Il-12 stability variants

Abstract

The present disclosure provides IL-12 stability variants, nucleic acids encoding the variants, vectors comprising the nucleic acids, pharmaceutical compositions comprising the variants, therapeutic cells expressing the variants, and methods or uses of the same for the treatment or prevention of a disease or disorder.

Description

IL-12 STABILITY VARIANTS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application 63 / 510,579, filed June 27, 2023, the content of which is incorporated by reference herein in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 27, 2024, is named 026225_WO032_SL.xml and is 36,143 bytes in size. BACKGROUND OF THE INVENTION

[0003] Interleukin-12 (IL-12) is a pleiotropic proinflammatory cytokine that can stimulate the proliferation of natural killer (NK) and T cells and drive secretion of IFN-γ and TNF-α (Tugues et al., Cell Death Differ. (2015) 22(2):237-46). IL-12 is a heterodimer composed of two subunits, p35 (a.k.a. IL-12A) and p40 (aka. IL-12B). The heterodimer is also called p70. IL-12 binds to IL-12 receptor (IL-12R), which in turn is composed of two subunits, IL- 12Rβ1 and IL-12Rβ2. IL-12Rβ1 primarily binds the IL-12 p40 subunit, while IL-12Rβ2 primarily binds the IL-12 p35 subunit (Presky et al., J Immunol. (1998) 160(5):2174-9). There is no complete co-crystal structure of IL-12 bound to IL-12R. Simultaneous binding of IL-12 to both IL-12Rβ1 and IL-12Rβ2 may be required to drive intracellular signaling (Presky et al., PNAS (1996) 93(24):14002-7; Presky et al., Ann NY Acad Sci. (1996) 795:390- 3; Robinson, Cytokine (2015) 71(2):348-59). Recent cryogenic electron microscopy studies produced a structure of IL-12 in complex with IL-12R, but they were unable to solve the detailed interactions between p35 and IL-12Rβ2 (Glassman et al., Cell (2021) 184:983-99).

[0004] IL-12 is a key cytokine in the initiation of a Th1 response and has been explored as a potential therapy to treat cancer (Lasek et al., Cancer Immunol Immunother. (2014) 63(5):419-35). But due to significant systemic toxicity, the approaches to IL-12-based immunotherapy have been focused on direct injections of IL-12 to tumor sites and on fusions of IL-12 to tumor-targeting moieties. Some researchers have attempted to use a cell-based approach to deliver IL-12 in which cells engineered to express IL-12 are administered in vivo(Wei et al., J Cell Mol Med. (2013) 17(11):1465-74; Zhang et al., Clin Cancer Res. (2015) 21(10):2278-88). However, this approach is also challenging because IL-12 activates proinflammatory signaling even at very low concentrations in circulation. Furthermore, efficacy is still a critical consideration as the immunosuppressive TME can restrain cell activity even in the presence of IL-12 (Lasek, supra).

[0005] Thus, there is a long-felt and unmet need for a safe and effective form of IL-12 for stimulation of the immune system, either as a monotherapy or as a combination therapy. SUMMARY OF THE INVENTION

[0006] The present disclosure provides an IL-12 variant comprising: a p40 subunit sequence with an amino acid sequence at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO:1, and a p35 subunit sequence with an amino acid sequence at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO:2, wherein the IL-12 variant lacks a native disulfide bond between the p40 subunit sequence and the p35 subunit sequence.

[0007] In some embodiments, the IL-12 p40 subunit sequence comprises a mutation removing a cysteine residue at a position corresponding to C177 of SEQ ID NO:1, optionally wherein the mutation is a C-to-S mutation; and / or the p35 subunit sequence comprises a mutation removing a cysteine residue at a position corresponding to C74 of SEQ ID NO:2, optionally wherein the mutation is a C-to-S mutation. In some embodiments, the IL-12 variant comprises mutations at both positions (e.g., both to a serine).

[0008] In some embodiments, the IL-12 variant p35 subunit sequence comprises one or more of: a mutation at a position corresponding to R181 of SEQ ID NO:2 (e.g., a R-to-A mutation); a mutation at a position corresponding to R183 of SEQ ID NO:2; a mutation at a position corresponding to V185 of SEQ ID NO:2 (e.g., a V-to-A mutation); and a mutation at a position corresponding to R189 of SEQ ID NO:2 (e.g., a R-to-K mutation).

[0009] In some embodiments, the IL-12 variant p40 subunit sequence further comprises a substitution to a C at a position corresponding to A179 of SEQ ID NO:1, and the p35 subunit sequence further comprises a substitution to a C at a position corresponding to I52 of SEQ ID NO:2.

[0010] In some embodiments, the IL-12 variant p40 subunit sequence comprises SEQ ID NO:12, and the p35 subunit sequence comprises SEQ ID NO:13.

[0011] In some embodiments, the IL-12 variant p40 subunit sequence comprises SEQ ID NO:12, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:13 but for an R181A mutation.

[0012] In some embodiments, the IL-12 variant p40 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:12 but for an A179C mutation, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:13 but for an I52C mutation.

[0013] In some embodiments, the IL-12 variant p40 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:12 but for an A179C mutation, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:13 but for an I52C mutation and an R181A mutation.

[0014] In some embodiments, the IL-12 variant p40 subunit sequence comprises SEQ ID NO:10, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:11 but for a mutation at position C70 (e.g., a C70S mutation).

[0015] In some embodiments, the IL-12 variant p40 subunit sequence comprises SEQ ID NO:10, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:11 but for a mutation at position C70 (e.g., a C70S mutation) and a mutation at position S177 (e.g., an S177R mutation).

[0016] In some embodiments, the IL-12 variant p40 subunit sequence comprises SEQ ID NO:10, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:11 but for a mutation at position N184 (e.g., an N184A mutation).

[0017] In some embodiments, the IL-12 variant is a fusion protein in which the p40 subunit sequence and the p35 subunit sequence are linked by a cleavable peptide linker.

[0018] In some embodiments, the IL-12 variant cleavable peptide linker comprises a furin-cleavage site, optionally wherein the cleavable linker comprises SEQ ID NO:5.

[0019] In some embodiments, the IL-12 variant comprises an amino acid sequence at least 90% sequence identical to amino acid residues 22-533 of SEQ ID NO:6.

[0020] In one aspect, the present disclosure provides an isolated nucleic acid molecule or isolated nucleic acid molecules encoding the IL-12 variant (e.g., mRNA or DNA).

[0021] In another aspect, the present disclosure provides an expression vector or expression vectors comprising the isolated nucleic acid molecule(s) encoding the IL-12 variant. In some embodiments, the expression vector(s) is / are selected from lentiviral vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors. In some embodiments, the expression vector(s) further comprising a tissue-specific or an induciblepromoter. In certain embodiments, the inducible promoter comprises a combination of one or more nuclear factor of activated T cells (NFAT) response element domains and a human beta-globin (hBG) minimal promoter domain, optionally wherein the inducible promoter comprises SEQ ID NO:14. In some embodiments, the inducible promoter comprises a combination of one or more nuclear factor of activated T cells (NFAT) response element domains and a YB_TATA minimal promoter domain

[0022] In yet another aspect, the present disclosure provides a method of producing an IL- 12 variant, comprising: culturing the mammalian cell comprising the nucleic acid molecule(s) or vector(s) herein under conditions that allow expression of the IL-12 variant; and isolating the IL-12 variant from the culture.

[0023] In one aspect, the present disclosure provides a mammalian cell comprising the nucleic acid molecule(s) encoding the IL-12 variant or the expression vector(s) comprising the isolated nucleic acid molecule(s) encoding the IL-12 variant. In some embodiments, the mammalian cell is a human immune cell. In some embodiments, the human immune cell is a T cell, a NK cell, or a TIL, optionally engineered to express a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), or a chimeric costimulatory receptor (CCR), further optionally wherein the CAR, engineered TCR, or CCR targets a tumor antigen.

[0024] In another aspect, the present disclosure provides a pharmaceutical composition comprising the IL-12 variant, nucleic acid molecule(s) encoding the IL-12 variant, the expression vector(s) comprising the isolated nucleic acid molecule(s) encoding the IL-12, or a mammalian cell comprising the nucleic acid molecule(s) encoding the IL-12 variant or the expression vector(s) comprising the isolated nucleic acid molecule(s) encoding the IL-12 variant; and a pharmaceutically acceptable carrier.

[0025] In another aspect, the present disclosure provides a method of stimulating the immune system, or treating cancer, in a human subject in need thereof, comprising administering the pharmaceutical composition herein to the human subject. Also provided is use of the IL-12 variant, nucleic acid molecule(s) encoding the IL-12 variant, the expression vector(s) comprising the isolated nucleic acid molecule(s) encoding the IL-12, or a mammalian cell comprising the nucleic acid molecule(s) encoding the IL-12 variant or the expression vector(s) comprising the isolated nucleic acid molecule(s) encoding the IL-12 variant for the manufacture of a medicament for stimulating the immune system or treating cancer in a human subject in need thereof. Also provided is the IL-12 variant, nucleic acid molecule(s), expression vector(s), the mammalian cell, or the pharmaceutical composition for use in stimulating the immune system or treating cancer in a human subject in need thereof.

[0026] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG.1 is a pair of schematic diagrams depicting the protein structure of scIL-12 in which each subunit (p35 and p40) comprise wildtype (WT) IL-12 sequence (referred to as WT scIL-12) (left) and the proteins structure of exemplary IL-12 stability variants of the disclosure, in which the disulfide bridge present in the WT structure has been replaced with a furin-cleavable linker (right). The sequence for the G6S peptide linker is set forth in SEQ ID NO:17.

[0028] FIG.2 is a legend and a corresponding photograph of an automated Western Blot, in which WT scIL-12 and exemplary IL-12 stability variants of the disclosure have been immunoprecipitated from the supernatant of T cells modified to express each variant, using a rabbit anti-p40 primary antibody and detected using an anti-rabbit Horseradish Peroxidase (HRP) labeled secondary antibody.

[0029] FIG.3 is a series of graphs demonstrating efficient furin cleavage of exemplary IL-12 stability variants (human and mouse) secreted from primary T cells modified to express the IL-12 stability variant. Top Row: this pair of graphs depicts the relative concentrations (measured in pg / mL) of exemplary IL-12 stability variants secreted (left plot, p40) and the dimerization state of these exemplary IL-12 stability variants (right plot, p70) in samples obtained from normalized T-cell supernatants. Bottom row: the left plot depicts the relative concentrations (measured in pg / mL) of IFN-γ secreted by proximal bystander CD19+T cells in response to secretion of an exemplary IL-12 stability variant from modified T cells in co- culture, when the IFN-γ measurement is taken at the same point in time as the p40 and p70 measurements depicted in the top row (the sample obtained from the normalized T-cell supernatant); the right plot depicts the relative concentrations (measured in pg / mL) of IFN-γ secreted by proximal bystander mock transfected CD19+T cells in response to secretion of an exemplary IL-12 stability variant from modified T cells in co-culture, when the IFN-γ measurement is taken at a later timepoint than the left plot (sample obtained from a supernatant transfer assay). In this experiment, IFN-γ secretion is an indication of activity of IL-12 stability variants. The experimental conditions, from top to bottom of the key and fromleft to right in each plot are: hscIL12-p40-furin-p35 and mscIL12-p40-furin-p35 (human and mouse forms, respectively, of scIL-12 having an intact inter-subunit disulfide bond; the term “furin” as used in any construct name refers to a furin-cleavable peptide linker); hscIL12- p40-C177S-furin-p35-C74S and mscIL12-p40-C175S-furin-p35-C70S (human and mouse forms, respectively, of scIL-12 having mutations to disrupt the disulfide bond); a negative control scIL12-p40-C177S-furin-p35-C74S-Y40A-R189K (a human form of scIL-12 having mutations to disrupt the disulfide bond, as well as a Y40A mutation to weaken affinity to IL- 12Rb2 and an R189K mutation to strongly destabilize p70) and a mock transfection control (mock).

