IL-12 stability variant

Abstract

This disclosure provides IL-12 stability variants, nucleic acids encoding the variants, nucleic acid-containing vectors, pharmaceutical compositions containing the variants, therapeutic cells expressing the variants, and methods or uses thereof for treating or preventing diseases or disorders.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 510,579, filed on 27 June 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Array List This application includes a sequence listing, which is submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on 27 June 2024, is named 026225_WO032_SL.xml and has a size of 36,143 bytes. [Background technology]

[0003] Interleukin-12 (IL-12) is a pleotropic pro-inflammatory cytokine that stimulates the proliferation of natural killer (NK) and T cells and can drive the secretion of IFN-γ and TNF-α (Non-Patent Literature 1). IL-12 is a heterodimer composed of two subunits, p35 (also known as IL-12A) and p40 (also known as IL-12B). The heterodimer is also called p70. IL-12 binds to the IL-12 receptor (IL-12R), which then forms two subunits, IL-12Rβ1 and IL-12Rβ2. IL-12Rβ1 primarily binds to the IL-12 p40 subunit, while IL-12Rβ2 primarily binds to the IL-12 p35 subunit (Non-Patent Literature 2). There is no complete co-crystal structure of IL-12 bound to IL-12R. The simultaneous binding of IL-12 to both IL-12Rβ1 and IL-12Rβ2 is necessary to drive intracellular signal transduction (Non-Patent Literature 3; Non-Patent Literature 4; Non-Patent Literature 5). Recent cryo-electron microscopy studies have created structures of IL-12 complexed with IL-12R, but the detailed interaction between p35 and IL-12Rβ2 could not be elucidated (Non-Patent Literature 6).

[0004] IL-12 is a cytokine crucial for initiating the Th1 response and has been explored as a potential therapeutic agent for treating cancer (Non-Patent Literature 7). However, due to significant systemic toxicity, approaches to IL-12-based immunotherapy have focused on direct injection of IL-12 into tumor sites and fusion of IL-12 into tumor-targeting regions. Some researchers have attempted to use cell-based approaches to deliver IL-12, in which cells engineered to express IL-12 are administered in vivo (Non-Patent Literature 8; Non-Patent Literature 9). However, this approach is also challenging because IL-12 activates inflammatory signaling even at very low concentrations in circulation. Furthermore, efficacy remains a significant concern because immunosuppressive TMEs can suppress cellular activity even in the presence of IL-12 (Non-Patent Literature 7, above).

[0005] Therefore, there has been a long-standing, unmet need for a safe and effective form of IL-12 for stimulating the immune system, either as monotherapy or in combination therapy. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Tugues et al., Cell Death Differ. (2015) 22(2): pp. 237-46 [Non-Patent Document 2] Presky et al., J Immunol. (1998) 160(5):2174~9 [Non-Patent Document 3] Presky et al., PNAS (1996) 93(24):14002~7 [Non-Patent Document 4] Presky et al., Ann NY Acad Sci. (1996) 795:390~3 [Non-Patent Document 5] Robinson, Cytokine (2015) 71(2): pp. 348-59 [Non-Patent Document 6] Glassman et al., Cell (2021) 184:983~99 [Non-Patent Document 7] Lasek et al., Cancer Immunol Immunother. (2014) 63(5): pp. 419-35 [Non-Patent Document 8] Wei et al., J Cell Mol Med. (2013) 17(11):1465~74 [Non-Patent Document 9] Zhang et al., Clin Cancer Res. (2015) 21(10):2278~88 [Overview of the project] [Problems that the invention aims to solve]

[0007] This disclosure provides an IL-12 variant comprising a p40 subunit sequence having an amino acid sequence identical to at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of SEQ ID NO: 1, and a p35 subunit sequence having an amino acid sequence identical to at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of SEQ ID NO: 2, wherein the variant lacks a native disulfide bond between the p40 subunit sequence and the p35 subunit sequence. [Means for solving the problem]

[0008] In some embodiments, the IL-12 p40 subunit sequence includes a mutation that removes a cysteine ​​residue at the position corresponding to C177 in SEQ ID NO: 1, and optionally the mutation is a C-to-S mutation; and / or the p35 subunit sequence includes a mutation that removes a cysteine ​​residue at the position corresponding to C74 in SEQ ID NO: 2, and optionally the mutation is a C-to-S mutation. In some embodiments, the IL-12 variant includes mutations at both positions (for example, both to serine).

[0009] 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 mutation from R to A); 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 mutation from V to A); and a mutation at a position corresponding to R189 of SEQ ID NO: 2 (e.g., a mutation from R to K).

[0010] In some embodiments, the IL-12 variant p40 subunit sequence further comprises a substitution to C at a position corresponding to A179 of SEQ ID NO: 1, and the p35 subunit sequence further comprises a substitution to C at a position corresponding to I52 of SEQ ID NO: 2.

[0011] 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.

[0012] 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 that is identical to SEQ ID NO: 13 except for the R181A mutation.

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

[0014] In some embodiments, the IL-12 variant p40 subunit sequence comprises an amino acid sequence that is identical to SEQ ID NO: 12 except for the A179C mutation, and the p35 subunit sequence comprises an amino acid sequence that is identical to SEQ ID NO: 13 except for the I52C mutation and the R181A 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 that is identical to SEQ ID NO: 11 except for a mutation at position C70 (e.g., C70S 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 that is identical to SEQ ID NO: 11 except for mutations at positions C70 (e.g., C70S mutation) and S177 (e.g., S177R mutation).

[0017] 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 that is identical to SEQ ID NO: 11 except for a mutation at position N184 (e.g., N184A mutation).

[0018] 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.

[0019] In some embodiments, the IL-12 variant cleavable peptide linker comprises a furin cleavage site, and optionally, the cleavable linker comprises SEQ ID NO: 5.

[0020] In some embodiments, the IL-12 variant comprises an amino acid sequence that is at least 90% identical to amino acid residues 22 - 533 of SEQ ID NO: 6.

[0021] In one aspect, the present disclosure provides an isolated nucleic acid molecule (e.g., mRNA or DNA) encoding an IL-12 variant.

[0022] In another embodiment, the disclosure provides an expression vector comprising an isolated nucleic acid molecule encoding an IL-12 variant. In some embodiments, the expression vector is selected from lentiviral vectors, adenovirus vectors, and adeno-associated virus (AAV) vectors. In some embodiments, the expression vector further comprises a tissue-specific or inducible promoter. In certain embodiments, the inducible promoter comprises one or more combinations of an activated T cell nuclear factor (NFAT) response element domain and a human beta-globin (hBG) minimal promoter domain, and optionally the inducible promoter comprises SEQ ID NO: 14. In some embodiments, the inducible promoter comprises one or more combinations of an activated T cell nuclear factor (NFAT) response element domain and a YB_TATA minimal promoter domain.

