand is administered when more activity of the payload is desired, and a lowering or elimination of the ligand dose is selected when less activity is desired. The dose and duration of ligand administration, as well as the resulting activity of the payload, can also be selected to avoid unacceptable or undesirable side effects or toxicity in the subject. The dosage of the ligand and the schedule for administering the dosage of the ligand can be empirically determined by those skilled in the art based on the amount of payload obtained, the activity of the payload, or based on one or more indications of the effect of the payload activity. The dosage range of the ligand can range from any value greater than zero to a saturating dose, and the resulting payload activity can range from a basal level to a maximum level, optionally with a sufficient dynamic range and combined activity range of the payload to allow a desired dose response to the ligand (e.g., for a given ligand and payload, the range of difference between the off-state and maximum payload activity will occur from at least a 10-fold range of the ligand).This sufficient dynamic range allows for fine tuning and a dose-response curve that is not unacceptably steep. In embodiments, the dosage or frequency of administration of the ligand, as well as the abundance and activity of the resulting payload, are selected to avoid, mitigate, or limit unacceptable or undesirable adverse side effects, and will vary depending on the age, condition, and / or sex of the subject, the type of condition being treated, the extent of the condition, or whether other therapeutic agents are included in the treatment regimen. Guidance on appropriate dosages for a given class of ligand can be found in the literature.

[0083] As a further example, for a subject having cancer, a nucleic acid construct or vector according to the present disclosure is provided to the subject, where the nucleic acid construct or vector encodes a payload operably linked to a regulatory hub, where the payload targets tumor cells or immune cells that target tumor cells. Such methods optionally include administering to the subject a nucleic acid construct or vector according to the present disclosure, where the payload is operably linked to a regulatory hub, where the payload is an immune checkpoint inhibitor, a cytokine, a CAR, or a TCR.

[0084] Also provided herein is a method of delivering a payload operably linked to a regulatory hub to a subject by administering to the subject cells containing a nucleic acid or vector described herein. Such a method may further include isolating cells from a subject, transducing the isolated cells with a nucleic acid or vector encoding a monomer described in this disclosure (which assembles into a regulatable oligomer when expressed) or a regulatable polypeptide, expanding the cells in vitro, and providing the cells to the same or a different subject. The payload is selected to treat the subject receiving the cells. The subject may have cancer, an autoimmune disease, a genetic mutation, a deficiency of an essential polypeptide, and the like. The transduced cells may be, for example, immune effector cells (e.g., NK cells, iNKT, αβT cells, γδT cells, tumor infiltrating lymphocytes (TIL), macrophages, B cells), or stem cells). The cells may be isolated from the same subject that receives the transduced cells (autologous source), or the cells may be isolated from a different subject (e.g., allogeneic source). By way of example, if a subject has cancer, T cells or TILs can be isolated from the subject and engineered to express a cytokine, a CAR, and / or a TCR. For example, the cells can be CD19 CAR-T cells. The cells are optionally expanded, and the cells or the expanded cell population are administered to the same or a different subject. In certain embodiments, T cells, CAR-T cells, NK cells, or TILs can be administered at about 1000 cells / injection up to about 10 billion cells / injection, e.g., 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3 1×10 cells / injection, or any range between any two of the numbers inclusive. 8 ~1×1010 The cells are administered to the subject. Optionally, the cells are administered 1, 2, 3, or 4 times, as needed.

[0085] In the therapeutic method, a ligand dosing regimen, including a selected amount of ligand for administration to a subject and a frequency of administration of the selected amount of ligand, is selected to result in modulation of the payload and / or desired outcome in the subject. The subject is optionally monitored for the outcome. Thus, for example, the number of malignant cells in a sample, circulating tumor DNA in a sample, or the size of a solid tumor upon imaging can be detected. If a desired endpoint is achieved (e.g., indicating successful treatment of cancer), the ligand can be reduced or discontinued to reduce or eliminate the payload, e.g., to reduce the abundance, availability, and / or activity of the payload below a predetermined threshold to eliminate or reduce undesirable or unwanted side effects. Similarly, if a subject develops a cytokine storm, allergic reaction, or other adverse effect from the payload, the ligand can be reduced or discontinued. Also disclosed herein are methods of modulating expression of a target protein downstream of a gene editing process. In some embodiments, such methods include engineering a cell to express an oligomer or engineered regulatable polypeptide that includes a payload, such as a CAS9 protein or a transcription factor protein, operably linked to a regulatory hub. In certain embodiments, a nucleic acid construct or vector according to the present disclosure is provided for delivering a payload that provides a nucleic acid editing polypeptide or system that is operably linked to a regulatory hub for a subject with a genetic mutation. A target cell in a subject is transduced with the nucleic acid construct or vector to allow the gene editing payload to modify the nucleic acid (e.g., genomic DNA or RNA) of the transduced cell. The activity level of the payload, and thus the expression of the target protein, can be regulated by administering a ligand to the subject. Systems for nucleic acid editing, such as CRISPR / Cas9 systems, TALENs, retrotransposons, and the like, are known in the art. A nucleic acid construct or vector can be engineered to encode one or more components of any one of the systems, such that one or more nucleic acids in the transduced cell of the subject are edited to provide the desired nucleic acid modification.

[0086] definition The terms "about" and "approximately," when used to refer to a measurable value, such as an amount, concentration, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, weight, location, length, and the like, are meant to include variations due to experimental error, which may 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, location, length, etc. 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. When the terms "about" and "approximately" are used in reference to the location or position of a region within a reference polypeptide, these terms encompass a variation 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.

[0087] In this specification, "basal activity level" is referred to. Basal level as used herein can be zero, near zero, or any amount in the absence of exogenous ligand. Basal activity can occur due to endogenous levels of the same or different ligand, or can occur due to resting levels of payload production in the absence of exogenous ligand.

[0088] References to "biological activity" should be understood to imply, even if not stated otherwise, under appropriate conditions.

[0089] As used herein, "contacting" is understood to mean providing an agent (such as a ligand) to a target (such as a DRD) so that the agent and the target can contact each other. For example, contacting includes providing a ligand to a cell in vitro (e.g., when the DRD is located outside the cell or on the cell surface). As another example, contacting also includes providing a ligand to a cell, where the DRD is located intracellularly, such that the ligand reaches the cytoplasm. Similarly, a cell can be contacted in vivo by administering a ligand to a subject so that the ligand reaches the cell or the DRD.

[0090] As used herein, the term "control construct" refers to a construct that is similar to a test engineered regulatable oligomer or engineered regulatable polypeptide ("test construct"), except that the control construct lacks an oligomerization domain and has only a single DRD.

[0091] "DRD" is understood to mean the ligand-responsive domain, even if not specified.

[0092] A "hetero-oligomerization domain" as described herein is a first oligomerization domain on a first monomer that promotes oligomerization via a second oligomerization domain on a second monomer, where the second oligomerization domain is different from the first oligomerization domain and the first oligomerization domain does not promote oligomerization with the same oligomerization domain.

[0093] The terms "ligand," "paired ligand," and "stabilizing ligand" are used interchangeably and mean the same thing when used in reference to a drug responsive domain ("DRD") and / or a regulatory hub.

[0094] As used herein, "operably linked" means that in the presence of a paired ligand, the regulatory hub or DRD is directly or indirectly linked to the payload to alter a measurable property of the payload (e.g., alter the activity level of the payload compared to the activity level in the absence of the paired ligand). In some embodiments, the amount and / or measured level of activity of the payload is increased in the presence of an effective amount of the ligand compared to the measured level of expression or activity in the absence of the ligand. An effective amount of the ligand refers to the amount of the ligand required to see an increase in the amount or measured activity of the payload. In some embodiments, the effective amount is not so large as to produce unacceptable toxicity or off-target effects. Optionally, the measurable property is a treatment outcome, an amount of the payload in a sample, or a biological activity level of the payload (for which measuring the amount of the payload can serve as a surrogate).