[0030] FIG.4 is a pair of graphs demonstrating that cleaved IL-12 stability variants dissociate over a time course of minutes. See Example 2 for a description of the terms used in this figure. The left plot depicts the off-rate (measured in 1 / s) of hscIL12-p40-C177S-p35- C74S (also referred to herein as the “DS” variant) and hscIL12-p40-C177S-p35-C74S- R181A (also referred to herein as the “DS+R181A” variant). The right plot depicts the molecule half-life (measured in seconds (s)) of hscIL12-p40-C177S-p35-C74S and hscIL12- p40-C177S-p35-C74S-R181A. For both plots, the error bars represent the standard deviation from two independent experiments. See FIG.9 for quantification of the data presented here graphically for IL-12 stability variants that signal through human IL-12 receptors.

[0031] FIG.5 is a pair of graphs demonstrating that mouse IL12 stability variants (variants that signal through mouse IL-12 Receptors) respond to p35 / p40 interface mutations (i.e., mutations in the p35 or p40 subunits at locations of interaction between the subunits when in heterodimer form; see e.g., FIG.1, right panel) differently than human IL12 stability variants (variants that signal through human IL-12 Receptors). Human IL12 stability variants are shown on the left plot while mouse IL12 stability variants are shown on the right plot. The presence of p70 (measured as picograms per milliliter) secreted from T cells modified to express each variant was measured in an MSD assay performed on samples from normalized T-cell supernatant.

[0032] FIG.6 is an alignment of mouse (top) and human (bottom) sequences of the p35 subunit in WT IL-12 and scIL-12 containing WT sequences depicting several positions appear to be important for either IL-12Rb2 or for dimer formation along a p35 / p40 interface. Tyrosine (Y) residues in alignment rows 2 and 4 are IL-12Rb2-interactive residues conserved in both human and mouse. The boxed position in row 3 of the alignment are IL-12Rb2- interactive residues that are not conserved in both human and mouse. Within row 4 of the alignment, the double-boxed positions (ST in mouse and RI in human) are important p35 / p40interface residues that are not conserved. Within row 4 of the alignment, the boxed valine (V) and arginine (R) residues are important p35 / p40 interface residues that are conserved. The figure discloses SEQ ID NOs:18 and 19, respectively, in order of appearance.

[0033] FIG.7 is an alignment of mouse (top) and human (bottom) sequences of helix 4 within the p35 subunit highlighting a position of interest at the p35 / p40 interface. The figure discloses SEQ ID NOs:20 and 21, respectively, in order of appearance.

[0034] FIG.8 is a series of graphs demonstrating that mscIL12-p35-C70S-furin-p40 (also referred to herein as the “mouse DS” variant) dissociates significantly faster than hscIL12- p40-C177S-p35-C74S (also referred to herein as the “Human DS” variant) but that the addition of an S177R mutation in mouse DS slows dissociation. Octet® data reflecting the activity of IL12 stability variants capable of signaling through human IL-12R (“human variants”) are shown in the top row while Octet® data reflecting the activity of IL-12 stability variants capable of signaling through mouse IL-12R (“mouse variants”) are shown in the bottom row.

[0035] FIG.9 is a table providing quantitative data for experiments described in FIGs.4, 8, and 10. *The noted error (±) represents the standard deviation from two independent experiments. **Only one measurement was performed for these samples.

[0036] FIG.10 is a pair of graphs demonstrating the off-rates and molecule half-life measurements for human variants and mouse variants. Mouse DS + S177R has comparable p35 / p40 dissociation kinetics to Human DS and provides a tool variant for syngeneic in vivo studies. See FIG.9 for quantification of the data presented here graphically for IL-12 stability variants that signal through human IL-12 receptors.

[0037] FIG.11 is a pair of graphs demonstrating localized IL-12 activity in vitro using a bystander assay. Left: The number of bystander cells (CD19+mock-transfected T-cells) that express IFN-γ intracellularly are shown for bystander cells either proximal (flat bottom) or distal (transwell) to modified T-cells modified to secrete IL-12 stability variants of the disclosure. Right: a corresponding plot to demonstrate, in each condition, the amount of IL- 12 stability variant available to proximal and distal bystander cells (measured in picograms per milliliter).

[0038] FIG.12 is a graph demonstrating a safety benefit of IL-12 stability variants of the disclosure. A worst-case-scenario equilibrium concentrations for active p70 in circulation were calculated for the human DS variant.

[0039] FIGs.13A-C are graphs demonstrating that IL-12 reduced stability variants enhanced tumor killing of antigen-specific TCR-expressing T cells against A375 target cellsover 4 rounds of co-culture with the target cells. The antigen-specific TCR was expressed in all conditions. In the sequential killing assay 25% of each co-culture was carried over to new target cells every 3 days, until differences in cytolysis between conditions were observed. “Furin-DS”: furin-cleaved disulfide mutants. “Furin-DS-R181”: Furin-DS + p70- destabilizing mutation R181A in the p35 subunit.

[0040] FIG.13D is a graph showing that all the TCR-expressing T cells (IL-12 reduced stability variants and wild-type) all demonstrated enhanced tumor cytolysis in the presence of IL-12 in the fourth round of co-culture with A375 cells. The Furin-DS, Furin-DS-I52C- A179C, and Furin-DS-I52C-A179C-R181A mutants and wildtype single-chain IL-12 (wt scIL-12) expressing T cells all performed significantly better than TCR alone while the Furin-DS-R181A variant trended towards significance (p = 0.07). No significant differences were seen in cytotoxicity between the wt scIL-12 and any of the IL-12 reduced stability variants. “Furin-DS-I52C-A179C”: Furin-DS + p70-stabilizing mutation I52C in the p35 subunit and A179C in the p40 subunit. “Furin-DS-I52C-A179C-R181A”: Furin-DS + p70- destabilizing mutation R181A in the p35 subunit, and p70-stabilizing mutations I52C and A179C noted above.

[0041] FIGs.14A-B are graphs showing tumor growths in NSG-MHC I / II double knockout mice (n = 8) implanted subcutaneously with 1.0 × 106A375 (human melanoma) cells for two studies using the same methodology. FIG.14A reflects “Study 1” and FIG. 14B reflects “Study 2.” After the tumors in each study reached the desired volume (average of 100 mm3), the animals were treated with 2.5 × 106antigen-specific TCR-expressing T cells co-transduced with IL-12 reduced stability variants under the control of engineered cell activation-inducible promoters (e.g., 4xNFAT-hBG). Mice receiving PBS (no T cells) were used as control. Tumor volumes were measured twice weekly following infusion. Mice treated with TCR T cells expressing the Furin-DS mutant (“Furin DS IL12 Mutant” in FIG. 14A and “Reduced Stability IL12” in FIG.14B) demonstrated deeper anti-tumor responses compared to those treated with wt-scIL-12 or TCR alone. In FIG.14A, the growth measured in mice treated with IL-12 between days 10-30 was determined to be due to expansion of T cells at the tumor site, and not true tumor progression.

[0042] FIG.14C is a Kaplan-Meier survival curve from Study 2 showing mice treated with TCR T cells expressing the Furin-DS mutant (“Reduced Stability IL12 Mutant”) demonstrated improved survival compared to those treated with T cells expressing wt-scIL- 12.

[0043] FIGs.15A-F are graphs showing the effects of the IL-12 reduced stability variants on T-cell proliferation, cytokine secretion in vivo, and safety as determined in Study 1 (FIGs. 15A and 15B) and Study 2 (FIGs.15C-15F). Blood was drawn from mice at 1, 7, 14, 21, 28, and 42 days after infusion with TCR-T cells, and analyzed via flow cytometry for blood PK, and cytokine levels. FIG.15A shows that TCR-T cells expressing wt scIL12 or Furin-DS mutant demonstrated a proliferative advantage over mice treated with TCR-T cells alone. FIG.15B shows that mice treated with TCR-T cells expressing wt scIL12 or Furin-DS also saw enhanced levels of IFN-γ secretion as compared to TCR-T cells alone. FIG.15C shows that the proliferation profiles of TCR-T cells expressing wt scIL12 were similar to those of TCR-T cells expressing the Furin-DS mutant. FIG.15D shows animals treated with T cells expressing the Furin-DS mutant (“Reduced Stability”) have higher plasma concentration of IFN-γ compared to animals treated with T cells expressing wild-type IL-12. FIGs.15E and 15F are graphs indicating that the IL-12 reduced stability variants demonstrate an improved safety profile. FIG.15E shows that that levels of IL-12 p40 were detected in the plasma of mice treated with TCR-T cells expressing the wt scIL-12 and in the plasma of mice treated with TCR-T cells expressing the reduced stability Furin-DS (“Reduced Stability”) variant, indicating that both IL-12 constructs are expressed in the mice. However, FIG.15F shows that levels of IL-12 p70 (the active, dimerized form) were only detected in the plasma of mice treated with TCR-T cells expressing wt scIL-12 and not in mice treated with TCR-T cells expressing the reduced stability Furin-DS IL-12 (“Reduced Stability”) variant. These results demonstrate the self-inactivating function of the Furin-DS IL-12 variant post expression. T his trend was replicated in a second mouse study, confirming the reproducibility of the self- inactivating function of the Furin-DS stability mutant (not shown).

[0044] FIG.16 is a graph showing mouse production of IL-12 p40 as determined by MSD from the highest expressing mouse B16-F10 cell lines. “trIL-12” refers to the murine equivalent of the Furin-DS mutant (“sc-mIL-12-p40-C175S-furin-p35-C70S-S177R”). These data confirm that mouse surrogate IL-12 stability variant mutation designs, such as sc-mIL- 12-p40-C175S-furin-p35-C70S-S177R, can be expressed in mouse cells.

[0045] FIGs.17A-17B are graphs showing the median tumor volume (FIG.17A) and the individual tumor growth curves (FIG.17B) of C57 / BL6 mice implanted with mixtures of B16F10 tumor cells, in which 10% of tumor cells express either wild-type murine single- chain IL-12 (“WT sc-mIL-12”) or a murine equivalent of the Furin-DS mutant (“sc-mIL-12- p40-C175S-furin-p35-C70S-S177R”).