[0023] In yet another aspect, the disclosure provides a method for producing an IL-12 variant, comprising the steps of: culturing mammalian cells containing the nucleic acid molecule or vector herein under conditions that enable the expression of an IL-12 variant; and isolating the IL-12 variant from the culture.

[0024] In one embodiment, the disclosure provides a mammalian cell containing a nucleic acid molecule encoding an IL-12 variant or an expression vector containing an isolated nucleic acid molecule encoding an 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, NK cell, or TIL, which is optionally engineered to express a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), or a chimeric costimulatory receptor (CCR), and which optionally targets a tumor antigen.

[0025] In another embodiment, the disclosure provides a pharmaceutical composition comprising an IL-12 variant, a nucleic acid molecule encoding the IL-12 variant, an expression vector comprising an isolated nucleic acid molecule encoding IL-12, or mammalian cells comprising a nucleic acid molecule coating the IL-12 variant or an expression vector comprising an isolated nucleic acid molecule encoding the IL-12 variant; and a pharmaceutically acceptable carrier.

[0026] In another aspect, the Disclosure provides a method for stimulating the immune system or treating cancer in a human subject that needs it, comprising the step of administering the pharmaceutical composition of the Specified to a human subject. It also provides the use of an expression vector comprising an IL-12 variant, a nucleic acid molecule encoding the IL-12 variant, an isolated nucleic acid molecule encoding IL-12, or mammalian cells comprising an IL-12 variant, or an expression vector comprising an isolated nucleic acid molecule encoding the IL-12 variant, for the manufacture of a pharmaceutical for stimulating the immune system or treating cancer in a human subject that needs it. Furthermore, it provides an IL-12 variant, a nucleic acid molecule, an expression vector, mammalian cells, or a pharmaceutical composition for use in a process of stimulating the immune system or treating cancer in a human subject that needs it.

[0027] Other features, purposes, and advantages of the present invention will become apparent in the following detailed description. However, it should be understood that the detailed description illustrates embodiments and aspects of the present invention, but is provided for illustrative purposes only and is not limiting. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawing]