[0095] The term "payload" refers to an agent whose abundance, activity, availability, expression, function or other property is desired to be modulated by a DRD and / or regulatory hub.

[0096] Whenever a phrase such as "linked" or "coupled" is used, the phrase "directly or indirectly" is understood to follow unless expressly stated otherwise or meaningless in the context. Thus, the phrase "DRD linked to another DRD" or "two linked DRDs" means in both cases that the first DRD is directly or indirectly linked to the second DRD. For example, "two linked DRDs" etc. encompasses the situation where the two DRDs are associated with each other, rather than being directly linked to each other, because each is connected to an oligomerization domain and each oligomerization domain is linked, for example, via a non-covalent bond.

[0097] 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 may be made without departing from the scope of this disclosure. In other words, exemplary embodiments and aspects are described below. It is to be understood, however, that in the development of such an actual embodiment, numerous implementation-specific decisions may be made to achieve the developer's particular goals, such as compliance with system-related and business-related constraints, which may vary from implementation to implementation. It is further to be understood that such a development effort may 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.

[0098] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entireties.

[0099] The following examples 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. EXAMPLES

[0100] Example 1. Cloning of IL12-X and CD19-IL12-X constructs IL12-(241-252 and 254-262) and CD19-IL12-(297-316, 319-332) plasmids were each constructed in pELNS vector (third generation self-inactivating lentivirus expression vector) using standard molecular biology techniques. Gene fragments (Gblocks or stranded DNA) encoding IL12, glycine-serine linkers, various hinges, transmembrane domains, and cytoplasmic tails were purchased from Integrated DNA Technologies or Thermo-fisher scientific. The gene fragments were inserted into pELNS vector and placed under the control of EF1a promoter using Gibson assembly (NEBuilder Hifi). The assembled plasmids were transformed into E. coli (NEB stable) for amplification and sequence confirmation before proceeding with virus production.

[0101] Example 2. Regulation of IL12 from a tandem IL12 mCherry construct in HEK cells HEK-293 cells and T cells were cultured in standard medium (DMEM (Fisher Scientific Catalog No. 11-960-044) containing 10% FBS (Fisher Scientific Catalog No. 10-082-147) and 1% P / S (ThermoFisher Catalog No. 15140122). HEK293 cells and T cells were cultured in standard medium (DMEM (Fisher Scientific Catalog No. 11-960-044) containing 10% FBS (Fisher Scientific Catalog No. 10-082-147) and 1% P / S (ThermoFisher Catalog No. 15140122). Transiently transfected cells were incubated with 2000 and 1 μg of each of IL12-223, 241, 243, 244, 245, 247, 248, 249, 250, 251, 254, 255, 256, 258 and 260 plasmid DNA using the manufacturer's protocol. One day after transfection, 100 μM acetazolamide was added and cells were cultured for an additional day. mCherry was used as a transfection marker and anti-IL12p40 / 70-V450 Cat. No. 561380 was used. IL12 was stained using BD. Flow cytometry graphs in Figures 5A, 5B, and 5C show the surface abundance of IL12 on HEK cells in the presence or absence of ACZ. IL12-223, constructed without an oligomerization motif, showed a higher basal payload abundance on the cell surface than the constructs IL12-241, 245, 254, 256, 258, and 260 constructed with a trimerization motif.

[0102] Example 3. Regulation of IL12 from a tandem IL12 mCherry construct in Jurkat cells Jurkat (clone E6-1) cells were cultured in standard media (RPMI + GlutaMAX supplement: Life Technologies catalog number 61870127, fetal bovine serum (FBS) Life Technologies catalog number 10-082-147). Jurkat cells were transduced with lentiviruses produced with each of IL12-217, -241, -243, -245, -247, -248, -254, -256, and -258. The components of these constructs are shown in Figure 3. Jurkat cells were transduced to be 10-25% positive, as determined by using mCherry as a transduction marker. Five days after transduction, cells were treated with 100 μM acetazolamide or DMSO, and cells were cultured for an additional day. mCherry was used as a transduction marker and anti-IL12p40 / 70-V450 Cat. No. 561380 BD was used to stain for IL12. Figure 6 shows the geometric MFI showing IL12 construct expression on mCherry positive Jurkat cells in the presence and absence of ACZ. Note that cells transduced with trimerizing constructs (IL12-245, 247, 248, 254, 256) have low IL12 expression (off state) in the absence of ACZ. The numbers above ACZ treated cells represent the high fold change between the on and off states. MFI was calculated for cells within the mCherry gate.

[0103] Example 4. Regulation of IL12 export from tandem IL12 mCherry constructs in primary human T cells On day 0, primary human T cells were stimulated with Dynabeads (T-expander CD3 / CD28) at a bead:cell ratio of 3:1 in medium containing 10% fetal bovine serum (FBS). The next day, lentiviruses produced with the constructs (IL12-241, 245, 247, 256, 223, 260 and CD19-IL12-192) and controls were added in the presence of reduced serum (5% FBS). The components of these constructs are shown in Figure 3. On day 2, cells were diluted 1:2 in fresh 10% FBS medium. On day 5, cells were analyzed for mCherry transduction and replated to account for differences in transduction efficiency, medium was replaced and cells were treated with 100 μM acetazolamide or DMSO. Cytokines accumulated over 24 hours in culture supernatants (from 100,000 cells per 200 μL of medium) were measured using human IL12p70 (and / or human interferon-gamma) MSD V-plex assay kits (Meso Scale Discovery). Figure 7 shows the levels of IL12p70 secreted from T cells transduced with different constructs. The same constructs tested in Jurkat cells yielded similar results in transduced T cells. Note that only IL12 with a hinge with a defined efflux mechanism resulted in an amplified dynamic range for the regulation of the efflux IL12 fraction in the cell supernatant.

[0104] Example 5. Regulation of membrane-bound IL12 constructs in primary human T cells On day 0, primary human T cells were stimulated with Dynabeads (T-expander CD3 / CD28) at a bead:cell ratio of 3:1 in medium containing 10% fetal bovine serum (FBS). The next day, constructs expressing mCherry and membrane-bound trimerizing flexi-IL12 (IL12-241, 245, 247, 256, 260, and CD19-IL12-192), as well as control-produced lentiviruses, were added in the presence of reduced serum (5% FBS). On day 2, cells were diluted 1:2 in fresh 10% FBS medium. On day 5, cells were analyzed for mCherry transduction, medium was replaced, and cells were treated with 100 μM acetazolamide or DMSO. After 24 h, T cells were analyzed by FACs. mCherry was used as a transduction marker and anti-IL12p40 / 70-V450 Cat. No. 561380 BD was used to stain for IL12. Alternatively, cells were grown for a total of 10-11 days and then frozen in liquid nitrogen. T cells were then thawed and counted. 1-2×10 5 Cells were plated per well in 96-well V-bottom plates, restimulated with soluble CD3 / CD28 Immunocult reagent (Stem Cell Technologies) and treated with a dose response of acetazolamide ranging from 0 to 100 μM. After overnight incubation, transduction efficiency was analyzed by flow cytometry using mCherry. Surface-expressed IL12 was detected with an anti-IL12p70 antibody (BD). Figure 8 shows the geometric MFI of surface IL12p70 expression on mCherry or CAR+ cells. Ligand concentration dose-response curves were fitted using Prism Software. [Table 2]