[0046] FIGs.18A-18C are three graphs showing the plasma cytokine concentrations of murine IL-12 p40 (FIG.18A), murine IL-12 p70 (FIG.18B), and murine IFN- ^ ^ ^FIG.18C) as determined by MSD from C57 / BL6 mice implanted with mixtures of B16F10 tumor cells, in which 10% of tumor cells express either wild-type murine single-chain IL-12 (“WT sc- mIL-12”) or a murine equivalent of the Furin-DS mutant (“sc-mIL-12-p40-C175S-furin-p35- C70S-S177R”). DETAILED DESCRIPTION OF THE INVENTION

[0047] The present disclosure describes IL-12 stability variants that avoid the systemic toxicity of prior IL-12 therapies. These stability variants have the same or similar biological activities of wildtype IL-12 but lack the wildtype protein’s stability. The variants rapidly inactivate after secretion from engineered cells (e.g., immune cells such as T cells) in vivo. The variants are less stable than wildtype IL-12 because they lack the native disulfide bond between the two subunits of IL-12 (e.g., the p35 or alpha subunit, and p40 or the beta subunit) due to mutations to residues that form or facilitate forming of the native disulfide bond.

[0048] In some embodiments, the engineered cells are tumor-specific T cells and this design approach achieves safe, local delivery of IL-12 activity from the engineered tumor- specific T cells. The locally active IL-12 variants modulate the tumor microenvironment (TME) to promote innate and adaptive immune responses and support cytotoxic activity of T and NK cells. IL-12 variants whose expression is substantially limited to TME are also termed tumor-restricted IL-12 (trIL-12) herein.

[0049] Existing approaches to using IL-12 as a therapeutic agent pose substantial risk because the potent activity of IL-12 even at very low concentrations. Even for local administration, small amounts of IL-12 that diffuse from sites of interest can drive unwanted activity and cause toxicity. The IL-12 stability variants of the present disclosure circumvent this problem because they have a shorter half-life due to the less stable complexing between the two subunits of IL-12. This short half-life allows the IL-12 variants act locally without posing toxicity to tissues distant from the site of secretion. I. IL-12 Stability Variants

[0050] The IL-12 variants of the present disclosure encompass only non-naturally occurring IL-12 molecules. It is believed that the IL-12 variants disclosed herein are non-naturally occurring. The IL-12 variants herein have reduced stability (reduced half-life) as compared to their wildtype counterpart, and thus are also called “IL-12 stability variants.” In some embodiments, the IL-12 stability variants herein have comparable binding affinity for IL-12R as compared to their wildtype counterpart.

[0051] The IL-12 stability variants comprise mutations in the p35 sequence, the p40 sequence, or both sequences. These mutations remove or prevent formation of the single native disulfide bond between the p35 and p40 subunits.

[0052] In some embodiments, the stability variant is a fusion of the p35 and p40 subunits linked by a cleavable peptide linker (e.g., a flexible, cleavable peptide linker) where the cleavable linker holds the two subunits together to form a functional IL-12, but can be cleaved by a protease in the local extracellular environment such that the IL-12 variant secreted from the engineered cell is only active for a limited period of time in vivo. The IL- 12 variant’s effect is therefore local without causing systemic toxicity.

[0053] In some embodiments, the IL-12 stability variant is a heterodimer and, while not having the native disulfide bond, contains a nonnative, weaker inter-subunit disulfide bond that is engineered into the protein recombinantly, leading to less stable coupling between p35 and p40. These stability variants retain highly effective signaling capacity, but on a limited temporal and thus spatial scale, thereby avoiding systemic toxicity. In some embodiments, the IL-12 stability variant is a heterodimer that lacks the native disulfide bond or an alternative disulfide bond structure, but rather the mutated p35 and / or p40 subunits maintain a less stable coupling by non-covalent bond attractions and as a result, the p35 and p40 subunits maintain a temporally limited heterodimeric configuration.

[0054] In some preferred embodiments, the IL-12 variants are variants of human IL-12. By “variants of human IL-12” or “human IL-12 variants’ is meant an IL-12 molecule comprising sequences of human IL-12 p35 and p40 subunits where one or both of the p35 and p40 sequences contain one or more amino acid mutations relative to wildtype human sequences.

[0055] In other preferred embodiments, the IL-12 variants are variants of mouse IL-12. By “variants of mouse IL-12” or “mouse IL-12 variants” is meant an IL-12 molecule comprising sequences of mouse IL-12 p35 and p40 subunits where one or both of the p35 and p40 sequences contain one or more amino acid mutations relative to wildtype mouse sequences.A. p40 Subunit

[0056] The p40 subunit of IL-12 is also known as the β subunit. An exemplary mature wildtype human p40 sequence is shown below: IWELKKDVYV VELDWYPDAP GEMVVLTCDT PEEDGITWTL DQSSEVLGSG KTLTIQVKEF GDAGQYTCHK GGEVLSHSLL LLHKKEDGIW STDILKDQKE PKNKTFLRCE AKNYSGRFTC WWLTTISTDL TFSVKSSRGS SDPQGVTCGA ATLSAERVRG DNKEYEYSVE CQEDSACPAA EESLPIEVMV DAVHKLKYEN YTSSFFIRDI IKPDPPKNLQ LKPLKNSRQV EVSWEYPDTW STPHSYFSLT FCVQVQGKSK REKKDRVFTD KTSATVICRK NASISVRAQD RYYSSSWSEW ASVPCS (SEQ ID NO:1) An unprocessed wildtype human p40 sequence (UniProt ID No. P29460) with the signal peptide still attached is provided herein as SEQ ID NO:8. The cysteine for forming the native inter-subunit disulfide bond is C177, boxed in the above sequence.

[0057] An exemplary wildtype mouse p40 mature amino acid sequence (UniProt ID No. P43432) is shown below: MWELEKDVYV VEVDWTPDAP GETVNLTCDT PEEDDITWTS DQRHGVIGSG KTLTITVKEF LDAGQYTCHK GGETLSHSHL LLHKKENGIW STEILKNFKN KTFLKCEAPN YSGRFTCSWL VQRNMDLKFN IKSSSSSPDS RAVTCGMASL SAEKVTLDQR DYEKYSVSCQ EDVTCPTAEE TLPIELALEA RQQNKYENYS TSFFIRDIIK PDPPKNLQMK PLKNSQVEVS WEYPDSWSTP HSYFSLKFFV RIQRKKEKMK ETEEGCNQKG AFLVEKTSTE VQCKGGNVCV QAQDRYYNSS CSKWACVPCR VRS (SEQ ID NO:10) The cysteine for forming the native inter-subunit disulfide bond is C175, boxed in the above sequence.

[0058] As used herein, the term “p40 subunit” encompasses mutant forms of the subunit (e.g., mutated, truncated or extended forms). In some embodiments, the mutant forms can have molecule weights that diverge from the molecule weight of a wildtype p40 subunit, e.g., with the amino acid sequence set forth in SEQ ID NO:10.

[0059] As used herein, a “corresponding” amino acid residue or position means an amino acid residue that aligns with (though not necessarily identical to) the reference residue, or an amino acid position that aligns with the reference position, when the subject sequence and the reference sequence containing the residues are aligned to achieve maximum homology (allowing gaps that are recognized in the art). For example, a position corresponding to an amino acid residue in SEQ ID NO:1 encompasses a position in an orthologous sequence (e.g., mouse p40) where the amino acid residue is conserved (e.g., the same as the human residue). The sequence alignment of human p40 to mouse p40 as performed by BLAST® is shown below; as apparent from this alignment, a position corresponding to C177 (boxed) of SEQ IDNO:4 (reflecting SEQ ID NO:1 minus the C-terminal serine) in mouse p40 is C175 (boxed) of SEQ ID NO:16 (reflecting SEQ ID NO:10 minus the four C-terminal residues):Human 240 WSTPHSYFSLTFCVQVQGKSKR--------EKKDRVFTDKTSATVICRKNASISVRAQDR 291 WSTPHSYFSL F V++Q K ++ +K +KTS V C K ++ V+AQDR Mouse 237 WSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQC-KGGNVCVQAQDR 295 Human 292 YYSSSWSEWASVPC 305 (SEQ ID NO:4) YY+SS S+WA VPC Mouse 296 YYNSSCSKWACVPC 309 (SEQ ID NO:16) B. p35 Subunit

[0060] The p35 subunit of IL-12 is also known as the α subunit. An exemplary mature wildtype human p35 sequence is shown below: RNLPVATPDP GMFPCLHHSQ NLLRAVSNML QKARQTLEFY PCTSEEIDHE DITKDKTSTV EACLPLELTK NESCLNSRET SFITNGSCLA SRKTSFMMAL CLSSIYEDLK MYQVEFKTMN AKLLMDPKRQ IFLDQNMLAV IDELMQALNF NSETVPQKSS LEEPDFYKTK IKLCILLHAF RIRAVTIDRV MSYLNAS (SEQ ID NO:2) An unprocessed wildtype human p35 sequence (UniProt ID No. P29459) with the signal peptide still attached is provided herein as SEQ ID NO:9. The cysteine for forming the native inter-subunit disulfide bond is C74, boxed in the above sequence.

[0061] An exemplary wildtype mouse IL-12 p35 mature amino acid sequence (UniProt ID No. P43431) is shown below: RVIPVSGPAR CLSQSRNLLK TTDDMVKTAR EKLKHYSCTA EDIDHEDITR DQTSTLKTCL PLELHKNESC LATRETSSTT RGSCLPPQKT SLMMTLCLGS IYEDLKMYQT EFQAINAALQ NHNHQQIILD KGMLVAIDEL MQSLNHNGET LRQKPPVGEA DPYRVKMKLC ILLHAFSTRV VTINRVMGYL SSA (SEQ ID NO:11)The cysteine for forming the native inter-subunit disulfide bond is C70, boxed in the above sequence.

[0062] As used herein, the term “p35 subunit” encompasses mutant forms of the subunit (e.g., mutated, truncated or extended forms). In some embodiments, the mutant forms can have molecule weights that diverge from the molecule weight of a wildtype p35 subunit, e.g., with the amino acid sequence set forth in SEQ ID NO:11.

[0063] A position corresponding to an amino acid residue in SEQ ID NO:2 encompasses a position in an orthologous sequence (e.g., mouse p35) when the subject sequence and orthologous sequence are aligned to maximize homology. In some embodiments, the corresponding positions contain residues that are conserved (e.g., the residue in the orthologous sequence (e.g., a mouse residue) is the same as the subject residue (e.g., a human residue)). The sequence alignment of human p35 to mouse p35 as performed by BLAST® is shown below; as apparent from this alignment, a position corresponding to C74 of SEQ ID NO:2 in mouse p35 is C70 of SEQ ID NO:11: Human 1 RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTV 60 R +PV+ P CL S+NLL+ +M++ AR+ L+ Y CT+E+IDHEDIT+D+TST+ Mouse 1 RVIPVSGP----ARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTL 56 Human 61 EACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMN 120 + CLPLEL KNESCL +RETS T GSCL +KTS MM LCL SIYEDLKMYQ EF+ +N Mouse 57 KTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAIN 116 Human 121 AKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAF 180 A L +QI LD+ ML IDELMQ+LN N ET+ QK + E D Y+ K+KLCILLHAF Mouse 117 AALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAF 176C. Mutations Removing Native Inter-Subunit Disulfide Bond

[0064] The IL-12 stability variants herein lack the native inter-subunit disulfide bond due to one or more mutations made to one or both of the two subunits. In some embodiments, the mutations are deletions or substitutions. In some embodiments, the mutation can reflect an inversion, transposition, or recombination.