[0028] [Figure 1]Figure 1 shows a pair of schematic diagrams illustrating the protein structure of scIL-12 (referred to as WT scIL-12) (left), in which each subunit (p35 and p40) contains the wild-type (WT) IL-12 sequence, and the protein structure of an exemplary IL-12 stability variant of this disclosure (right), in which the disulfide bonds present in the WT structure are replaced by a Hurin-cleavable linker. The sequence of the G6S peptide linker is described in SEQ ID NO: 17. [Figure 2] Figure 2 shows the legend and corresponding photographs of the Automated Western Blot, in which WT scIL-12 and exemplary IL-12 stable variants of this disclosure were 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. [Figure 3]Figure 3 is a series of graphs demonstrating effective Hurin cleavage of exemplary IL-12 stable variants (human and mouse) secreted from primary T cells modified to express IL-12 stable variants. Top row: This pair of graphs shows the relative concentrations (measured in pg / mL) of secreted exemplary IL-12 stable variants (left plot, p40) and the dimerized state of these exemplary IL-12 stable variants (right plot, p70) in samples obtained from normalized T cell supernatant. Bottom row: The left plot shows the relative concentration (measured in pg / mL) of IFN-γ secreted by proximal bystander CD19+ T cells in response to the secretion of exemplary IL-12 stable variants from co-cultured modified T cells, when IFN-γ measurements are performed at the same time as the p40 and p70 measurements shown in the top row (samples obtained from normalized T cell supernatant); the right plot shows the relative concentration (measured in pg / mL) of IFN-γ secreted by proximal bystander mock-transfected CD19+ T cells in response to the secretion of exemplary IL-12 stable variants from co-cultured modified T cells, when IFN-γ measurements are performed at a later time than the left plot (samples obtained from supernatant transfer assay). In this experiment, IFN-γ secretion is an indicator of IL-12 stable variant activity.Experimental conditions, legend from top to bottom and left to right for each plot: hscIL12-p40-furin-p35 and mscIL12-p40-furin-p35 (human and mouse forms, respectively, of scIL-12 with intact intersubunit disulfide bonds; the term "furin" used in any construct name refers to the furin-cleavable peptide linker); hscIL12-p40-C177S-furin-p35-C74S and mscIL12-p40-C175S-fu The control groups are rin-p35-C70S (human and mouse morphologies of scIL-12 with a disulfide bond disruption mutation); the negative control scIL12-p40-C177S-furin-p35-C74S-Y40A-R189K (human morphology of scIL-12 with a disulfide bond disruption mutation, as well as the Y40A mutation which weakens affinity for IL12Rb2 and the R189K mutation which strongly destabilizes p70); and a mock transfection control (mock). [Figure 4] Figure 4 is a pair of graphs showing the dissociation of truncated IL-12 stability variants over time. See Example 2 for an explanation of the terms used in this figure. The plot on the left shows the off-rate (measured in 1 / second) 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 plot on the right shows the molecular half-life (measured in seconds (s)) of hscIL12-p40-C177S-p35-C74S and hscIL12-p40-C177S-p35-C74S-R181A. In both plots, the error bars represent the standard deviation of two independent experiments. For a graphical representation of the quantification of the data presented here regarding IL-12 stability variants that signal through the human IL-12 receptor, please refer to Figure 9. [Figure 5]Figure 5 is a pair of graphs demonstrating that mouse IL-12 stable variants (variants that signal through the mouse IL-12 receptor) respond differently from human IL-12 stable variants (variants that signal through the human IL-12 receptor) to p35 / p40 interface mutations (i.e., mutations in the p35 or p40 subunit at the site of the interaction between subunits in the case of heterodimerization; see, e.g., Figure 1, right panel). Human IL-12 stable variants are shown in the left plot, while mouse IL-12 stable variants are shown in the right plot. The presence of p70 secreted from T cells modified to express each variant (measured as picograms per milliliter) was measured by an MSD assay performed on samples from normalized T cell supernatant. [Figure 6] Figure 6 shows alignments of mouse (top) and human (bottom) sequences of the p35 subunit of WT IL-12 and scIL-12 containing the WT sequence, indicating several positions that appear to be important for either dimerization along the IL-12Rb2 or p35 / p40 interface. The tyrosine (Y) residues in alignment columns 2 and 4 are IL-12Rb2 interaction residues conserved in both humans and mice. The boxed positions in alignment column 3 are IL-12Rb2 interaction residues that are not conserved in both humans and mice. The double-boxed positions (ST in mouse and RI in human) within alignment column 4 are important p35 / p40 interface residues that are not conserved. The boxed valine (V) and arginine (R) residues within alignment column 4 are important p35 / p40 interface residues that are conserved. The figure discloses sequence numbers 18 and 19, respectively, in order of appearance. [Figure 7] Figure 7 shows the alignment of mouse (top) and human (bottom) sequences of helix 4 within the p35 subunit, highlighting the desired location at the p35 / p40 interface. The figure discloses sequence numbers 20 and 21, respectively, in order of appearance. [Figure 8]Figure 8 is a series of graphs showing 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 the addition of the S177R mutation in mouse DS slows down dissociation. The Octer® data reflecting the activity of IL12 stable variants that can signal through human IL-12R (“human variants”) are shown in the upper row, while the Octer® data reflecting the activity of IL-12 stable variants that can signal through mouse IL-12R (“mouse variants”) are shown in the lower row. [Figure 9] Figure 9 is a table providing quantitative data for the experiments described in Figures 4, 8, and 10. *Note: The error (±) indicates the standard deviation from two independent experiments. **Only one measurement was performed on these samples. [Figure 10] Figure 10 shows a pair of graphs demonstrating off-rate and molecular half-life measurements for human and mouse variants. Mouse DS+S177R has a p35 / p40 dissociation rate comparable to human DS and provides a tool variant for in vivo testing of the same strain. See Figure 9 for quantification of the data shown here by graph of IL-12 stable variants that signal through the human IL-12 receptor. [Figure 11] Figure 11 shows 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) expressing IFN-γ intracellularly is shown for proximal (flat-bottomed) or distal (Transwell) bystander cells to modified T cells modified to secrete the IL-12 stability variant of this disclosure. Right: Corresponding plots (measured in picograms per milliliter) demonstrating the amount of IL-12 stability variant that can be obtained for proximal and distal bystander cells under each condition. [Figure 12]Figure 12 is a graph demonstrating the safety advantages of the IL-12 stability variant of this disclosure. The worst-case scenario for the equilibrium concentration of circulating active p70 was calculated for the human DS variant. [Figure 13A-13C] Figures 13A-C are graphs demonstrating that IL-12 stability-reduced variants enhanced tumor death of antigen-specific TCR-expressing T cells against A375 target cells over four co-culture cycles with target cells. Antigen-specific TCRs were expressed under all conditions. In the sequential killing assay, 25% of each co-culture was carried over to new target cells every three days until differences in cell lysis between conditions were observed. "Furin-DS": Furin cleavage disulfide mutant. "Furin-DS-R181": Furin-DS + destabilization mutation R181A in the p70-p35 subunit. [Figure 13D] Figure 13D is a graph showing that all TCR-expressing T cells (IL-12 stability-reduced variant and wild-type) demonstrated enhanced tumor cell lysis in the presence of IL-12 in the fourth co-culture with A375 cells. Furin-DS, Furin-DS-I52C-A179C, and Furin-DS-I52C-A179C-R181A mutants and wild-type single-stranded IL-12 (wt scIL-12)-expressing T cells all performed significantly better than TCR alone, although the Furin-DS-R181A variant showed a tendency toward a significant difference (p=0.07). No significant difference in cytotoxicity was observed between either wt scIL-12 or the IL-12 stability-reduced variant. "Furin-DS-I52C-A179C": Stabilizing mutation I52C in the p70-p35 subunit and A179C in the p40 subunit of Furin-DS. "Furin-DS-I52C-A179C-R181A": Destabilizing mutation R181A in the p70-p35 subunit of Furin-DS, and the p70-stabilizing mutations I52C and A179C mentioned above. [Figure 14A]Figures 14A and 14B are graphs showing tumor growth in NSG-MHC I / II double knockout mice (n=8) subcutaneously transplanted with 1.0 × 10⁶ A375 (human melanoma) cells for two trials using the same method. Figure 14A reflects "Trial 1," and Figure 14B reflects "Trial 2." After the tumors in each trial reached the desired volume (mean 100 mm³), the animals were treated with 2.5 × 10⁶ antigen-specific TCR-expressing T cells co-transduced with an IL-12 stability-reduced variant under the control of an engineered cell activation-inducible promoter (e.g., 4xNFAT-hBG). Mice receiving PBS (without T cells) were used as controls. Tumor volume was measured twice weekly after injection. Mice treated with TCR T cells expressing Furin-DS mutants ("Furin DS IL12 mutant" in Figure 14A and "stability-reduced IL12" in Figure 14B) demonstrated a deeper antitumor response compared to mice treated with wt-scIL-12 or TCR alone. In Figure 14A, the proliferation measured in IL-12-treated mice between 10 and 30 days was determined not to be true tumor progression due to the expansion of T cells at the tumor site. [Figure 14B] Same as above. [Figure 14C] Figure 14C shows the Kaplan-Meier survival curve from Experiment 2, demonstrating improved survival in mice treated with TCR T cells expressing the Furin-DS mutant ("stability-reduced IL12 mutant") compared to mice treated with T cells expressing wt-scIL-12. [Figure 15A]Figures 15A–F are graphs showing the effects of IL-12 stability-reducing variants on T cell proliferation, in vivo cytokine secretion, and safety as determined in Experiment 1 (Figures 15A and 15B) and Experiment 2 (Figures 15C–F). Blood was collected from mice 1, 7, 14, 21, 28, and 42 days after TCR-T cell infusion and analyzed by flow cytometry of blood PKs and cytokine levels. Figure 15A shows that TCR-T cells expressing wt scIL12 or Furin-DS variants demonstrated enhanced proliferation compared to mice treated with TCR-T cells alone. Figure 15B also shows that mice treated with TCR-T cells expressing wt scIL12 or Furin-DS showed enhanced levels of IFN-γ secretion compared to TCR-T cells alone. Figure 15C shows that the proliferation profile of TCR-T cells expressing wt scIL12 was similar to that of TCR-T cells expressing the Furin-DS variant. Figure 15D shows that animals treated with T cells expressing the Furin-DS variant ("reduced stability") had higher plasma concentrations of IFN-γ compared to animals treated with T cells expressing wild-type IL-12. Figures 15E and 15F are graphs demonstrating that the IL-12 reduced stability variant improves the safety profile. Figure 15E shows that IL-12 p40 levels were detected in the plasma of mice treated with TCR-T cells expressing wt scIL-12 and in the plasma of mice treated with TCR-T cells expressing the reduced stability Furin-DS ("reduced stability") variant, demonstrating that both IL-12 constructs are expressed in mice. However, Figure 15F shows that levels of IL-12 p70 (active, dimeric form) were detected only 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 destabilized Furin-DS IL-12 ("destabilized") variant. These results demonstrate the autoinactivating function of the Furin-DS-IL-12 variant after expression.This trend was repeated in the second mouse study, confirming (or not demonstrating) the reproducibility of the self-inactivation function of the Furin-DS stable mutant. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 15F] Same as above. [Figure 16] Figure 16 is a graph showing mouse production of IL-12 p40 determined by MSD from the highest-expressing mouse cell line B16-F10. "trIL-12" refers to the mouse equivalent of the Furin-DS mutant ("sc-mIL-12-p40-C175S-furin-p35-C70S-S177R"). These data confirm that mouse surrogate IL-12 stable variant mutation designs, such as sc-mIL-12-p40-C175S-furin-p35-C70S-S177R, are expressed in mouse cells. [Figure 17] Figures 17A and 17B are graphs showing the mean tumor volume (Figure 17A) and individual tumor growth curves (Figure 17B) of C57 / BL6 mice transplanted with a mixture of B16F10 tumor cells in which 10% of the tumor cells express either wild-type mouse single-stranded IL-12 ("WT sc-mIL-12") or the mouse equivalent of the Furin-DS mutant ("sc-mIL-12-p40-C175S-furin-p35-C70S-S177R"). [Figure 18-1] Figures 18A–18C are three graphs showing the plasma cytokine concentrations of mouse IL-12 p40 (Figure 18A), mouse IL-12 p70 (Figure 18B), and mouse IFN-γ (Figure 18C), determined by MSD from C57 / BL6 mice transplanted with a mixture of B16F10 tumor cells in which 10% of the tumor cells expressed either wild-type mouse single-stranded IL-12 ("WT sc-mIL-12") or the mouse equivalent of the Furin-DS mutant ("sc-mIL-12-p40-C175S-furin-p35-C70S-S177R"). [Figure 18-2]Same as above. [Modes for carrying out the invention]