[0105] Example 6. Measurement of membrane-bound IL12 on T cells after construct transduction For an overview of the study schematic see Figure 4. On day 0, primary human T cells were stimulated with Dynabeads (T-expander CD3 / CD28) at a bead:cell ratio of 3:1 in medium containing 10% fetal bovine serum (FBS). The next day, either one of the constructs derived from CD19-IL12-192 (SEQ ID NO: 124) and CD19-IL12(297-316 or 319-332) (SEQ ID NOs: 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232) were packaged as lentivirus. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0106] On day 2, cells were diluted 1:2 in fresh 10% FBS medium. On day 3, cells were expanded 1:3 in fresh medium. On day 6, cells were assessed for transduction efficiency using flow cytometry. As a readout for cell transduction, CD19-CAR expression was detected using CD19-Fc reagent followed by fluorescently labeled anti-human Fc antibody. Dynabeads were removed using magnetic selection and cells were split 1:2 into fresh medium. On day 8, normalized numbers of transduced (CAR+) cells were plated on 96-well plates and treated with either 20 μM ACZ or an equal volume of DMSO vehicle control in the absence or presence of antigen restimulation. Stimulation conditions were either human Immunocult soluble CD3 / CD28 reagent (StemCell Technologies, Cat. No. 10971), K562 cells stably expressing the CAR antigen CD19 at an E:T ratio of 1:1, or parental control K562 cells. After overnight incubation, mbIL12 abundance on transduced T cells was analyzed by flow cytometry using CD19-Fc followed by fluorescently labeled anti-human Fc antibodies to detect surface CAR expression, anti-IL12p70, anti-CD45, anti-CD3, anti-CD4, and anti-CD8 antibodies. The geometric mean fluorescence intensity (GMFI) of IL12p70 on transduced cells was normalized to the gMFI of mbIL12 from identically treated (ligand + antigen restimulated) non-transduced cells, with values ​​close to 1 reflecting undetectable amounts of mbIL12. Table 4 shows the fold change of mbIL12 within the restimulation conditions, calculated as the gMFI of mbIL12 with ligand divided by the gMFI without ligand for a given construct. [Table 4-1] [Table 4-2]

[0107] Cytokines accumulated in overnight culture supernatants from 40,000 transduced cells (CD19-CAR+) per 200 μL of media were measured using human IL12p70 and / or human interferon-gamma MSD V-plex assay kits (Meso Scale Discovery). Fold change in IL12 within restimulation conditions was calculated as the concentration of IL12 with ligand divided by the concentration without ligand for a given construct. All constructs were efficiently transduced and expanded in primary human T cells. Figure 3, Figure 9 and Table 3 provide a list and schematic of all constructs generated with oligomerization domains. A subset of constructs (Figure 9 and Table 3) were advanced to evaluation of expression and activity in T cells based on achieving low off-state and high regulation in HEK cells and T cells. All constructs containing the oligomerization domain reduced the abundance of the off state (no ligand) of mbIL12 compared to a similar construct without the oligomerization domain (construct CD19-IL12-192) (Figures 10A and 10B). This regulation was largely maintained when looking at the fraction of IL12 excreted in the supernatant (Figures 11A and 11B).

[0108] Example 7. Regulation of IL12 activity assessed using STAT4 phosphorylation in bystander natural killer cells Figure 12 describes the procedure to test the engagement of IL12 and its function with its receptor by measuring the phosphorylation of STAT4. Supernatant (100 μL) from cells plated on day 8 and harvested on day 9 was added to 40,000 natural killer (NK) cells (in 100 μL of medium) derived from peripheral blood of a matched donor. NK cells were thawed from liquid nitrogen and allowed to rest overnight before starting the experiment. After 1 h, NK cells were then stained with anti-CD56, anti-CD16, anti-CD45, and anti-CD3 and assessed for STAT4 phosphorylation. All constructs were efficiently transduced and expanded in primary human T cells (as described in Example 6). A list of constructs generated with the CD19-CAR oligomerization domain is shown in Figures 2C-2E. Construct CD19-IL12-192 does not contain an oligomerization domain. Other controls include CD19-CAR only (063), empty vector (EV), and untransduced (UT) cells. For non-efflux constructs (303-305), transduced cells were incubated with donor-matched NK cells (pellet). All other samples tested were supernatants of cultured T cells. The modulation of pSTAT4 shown in Figure 13A suggests that modulation of the efflux moiety is functionally significant. Figure 13B shows a standard curve depicting pSTAT MFI with respect to IL12 concentration.

[0109] Example 8. Oligomerization in a cytoplasmic setting: trimerization and tetramerization of FoxP3 HEK-293T cells are cultured in standard medium (DMEM (Fisher Scientific Catalog No. 11-960-044) containing 10% FBS (Fisher Scientific Catalog No. 10-082-147) and 1% penicillin / streptomycin (P / S, ThermoFisher Catalog No. 15140122). As shown in FIG. 14, HEK293T cells are transiently transfected with Lipofectamine 2000 and 1 μg of plasmid DNA of each of the FOXP3-X constructs (X varies) using the manufacturer's protocol. One day after transfection, 100 μM acetazolamide was added and the cells were cultured for another day. mCherry was used as a transfection marker and expression levels were checked by FAC, after which equal numbers of transfected cells were pelleted and T-PERC were cultured. Lysates are lysed using Tissue Protein Extraction Reagent (ThermoFisher Catalog No. 78510). Lysates are run on NuPAGE 4-12% Bis-Tris gels (ThermoFisher Catalog No. NP0321BOX) and transferred onto nitrocellulose membranes (iBLOT2 NC Regular Stack (ThermoFisher IB23001)). FoxP3 is detected using anti-FOXP3 monoclonal antibody (ThermoFisher Catalog No. MA5-16222) and IRDye 800CW goat anti-mouse IgG (LI-COR P / N 926-32210).

[0110] Example 9. Multiple TNFSF cytoplasmic tail-TM-hinge combinations To fine-tune the regulation of IL12, multiple TNFSF cytoplasmic tail-TM-hinge combinations (from TWEAK, TRAIL, BAFF, GITRL and APRIL) trimerized using the trimerizing domain of collectin 7 are used. HEK-293T cells are cultured in standard medium (DMEM (Fisher Scientific, Catalog No. 11-960-044) containing 10% FBS (Fisher Scientific, Catalog No. 10-082-147) and 1% P / S (ThermoFisher Catalog No. 15140122). As shown in FIG. 14, HEK293T cells are transiently transfected with Lipofectamine 2000 and 1 μg of each of the plasmid DNAs of IL12-X (X varies) using the manufacturer's protocol. One day after transfection, 100 μM acetazolamide is added and the cells are cultured for an additional day. mCherry is used as a transfection marker and anti-IL12p40 / 70-V450 Catalog No. 561380 BD is used to stain for IL12.

[0111] Example 10. Use of other oligomerization domains such as tetramerization domains from hnRNPs HEK-293T cells are cultured in standard medium (DMEM (FisherScientific Catalog No. 11-960-044) containing 10% FBS (FisherScientific Catalog No. 10-082-147) and 1% P / S (ThermoFisher Catalog No. 15140122). As shown in Figure 14, HEK293T cells are transiently transfected with Lipofectamine 2000 and 1 μg of each of the plasmid DNAs of FOXP3-X (X varies) using the manufacturer's protocol. One day after transfection, 100 μM acetazolamide was added and the cells were cultured for another day. mCherry was used as a transfection marker and expression levels were checked by FAC, after which equal numbers of transfected cells were pelleted and lysed using T-PER Tissue protein extraction reagent (ThermoFisher Catalog No. 78510). Lysates were analyzed by NuPAGE. Run on a 4-12% Bis-Tris gel (ThermoFisher Cat. No. NP0321BOX) and transfer onto a nitrocellulose membrane (iBLOT2 NC Regular Stack (ThermoFisher IB23001)). FoxP3 is detected using anti-FOXP3 monoclonal antibody (ThermoFisher Cat. No. MA5-16222) and IRDye 800CW goat anti-mouse IgG (LI-COR P / N 926-32210).