[0065] In some embodiments, a p40 variant contains a mutation at a position corresponding to C177 of SEQ ID NO:1 (e.g., C175 of SEQ ID NO:10), e.g., a mutation removing a cysteine from a position corresponding to position 177 of SEQ ID NO:1 or from a position corresponding to position 175 of SEQ ID NO:10. In some embodiments, the mutation may be a substitution of the cysteine by a serine (S), which is illustrated inexemplary sequences set forth in SEQ ID NO:12. In some embodiments, the p40 sequence in a human IL-12 stability variant comprises SEQ ID NO:12 (i.e., SEQ ID NO:1 with a C177S mutation).

[0066] In some embodiments, a p35 variant contains a mutation at a position corresponding to C74 of SEQ ID NO:2 (e.g., C70 of SEQ ID NO:11), e.g., a mutation removing a cysteine from a position corresponding to position 74 of SEQ ID NO:2 or from a position corresponding to position 70 of SEQ ID NO:11. In some embodiments, the mutation may be a substitution of the cysteine by a serine (S), which is illustrated in exemplary sequences set forth in SEQ ID NO:13. In some embodiments, the p35 sequence in a human IL-12 stability variant comprises SEQ ID NO:13 (i.e., SEQ ID NO:2 with a C74S mutation).

[0067] In some embodiments, the IL-12 stability variant comprises both a mutation at a position corresponding to C177 of SEQ ID NO:1 and a mutation at a position corresponding to C74 of SEQ ID NO:2. For example, the IL-12 stability variant is a human IL-12 variant comprising SEQ ID NOs:12 and 13. In another example, the IL-12 stability variant is a mouse IL-12 variant comprising a C175S mutation in the p40 subunit and a C70S mutation in the p35 subunit.

[0068] In some embodiments, the p40 subunit and / or the p35 subunit comprises other mutations that weaken the coupling of the two subunits, optionally without significantly altering the biological activity (e.g., binding affinity for IL-12R) when the two subunits do couple. For example, the p35 subunit may contain one or more mutations at a position corresponding to residue R181 of SEQ ID NO:2 (e.g., R181A), a position corresponding to R183 of SEQ ID NO:2 (e.g., R179 of SEQ ID NO:11), a position corresponding to V185 of SEQ ID NO:2 (e.g., V181 of SEQ ID NO:11) (e.g., a V185A mutation), or a position correspond to R189 of SEQ ID NO:2 (e.g., R185 of SEQ ID No:11) (e.g., an R189K mutation).

[0069] In some embodiments, the IL-12 stability variant, while lacking the native inter- subunit disulfide bond, is modified to have a new, weaker disulfide bond between the subunits. For example, the p40 contains a mutation to a cysteine (C) at a position corresponding to A179 of SEQ ID NO:2 (e.g., a A179C mutation) and the p35 contains a mutation to a C at a position corresponding to I52 of SEQ ID NO:2 (e.g., an I52C mutation). This pair of engineered-in cysteine residues can form a weak inter-subunit disulfide bond. In some embodiments, the p40 contains a mutation to a cysteine (C) at a position corresponding to A179 of SEQ ID NO:2 (e.g., a A179C mutation) and the p35 contains a mutation to a C ata position corresponding to I52 of SEQ ID NO:2 (e.g., an I52C mutation) and a mutation at a position corresponding to residue R181 of SEQ ID NO:2 (e.g., R181A).

[0070] In some embodiments, the IL-12 stability variant comprises a p40 sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:1. In some embodiments, the IL-12 stability variant comprises a p35 sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:2. In some embodiments, the IL-12 stability variant comprises a p40 sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:1 and a p35 sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:2.

[0071] The percent identity of two amino acid sequences (or of two nucleic acid sequences) may be obtained by, e.g., BLAST® using default parameters (available at the U.S. National Library of Medicine’s National Center for Biotechnology Information website). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the reference sequence.

[0072] The present disclosure also provides IL-12 stability variants that are not derived from wildtype human or mouse sequences but contain mutations relative to their cognate wildtype sequences at positions that correspond to the numbered positions disclosed herein in relation to SEQ ID NO:1 or 2. D. Single-Chain IL-12

[0073] In some embodiments, the present IL-12 variants are single-chain IL-12 (scIL-12) fusion proteins comprising a p35 sequence fused to a p40 sequence. The p35 sequence may be either N-terminal, or C-terminal, to the p40 sequence. The p35 sequence and the p40 sequence may be linked by a cleavable peptide linker, optionally a flexible, cleavable peptide linker. A flexible linker may have, for example, glycine / serine rich sequences.

[0074] In some embodiments, the cleavable linker comprises a substrate sequence for furin, such as R-X-[K / R]-R, or a TEV cleavage site. For example, the linker may comprise the sequence GSRRRR*R*R*S (SEQ ID NO:5), where “*” represents a potential cleavage point by furin.

[0075] In some embodiments, the cleavable linker comprises a substrate sequence of urokinase-type plasminogen activator (uPA), matrix metallopeptidase (MMP) 2, MMP9, or a combination thereof.

[0076] In particular embodiments, the IL-12 variant is single-chain human IL-12 (sc-hIL- 12) comprising a C177S mutation in the p40 sequence or a C74S mutation in the p35sequence. In particular embodiments, the IL-12 variant is single-chain human IL-12 (sc-hIL- 12) comprising a C177S mutation in the p40 sequence and a C74S mutation in the p35 sequence. In certain embodiments, the variant comprises residues 22-533 of SEQ ID NO:6 (where residues 1-21 are a signal sequence and cleaved off in the mature protein). This variant is referred to as “DS-furin variant”, “furin-DS variant” or “p40-C177S-p35-C74S” in the Working Examples below. The term “furin” as it appears in any construct name refers to a furin-cleavable domain, e.g., with a sequence set forth in SEQ ID NO:5.

[0077] In particular embodiments, the IL-12 variant is an sc-hIL-12 DS-furin variant but additionally contains an A179C mutation in the p40 sequence and an I52C mutation in the p35 sequence. This variant is referred to as “DS-furin-I52C-A179C variant” in the Working Examples below.

[0078] In particular embodiments, the IL-12 variant is an sc-hIL-12 DS-furin-I52C- A179C variant but additionally contains an R181A mutation in the p35 sequence. This variant is referred to as “DS-furin-I52C-A179C-R181A variant” in the Working Examples below.

[0079] In particular embodiments, the IL-12 variant is an sc-hIL-12 p40-C177S-p35-C74S but additionally contains an R181A mutation in the p35 sequence. This variant is referred to as “p40-C177S-R181A-p35-C74S variant” in the Working Examples below.

[0080] In particular embodiments, the IL-12 variant is a single-chain mouse IL-12 (sc- mIL-12) comprising a C70S mutation in the p35 sequence. This variant is referred to as “sc- mIL-12-p35-C70S variant” in the Working Example below.

[0081] In particular embodiments, the IL-12 variant is an sc-mIL-12-p35-C70S variant, as described above, but additionally contains a S177R mutation in the p35 sequence. This variant is referred to as “sc-mIL-12-p35-C70S-S177R variant” in the Working Examples below.

[0082] In some embodiments, the scIL-12 variant herein comprises an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the scIL- 12 exemplified herein (e.g., SEQ ID NO:6 and the further variants thereof). II. Expression of IL-12 Variants

[0083] The present disclosure provides nucleic acid molecules encoding the IL-12 stability variants herein and any vector or combination of vectors comprising the nucleic acid molecules encoding the subunits of the IL-12 stability variants. If the variant is a heterodimer, the coding sequences for the two IL-12 subunits may be placed on separateexpression vectors; alternatively, the two coding sequences may be placed on the same vector, with one polycistronic expression cassette, or with two separate expression cassettes. Where the IL-12 variant is a heterodimer, the coding sequences for the two subunits may be placed in a single expression cassette, separated by an IRES, or separated in frame by a coding sequence for a self-cleaving peptide (e.g., a 2A peptide, such as T2A, E2A, P2A, and F2A), such that the two subunits are expressed as separate polypeptides. Polycistronic configurations to facilitate expression of multiple proteins from the same promoter / expression cassette are described in more detail in WO 2021 / 119539. The two translated protein subunits can then heterodimerize to form a functional IL-12 variant with temporally limited stability outside the cell due to the lack of stabilizing covalent bonds.

[0084] The IL-12 stability variants (e.g., heterodimeric IL-12 or scIL-12 proteins having the mutations described herein) can be expressed in cells of interest, such as eukaryotic host cells where purified proteins are desired, or cells (e.g., immune cells) where cell-based therapies are desired.

[0085] The nucleic acid molecules (e.g., the encoding nucleic acid molecule or DNA or RNA vectors containing the encoding nucleic acid molecules) may be introduced into the cells by well-known techniques, including without limitation, electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, colloidal dispersion systems (e.g., as macromolecule complexes, nanocapsules, microspheres, and beads), and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, and liposomes). Alternatively, the nucleic acid molecules may be introduced into the cells by transduction of recombinant viruses whose genomes comprise the nucleic acid molecules. Examples of viral vectors include, without limitation, vectors derived from lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus, and vaccinia virus. In certain embodiments, the recombinant virus is pseudotyped with a heterologous envelope protein. In one embodiment, the recombinant virus is a lentivirus pseudotyped with an envelope glycoprotein derived from vesicular stomatitis virus (VSV), measles virus, or another virus (see e.g., Cronin et al., Curr Gene Ther. (2005) 5(4):387-98; Gutierrez-Guerrero et al., Viruses (2020) 12(9):1016).

[0086] Mammalian host cells for producing IL-12 stability variants include, without limitations, CHO cells, NS0 cells, HEK cells, 293 cells, and the like. Methods of producing therapeutic proteins in mammalian host cells are well known in the art. Insect cells such as Sf9 cells and Sf21 cells may also be used.

[0087] In the context of cell-based therapy, the IL-12 variant herein may be expressed in allogeneic or autologous cells. In some embodiments, coding sequences for the IL-12 variant may be introduced into immune cells such as T cells (e.g., CD4+T cells and CD8+T cells) and natural killer (NK) cells, or their precursor cells (e.g., hematopoietic stem or progenitor cells). Coding sequences for the variant may be introduced through an expression construct (e.g., a viral vector) or through mRNA directly (e.g., delivered by lipid nanoparticles to the cells through local or systemic administration).

[0088] For therapy of human patients, human immune cells can be used. In some embodiments, the human immune cells are T cells (e.g., CD4+T cells and / or CD8+T cells). In some embodiments, the human immune cells are derived from peripheral blood mononuclear cells (PBMCs) derived from a subject. In some embodiments, the T cells are engineered T cells. In some embodiments, the engineered T cells further comprise nucleic acids that encode a chimeric antigen receptor (CAR) specific for an antigen of interest. In some embodiments, the engineered T cells further comprise a nucleic acid encoding an engineered T cell receptor (TCR). In some embodiments, the T cells are tumor-infiltrating T cells (TILs).

[0089] The combination of an IL-12 stability variant with control strategies that localize the expression of the variant to a site of interest (e.g., a tumor or a tumor microenvironment (TME)) maintains IL-12R signaling by increased localized concentrations at the site of interest, while avoiding unwanted signaling activity at lower concentrations away from the site of interest. Thus, expression of the IL-12 stability variant in therapeutic immune cells homed to a target site provides an unprecedented method of tightly regulating IL-12 activity and balancing safety with efficacy.