[0029] This disclosure describes IL-12 stable variants that avoid the systemic toxicity of prior IL-12 therapies. These stable variants have the same or similar biological activity as wild-type IL-12 but lack the stability of the wild-type protein. The variants are rapidly inactivated after secretion from engineered cells (e.g., immune cells such as T cells) in vivo. The variants are less stable than wild-type IL-12 because they lack the native disulfide bond between the two subunits of IL-12 (e.g., p35 or alpha subunit and p40 or beta subunit) due to mutations in residues that form or promote the formation of the native disulfide bond.

[0030] In some embodiments, the manipulated cells are tumor-specific T cells, and this design approach achieves safe, local delivery of IL-12 activity from the manipulated tumor-specific T cells. Locally active IL-12 variants modulate the tumor microenvironment (TME), promote innate and adaptive immune responses, and support the cytotoxic activity of T cells and NK cells. IL-12 variants whose expression is substantially restricted to the TME are also referred to herein as tumor-restricted IL-12 (trIL-12).

[0031] Existing approaches using IL-12 as a therapeutic agent carry considerable risk due to its potent activity even at very low concentrations. Even with topical administration, small amounts of IL-12 diffusing from the site of interest can drive undesirable activity and cause toxicity. The IL-12 stable variant of this disclosure avoids this problem by having a short half-life due to the unstable complexation between the two subunits of IL-12. This short half-life allows the IL-12 variant to act locally without toxicity to tissues far from the secretory site.

[0032] I. IL-12 Stability Variant The IL-12 variants disclosed herein encompass only non-naturally occurring IL-12 molecules. The IL-12 variants disclosed herein are considered to be non-naturally occurring. The IL-12 variants herein have reduced stability (reduced half-life) compared to their wild-type counterparts and are therefore also called “IL-12 stable variants.” In some embodiments, the IL-12 stable variants herein have binding affinity to IL-12R comparable to their wild-type counterparts.

[0033] IL-12 stability variants contain mutations in the p35 sequence, the p40 sequence, or both sequences. These mutations remove or prevent the formation of a single native disulfide bond between the p35 and p40 subunits.

[0034] In some embodiments, the stable variant is a fusion of p35 and p40 subunits linked by a cleavable peptide linker (e.g., a mobile, cleavable peptide linker), which keeps the two subunits together to form functional IL-12, but is cleaved by proteases in the local extracellular environment so that the IL-12 variant secreted from the engineered cells is active only for a limited period in vivo. The effect of the IL-12 variant is therefore local without causing systemic toxicity.

[0035] In some embodiments, IL-12 stability variants are heterodimers and lack the native disulfide bond, but contain a non-native, weak intersubunit disulfide bond engineered into the protein by recombination, resulting in a less stable coupling between p35 and p40. These stability variants retain highly effective signaling ability, but it is limited in time and therefore spatial scale, thereby avoiding systemic toxicity. In some embodiments, IL-12 stability variants are heterodimers and lack the native disulfide bond or alternative disulfide bond structure, but the mutant p35 and / or p40 subunits maintain a weak stable coupling by non-covalent attraction, and as a result, the p35 and p40 subunits maintain a transiently limited heterodimer structure.

[0036] In some preferred embodiments, the IL-12 variant is a variant of human IL-12. By “human IL-12 variant” or “human IL-12 variant,” we mean an IL-12 molecule containing sequences of human IL-12 p35 and p40 subunits, wherein one or both of the p35 and p40 sequences contain one or more amino acid mutations compared to the wild-type human sequence.

[0037] In another preferred embodiment, the IL-12 variant is a variant of mouse IL-12. By “mouse IL-12 variant” or “mouse IL-12 variant,” we mean an IL-12 molecule containing sequences of mouse IL-12 p35 and p40 subunits, wherein one or both of the p35 and p40 sequences contain one or more amino acid mutations compared to the wild-type mouse sequence.

[0038] A.p40 Subunit The p40 subunit of IL-12 is also known as the β subunit. An exemplary mature wild-type human p40 sequence is shown below: [ka] The unprocessed wild-type human p40 sequence with the signal peptide attached (UniProt ID No. P29460) is provided herein as Sequence ID No. 8. The cysteine ​​for forming the natural intersubunit disulfide bond is C177, which is enclosed in a box in the sequence above.

[0039] An exemplary wild-type mouse p40 mature amino acid sequence (UniProt ID No. P43432) is shown below: [ka] The cysteine ​​responsible for forming the natural intersubunit disulfide bond is C175, which is enclosed in a box in the sequence above.

[0040] As used herein, the term “p40 subunit” encompasses variant forms of the subunit (e.g., mutant, truncated, or elongated forms). In some embodiments, the variant forms may have a molecular weight different from that of the wild-type p40 subunit having, for example, the amino acid sequence described in SEQ ID NO: 10.