[0112] Example 11. hDHFR-IL12 and hDHFR-IL2 constructs showing the advantages of trimerization with the existing oligomerization domain and the alternative DRD (hDHFR) HEK-293T cells are cultured in standard medium (DMEM (Fisher Scientific, Catalog No. 11-960-044) containing 10% FBS (Fisher Scientific, Catalog No. 10-082-147) and 1% P / S (ThermoFisher Catalog No. 15140122). As shown in FIG. 14, HEK293T cells are transiently transfected with Lipofectamine 2000 and 1 μg of each of the plasmid DNAs of IL12-X (X varies) using the manufacturer's protocol. One day after transfection, 100 μM acetazolamide is added and the cells are cultured for an additional day. mCherry is used as a transfection marker and anti-IL12p40 / 70-V450 Catalog No. 561380 BD is used to stain for IL12.

[0113] In a similar experiment, HEK-293T cells are cultured in standard medium (DMEM (Fisher Scientific, Catalog No. 11-960-044) containing 10% FBS (Fisher Scientific, Catalog No. 10-082-147) and 1% P / S (ThermoFisher Catalog No. 15140122). As shown in FIG. 14, HEK293T cells are transiently transfected with Lipofectamine 2000 and 1 μg of each of the plasmid DNAs of IL2-X (X varies) using the manufacturer's protocol. One day after transfection, 100 μM acetazolamide is added and the cells are cultured for an additional day. mCherry is used as a transfection marker and anti-IL2 antibody Biolegend Catalog No. 500311 is used to stain for IL2.

[0114] Example 12. Trimerization motif with IL2 payload HEK-293T cells are cultured in standard medium (DMEM (Fisher Scientific, catalog number 11-960-044) containing 10% FBS (Fisher Scientific, catalog number 10-082-147) and 1% P / S (ThermoFisher catalog number 15140122). HEK293T cells are transiently transfected with Lipofectamine 2000 and 1 μg of each of the plasmid DNAs of IL2-X (X varies) using the manufacturer's protocol. One day after transfection, 100 μM acetazolamide is added and the cells are cultured for an additional day. mCherry is used as a transfection marker and an anti-IL2 antibody (Biolegend catalog number 500311) is used to stain for IL2.

[0115] Example 13. Trimerization motif with IL15 payload HEK-293T cells are cultured in standard medium (DMEM (catalog no. 11-960-044) containing 10% FBS (catalog no. 10-082-147) and 1% P / S (ThermoFisher catalog no. 15140122). HEK293T cells are transiently transfected with Lipofectamine 2000 and 1 μg of each of the plasmid DNAs of IL15-X (where X varies) using the manufacturer's protocol. One day after transfection, 100 μM acetazolamide is added and the cells are cultured for an additional day. mCherry is used as a transfection marker and IL15 is stained using IL15Ra-Fc-biotin (catalog no. ILA-82F4 Acrobiosystems) followed by streptavidin BV421 (Biolegend, catalog no. 405225).

[0116] Example 14. Use of heterotrimerization to modulate a single copy of a payload Lentiviruses generated using constructs IL12-266, 267 and 268 shown in Figure 20 were used to transduce Jurkat cells to 10-15% positivity as determined by using RQR8 as a transduction marker. Five days after transduction, cells were treated with 100 μM acetazolamide or DMSO and cells were cultured for an additional day. RQR8 was detected using anti-CD34 clone QBEND10 (R&D catalog number FAB7227A) and IL12 was detected using anti-IL12p40 / 70-V450 (BD catalog number 561380). Figure 21A shows histograms showing IL12 levels in the presence and absence of acetazolamide. Figure 21B is a schematic showing the assembly and oligomeric state of the DRD in IL12-266, 267 and 268. Figure 21C shows the fold change in geometric MFI of IL12 following drug addition for each of IL12-266, 267, and 268. Transduced cells show a pattern of increasing fold regulation as they progress from monomers, dimers to trimers.

[0117] Example 15. Regulation of membrane-bound IL23 constructs in primary human CAR-T cells On day 0, primary human T cells were stimulated with Dynabeads (T-expander CD3 / CD28) at a bead:cell ratio of 3:1 in medium containing 10% fetal bovine serum (FBS). The next day, lentiviruses produced with constructs expressing CD19 CAR and membrane-bound oligomerizing flexi-IL23 (CD19-IL23-002, CD19-IL23-003, CD19-IL23-004, CD19-IL23-005, CD19-IL23-006, CD19-IL23-007, CD19-IL23-008) and control constructs (CD19-IL23-001, expressing secreted flexi-IL23; CD19-IL23-009, expressing flexi-IL23 without any oligomerizing regulatory hub) were added in the presence of reduced serum (5% FBS). On day 2, cells were diluted 1:2 with fresh 10% FBS medium. On day 5, cells were analyzed for CD19 CAR expression using CD19-Fc (R&D Systems, Minneapolis, MN; Part #9269-CD-050), followed by anti-human Alexa 647 anti-human IgG (Jackson ImmunoResearch, West Grove, PA; Part #109-607-003). Media was replaced and cells were treated with 100 μM acetazolamide or DMSO. After 24 hours, T cells were analyzed by FAC. CD19 CAR was used as a transduction marker and IL23 expression was stained using anti-IL12 / IL23p40-PE (Biolegend, San Diego, CA, Cat #501807). The geometric MFI of surface IL23p40 expression on CAR+ cells was plotted using Prism Software. The results are shown in FIG. 22. IL23 abundance was analyzed in the presence and absence of ACZ. Cells transduced with several oligomerization constructs have low IL23 abundance in the absence of ACZ (off state). The numbers above ACZ-treated cells represent the fold change between the on and off states. Oligomerization constructs with multiple DRDs reduced the basal abundance and enhanced the fold change between the on and off states of IL23.

[0118] In this example, the following constructs were used: [Table 32] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTF LRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSW EYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEG DEETTNDVPHIQCGGGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP [Table 33]

Table 34

Table 35

Table 36

Table 37

Table 38

Table 39

Table 13

[0119] Example 16. Regulation of membrane-bound IFNα constructs in primary human CAR-T cells Primary human T cells were stimulated with Dynabeads (T-expander CD3 / CD28) at a bead:cell ratio of 3:1 in medium containing 10% fetal bovine serum (FBS) on day 0. The following day, lentiviruses produced with constructs expressing CD19 CAR and membrane-bound oligomerizing IFNα (CD19-IFNα-002, CD19-IFNα-003, CD19-IFNα-004, CD19-IFNα-005, CD19-IFNα-006, CD19-IFNα-007, CD19-IFNα-008), and control constructs (CD19-IFNα-001, expressing secreted IFNα; CD19-IFNα-009, expressing monomeric IFNα without the oligomerizing regulatory hub) were added in the presence of reduced serum (5% FBS). On day 2, cells were diluted 1:2 with fresh 10% FBS medium. On day 5, cells were analyzed for CD19 CAR expression using CD19-Fc (R&D Systems, Part number 9269-CD-050), followed by anti-human Alexa 647 anti-human IgG (Jackson ImmunoResearch, Part number 109-607-003). Media was replaced and cells were treated with 100 μM acetazolamide or DMSO. After 24 hours, T cells were analyzed by FAC. CD19 CAR was used as a transduction marker and IFNα expression was stained using anti-IFNα PE (BD Biosciences, Catalog number 560097). The geometric MFI of surface IFNα expression on CAR+ cells was plotted using Prism Software. The results are shown in Figure 23. IFNα abundance was analyzed in the presence and absence of ACZ. Cells transduced with several oligomerized constructs have low IFNα abundance in the absence of ACZ (off state). The numbers above ACZ-treated cells represent the fold change between the on and off states. Oligomerized constructs with multiple DRDs lowered the basal abundance and enhanced the fold change between the on and off states of IFNα. Of note, CD19-IFNα-009 configured as a non-oligomerized construct is robustly abundant on the cell surface after stabilization with ACZ, but does not have the same low basal abundance as the oligomerized construct.