[0090] In some embodiments, the IL-12 stability variants are expressed under the control of a constitutive promoter, such as a tissue-specific or cell-specific promoter. In alternative embodiments, the IL-12 stability variants are expressed under the control of an inducible promoter. In some embodiments, the inducible promoter responds to an extracellular signal, e.g., a stimulatory or inhibitory immunomodulatory signal. In some embodiments, the inducible promoter responds to an intracellular signal. In some embodiments, the inducible promoter is inducible in a T cell context, e.g., can be a T cell activation inducible promoter. For example, the promoter herein can respond directly or indirectly to a CD3-mediated signal, e.g., a CD3-mediated signal triggered by CAR or TCR stimulation. Examples of promoters useful herein include, without limitation, an immediate early cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an elongation factor-1α (EF-1α) promoter, an MND promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. In some embodiments, the IL-12 stability variants are under the control of inducible promoters that incorporate core or minimal promoters, including core or minimal promoters derived from the aforementioned promoters. Core or minimal promoters suitable for the inducible promoter are described in the art and can be incorporated to minimize the basal level of transcription in cells when the appropriate transcription factors are not present and yet facilitate elevated expression when appropriate transcription factors are present. In non-limiting examples, in some embodiments the inducible promoters of the present disclosure include a core promoter selected from human beta globin (hBG), pJB42CAT5, MiniTK, YB_TATA, minCMV, minSV40, CMV53, or MLP, or functional variants thereof. Exemplary sequences of the aforementioned functional core or minimal promoters are set forth in SEQ ID NOS: 22-29. See also Ede et al., ACS Synth Biol. (2016) 5(5):395-404 (including supplemental information), WO 2021 / 244654, and WO 2020 / 206149, each of which is incorporated herein by reference in its entirety. Additional exemplary core promoters useful in the inducible promoters of the disclosure include minAdeP and minIL2 promoters, and functional variants thereof. “Functional variants” include minor sequence variants, e.g., changes or deletions of ~1, 2, 3, etc., nucleic acid residues of the sequence, where the core promoter retains its desired function to facilitate transcription in the presence of appropriate transcription factors.

[0091] Inducible promoter systems useful for expressing the disclosed IL-12 stability variants include, without limitation, hormone-regulated elements, synthetic ligand-regulated elements, ionizing radiation-regulated elements, and tetracycline (Tet) systems (e.g., “Tet- Off” and “Tet-On” systems). In some embodiments, the inducible promoter comprises transcription factor response elements to facilitate condition-dependent induction of transcription. In some embodiments, the inducible promoters further comprise transcription factor response elements, for example, in combination with a core or minimal promoter sequence, as described above. Response elements are segments of nucleic acid with sequences specifically recognized by transcription factors. Exemplary response elements can be or comprise sequences that are responsive to the activation state of an immune cell.

[0092] For example, when an immune cell is activated (e.g., by antigen engagement), nuclear translocation of certain transcription factors, such as activator protein-1 (AP-1), nuclear factor of activated T-cells (NFAT), nuclear factor-κ-light chain enhancer of activatedB cells (NF-κB), and IRF4, occurs. Such transcription factors bind to their respective response elements. Accordingly, in some illustrative embodiments, the inducible promoter may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) copies of transcription factor response elements, such as AP-1, NFAT, NF-κB response elements, iron-responsive element (IRE), interferon-stimulated response element (ISRE), or any combination thereof. For example, illustrative NFAT response systems relevant to the present disclosure are described in, e.g., Kallunki et al., Cells (2019) 8(8):796; Uchibori et al., Mol Ther Oncolytics. (2018) 12:16-25.

[0093] In some embodiments, the inducible promoter comprises four copies of NFAT response elements. The sequence of an exemplary NFAT response element is set forth in SEQ ID NO:30. In some embodiments, the T cell activation inducible promoter comprises one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc., NFAT response elements) in functional combination with an hBG promoter sequence (see, e.g., Na et al., Blood (2010) 116(11):e18-e25; Szyska et al., Cancer Immunol Res. (2018) 6 (1): 110-120 and GenBank Accession No. MF462285.1 dated September 13, 2017). In some embodiments, the T cell activation inducible promoter comprises four repeats of an NFAT response element (e.g., SEQ ID NO:30) functionally linked directly or with minor intervening sequence to an hBG core promoter (e.g., SEQ ID NO:22). In further embodiments, the promoter comprises SEQ ID NO:14 (see, e.g., Szyska et al., supra and GenBank Accession No. MF462285.1, supra), or a nucleotide sequence that is at least 80% (e.g., at least 85, 90, 95, 96, 97, 98, or 99%) identical thereto and retains T cell activation inducibility. The sequence of an exemplary expression cassette containing such a promoter for expressing a DS-Furin variant is shown in SEQ ID NO:15.

[0094] In some embodiments, the T cell activation inducible promoter comprises one or multiple NFAT response elements in functional combination with a YB_TATA synthetic core promoter sequence (see, e.g., Ede et al., ACS Synth Biol. (2016) 5(5):395-404 (including supplemental information), WO 2021 / 244654, and WO 2020 / 206149). The sequence of an exemplary YB_TATA core promoter is set forth in SEQ ID NO:25. WO 2020 / 206149 discloses combinations of three and six repeats of NFAT response element domains linked to a YB_TATA core promoter domain. In some embodiments, the T cell activation inducible promoter comprises SEQ ID NO:31.

[0095] In some embodiments, the T cell activation inducible promoter comprises one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc., NFAT response elements (e.g., SEQ ID NO:30) in functional combination with a pJB42CAT5 core promoter sequence (e.g., SEQID NO:23). In some embodiments, the T cell activation inducible promoter comprises one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc., NFAT response elements (e.g., SEQ ID NO:30) in functional combination with a MiniTK core promoter sequence (e.g., SEQ ID NO:24). In some embodiments, the T cell activation inducible promoter comprises one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc., NFAT response elements (e.g., SEQ ID NO:30) in functional combination with a minCMV core promoter sequence (e.g., SEQ ID NO:26). In some embodiments, the T cell activation inducible promoter comprises one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc., NFAT response elements (e.g., SEQ ID NO:30) in functional combination with a minSV40 core promoter sequence (e.g., SEQ ID NO:27). In some embodiments, the T cell activation inducible promoter comprises one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc., NFAT response elements (e.g., SEQ ID NO:30) in functional combination with a CMV53 core promoter sequence (e.g., SEQ ID NO:28). In some embodiments, the T cell activation inducible promoter comprises one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc., NFAT response elements (e.g., SEQ ID NO:30) in functional combination with an MLP core promoter sequence (e.g., SEQ ID NO:29). In some embodiments, the T cell activation inducible promoter comprises one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc., NFAT response elements (e.g., SEQ ID NO:30) in functional combination with a minAdeP core promoter sequence. In some embodiments, the T cell activation inducible promoter comprises one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc., NFAT response elements (e.g., SEQ ID NO:30) in functional combination with a minIL2 core promoter sequence.

[0096] In some embodiments, the expression cassettes also include Kozak sequences, polyadenylation sites, and other elements that facilitate transcription and / or translation of the coding sequences. For example, a woodchuck hepatitis virus post-transcriptional response element (WPRE) or variants thereof may be included at the 3’ untranslated region of the expression cassette.

[0097] In the expression cassettes, the transcription / translation regulatory elements such as the promoters, any enhancers, and the like are operably linked to the coding sequences so as to allow efficient expression of the coding sequences and efficient translation of the RNA transcripts. III. Use of the IL-12 Stability Variants

[0098] The IL-12 stability variants herein can be used in a cytokine therapy and delivered in a pharmaceutical composition systemically (e.g., through intravenous injection or infusion)or locally (e.g., intratumorally) to subjects in need of immune stimulation, such as subjects with cancer (e.g., hematological cancer or a solid tumor) or a compromised immune system. For example, the pharmaceutical composition may be injected directly to a tumor site. Pharmaceutical compositions herein may comprise a pharmaceutically acceptable carrier or excipients. Examples of such carriers and excipients include water, saline, phosphate- buffered saline, sodium chloride, sodium phosphate, polyols (e.g., sucrose, mannitol, and trehalose), methionine, albumin, chelating agents, and the like.

[0099] The IL-12 stability variants herein may also be used in a cell-based therapy. As described above, immune cells such as T cells may be engineered to express the variants, for example, be transduced with an appropriate vector (or vectors) comprising the nucleic acid(s) encoding the IL-12 stability variant. In some embodiments, the T cells also express recombinant antigen receptor. As used herein, a “recombinant antigen receptor” refers to an antigen receptor that is not natively expressed by the T cells. A recombinant antigen receptor may be a cell surface molecule that binds to an antigen of interest on another cell (e.g., a tumor cell), and may, for example, be derived from a T cell receptor or an antibody. The recombinant antigen receptor may be, for example, an antibody, an engineered antibody such as an scFv, a CAR, an engineered TCR, a TCR mimic (e.g., an antibody-T cell receptor (abTCR) or a chimeric antibody-T cell receptor (caTCR)), a chimeric signaling receptor (CSR), TCR mimics (e.g., antibodies that recognize epitopes similar to those recognized by TCRs), TCR fusion constructs (TRuCs). See, e.g., EP340793B1, WO 2017 / 070608, WO 2018 / 200582, WO 2018 / 200583, WO 2018 / 200585, Xu et al., Cell Discovery (2018) 4:62, Baeuerle et al., Nat Comm. (2019) 10:2087.

[0100] By way of example, a CAR may comprise an extracellular antigen-binding domain (e.g., a scFv domain), a transmembrane domain, and intracellular signaling domains, optionally peptide stretches linking the domains (e.g., a hinge region linking the antigen- binding domain and the transmembrane domain). In some embodiments, the transmembrane domain may be derived from a natural source, for example, the TCR alpha, beta, gamma, or delta chain, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or 4-1BB. Alternatively, the transmembrane domain may be synthetic and may comprise predominantly hydrophobic residues (e.g., alanine, leucine, valine, glycine, isoleucine, proline, phenylalanine, and tryptophan). In some embodiments, the intracellular signaling domains are those that provide a signal similar to that from a natural antigen receptor and maycomprise, for example, a costimulatory domain (e.g., one derived from CD28, 4-1BB, OX40, DAP10, or ICOS) and a primary signaling domain (e.g., one derived from CD3 zeta chain).

[0101] In some embodiments, an abTCR may comprise an engineered TCR in which the antigen-binding domain of a TCR (e.g., an alpha / beta TCR or a gamma / delta TCR) has been replaced by that of an antibody (with or without the antibody’s constant domains); the engineered TCR then becomes specific for the antibody’s antigen while retaining the TCR’s signaling functions.

[0102] In some embodiments, a CSR may comprise (1) an extracellular binding domain (e.g., natural / modified receptor extracellular domain, natural / modified ligand extracellular domain, scFv, nanobody, Fab, DARPin, and affibody), (2) a transmembrane domain, and (3) an intracellular signaling domain (e.g., a domain that activates transcription factors, or recruits and / or activates JAK / STAT, kinases, phosphatases, and ubiquitin; SH3; SH2; and PDZ).