[0041] As used herein, “corresponding” amino acid residue or position means an amino acid residue that aligns (but is not necessarily identical) with a reference residue, or an amino acid position that aligns with a reference position, when the target sequence and the reference sequence containing the residue are aligned to achieve maximum homology (allowing gaps as recognized in the art). For example, the position corresponding to the 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). Sequence alignment of human p40 to mouse p40, as performed by BLAST®, is shown below; as is evident from this alignment, the position corresponding to C177 (boxed) in SEQ ID NO: 4 (sequence ID: 1 minus the C-terminal serine) in mouse p40 is C175 (boxed) in SEQ ID NO: 16 (sequence ID: 10 minus the four C-terminal residues). [ka]

[0042] B.p35 Subunit The p35 subunit of IL-12 is also known as the α subunit. An exemplary mature wild-type human p35 sequence is shown below: [ka] The unprocessed wild-type human p35 sequence with the signal peptide attached (UniProt ID No. P29459) is provided herein as Sequence ID No. 9. The cysteine ​​for forming the natural intersubunit disulfide bond is C74, which is enclosed in a box in the sequence above.

[0043] An exemplary wild-type mouse IL-12 p35 mature amino acid sequence (UniProt ID No. P43431) is shown below: [ka] The cysteine ​​responsible for forming the natural intersubunit disulfide bond is C70, which is enclosed in a box in the sequence above.

[0044] As used herein, the term “p35 subunit” encompasses variant forms of the subunit (e.g., mutant, truncated, or elongated forms). In some embodiments, the variant forms may have a molecular weight different from that of the wild-type p35 subunit having, for example, the amino acid sequence described in SEQ ID NO: 11.

[0045] The position corresponding to the amino acid residue in SEQ ID NO: 2 encompasses the position in the orthologous sequence (e.g., mouse p35) when aligned to maximize homology between the target sequence and the orthologous sequence. In some embodiments, the corresponding position contains a conserved residue (e.g., the residue in the orthologous sequence (e.g., mouse residue) is the same as the target residue (e.g., human residue)). Sequence alignment of human p35 to mouse p35, as performed by BLAST®, is shown below; as is evident from this alignment, the position corresponding to C74 in SEQ ID NO: 2 in mouse p35 is C70 in SEQ ID NO: 11: [ka]

[0046] C. Mutation that eliminates the natural intersubunit disulfide bond. The IL-12 stability variants described herein lack the native intersubunit disulfide bond due to one or more mutations made in one or both of the two subunits. In some embodiments, the mutation is a deletion or substitution. In some embodiments, the mutation may be an inversion, transposition, or recombination.

[0047] In some embodiments, the p40 variant contains a mutation at the position corresponding to C177 in SEQ ID NO: 1 (e.g., C175 in SEQ ID NO: 10), for example, a mutation that removes cysteine ​​from the position corresponding to position 177 in SEQ ID NO: 1, or from the position corresponding to position 175 in SEQ ID NO: 10. In some embodiments, the mutation may be a serine (S) substitution of cysteine, as shown in the exemplary sequence described in SEQ ID NO: 12. In some embodiments, the p40 sequence of the human IL-12 stable variant includes SEQ ID NO: 12 (i.e., SEQ ID NO: 1 with the C177S mutation).

[0048] In some embodiments, the p35 variant contains a mutation at the position corresponding to C74 in SEQ ID NO: 2 (e.g., C70 in SEQ ID NO: 11), for example, a mutation that removes cysteine ​​from the position corresponding to position 74 in SEQ ID NO: 2, or from the position corresponding to position 70 in SEQ ID NO: 11. In some embodiments, the mutation may be a serine (S) substitution of cysteine, as shown in the exemplary sequence described in SEQ ID NO: 13. In some embodiments, the p35 sequence of the human IL-12 stable variant includes SEQ ID NO: 13 (i.e., SEQ ID NO: 2 with the C74S mutation).

[0049] In some embodiments, the IL-12 stability variant includes both a mutation at the position corresponding to C177 in SEQ ID NO: 1 and a mutation at the position corresponding to C74 in SEQ ID NO: 2. For example, the IL-12 stability variant is a human IL-12 variant including SEQ ID NOs: 12 and 13. In another embodiment, the IL-12 stability variant is a mouse IL-12 variant including a C175S mutation in the p40 subunit and a C70S mutation in the p35 subunit.

[0050] In some embodiments, the p40 subunit and / or p35 subunit may include other mutations that weaken the coupling of the two subunits without significantly altering the biological activity (e.g., IL-12R binding affinity) when the two subunits are coupled. For example, the p35 subunit may contain one or more mutations at the position corresponding to residue R181 in SEQ ID NO: 2 (e.g., R181A), the position corresponding to R183 in SEQ ID NO: 2 (e.g., R179 in SEQ ID NO: 11), the position corresponding to V185 in SEQ ID NO: 2 (e.g., V181 in SEQ ID NO: 11) (e.g., V185A mutation), or the position corresponding to R189 in SEQ ID NO: 2 (e.g., R185 in SEQ ID NO: 11) (e.g., R189K mutation).

[0051] In some embodiments, IL-12 stability variants are modified to lack the native intersubunit disulfide bond but to have a new, weak disulfide bond between subunits. For example, p40 contains a mutation to cysteine ​​(C) at the position corresponding to A179 in SEQ ID NO: 2 (e.g., A179C mutation), and p35 contains a mutation to C at the position corresponding to I52 in SEQ ID NO: 2 (e.g., I52C mutation). This pair, manipulated to cysteine ​​residues, can form a weak intersubunit disulfide bond. In some embodiments, p40 contains a mutation to cysteine ​​(C) at the position corresponding to A179 in SEQ ID NO: 2 (e.g., A179C mutation), and p35 contains a mutation to C at the position corresponding to I52 in SEQ ID NO: 2 (e.g., I52C mutation), and a mutation at the position corresponding to residue R181 in SEQ ID NO: 2 (e.g., R181A).

[0052] In some embodiments, the IL-12 stability variant includes a p40 sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 1. In some embodiments, the IL-12 stability variant includes a p35 sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 2. In some embodiments, the IL-12 stability variant includes a p40 sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 1 and a p35 sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 2.

[0053] The percentage of identity between two amino acid sequences (or two nucleic acid sequences) is obtained, for example, by BLAST® using default parameters (available on the US National Library of Medicine's National Center for Biotechnology Information website). In some embodiments, the length of the aligned reference sequence 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.

[0054] This disclosure also provides IL-12 stability variants that are not derived from wild-type human or mouse sequences, but that contain mutations compared to their related wild-type sequences at positions corresponding to the numbered positions disclosed herein with respect to Sequence ID No. 1 or 2.

[0055] D. Single-chain IL-12 In some embodiments, the IL-12 variant is a single-stranded IL-12 (scIL-12) fusion protein containing a p35 sequence fused to a p40 sequence. The p35 sequence may be either the N-terminus or C-terminus of the p40 sequence. The p35 and p40 sequences are linked by a cleavable peptide linker, optionally a mobile, cleavable peptide linker. The mobile linker may, for example, have a glycine / serine-rich sequence.

[0056] In some embodiments, the cleavable linker includes a substrate sequence of hurin, e.g., RX-[K / R]-R, or a TEV cleavage site. For example, the linker may have the sequence GSRRRR * R * R * S (sequence number 5) is included, and here " * " represents the point at which the section can be cut by Hurin.