[0120] In this example, the following constructs were used: [Table 14] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPGSMALTFALLVALLV LSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE [Table 15]

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

[0121] Example 17. Regulation of IL2 from tandem CD19CAR-p2a-IL2 constructs in Jurkat cells Jurkat (clone E6-1) cells were cultured in standard medium (RPMI + GlutaMAX supplement (Life Technologies catalog number 61870127), fetal bovine serum (FBS) (Life Technologies catalog number 10-082-147)). Jurkat cells were transduced with lentiviruses produced in each of the following strains: CD19CAR-IL2-001, CD19CAR-IL2-002, CD19CAR-IL2-003, CD19CAR-IL2-004, CD19CAR-IL2-005, CD19CAR-IL2-006, CD19CAR-IL2-007, CD19CAR-IL2-008, and IL2-017. Jurkat cells were transduced to be 10-25% positive, as determined by using CD19 CAR as a transduction marker. CD19 CAR expression was assayed using CD19-Fc (R&D Systems, Part No. 9269-CD-050), followed by anti-human Alexa 647 anti-human IgG (Jackson ImmunoResearch, Part No. 109-607-003). Five days after transduction, cells were treated with 100 μM acetazolamide or DMSO and cells were cultured for an additional day. IL2 expression was stained using PE anti-human IL2 antibody (Biolegend, Cat No. 500307). Figure 24 shows the geometric MFI indicating IL2 abundance on CD19CAR positive Jurkat cells in the presence and absence of ACZ. Note that cells transduced with several oligomerization constructs (CD19CAR-IL2-001, CD19CAR-IL2-002, CD19CAR-IL2-003, CD19CAR-IL2-004, CD19CAR-IL2-005, CD19CAR-IL2-006) had low IL2 abundance in the absence of ACZ (off state), while the concept of oligomerization explored with CD19CAR-IL2-007, CD19CAR-IL2-008 did not result in significant improvement compared to the non-oligomerization DRD-regulated construct, IL2-017. Numbers above ACZ-treated cells represent fold change between on and off states. MFI was calculated for cells within the CD19CAR positive gate.Oligomerization constructs with multiple DRDs reduced the basal abundance and enhanced the fold change between IL2 on and off states.

[0122] In this example, the following constructs were used: [Table 23] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPGGSMSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATNLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFKGGSGGGSGGGSGSKQRGTFSEVSLAQDARQKLQNLFINFCLILICLLLICIIVMLLIPFLEQNNSSPNTRTQKSFEMQKGSGSGSGSGSGSGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

Table 24

Table 25

Table 26

Table 27

Table 28

Table 29

Table 30

[0123] Example 18. Oligomerization of IL18 constructs [Table 5-1] [Table 5-2]

[0124] T cell transduction On day 1, primary human T cells were stimulated with Dynabeads (T-expander CD3 / CD28) at a bead:cell ratio of 3:1 in medium containing 10% fetal bovine serum (FBS). The following day, lentiviral vectors were used to transduce T cells with IL18 oligomerizing constructs. On day 3, cells were diluted 1:2 in fresh 10% FBS medium. On day 4, cells were split 1:4 in fresh 10% FBS medium.

[0125] On day 6, de-bead the cells and dilute them to 1 x 10 6 Cells were then replated at 1000 cells / mL. The next day, cells were left unstimulated or activated with soluble CD3 / CD28 (StemCell Technologies) and treated with either 50 μM acetazolamide (ACZ) or DMSO. After 24 hours, membrane-bound IL18 was detected by flow cytometry using anti-IL18 (clone 159-12B, MBL Int Cat. No. D045-3) and anti-rat IgG2a PE conjugate (Biolegend, Cat. No. 407508). Cytokines accumulated over 24 hours in culture supernatants (from 200,000 cells per 200 μL of medium) were measured using the human IL18 MSD V-plex assay kit (Meso Scale Discovery).

[0126] Figure 25 is a schematic diagram showing the experimental design for determining IL18 expression, and Figure 26 is a schematic diagram showing the experimental design for determining IFNγ release. Figures 27, 28, 31-32, 33-35, 38-45, 52-53 show the frequency of IL18-expressing T cells in the presence and absence of ACZ. For Figures 38-40, the numbers above the cells treated with ACZ represent the fold change between the on and off states in the presence or absence of ACZ. In Figures 27-33, 35-37, and 41-54, the diagrams with white circles refer to the left y-axis and show the IL18 released into the supernatant. The IL18 constructs containing hinges with defined efflux sites resulted in an amplified dynamic range for the regulation of the efflux IL18 fraction in the cell supernatant.

[0127] Antigen-independent interferon-gamma release On day 1, primary human T cells were stimulated with Dynabeads (T-expander CD3 / CD28) at a bead:cell ratio of 3:1 in medium containing 10% fetal bovine serum (FBS). The following day, lentiviral vectors were used to transduce T cells with IL18 oligomerizing constructs. On day 3, cells were diluted 1:2 in fresh 10% FBS medium. On day 4, cells were split 1:4 in fresh 10% FBS medium.

[0128] On day 6, de-bead the cells and dilute them to 1 x 10 6 Cells were replated at 200,000 cells / mL and allowed to rest for 48 hours. On day 8, cells were replated at 200,000 cells per 200 μL medium and treated or not with recombinant 20 ng / mL human IL15 (Biolegend, Cat. No. 570304). Interferon (IFN)-γ and IL18 accumulated in the culture supernatant over 48 hours were measured using a customized human IFNγ / IL18 MSD V-plex assay kit (Meso Scale Discovery).

[0129] Figures 29, 30, 36, 37, 46-51, and 54 are graphs showing the amount of IFNγ released from transduced T cells treated or not with recombinant IL15. The circles indicate the right y-axis and represent the amount of IL18 released into the supernatant.

[0130] Jurkat transduction Jurkat (clone E6-1) cells (ATCC) were cultured in standard medium (RPMI + GlutaMAX supplement: Life Technologies catalog number 61870036, fetal bovine serum (FBS) Life Technologies catalog number A38400-01). Jurkat cells were transduced with lentiviral vectors containing IL18 oligomerizing constructs. Five days after transduction, cells were treated with 50 μM acetazolamide or DMSO and cells were cultured for an additional day. Membrane-bound I-18 was detected by flow cytometry using anti-IL18 (clone 159-12B, MBL Int catalog number D045-3) and anti-rat IgG2a PE conjugate (Biolegend, catalog number 407508).

[0131] Figure 55 shows flow cytometry histograms showing the change in mbIL18 expression levels on Jurkat cells in the presence and absence of ACZ. Numbers above ACZ treated cells represent the fold change between IL18 levels observed in the presence and absence of ACZ (on vs. off states). MFI was calculated for cells within the mCherry gate.

[0132] In summary, mbIL18 synergizes with mbIL15 or recombinant IL15 to induce IFNγ release in T cells. IL18 constructs IL18-037 and IL18-042 show 2-3 fold modulation and ability to synergize with recombinant IL15 to induce IFNγ release in T cells. The IL18-042 construct also shows IL18 activity in media collected from transduced activated cells, supporting a potential pleiotropic effect.