[0103] The recombinant antigen receptor may target an antigen of interest (e.g., a tumor antigen or an antigen of a pathogen). The antigens may include, without limitation, AFP (alpha-fetoprotein), αvβ6 or another integrin, BCMA, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (C-C motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD98, CD123, CD138, CD171, CD352, CEA (carcinoembryonic antigen), Claudin 18.2, Claudin 6, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrinB2, EPHa2 (ephrine receptor A2), ERBB dimers, estrogen receptor, ETBR (endothelin B receptor), FAP-α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (a folate binding protein), FCRL5, FR-α (folate receptor alpha), GCC (guanyl cyclase C), GD2, GD3, GPC2 (glypican- 2), GPC3, gp100 (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G Protein Coupled Receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA-A1 (human leukocyte antigen Al), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight-melanoma-associated antigen), IGF1R (insulin-like growth factor 1 receptor), Ig kappa, Ig lambda, IL-22Ra (IL-22 receptor alpha), IL-13Ra2 (IL-13 receptor alpha 2), KDR (kinase insert domain receptor), KLK2, LI cell adhesion molecule (LI -CAM), Liv-1, LRRC8A (leucine rich repeat containing 8 Family member A), Lewis Y, melanoma- associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1 (melan A), murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan),mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complexes (e.g., HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE-A, and WT1), NCAM (neural cell adhesion molecule), Nectin-4, NKG2D (natural killer group 2 member D) ligands, NY-ESO, oncofetal antigen, PD-1, PD-L1, PRAME (preferentially expressed antigen of melanoma), progesterone receptor, PSA (prostate specific antigen), PSCA (prostate stem cell antigen ), PSMA (prostate specific membrane antigen), ROR1, ROR2, SIRPα (signal-regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (six transmembrane epithelial antigen of the prostate 1), STEAP2, survivin, TAG72 (tumor-associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens from HIV, HBV, HCV, HPV, and other pathogens.

[0104] In some embodiments, the antigen receptor may be bispecific and target two different antigens, such as two of the antigens listed above. For example, the antigen receptor, such as a CAR, targets CD19 and CD20, or CD19 and CD22.

[0105] The present pharmaceutical compositions and engineered immune cells may be used to prevent a disease or disorder by being administered in a therapeutically effective amount, wherein an onset, progression, or a relapse of a sign or symptom of the disease or disorder is delayed or inhibited, thereby preventing the disease or disorder. In some embodiments, the disease or disorder is a cancer.

[0106] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of immunology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents formspart of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0107] According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Any molecule disclosed herein can be used in any of the treatment method here, wherein the individual to be treated is as defined anywhere herein. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any manner.

[0108] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. EXAMPLES Example 1: Generation of IL-12 Stability Variant Mutations and Characterization of Efficient Linker Cleavage in Primary T Cells A. Methods and Materials

[0109] T cell supernatants were harvested from primary donor T cells each transduced with an IL-12 constructs. Transduced cells were incubated for six days. The expression of a cellular marker (LNGFR) was normalized in these T cells (with or without 1:100 transact for stimulation), which were incubated in a 24 well plate for 20 hours prior to harvest. P40, P70 and interferon gamma (INFg) were measured by a Meso Scale Discovery (MSD) assay in supernatants unstimulated for 24 hours prior to the assay. In this assay, co-cultured with the modified T cells described above, mock-transected CD19+ T cells were included as “bystander cells”. IL-12 Persistent Activation Assay

[0110] Continuing from the co-culture described above, the co-culture was depleted of INFg and then supplemented with 20 microliters (mL) of supernatant for stimulation with transact (1:500). After a 20-hour incubation, the bystander cells were harvested and INFg secretion from these cells determined by MSD.B. Results and Discussion

[0111] As shown in FIG.1, IL-12 stability variants were designed to eliminate the disulfide bridge present between the two subunits of wildtype (WT) IL-12 and to introduce a furin-cleavable linker not present in either of WT or of any naturally occurring variant of IL- 12. To eliminate the disulfide bridge present in WT IL-12, IL-12 stability variants are designed to lack a cystine (C) residue at each of position 177 in the p35 subunit (also referred to herein as a first subunit) and position 74 in the p40 subunit (also referred to herein as a second subunit). In place of a cysteine residue, a preferred IL-12 stability variant of the disclosure comprises a serine (S) residue at each of position 177 in the p35 subunit (also referred to herein as a first subunit) and position 74 in the p40 subunit (also referred to herein as a second subunit). IL-12 stability variants of the disclosure may comprise additional mutations as disclosed herein.

[0112] T cells were transduced with nucleic acids encoding a one of several IL-12 variants that comprised furin-cleavable linker and wild-type single chain IL-12 as control. The IL12 was immunoprecipitated from the cultures and assayed by Western Blot to determine the stability of the IL-12 dimer configuration. FIG.2 confirms that the IL-12 stability variants were cleaved at approximately 100% efficiency when produced by and secreted from T cells.

[0113] As shown in FIG.3, the presence of p40 indicates that the molecules are being secreted from all constructs. The absence of measurable p70 indicates that the molecules are cleaved and dissociated at equilibrium. The presence of proximal bystander T cell activity (measured by IFN- ^ production) soon after secretion indicates that the secreted IL-12 stability variants maintain the ability to signal through IL-12R, and the absence of proximal bystander activity after a period of incubation indicates that the secreted IL-12 stability variants become inactive over time. Example 2: Determination of the Disassociation Rates of IL-12 Stability Variants A. Methods and Materials Octet® Analysis

[0114] For each IL-12 stability variant, a biotinylated p35 subunit (p35-biotin) was captured onto a streptavidin-coated surface. Captured p35 was allowed to associate with its corresponding p40 subunit in solution. Dissociation of the two subunits was measured as a function of time (in seconds) by transferring the surface into solution that did not contain p40. Over the time course of the assay, each phase of protein subunit interaction (capture,association and dissociation) of the exemplary IL-12 stability variants was marked by p35- p40 binding as a function of time (seconds). Octet®-mediated analysis involves bio-layer interferometry (BLI) technology to evaluate biomolecular interactions in microwell plates. Octet® systems are commercially available. B. Results and Discussion

[0115] As used in this disclosure and as depicted in FIG.4, the term “half-life” is distinct form the pharmacokinetic use of the term “half-life”. In this experiment, the term “half-life” is meant to describe the half-life of each p70 form of a reduced stability variant after that variant has been secreted and diluted to concentrations comparable to physiological concentrations of WT IL-12 observed from samples obtained from peripheral circulating blood. At this time point, p35 and p40 begins dissociating at the rates shown in FIG.9. The measured half-life of the hscIL12-p40-C177S-p35-C74S (also referred to herein as the “DS” variant) is on the order of approximately ten minutes. This half-life profile is in contrast to the “DS + R181A” variant (hscIL12-p40-C177S-p35-C74S-R181A), which has a considerably shorter measured half-life on the order of approximately one minute. While not intending to be bound by theory, the preference for one form over another in this experiment can be based on a goal to achieve a dissociation rate slow enough to allow the active molecule to diffuse 1-2 cell diameters from a therapeutic cell modified to secrete the variant IL-12. Thus, a preferred time course of dissociation occurs over the course of minutes. The particular selection of mutations can be made to tune the stability of the IL-12 variant and achieve the desired half-life. Example 3: Demonstration of Localized IL-12 Stability Variant Activity in Vitro A. Methods and Materials Bystander Assay

[0116] Modified T cells secreting an IL-12 stability variant (Modified cells) or Bystander cells, each transduced or mock-transduced, respectively, were incubated in separate cultures for six days. The expression of a cellular marker (LNGFR) was normalized in each population (with 1:100 transact for stimulation), which were incubated for 20 hours prior to initiating the bystander study. Modified T cells (at a concentration of 200,000 cells per well) and bystander cells (e.g., mock-transfected CD19-expressing T cells) (at a concentration of 100,000 cells / well) were stimulated by transact 1:100 (bystander cells) or 1:1000 (Modified cells) for 8 hours and combined into a co-culture for 10 hours. The co-cultures in flat-bottom wells placed the bystander cells in a position proximal to the modified cells while the co-cultures established in transwells placed the bystander cells in a position distal to the modified cells, separated by a membrane that allowed proteins (such as the IL-12 stability variants), but not cells, to diffuse across it. Each co-culture form was then contacted with a Protein Transport Inhibitor Cocktail for 2 hours prior to harvesting cells and supernatant for analysis. Bystander cells were collected for analysis of Intracellular IFN-γ expression by ICS. Bystander cell supernatant was collected for detection of p40 by MSD. B. Results and Discussion

[0117] As shown in FIG.11, exemplary IL-12 stability variants of the disclosure exhibit localized activity in vitro. Bystander cells proximal to modified T cells secreting an IL-12 stability variant, but not bystander cells distal to the IL-12 stability variant secreting T cells, demonstrate activation as indicated by intracellular expression of IFN-γ. Proximal and distal bystander cells were exposed to similar levels of the IL-12 stability variant across either the flat-bottom well or transwell conditions, respectively. These results indicate localized activity, which is an important indicator of safety in vivo.

[0118] These results show that the inducible promoter (e.g., 4xNFAT-hBG) allowed minimal basal IL-12 production from T cells and substantially increased IL-12 production after T-cell activation. The study also shows that the IL-12 stability variants dissociated into an inactive state post-cleavage with a half-life of about 10 minutes, and it activated proximal but not distal bystander T cells, demonstrating that function was restricted to the site of induced expression. Example 4: Effects of IL-12 Stability Variants on T-cell Mediated Cytolysis of Tumor Cells

[0119] As demonstrated in the above Examples, single-chain IL-12 variants were designed, where each variant was a fusion protein comprising the two IL-12 subunits linked by a furin-cleavable peptide linker and the sequences of the two subunits altered to remove the intermolecular disulfide bond between them (a C177S substitution in p40 and a C74S substitution in p35). The dissociation rate of the cleaved IL-12 heterodimers were measured using bio-layer interferometry. T cells were engineered with lentiviral vectors expressing either a wildtype (wt) scIL-12 (an scIL-12 in which the p40 and p35 subunits are not altered) or the scIL-12 variants under the control of an engineered inducible promoter (e.g., 4xNFAT- hBG); the T cells were also engineered to constitutively express an TCR specific for NY- ESO-1, a tumor antigen. IL-12 activity on proximal or distal bystander immune cells wasmeasured by IFN-γ production in T cells co-cultured with engineered T cells producing IL- 12, either directly or separated by a trans-well membrane.

[0120] This Example describes additional functional testing of the exemplary IL-12 stability variants. Engineered T cells were functionally assessed in vitro by repeat challenge with NY-ESO-1+A375 tumor (melanoma) cells. Details of the study are described below. A. Methods and Materials

[0121] T cell productions were carried out over 7 days using TCM medium (CTS™ OpTmizer™ medium, supplemented with L-glutamine, GlutaMAX™, OpTmizer™ cell supplement, Immune Cell Serum Replacement, and supplemented with 200 IU / mL of IL-2, 1,200 IU / mL of IL-7, and 200 IU / mL of IL-15). On day 0, CD4+and CD8+T cells were thawed into TCM medium, mixed at a 1:1 ratio at a concentration of 2 x 106cells / mL, and activated for 24-28 hours with a 1:100 dilution of TransACT™.