[0057] In some embodiments, the cleavable linker includes a substrate sequence of urokinase-type plasminogen activator (uPA), matrix metallopeptidase (MMP) 2, MMP9, or a combination thereof.

[0058] In certain embodiments, the IL-12 variant is single-stranded human IL-12 (sc-hIL-12) containing a C177A mutation in the p40 sequence or a C74S mutation in the p35 sequence. In certain embodiments, the IL-12 variant is single-stranded human IL-12 (sc-hIL-12) containing a C177S mutation in the p40 sequence and a C74S mutation in the p35 sequence. In certain embodiments, the variant contains residues 22-533 of SEQ ID NO: 6 (where residues 1-21 are a signal sequence that is cleaved in the mature protein). This variant is referred to as the "DS-furin variant," "furin-DS-variant," or "p40-C177S-p35-C74S" in the following examples. The term "furin" as seen in the construct name refers to a furin-cleavable domain having, for example, the sequence described in SEQ ID NO: 5.

[0059] In certain embodiments, the IL-12 variant is the sc-hIL-12 DS-furin variant, which further contains an A179C mutation in the p40 sequence and an I52C mutation in the p35 sequence. This variant is referred to as the "DS-furin-I52C-A179C variant" in the following examples.

[0060] In certain embodiments, the IL-12 variant is the sc-hIL-12 DS-furin-I52C-A179C variant, which further contains the R181A mutation in the p35 sequence. This variant is referred to as the "DS-furin-I52C-A179C-R181A variant" in the following examples.

[0061] In certain embodiments, the IL-12 variant is sc-hIL-12 p40-C177S-p35-C74S, but further contains the R181A mutation in the p35 sequence. This variant is referred to as the "p40-C177S-R181A-p35-C74S variant" in the following examples.

[0062] In certain embodiments, the IL-12 variant is single-stranded mouse IL-12 (sc-mIL-12) containing a C70S mutation in the p35 sequence. This variant is referred to as the "sc-mIL-12-p35-C70S variant" in the following embodiments.

[0063] In certain embodiments, the IL-12 variant is the sc-mIL-12-p35-C70S variant as described above, but further contains an S177R mutation in the p35 sequence. This variant is referred to as "sc-mIL-12-p35-C70S-S177R" in the following examples.

[0064] In some embodiments, the scIL-12 variants herein include amino acid sequences that are 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 its further variants).

[0065] II. Expression of IL-12 variants This disclosure provides any vector or combination of vectors comprising nucleic acid molecules encoding IL-12 stable variants and nucleic acid molecules encoding subunits of the IL-12 stable variants as herein. If the variant is a heterodimer, the coding sequences of the two IL-12 subunits are placed in separate expression vectors, or the two coding sequences are placed in the same vector having one polycistronic expression cassette or two separate expression cassettes. If the IL-12 variant is a heterodimer, the coding sequences of the two subunits are placed in a single expression cassette separated by IRES, or separated in frame by the coding sequences of self-cleaving peptides (e.g., 2A peptides, e.g., T2A, E2A, P2A, and F2A) so that the two subunits are expressed as separate polypeptides. Polycistronic structures that facilitate the expression of multiple proteins from the same promoter / expression cassette are described in detail in WO2021 / 119539. The two translated protein subunits can then heterodimerize to form a functional IL-12 variant with transiently limited stability outside the cell due to the absence of stabilizing covalent bonds.

[0066] IL-12 stability variants (e.g., heterodimeric IL-12 or scIL-12 proteins having the mutations described herein) are expressed in target cells, such as eukaryotic host cells where purified protein is desired, or cells where cell-based therapy is desired (e.g., immune cells).

[0067] Nucleic acid molecules (e.g., coding nucleic acid molecules or DNA or RNA vectors containing coding nucleic acid molecules) are introduced into cells by known techniques, but are not limited to, electroporation, calcium phosphate precipitation, lipofection, particulate guns, microinjection, colloidal dispersion systems (e.g., macromolecular complexes, nanocapsules, microspheres, and beads), and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, and liposomes). Alternatively, nucleic acid molecules are introduced into cells by transduction of recombinant viruses containing nucleic acid molecules in their genome. Examples of viral vectors, but are not limited to, vectors derived from lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, Sendai viruses, and vaccinia viruses. In certain embodiments, the recombinant virus is pseudotyped with heterologous envelope proteins. In one embodiment, the recombinant virus is a pseudotype lentivirus with an envelope glycoprotein derived from vesicular stomatitis virus (VSV), measles virus, or another virus (see, for example, Cronin et al., Curr Gene Ther. (2005) 5(4):387-398; Gutierrez-Guerrero et al., Viruses (2020) 12(9):1016).

[0068] Mammalian host cells that produce IL-12 stable variants include, but are not limited to, CHO cells, NS0 cells, HEK cells, 293 cells, etc. Methods for producing therapeutic proteins in mammalian host cells are well known in the art. Insect cells such as Sf9 cells and Sf21 cells are also used.

[0069] In the context of cell-based therapy, the IL-12 variants described herein are expressed in allogeneic or autologous cells. In some embodiments, the coding sequence of the IL-12 variant is expressed in T cells (e.g., CD4). + T cells and CD8 +It is introduced into immune cells such as T cells and natural killer (NK) cells, or their progenitor cells (e.g., hematopoietic stem cells or progenitor cells). The variant coding sequence is introduced by an expression construct (e.g., a viral vector) or directly by mRNA (e.g., delivered to cells by lipid nanoparticles via local or systemic administration).

[0070] Human immune cells are used to treat human patients. In some embodiments, human immune cells are T cells (e.g., CD4 + T cells and / or CD8 + The T cells are, in some embodiments, human immune cells are derived from peripheral blood mononuclear cells (PBMCs) of the target. In some embodiments, the T cells are engineered T cells. In some embodiments, the engineered T cells further comprise nucleic acids encoding a chimeric antigen receptor (CAR) specific to the antigen of interest. In some embodiments, the engineered T cells further comprise nucleic acids encoding an engineered T cell receptor (TCR). In some embodiments, the T cells are tumor-infiltrating T cells (TILs).

[0071] Combining IL-12 stable variants with regulatory strategies that localize variant expression to a target site (e.g., tumor or tumor microenvironment (TME)) maintains IL-12R signaling by increasing local concentrations at the target site, while avoiding undesirable signaling activity at low concentrations away from the target site. Therefore, the expression of IL-12 stable variants in therapeutic immune cells migrating to a target site provides an unprecedented method for tightly controlling IL-12 activity while maintaining a balance between efficacy and safety.