[0133] Example 19. Pharmacologically controlled expression of membrane-bound IL12 results in T cell therapy with enhanced efficacy in preclinical solid tumor models. [Table 6]

[0134] Constructs 333 and 345 were characterized in vitro. Figures 56-61 show the geometric MFI indicating IL12 abundance on the surface of CAR-T cells transduced with control (192) and IL12 constructs (302, 333, 345), indicating modulation of IL12 function and lower basal off-state levels of IL12 constructs containing collectin 7 oligomerization domains compared to control monomeric constructs. Also shown are IL12 efflux levels detected using a mesoscale discovery (MSD) assay and activity of efflux components on NK cells. Figures 56-58 are graphs showing that collectin-7 trimerizing constructs have a lower off-state in resting T cells (Figure 57) with modulation of efflux IL12 levels (Figure 58) indicating functional activity on bystander natural killer (NK) cells. Figures 59-61 are graphs showing that in activated T cells stimulated with CD3 / CD28 for 20 hours, IL12 constructs containing collectin-7 trimerizing domains show a low basal off state with regulated shed IL12 levels (Figure 60) and in vitro functional activity (phosphorylation of STAT4) on bystander NK cells (Figure 61).

[0135] For in vivo studies, NOD SCID gamma mice were implanted intravenously with 100,000 cells from the CD19+ Raji Burkitt's lymphoma cell line stably expressing luciferase to track tumor growth. Mice were randomized into treatment groups for equal tumor burden 3 days after tumor implantation. Four days after tumor implantation, CD19-CAR-T cells with different cell doses bearing various IL12 constructs were treated via adoptive cell transfer (ACT). Mice were bled 7 days after ACT to provide a baseline of cytokine levels and cell proliferation before ACZ administration by oral gavage. ACZ administration and blood collection continued until the end of the study 16 days after ACT, as described in Figure 62. IL12 is regulated on the surface of CD19+ cells, and constructs 302, 333, and 345 show a greater ability to regulate surface abundance than the control monomeric construct 192. Similarly, IL12 in plasma was modulated by the 302, 333 and 345 constructs, but not the control construct, showing up to 54-fold modulation with ACZ.

[0136] As shown in Figures 63 and 64, IL12 is regulated in vivo. Figure 63 shows that IL12 is regulated on the surface, and Figure 64 shows that IL12 is also regulated in plasma. Figure 63 shows that IL12 is regulated on the surface of CD19+ cells, showing that constructs 302, 333, and 345 have a greater ability to regulate surface abundance than the control monomeric construct 192. Similarly, Figure 64 shows that IL12 in plasma was regulated with the 302, 333, and 345 constructs, but unlike CAR alone (063 construct) or the monomeric control (192 construct), which showed up to 54-fold regulation using ACZ.

[0137] In the scRaji study, NOD SCID gamma mice were subcutaneously implanted with 5 million Raji cells, a CD19+ Burkitt's lymphoma cell line. Mice were randomized into treatment groups for equal tumor volumes (measured using digital calipers) 13 days after tumor implantation. 14 days after tumor implantation, control construct 063 (CAR alone), EV (empty vector) or construct 345 modified CD19-CAR-T cells were administered for adoptive cell transfer (ACT). Mice received ACZ or vehicle consecutively daily starting on the same day as ACT. Blood was collected from mice and measured as described in the study design (Figure 65). Cell proliferation and phenotype in blood were measured using flow cytometry, and cytokine levels in plasma were determined using the Meso Scale Discovery assay. Mice were euthanized upon reaching the endpoints of tumor volume (2000mm3), hind leg paralysis, or evidence of graft-versus-host disease. Animals administered CAR-T cells containing construct 345 demonstrated efficacy in the on state and the ability to control large solid tumors. Subcutaneous Raji tumors were measured twice weekly using digital calipers. Mice treated with CAR-T cells modified with construct 345 demonstrated ACZ-modulated efficacy (in terms of tumor burden) at a 10-fold lower dose than those modified with the CAR-only control construct 063 (Figures 66-68). Figures 66-68 show that injection of regulatory hub IL12-expressing CAR-T cells (construct 345) provided modulated anti-tumor activity at suboptimal doses of CAR-T cells.

[0138] In the PMEL study (Figures 69-78), C57BL / 6 mice were subcutaneously implanted with 500,000 B16-F10 (PMEL antigen positive) melanoma cells. Mice were randomized into treatment groups for equal tumor burden (measured using digital calipers) 13 days after tumor implantation and endogenous immune system depletion was performed using intravenous cytoxan. 14 days after tumor implantation, PMEL antigen-specific T cells were treated via adoptive cell transfer (ACT) with surrogate murine IL12 constructs with or without IL12 armouring. Mice received continuous daily dosing of ACZ or vehicle starting on the day of ACT. Blood was collected from mice and measured as shown in Figure 65. Cell proliferation and phenotype in blood was measured using flow cytometry and cytokine levels were determined using MSD assay. Seven days after ACT, tumors, spleens, and tumor-draining lymph nodes (TDLNs) were harvested, digested, and analyzed using flow cytometry. Cytokine levels in tumors after digestion were determined using the Meso Scale Discovery assay. The regulated mbIL12 construct, a surrogate mouse analog of human construct 333, showed localization of IL12 and IFNγ to tumors, as well as regulation in plasma (Figures 69-72). The regulatory hub IL12 construct allowed for regulation of IL12 in plasma and tumors. In animals receiving Pmel cells engineered with the regulatory hub IL12 construct, IL12 and IFNγ levels showed a greater degree of localization (greater tumor to plasma cytokine ratios) than unregulated (lacking DRD CA2) secreted or membrane-bound IL12. This ACZ-induced modulation of IL12 using a regulatory hub IL12 construct had functional impact in terms of modulating the frequency of circulating dendritic cells (Figure 73), the phenotype of activated monocytes in the circulation (Figure 74), and demonstrated secretion of IL12 and IFNγ levels in tumors, but not systemically (Figures 69-72). As shown in Figures 73 and 74, MHC II monocytes 14 days post-ACT from the study demonstrated that ACZ-induced modulation of IL12 using a construct with a regulatory hub resulted in a modulated phenotype (MHCII+ activated monocytes) in the circulation.As shown in Figures 75-78, the phenotype of myeloid populations in the tumor and systemically in animals receiving Pmel cells transduced with the regulatory hub IL12 construct shows the same M1 to M2 and phenotypic switch in the tumor microenvironment as animals receiving Pmel cells transduced with the secreted non-regulated IL12 construct. These phenotypic changes were more localized for regulatory hub IL12 than secreted mbIL12 or constitutive mbIL12, as evidenced by PD-L1 upregulation on myeloid cell populations in the spleen and TDLN (occurred more at the tumor site than in the spleen or lymph nodes), although the extent of PD-L1 upregulation in the spleen was similar.

[0139] In summary, IL12 was mostly localized in the tumor when using the membrane-bound regulated construct, whereas regulated IL12 and IFNy were observed systemically. In the blood, regulated regulatory hub IL12 was found to have functional impact. On-state IL12 levels (in plasma and tumor) produced by adoptively transferred Pmel cells engineered with the regulatory hub IL12 construct were closer to IL12 levels in animals receiving Pmel cells expressing the constitutive mbIL12 construct than to IL12 levels in animals in which the regulatory hub IL12 remained in the off-state (without ACZ dosing). Similarly, phenotypes such as MHCII expression on circulating monocytes were regulated by ACZ in animals receiving Pmel cells engineered with the regulatory hub IL12 (Figure 74). Functional impacts were also observed in the tumor microenvironment (TME). For example, the regulatory hub IL12 promoted the M1-M2 phenotype switch of macrophages to a similar extent as the unregulated secreted IL12 (Figure 77). However, the unregulated secreted IL12 produced different effects systemically than the regulatory hub IL12. For example, the unregulated secreted IL12 produced significantly greater PD-L1 expression on dendritic cells and myeloid-derived suppressor cells (MDSCs) in the spleen and lymph nodes than the regulatory hub IL12 (Figure 78).