[0122] On day 1, T cells were co-transduced with two lentiviral vectors. The first vector encoded an antigen-specific TCR expressed off a constitutive promoter, and the second vector encoded a human LNGFR expressed off a constitutive promoter and an IL-12 mutein expressed off a T-cell activation-induced promoter that four NFAT domains linked to an hBG minimal promoter domain.

[0123] On day 2, at least 24 hours post-transduction, T cells were scaled up from 96-well plates to 24-well G-Rex® plates, or from 24-well plates to 6- well G-Rex® plates.

[0124] On day 7, cells were counted and assessed for viability by AOPI staining, and then harvested and assessed 1) phenotypically by flow cytometry for activation markers, memory markers, and transduction efficiency of both TCR and IL-12 (via the LNGFR marker), and 2) functionally by repeat challenge with target cell lines (sequential kill assays).

[0125] Sequential kill assays were carried out in RPMI 1640 + 10% FBS media. On the first day of stimulation, target cells (A375-NLR) were counted, seeded into the appropriate plates, and incubated at 37oC for 2 hours to allow for adherence. Effector T cells (fresh or frozen from the above production) were then co-cultured with target cells at an E:T ratio of 1:5. E:T ratios were based on the number of live, TCR+T cells. IL-12 expression was characterized by the constitutive LNGFR tag, and expression variance was kept within 10% across all samples. These co-culture plates were then placed in an IncuCyte® with readings every 2 hours, to measure changes in target cells over time (via NLR); decreases in NLR over time indicate clearance of target cells. A small portion of the supernatant was collected at 24 hours post-seeding of effector cells, and frozen at -80oC for later analysis of cytokine levels.

[0126] Three days after initiation of the co-culture, new plates of target cells were seeded as above, and 25% of the previous culture was transferred to this new plate, initiating a repeat challenge / second stimulation of the effector cells in the sequential kill assay. The remaining cells in the initial plate were then assessed phenotypically by flow cytometry for cell expansion, activation markers, memory markers, and transduction efficiency of both TCR and IL-12 (LNGFR).

[0127] Supernatant was again collected at 24 hours post-seeding of effector cells as before, and frozen as before for later analysis of cytokine levels. Cells continued to be re- plated every 3 days until the repeat challenges provided the resolution required to differentiate between the function of various IL-12 constructs (as determined by NLR- reductions in the IncuCyte®). Following termination of the sequential kill assays, the collected supernatants were analyzed by MSD at 1:5 and 1:20 dilutions (due to concentration differences between IL-12 and IFN-γ) according to the manufacturer’s instructions. B. Results and Discussion

[0128] Four single-chain human IL-12 variants were made and tested: (1) “DS-furin variant”: the variant contains a C177S mutation in the p40 sequence and C74S mutation in the p35 sequence; (2) “DS-furin-I52C-A179C variant”: the variant contains C177S and A179C mutations in the p40 sequence, and C74S and I52C mutations in the p35 sequence, such that the native disulfide bond between C177 in p40 and C74 in p35 is removed and instead a new disulfide bond can form between 179C in p40 and 52C in p35; (3) “DS-furin-I52C-A179C-R181A variant”: same as (2) except that the p35 sequence additionally contains an R181A mutation; and (4) “p40-C177S-R181A-p35-C74S” variant: same as (1) except that the p35 sequence additionally contains an R181A mutation.

[0129] T cells were transduced with lentiviral vectors for expressing the variants and wt scIL-12 under the control of an engineered inducible promoter (e.g., 4xNFAT-hBG). The T cells also were transduced to constitutively express NY-ESO-1-specific TCR. Transduced T cells were sequentially co-cultured with A375 target cells to compare the effects of these IL- 12 variants on T cell-mediated cytolysis of the A375 tumor cells (see Materials and Methods above). TCR-expressing T cells all demonstrated enhanced tumor cytolysis in the presence of IL-12 (representative variants and wildtype single chain IL-12, FIGs.13A-C). Analysis of the final repeat stimulation demonstrated that T cells expressing the DS-furin variant, DS- furin-I52C-A179C variant, DS-furin-I52C-A179C-R181A variant, or wildtype scIL-12 allperformed significantly better than T cells expressing TCR alone (p < 0.05, FIG.13D), while the p40-C177S-R181A-p35-C74S mutant trended towards significance (p = 0.07). At the same time, no significant differences were seen in cytotoxicity between the wildtype scIL-12 and any of the IL-12 variants.

[0130] These results show that the cell activation-induced expression of the IL-12 stability variants exhibit proximal cell cytotoxicity effects similar to wild-type IL-12. Example 5: Effects of IL-12 Stability Variants on T-cell Mediated Efficacy in vivo

[0131] This Example describes the functional testing of IL-12 stability variants in vivo. T- cell efficacy and systemic IL-12 exposure in vivo were evaluated using mice with A375 tumors and treated intravenously with engineered T cells. A. Methods and Materials

[0132] T cell productions were carried out as described above in Example 4. Following production, IL-12 expressing T cells underwent magnetic selection for LNGFR, following the manufacturer’s (StemCell) instructions, prior to freezing. An aliquot of cells from each condition was then thawed and assessed for cytotoxicity (IncuCyte®), phenotype (flow cytometry), and viability (AOPI) to insure functionality prior to in vivo testing. Functional assessment of T cell efficacy in vivo was then carried out in an A375 xenograft model using NSG-MHC I / II DKO mice (Jackson Labs).

[0133] A375 cell productions were carried out in RPMI 1640 media supplemented with 10% FBS. Cells were passaged / expanded every 2-3 days, and harvested once the requisite cell numbers were reached. Cells were first washed with PBS, then treated with TrypLE for 5 minutes at 37oC. The detached cells were then washed with RPMI 1640, and then counted and resuspended at the proper concentration in HBSS. Either 1 x 106or 2.5 x 106cells resuspended in 100 µL were then mixed 1:1 with Matrigel™ before being injected subcutaneously into the flank of mice. Tumors were measured thrice weekly until they reached the desired volume (average of 100 mm3). Mice were then randomized into various treatment conditions, and injected with either 1 x 106or 2.5 x 106antigen-specific TCR- expressing T cells co-transduced with various IL-12 variants.

[0134] Readouts for T cell and IL-12 efficacy included measurements of tumor volume, persistence of T cells via blood PK, and plasma cytokine levels of IL-12 and IFN-γ. Tumor measurements were taken twice a week by caliper, and blood was drawn for measurement of blood PK and plasma cytokine levels at Days 1, 7, 14, 21, 28, and 42 post-T cell infusion. 30 µL of blood was diluted 1:5 in a solution of PBS + 0.1% BSA, and then centrifuged at 2,000x g for 15 minutes at 4oC to separate the plasma from red blood cells. Plasma was collected from the supernatant, and used at a 1:10 dilution in MSD assays, which were carried out according to the manufacturer’s instructions. 50 µL of blood was then treated thrice with ACK lysis buffer to reduce red blood cell contamination, with washes using cell staining buffer (BioLegend) between each treatment. The remaining cells were then blocked with 20% normal mouse serum in cell staining buffer, stained for antibodies against our constitutively expressed TCR, human CD3, human CD4, human CD8, human CD271 (LNGFR), and mouse CD45 (to exclude mouse immune cells), then analyzed by flow cytometry. B. Results and Discussion

[0135] TCR T cells + / - IL-12 (and IL-12 variants) were produced and infused into NSG- MHC I / II DKO mice carrying A375 xenografts as described in Materials and Methods above. Tumor volumes were measured twice weekly following infusion to assess treatment efficacy. Mice treated with TCR T cells expressing the p40-C177S-p35-C74S variant (i.e., DS-furin or Reduced Stability variant) demonstrated deeper anti-tumor responses and improved survival compared to those treated with T cells expressing wt scIL-12 or the TCR alone (FIGs.14A- C). In one study (FIG.14A), the growth measured in mice treated with IL-12 between Days 10-30 was determined to be due to massive expansion of T cells at the tumor site, and not true tumor progression. The data show that DS-furin variant enhanced T-cell cytotoxicity in vitro (Example 4) and exhibited potent and comparable anti-tumor efficacy in vivo as well. Example 6: Effects of IL-12 on T-cell Proliferation and Cytokine Secretion in vivo; Safety Assessment

[0136] This Example describes a further study of the biological activities of IL-12 stability variants in vivo.

[0137] In this study, blood was drawn from mice once a week at Days 1, 7, 14, 21, 28, and 42 post-infusion with NY-ESO-1 TCR-T cells (“TCR-T cells”), and analyzed via flow cytometry for blood PK, and cytokine levels via MSD as described above. A proliferative advantage of TCR-T cells was observed in mice treated with TCR-T cells expressing IL-12 (wildtype or stability variant) as compared to mice treated with TCR-T cells alone (FIG. 15A). Proliferation profiles of TCR-T cells expressing wt scIL12 were similar to those of TCR-T cells expressing the p40-C177S-p35-C74S (“Furin-DS”) mutant (FIG.15A and FIG. 15C). Mice treated with TCR-T cells expressing IL-12 (wt or ”Furin-DS Mutant” a reduced stability variant) also saw enhanced levels of IFN-γ secretion as compared to TCR-T cellsalone (FIG.15B), with animals treated with TCR-T cells expressing the p40-C177S-p35- C74S mutant (“Furin-DS Mutant”) demonstrating either similar or higher levels of IFN-γ (FIG.15B and FIG.15D).

[0138] Additionally, to address whether the expressed IL-12 stability variants may demonstrate an improved safety profile, levels of the IL-12p70 (active, dimerized form) and IL-12p40 (subunit of the active single-chain IL-12 or the inactive free subunit post cleavage of the p40-C177S-p35-C74S mutant) were assessed. Levels of IL-12p40 were readily detected in the plasma of mice treated with TCR-T cells expressing IL-12 (wt or stability variant, FIG.15E). However, levels of IL-12p70 were only detected in mice treated with TCR-T cells expressing wt scIL12 (FIG.15F), empirically demonstrating the self- inactivating function of the p40-C177S-p35-C74S mutant. This trend was replicated in a second mouse study, confirming the reproducibility of the self-inactivating function of the p40-C177S-p35-C74S stability mutant (not shown).

[0139] To further demonstrate a safety benefit of IL-12 stability variants of the disclosure, worst-case-scenario equilibrium concentrations for active p70 in circulation were calculated for the human DS variant using measured p40 concentrations in circulation of an exemplary donor and the measured p35 / p40 dissociation rates from Octet®, which showed a dissociation constant (KD) of approximately 60 nM (6E-08 M) (FIG.9). As shown in FIG. 12, the measured mean p70 at Day 42 for WT IL-12 was 5951 pg / mL and the measured maximum p70 for WT IL-12 was 26,562 pg / mL. The calculated mean p70 at Day 42 for the human DS variant is 0.03 pg / mL and the calculated maximum p70 for the human DS variant is 0.17 pg / mL. These calculated maximal levels of circulating p70 are well below levels considered to be dangerous in vivo.

[0140] These results demonstrate that only wt scIL-12, but not DS-furin variant, was detectable in an active state in peripheral blood, thus demonstrating the variant’s localized activity that may improve the safety profile.