[0072] In some embodiments, the IL-12 stable variant is expressed under the control of a constitutive promoter, such as a tissue-specific or cell-specific promoter. In alternative embodiments, the IL-12 stable variant is expressed under the control of an inductive promoter. In some embodiments, the inductive promoter responds to extracellular signals, e.g., stimulating or inhibitory immunomodulatory signals. In some embodiments, the inductive promoter responds to intracellular signals. In some embodiments, the inductive promoter is induced in a T cell context and may be, for example, a T cell activation inductive promoter. For example, the promoters herein may respond directly or indirectly to CD3-mediated signals, e.g., CD3-mediated signals triggered by CAR or TCR stimulation. Examples of useful promoters in this specification include, but are not limited to, the earliest cytomegalovirus (CMV) promoter, the earliest monkey virus 40 (SV40) promoter, the human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, the earliest Epstein-Barr virus promoter, the Roussarcoma virus promoter, the elongation factor-1α (EF-1α) promoter, the MND promoter, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In some embodiments, the IL-12 stable variant is under the control of an inducible promoter incorporating a core or minimal promoter, which includes a core or minimal promoter derived from one of the aforementioned promoters. Suitable core or minimal promoters for inducible promoters are described in the Art and are incorporated to minimize the baseline level of transcription in cells when suitable transcription factors are not present, and to promote increased expression when suitable transcription factors are present. In non-limiting examples, in some embodiments, the inducible promoters of this 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 given in SEQ ID NOs. 22-29.See also Ede et al., ACS Synth Biol. (2016) 5(5):395-404 (including supplementary information), WO2021 / 244654, and WO2020 / 206149, respectively, which are incorporated herein by reference in their entirety. Further exemplary core promoters useful for the inducible promoters of this disclosure include the minAdeP and minIL2 promoters, as well as functional variants thereof. A “functional variant” includes a minor sequence variant, e.g., a modification or deletion of approximately one, two, or three nucleic acid residues in the sequence, and the core promoter retains its desired function of promoting transcription in the presence of a suitable transcription factor.

[0073] Inducible promoter systems useful for expressing the IL-12 stability variants of this disclosure include, but are not limited to, hormone-regulating elements, synthetic ligand-regulating elements, ionization radiation-regulating elements, and tetracycline (Tet) systems (e.g., "Tet-Off" and "Tet-On" systems). In some embodiments, the inducible promoter includes a transcription factor response element that facilitates condition-dependent induction of transcription. In some embodiments, the inducible promoter further includes a transcription factor response element combined with, for example, a core or minimal promoter sequence as described above. The response element is a segment of nucleic acid having a sequence that is specifically recognized by a transcription factor. Exemplary response elements are sequences that are sensitive to the activation state of immune cells or include such sequences.

[0074] For example, when immune cells are activated (e.g., by antigen binding), nuclear translocation of specific transcription factors occurs, such as activating protein-1 (AP-1), activated T cell nuclear factor (NFAT), activated B cell nuclear factor-κ-light chain enhancer (NF-κB), and IRF4. Such transcription factors bind to their respective response elements. Thus, in some exemplary embodiments, the inducible promoter may include 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 element, iron response element (IRE), interferon-stimulated response element (ISRE), or any combination thereof. For example, exemplary NFAT response systems related to this disclosure are described, for example, Kallunki et al., Cells (2019) 8(8):796; Uchibori et al., Mol Ther Oncolytics (2018) 12:16-25.

[0075] In some embodiments, the inducible promoter includes four copies of an NFAT response element. The sequence of an exemplary NFAT response element is described in SEQ ID NO: 30. In some embodiments, the T cell activation inducible promoter includes one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc.) functionally combined with the hBG promoter sequence (see Na et al., Blood (2010) 116(11):e18-e25; Szyska et al., Cancer Immunol Res. (2018) 6(1):pp. 110-120 and GenBank Accession No. MF462285.1 dated September 13, 2017). In some embodiments, the T cell activation inducible promoter includes four repeats of an NFAT response element (e.g., SEQ ID NO: 30) functionally linked directly to the hBG core promoter (e.g., SEQ ID NO: 22) or by a minor intervening sequence. In further embodiments, the promoter includes a nucleotide sequence that is at least 80% identical (e.g., at least 85, 90, 95, 96, 97, 98, or 99%) to SEQ ID NO. 14 (see, e.g., Szyska et al. and GenBank Accession No. MF462285 above) and retains T cell activation-inducible properties. The sequence of an exemplary expression cassette containing such a promoter for expressing a DS-Furin variant is shown in SEQ ID NO. 15.

[0076] In some embodiments, the T cell activation-inducible promoter includes one or more NFAT response elements functionally combined with the YB_TATA synthetic core promoter sequence (see, e.g., Ede et al., ACS Synth Biol. (2016) 5(5):395-404 (including supplemental information), WO2021 / 244654, and WO2020 / 206149). An exemplary YB_TATA core promoter sequence is described in SEQ ID NO: 25. WO2020 / 206149 discloses combinations of 3- and 6-repeat NFAT response element domains linked to the YB_TATA core promoter domain. In some embodiments, the T cell activation-inducible promoter includes SEQ ID NO: 31.

[0077] In some embodiments, the T cell activation-inducible promoter includes one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc. NFAT response elements (e.g., SEQ ID NO: 30)) functionally combined with the pJB42CAT5 core promoter sequence (e.g., SEQ ID NO: 23). In some embodiments, the T cell activation-inducible promoter includes one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc. NFAT response elements (e.g., SEQ ID NO: 30)) functionally combined with the MiniTK core promoter sequence (e.g., SEQ ID NO: 24). In some embodiments, the T cell activation-inducible promoter includes one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc. NFAT response elements (e.g., SEQ ID NO: 30)) functionally combined with the minCMV core promoter sequence (e.g., SEQ ID NO: 26). In some embodiments, the T cell activation-inducible promoter includes one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc. NFAT response elements (e.g., SEQ ID NO: 30)) functionally combined with a minSV40 core promoter sequence (e.g., SEQ ID NO: 27). In some embodiments, the T cell activation-inducible promoter includes one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc. NFAT response elements (e.g., SEQ ID NO: 30)) functionally combined with a CMV53 core promoter sequence (e.g., SEQ ID NO: 28). In some embodiments, the T cell activation-inducible promoter includes one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc. NFAT response elements (e.g., SEQ ID NO: 30)) functionally combined with an MLP core promoter sequence (e.g., SEQ ID NO: 29). In some embodiments, the T cell activation-inducible promoter includes one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc. NFAT response elements (e.g., SEQ ID NO: 30)) functionally combined with the minAdeP core promoter sequence.In some embodiments, the T cell activation-inducible promoter includes one or more NFAT response elements (e.g., 1, 2, 3, 4, 5, 6, etc. NFAT response elements (e.g., SEQ ID NO: 30)) functionally combined with the minIL2 core promoter sequence.