[0140] Example 20. IL12 Constructs with PDE5 [Table 7]

[0141] Three oligomerization concepts were tested using PDE5 as DRD. Two control constructs, IL12-229 and IL12-235, were included in the study. In IL12-274, IL12 was directly fused to CD40L, abolishing the natural shedding site of CD40L. IL12-275 uses the DAP12 transmembrane hinge. IL12-276 was oligomerized with collectin 7 helix trimer, except collectin 7 was placed intracellularly. Flow cytometry analysis shown in Figures 79 and 80 shows that the performance is similar to the oligomerized CA2 DRD-IL12 fusion, with a reduced basal abundance.

[0142] Example 21. Regulation of IFNα in tumor-infiltrating lymphocytes in combination with constitutive membrane-bound IL15 (mbIL15) [Table 8]

[0143] Tumor infiltrating lymphocytes (TILs) were isolated from tumors, expanded, and transduced with a control construct (construct 292) containing only membrane-bound IL15 (mbIL15) and a construct containing both constitutive mbIL15 and IFNα regulated by the CA2 regulatory hub (construct 016). Transduced cells (500,000) were transferred to a GREX device and grown for 2 weeks following the rapid growth protocol with or without 25 μM ACZ. At the end of the 2-week period, TILs were analyzed for surface abundance of IFNα using flow cytometry (BD Biosciences Cat. No. 560097). The results are shown in Figures 81 and 82. Graph showing geometric mean fluorescence intensity (geoMFI) indicating membrane-bound IFNα abundance on the surface of tumor-infiltrating lymphocytes transduced with control (construct 292) and IFNα experimental (construct 016) constructs, showing modulation of IFNα by ACZ added throughout the rapid expansion protocol. Modulation is shown on the final day of TIL expansion.

[0144] For Figures 83-85, on day 7, primary human T cells were stimulated with Dynabeads (T-expander CD3 / CD28) at a bead:cell ratio of 3:1 in medium containing 10% fetal bovine serum (FBS). The next day, lentiviral vectors were used to transduce T cells with an IL15-IFNα oligomerizing construct. On day 5, cells were diluted 1:2 in fresh 10% FBS medium. On day 4, cells were split 1:2 in fresh 10% FBS medium. On day 1, cells were de-beaded and diluted to 1 x 10 6 Cells were replated at 500,000 cells / mL and allowed to rest for 24 hours. On day 0, cells were replated at 500,000 cells per 1000 μL medium and treated or not with 50 μM ACZ. Expression of membrane-bound IL15 and membrane-bound IFNα was measured on days 3, 7, 10, 14, and 20. As shown in Figures 83-85, mbIL15 expressing cells accumulated over time and IFNα was modulated approximately 20-fold with the IFNα-016 and -018 constructs.

[0145] In another round of experiments, human TILs were expanded and engineered with lentiviral vectors to express IFNα, IL18 (an IL1 family member) (see Example 18) or IL15 with TNFSF-X (a TNF superfamily member). Expanded TILs were immunophenotyped and assessed for polyfunctionality by flow cytometry after CD3 / CD28 stimulation. Engineered TILs were transferred into NSG mice to assess antigen-independent TIL persistence in the absence of exogenous IL2. Our carbonic anhydrase 2 (CA2)-based drug-responsive domain (DRD)-modified cytokines were assessed for regulation of protein levels by the CA2 ligand acetazolamide (ACZ). Cytokine expression was assessed by flow cytometry and Meso Scale Discovery assays.

[0146] Engineered TILs expressing IL15 and either IFNα, IL18 or TNFSF-X displayed similar fold expansion, immunophenotype and polyfunctionality in vitro as TILs expressing IL15 alone. Combinations of cytokine-expressing TILs displayed similar in vivo antigen-independent persistence in the absence of IL2 as TILs engineered with IL15 alone. Compared to control cells, suboptimal cell doses of T cells expressing both IL15 and either IFNα or IL18 showed improved efficacy and TME remodeling, while combining IL15 with TNFSF-X resulted in significant tumor growth arrest of B16 melanoma tumors without bypass.

[0147] IL15 induces the proliferation and persistence of IL2-free cytoTIL15TM cells and provides enhanced efficacy to patients with solid tumors marked by an immunosuppressive TME by adding polystimulatory and highly immunostimulatory members of the IFN, IL1, or TNF families.

[0148] Example 22. Jurkat transduced IL18 constructs. [Table 9]

[0149] The test protocol is shown in Figure 86. As shown in Figures 87-90, mbIL18 was modulated 10-fold in the IL18-080 construct. Released IL18 was modulated about 2-fold in constructs IL18-076 and IL18-082. All constructs with collectin 7 showed very low IL18 basal with no / low regulation, which may be due to low mbIL18 expression below our detection range. VEFGR-containing constructs showed good regulation, especially in 100 μl virus titration, with the lowest basal, and the regulation was 10-fold higher for IL18-080. [Table 10]

[0150] Example 23. Oligomerization in a cytoplasmic setting: trimerization and tetramerization of SOX2 HEK-293T cells were cultured in standard medium (DMEM (Fisher Scientific Catalog No. 11-960-044) containing 10% FBS (Fisher Scientific Catalog No. 10-082-147) and 1% penicillin / streptomycin (P / S, ThermoFisher Catalog No. 15140122). HEK293T cells were cultured in standard medium (DMEM (Fisher Scientific Catalog No. 11-960-044) containing 10% FBS (Fisher Scientific Catalog No. 10-082-147) and 1% penicillin / streptomycin (P / S, ThermoFisher Catalog No. 15140122) using Lipofectamine 1000 ng / ml 200 mL of 100% PBS (Table 12). 2000 and 1 μg of each plasmid DNA of SOX2-001 to SOX2-006X constructs (X varies) were used to transiently transfect cells using the manufacturer's protocol. Constructs SOX2-001 and SOX2-002 are not oligomerized. Constructs SOX2-003 and SOX2-005 are trimerized with collectin 7 located either at the N-terminus (SOX2-003) or C-terminus (SOX2-005). Similarly, SOX2-004 and SOX2- SOX2-006 was tetramerized with the helix from Kv7.4. One day after transfection, 100 μM acetazolamide was added and the cells were cultured for another day. mCherry was used as a transfection marker for SOX2-001 and SOX2-002, and GFP was used as a transfection marker for SOX-003 to SOX2-006. After checking the expression levels by FAC, equal numbers of transfected cells were pelleted and T-PER was analyzed. Lysates were lysed using Tissue Protein Extraction Reagent (ThermoFisher Cat. No. 78510). Lysates were run on NuPAGE 4-12% Bis-Tris gels (ThermoFisher Cat. No. NP0321BOX) and transferred onto nitrocellulose membranes (iBLOT2 NC Regular Stack (ThermoFisher IB23001)). SOX2 was detected using anti-FOXP3 SOX2 monoclonal antibody (RnD Cat. No. AF2018-SP) and IRDye 800CW goat anti-mouse IgG (LI-COR P / N 926-32210). Results are shown in Figure 91 and Table 11. Jurkat cells were successfully transduced with SOX2 constructs. [Table 11]

Table 12

Claims

1. 1. An engineered regulatable polypeptide monomer, comprising: (a) at least one payload having biological activity; (b) at least one drug responsive domain (DRD), wherein the DRD is operably linked to the payload, and the at least one DRD is responsive to a ligand; (c) an oligomerization domain configured to promote oligomerization of the polypeptide monomer.

2. 2. The engineered regulatable polypeptide monomer of claim 1, wherein the biological activity of the at least one payload is modulated by interaction between the at least one DRD and an effective amount of the ligand.