[0141] In conclusion, the results in the above Examples demonstrate that the IL-12 variants can deliver potent IL-12 stimulation at tumor sites while avoiding systemic exposure, potentially improving efficacy for T-cell therapies while maintaining a favorable safety profile that may finally allow effective administration of IL-12.Example 7: Design of Mouse Surrogate IL-12 Stability Variants with Similar Kinetics of Dissociation A. Methods and Materials

[0142] See Examples 1 and 2 for characterization and Octet® methods, respectively. B. Results and Discussion

[0143] As shown in FIG.5, mouse IL12 stability variants (variants that signal through mouse IL-12 Receptors) respond to p35 / p40 interface mutations differently than human IL12 stability variants (variants that signal through human IL-12 Receptors). Mouse p70 formation is practically eliminated when the p35-p40 inter-subunit disulfide is mutated (see right plot, asterisks). Certain mouse to human reversion mutations S177R and T178I appear to increase mouse p70 formation (see right plot, light bars) (see also, FIG.7).

[0144] Upon alignment of the mouse (top) and human (bottom) sequences of the p35 subunit in WT IL-12 and scIL-12 containing WT sequences, several positions appear to be important for either IL-12Rb2 or for dimer formation along a p35 / p40 interface. As shown in FIG.6, tyrosine (Y) residues in alignment rows 2 and 4 are IL-12Rb2-interactive residues conserved in both human and mouse. By contrast the boxed position in row 3 of the alignment are IL-12Rb2-interactive residues that are not conserved in both human and mouse. Within row 4 of the alignment, the double-boxed positions (ST in mouse and RI in human) are important p35 / p40 interface residues that are not conserved. By contrast, within row 4 of the alignment, the valine(v) and arginine (R) boxed residues are important p35 / p40 interface residues that are conserved.

[0145] FIG.8 shows Octet® data demonstrating that mscIL12-p35-C70S-furin-p40 (also referred to herein as the “mouse DS” variant) dissociates significantly faster than hscIL12- p40-C177S-p35-C74S (also referred to herein as the “Human DS” variant) but that the addition of an S177R mutation in mouse DS slows dissociation. Octet® data reflecting the activity of IL12 stability variants capable of signaling through human IL-12R (“human variants”) are shown in the top row while Octet® data reflecting the activity of IL12 stability variants capable of signaling through mouse IL-12R (“mouse variants”) are shown in the bottom row. Quantification of the data shown in FIG.8 is shown in FIG.9 and FIG.10, which demonstrate that mouse DS + S177R has comparable p35 / p40 dissociation kinetics to Human DS and provides a tool variant for syngeneic in vivo studies.Example 8: Mouse Syngeneic Study

[0146] This Example describes the functional testing of mouse IL-12 (mIL-12) stability variants in vivo.

[0147] B16F10 cells (from ATCC) were cultured in DMEM + 10%FBS + 1% Pen / Strep. B16F10 cells were seeded into 6-well dishes (XM cells / well), and transduced with lentivirus expressing either sc-mIL-12 under the control of a constitutive UBC promoter, sc-mIL-12- p40-C175S-furin-p35-C70S-S177R (mutations with respect to positions in SEQ ID NO:10 for mouse p40 and SEQ ID NO:11 for mouse p35) under the control of a constitutive mPGK promoter, or sc-mIL-12-p35-C70S under the control of the constitutive UBC promoter, to generate B16F10 cells capable of secreting mIL-12. Cell lines then underwent puromycin selection (2 µg / mL) over the course of 48 hours, then maintained in 1 µg / mL puromycin for 2 weeks, with cell passages as necessary, to generate pure mIL-12 expressing cell lines.

[0148] B16F10 cells expressing mIL-12 were then removed from puromycin selection, and passaged for 2 weeks to demonstrate stable mIL-12 expression in the absence of selection. At each passage (about every 3 days), 50,000 cells were plated in a 24-well plate in 1 mL of media. 24 hours after plating, supernatant was collected and analyzed for IL-12 p40 levels by MSD to determine levels of cytokine production (FIG.16). The secretion of IL-12 was stable for at least 10 days in culture.

[0149] A fresh aliquot of B16F10 cells expressing mIL-12 was then thawed and expanded. B16F10 cells expressing WT sc-mIL-12 or sc-mIL-12-p40-C175S-furin-p35- C70S-S177R were then mixed with parental B16F10 cells at various frequencies (10%, 50%, and 100% IL-12 expressing cells). 1 x 106of these B16F10 cell mixes were implanted into C57BL / 6 mice and allowed to expand in vivo.

[0150] Mice implanted with parental B16F10 tumors showed rapid tumor growth and all mice were taken off study within 15 days. Mice implanted with more than 10% B16F10 cells expressing IL-12 did not show tumor outgrowth. Mice implanted with tumors in which 10% B16F10 cells expressed either WT sc-mIL-12 or sc-mIL-12-p40-C175S-furin-p35-C70S- S177R showed significant delay in tumor outgrowth, with tumors expressing sc-mIL-12-p40- C175S-furin-p35-C70S-S177R showing a lower median tumor volume (FIG.17A) and reduced frequency of tumor outgrowth (FIG.17B). Four of 10 mice implanted with B16F10 expressing sc-mIL-12-p40-C175S-furin-p35-C70S-S177R remained tumor free, whereas 2 of 10 mice implanted with B16F10 expressing WT sc-mIL-12 remained tumor free.

[0151] Additionally, we detected higher levels of mIL-12p40, mIL-12-70 and mIFN-γ in the plasma of mice implanted with B16F10 cells expressing wt sc-mIL-12 compared to thoseexpressing sc-mIL-12-p40-C175S-furin-p35-C70S-S177R, suggesting that sc-mIL-12-p40- C175S-furin-p35-C70S-S177R may have improved safety compared to wildtype IL-12 (FIGs.18A-18C). Importantly, we did not detect mIL-12p70 in the plasma of mice with tumors expressing sc-mIL-12-p40-C175S-furin-p35-C70S-S177R.

[0152] Together, these data demonstrate the similar levels of efficacy between sc-mIL-12 and our stability mutant at a local level, while also exhibiting the potential of our stability mutant to decrease systemic IL-12 activity.SEQUENCES

[0153] The following table provides a list of the sequences disclosed herein (SEQ: SEQ ID NO).

Claims

CLAIMS 1. An IL-12 variant comprising: a p40 subunit sequence with an amino acid sequence at least 90% identical to SEQ ID NO:1, and a p35 subunit sequence with an amino acid sequence at least 90% identical to SEQ ID NO:2, wherein the IL-12 variant lacks a native disulfide bond between the p40 subunit sequence and the p35 subunit sequence.

2. The IL-12 variant of claim 1, wherein: the p40 subunit sequence comprises a mutation removing a cysteine residue at a position corresponding to C177 of SEQ ID NO:1, optionally wherein the mutation is a C-to-S mutation; and / or the p35 subunit sequence comprises a mutation removing a cysteine residue at a position corresponding to C74 of SEQ ID NO:2, optionally wherein the mutation is a C-to-S mutation.

3. The IL-12 variant of claim 1 or 2, wherein the p35 subunit sequence comprises one or more of: a mutation at a position corresponding to R181 of SEQ ID NO:2, optionally a R-to-A mutation, a mutation at a position corresponding to R183 of SEQ ID NO:2, a mutation at a position corresponding to V185 of SEQ ID NO:2, optionally a V-to-A mutation, and a mutation at a position corresponding to R189 of SEQ ID NO:2, optionally a R-to-K mutation.

4. The IL-12 variant of claim 2 or 3, wherein the p40 subunit sequence comprises a substitution to a C at a position corresponding to A179 of SEQ ID NO:1, and the p35 subunit sequence comprises a substitution to a C at a position corresponding5. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises SEQ ID NO:12, and the p35 subunit sequence comprises SEQ ID NO:

13.

6. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises SEQ ID NO:12, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:13 but for an R181A mutation.

7. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:12 but for an A179C mutation, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:13 but for an I52C mutation.

8. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:12 but for an A179C mutation, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:13 but for an I52C mutation and an R181A mutation.

9. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises SEQ ID NO:10, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:11 but for a mutation at position C70, optionally a C70S mutation.

10. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises SEQ ID NO:10, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:11 but for a mutation at position C70, optionally a C70S mutation and a mutation at position S177, optionally a S177R mutation.

11. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises SEQ ID NO:10, andthe p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:11 but for a mutation at position N184, optionally a N184A mutation.

12. The IL-12 variant of any one of claims 1-11, wherein the IL-12 variant is a fusion protein in which the p40 subunit sequence and the p35 subunit sequence are linked by a cleavable peptide linker.

13. The IL-12 variant of claim 12, wherein the cleavable peptide linker comprises a furin- cleavage site, optionally wherein the cleavable linker comprises SEQ ID NO:

5.

14. The IL-12 variant of claims 13, wherein the IL-12 variant comprises an amino acid sequence at least 90% sequence identical to amino acid residues 22-533 of SEQ ID NO:

6.

15. An isolated nucleic acid molecule or isolated nucleic acid molecules encoding the IL- 12 variant of any one of claims 1-14, optionally wherein the nucleic acid molecule(s) are mRNA or DNA.

16. An expression vector or expression vectors comprising the isolated nucleic acid molecule(s) of claim 15.

17. The expression vector(s) of claim 16, wherein the expression vector(s) is / are selected from lentiviral vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors.

18. The expression vector(s) of claim 16 or claim 17, further comprising a tissue-specific or inducible promoter.

19. The expression vector(s) of claim 18, wherein the inducible promoter comprises a combination of a nuclear factor of activated T cells (NFAT) domain and a human beta-globin (hBG) minimal promoter domain, optionally wherein the inducible promoter comprises SEQ ID NO:

14.

20. A mammalian cell comprising the nucleic acid molecule(s) of claim 15 or the expression vector(s) of any one of claims 16-19.

21. The mammalian cell of claim 20, wherein the mammalian cell is a human immune cell.

22. The mammalian cell of claim 21, wherein the human immune cell is a T cell, a NK cell, or a TIL, optionally engineered to express a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), or a chimeric costimulatory receptor (CCR), further optionally wherein the CAR, engineered TCR, or CCR targets a tumor antigen.

23. A pharmaceutical composition comprising the IL-12 variant, nucleic acid molecule(s), expression vector(s), or mammalian cell of any one of claims 1-22 and a pharmaceutically acceptable carrier.

24. A method of stimulating the immune system, or treating cancer, in a human subject in need thereof, comprising administering the pharmaceutical composition of claim 23 to the human subject.

25. Use of the IL-12 variant, nucleic acid molecule(s), expression vector(s), or mammalian cell of any one of claims 1-22 for the manufacture of a medicament for stimulating the immune system or treating cancer in a human subject in need thereof.

26. The IL-12 variant, nucleic acid molecule(s), expression vector(s), mammalian cell, or pharmaceutical composition of any one of claims 1-23 for use in stimulating the immune system or treating cancer in a human subject in need thereof.

27. A method of producing an IL-12 variant, comprising: culturing the mammalian cell of any one of claims 20-22 under conditions that allow expression of the IL-12 variant; and isolating the IL-12 variant from the culture.

Images