3. 2. The engineered regulatable polypeptide monomer of claim 1, wherein the oligomerization domain is selected from a dimerization domain, a trimerization domain, a tetramerization domain, a pentamerization domain, and a hexamerization domain.

4. 10. An oligomer comprising at least two engineered regulatable polypeptide monomers of claim 1, wherein the monomers are linked through the oligomerization domains of the monomers.

5. 5. The oligomer of claim 4, comprising at least three engineered regulatable polypeptide monomers of claim 1, wherein the monomers are linked through the oligomerization domains of the monomers.

6. 5. The oligomer of claim 4, wherein the monomers are non-covalently linked through the oligomerization domain of each of the monomers.

7. 5. The oligomer of claim 4, wherein the at least one payload of each of the monomers is one payload, the one payload of each of the monomers is the same, the DRD of each of the monomers is one DRD, and the one DRD of each of the monomers is responsive to the same ligand.

8. 5. The oligomer of claim 4, wherein the at least one payload is a first payload, the first payload of each of the monomers is the same, and the biological activity of the first payload in the oligomer ranges from a basal activity level in the absence of a ligand to a maximum activity in the presence of a saturating amount of a ligand, and the basal activity level of the first payload in the oligomer is lower than the basal activity level of the same payload in a control construct.

9. 5. The oligomer of claim 4, wherein the at least one payload is a first payload, the first payload of each of the monomers is the same, and the biological activity of the first payload in the oligomer ranges from a basal activity level in the absence of a ligand to a maximum activity in the presence of a saturating amount of a ligand, and the activity range of the first payload in the oligomer is higher than the activity range of the same payload in a control construct.

10. 1. An engineered regulatable polypeptide, comprising: (a) at least one payload having biological activity; (b) a regulatory hub comprising at least two drug responsive domains (DRDs), said regulatory hub being operably linked to said payload, and said at least two DRDs being responsive to a ligand.

11. 11. The engineered regulatable polypeptide of claim 10, wherein the biological activity of the payload is modulated by interaction between the at least two DRDs and an effective amount of the ligand.

12. 11. The engineered regulatable polypeptide of claim 10, wherein the at least two DRDs are responsive to the same ligand.

13. 13. The engineered regulatable polypeptide of claim 12, wherein the regulatory hub comprises three DRDs, and the three DRDs are responsive to the same ligand.

14. The engineered regulatable polypeptide of claim 10, wherein the biological activity of at least one payload ranges from a basal activity level in the absence of the ligand to a maximum activity level of the payload in the presence of a saturating amount of the ligand, and the range of activity levels of the payload in the engineered regulatable polypeptide is higher than the range of activity levels of the same payload in a control construct.

15. 11. The engineered regulatable polypeptide of claim 10, wherein the payload has a basal level of biological activity in the absence of a ligand that is lower than the basal level of biological activity of the same payload in a control construct.

16. An engineered tunable oligomer comprising at least two polypeptide monomers, each monomer comprising at least one payload or one DRD, said oligomer comprising at least one payload operably linked to at least two DRDs, said at least one payload having biological activity, and said at least two DRDs being responsive to a ligand.

17. 17. The engineered regulatable oligomer of claim 16, wherein the at least two polypeptide monomers each comprise an oligomerization domain, and the oligomerization domain of one of the polypeptide monomers comprises a hetero-oligomerization domain.

18. 17. The engineered tunable oligomer of claim 16, wherein the at least two DRDs are responsive to the same ligand.

19. 17. The engineered, tunable oligomer of claim 16, wherein the biological activity of the payload in the oligomer ranges from a basal activity level in the absence of a ligand to a maximal activity in the presence of a saturating amount of a ligand, and the basal activity level of the oligomer is lower than the basal activity level of the same payload in a control construct.

20. 18. The engineered regulatable oligomer of Claim 17, wherein said at least two polypeptide monomers comprise a first, second, and third polypeptide monomer, wherein said first polypeptide monomer and said second polypeptide monomer are the same, wherein said first polypeptide monomer and said second polypeptide monomer each comprise a DRD, and wherein said third polypeptide monomer comprises a hetero-oligomerization domain and a payload.

21. 17. The engineered, tunable oligomer of claim 16, wherein the ratio of the payload to DRD in the oligomer is 1:

1.

22. An expressible nucleic acid construct encoding one or more engineered regulatable polypeptide monomers of claim 1, one or more engineered regulatable polypeptides of claim 10, or one or more oligomeric polypeptide monomers of claim 16.

23. A plurality of expressible nucleic acid constructs, each construct encoding an engineered regulatable polypeptide monomer of claim 1, one or more engineered regulatable polypeptides of claim 10, or one or more oligomeric polypeptide monomers of claim 16.

24. A vector comprising one or more expressible nucleic acid constructs according to claim 16.

25. 17. A cell comprising one or more expressible nucleic acid constructs of claim 16.

26. 26. The cell of claim 25, wherein the cell expresses at least two polypeptide monomers, and the at least two polypeptide monomers form an oligomer.

27. 27. The cell of claim 26, wherein the oligomer comprises at least three polypeptide monomers.

28. A cell comprising the vector of claim 24.

29. The cell of claim 25 , wherein the cell is a human cell.

30. 30. The cell of claim 29, wherein the human cell is an immune cell.

31. 26. The engineered regulatable polypeptide monomer of claim 1, the engineered regulatable polypeptide of claim 10, the engineered expressible oligomer of claim 16, the expressible nucleic acid construct of claim 16, the vector of claim 24, or the cell of claim 25, wherein the DRD is selected from CA2 (SEQ ID NO: 1), ecDHFR (SEQ ID NO: 18), hDHFR (SEQ ID NO: 37), FKBP (SEQ ID NO: 93), PDE5 ligand binding domain (SEQ ID NO: 94), PDE5 (SEQ ID NO: 95), and ER (SEQ ID NO: 96), and / or an amino acid sequence having at least 85% identity to SEQ ID NOs: 1, 18, 37, 93, 94, 95, or 96.

32. 26. The engineered regulatable polypeptide monomer of claim 1, the engineered regulatable polypeptide of claim 10, the expressible nucleic acid construct of claim 16, the vector of claim 24, or the cell of claim 25, wherein the payload is selected from the group consisting of a cytokine, a cytokine receptor, a TCR, a CAR, an immunomodulatory protein, and combinations thereof.

33. 11. A composition comprising the oligomer of claim 4 or the engineered regulatable polypeptide of claim 10 for use in a method for modulating the amount or activity of a payload in the oligomer of claim 4 or the engineered regulatable polypeptide of claim 10, the method comprising contacting the DRD with a selected dose of the ligand, wherein the selected dose of the ligand results in a selected amount or activity level of the payload.

34. 17. A composition comprising the engineered tunable oligomer of claim 16 for use in a method of modulating the amount or activity of said at least two payloads in the engineered tunable oligomer of claim 16, said method comprising contacting said at least two DRDs with said ligand, wherein said selected dose of said ligand results in a selected amount or activity level of said at least two payloads.

35. 34. The composition of claim 33, wherein said contacting occurs in vivo.

36. 26. A composition for delivering a payload to a subject, the composition comprising the nucleic acid construct of claim 16 or the vector of claim 24.

37. 24. A composition for delivering a payload to a subject, the composition comprising a plurality of expressible nucleic acid constructs of claim 23.

38. 37. The composition of claim 36, wherein the composition is administered to the subject in combination with a selected amount of a ligand to deliver a selected amount or activity of the payload to the subject.

39. 37. The composition of claim 36, wherein expression of the two or more nucleic acid constructs in the subject's cells results in expression of two or more polypeptide monomers, and the two or more polypeptide monomers form an oligomer.

Images