[0078] In some embodiments, the expression cassette also includes other elements that facilitate the transcription and / or translation of the Kozak sequence, polyadenylation sites, and coding sequences. For example, the Woodchuck hepatitis virus post-transcriptional response element (WPRE) or a variant thereof is included in the 3' untranslated region of the expression cassette.

[0079] Within the expression cassette, transcription / translation control elements such as promoters and optional enhancers are operably ligated to the coding sequence to enable efficient expression of the coding sequence and efficient translation of the RNA transcript.

[0080] III. Use of IL-12 Stability Variants The IL-12 stable variants described herein are used in cytokine therapy and are delivered systemically (e.g., by intravenous injection or infusion) or locally (e.g., intratumor) in a pharmaceutical composition to subjects requiring immunostimulation, such as subjects with cancer (e.g., hematological malignancies or solid tumors) or immunodeficiency. For example, the pharmaceutical composition may be injected directly into the tumor site. The pharmaceutical compositions described herein may contain pharmaceutically acceptable carriers 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.

[0081] The IL-12 stable variants described herein are also used in cell-based therapies. As described above, immune cells such as T cells are engineered to express the variant and are transduced, for example, by a suitable vector containing a nucleic acid encoding the IL-12 stable variant. In some embodiments, T cells also express recombinant antigen receptors. As used herein, “recombinant antigen receptor” refers to an antigen receptor that is not naturally expressed by T cells. Recombinant antigen receptors are cell surface molecules that bind to an antigen of interest on another cell (e.g., a tumor cell) and may be derived, for example, from a T cell receptor or an antibody. Recombinant antigen receptors may be, for example, antibodies, engineered antibodies such as scFv, CARs, engineered TCRs, TCR mimics (e.g., antibody-T cell receptor (abTCR) or chimeric antibody-T cell receptor (caTCR)), chimeric signaling receptors (CSRs), TCR mimics (e.g., antibodies that recognize an epitope similar to that recognized by a TCR), or TCR fusion constructs (TRuCs). For example, see EP340793B1, WO2017 / 070608, WO2018 / 200582, WO2018 / 200583, WO2018 / 200585, Xu et al., Cell Discovery (2018) 4:62, Baeuerle et al., Nat Comm. (2019) 10:2087.

[0082] For example, a CAR may include an extracellular antigen-binding domain (e.g., an scFv domain), a transmembrane domain, and an intracellular signaling domain, and optionally a peptide stretch 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, e.g., 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 mainly consist of hydrophobic residues (e.g., alanine, leucine, valine, glycine, isoleucine, proline, phenylalanine, and tryptophan). In some embodiments, the intracellular signaling domain provides a signal similar to that from a native antigen receptor and may include, for example, a co-stimulatory domain (e.g., derived from CD28, 4-1BB, OX40, DAP10, or ICOS) and a primary signaling domain (e.g., derived from the CD3 zeta chain).

[0083] In some embodiments, abTCR may include an engineered TCR in which the antigen-binding domain of a TCR (e.g., alpha / beta TCR or gamma / delta TCR) is replaced by that of an antibody (with or without the antibody's constant domain), and the engineered TCR then becomes specific to the antibody's antigen while retaining the signaling function of the TCR.

[0084] In some embodiments, the CSR may include (1) an extracellular binding domain (e.g., a native / modified receptor extracellular domain, a native / 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).

[0085] Recombinant antigen receptors can target antigens of interest (e.g., tumor antigens or pathogen antigens). Antigens include, but are not limited to, AFP (alpha-fetoprotein), αvβ6 or other integrins, BCMA, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (CC 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, ephrin B2, EPHa2 (ephrin receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelin B receptor), FAP-α (fibroblast-activating protein α), fetal AchR (fetal Acetylcholine receptor, FBP (folate-binding protein), FCRL5, FR-α (folate receptor alpha), GCC (guanylic acid C), GD2, GD3, GPC2 (glypican-2), GPC3, gp100 (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G protein-binding receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA-A1 (human leukocyte antigen A1), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight melanoma-associated antigen), IGF 1R (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), mouse 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 (neuronal cell adhesion molecule), nectin-4, NKG2D (natural killer group 2 member D) ligand, NY-ESO, carcinoembryonic antigen, PD-1, PD-L1, PRAME (preferentially expressed antigen in melanoma), progesterone receptor, PSA (prostate-specific antigen) This may include PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), ROR1, ROR2, SIRPα (signal regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (prostate 6-transmembrane epithelial antigen 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.

[0086] In some embodiments, the antigen receptor may be bispecific, targeting two different antigens, such as the two antigens listed above. For example, an antigen receptor such as CAR may target CD19 and CD20, or CD19 and CD22.

[0087] This pharmaceutical composition and the manipulated immune cells are used to prevent disease or disorder by being administered in a therapeutically effective dose, thereby delaying or inhibiting the onset, progression, or recurrence of signs or symptoms of the disease or disorder, and thereby preventing the disease or disorder. In some embodiments, the disease or disorder is cancer.

[0088] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have meanings generally understood by those skilled in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used in the practice or testing of this disclosure. In case of any conflict, the definitions provided herein shall prevail. In general, the nomenclature used herein in connection with immunological techniques, pharmaceuticals, medicinal chemistry and medicinal chemistry, and cell biology is well known and commonly used in the art. Furthermore, unless otherwise required by context, singular terms include plural forms, and plural terms include singular forms. Throughout this specification and its embodiments, the phrases “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” are understood to indicate inclusion of the integer or group of integers described, but not exclusion of any other integer or group of integers. All papers and other references mentioned herein are incorporated by reference in their entirety. Many references are cited herein, but these citations do not constitute an acknowledgment that any of these references form part of the general knowledge of the art. Where used herein, the terms “approximately” or “about” applied to one or more values ​​of interest refer to values ​​similar to the reference values ​​stated. In certain embodiments, unless otherwise stated or otherwise evident from the context, the terms refer to a range of values ​​that are 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the reference values ​​stated.

[0089] In this disclosure, return references in dependent claims mean abbreviations of the direct and explicit disclosures of each and all combinations of claims indicated by the return reference. Any molecule disclosed herein can be used in any treatment method, and the individual being treated is defined in any of these. Furthermore, the headings herein are constructed for ease of organization and are not intended to limit the scope of the claimed invention in any way.

[0090] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. [Examples]

[0091] Characterization of IL-12 stability variant mutation generation and effective linker cleavage in primary T cells A. Methods and Materials T cell supernatants were collected from primary donor T cells transduced with IL-12 constructs. Transduced cells were incubated for 6 days. Cell marker (LNGFR) expression was normalized in these T cells (with or without 1:100 transacting for stimulation) incubated in 24-well plates for 20 hours prior to collection. P40, P70, and interferon-gamma (IFNg) were measured by Meso Scale Discovery (MSD) assay in supernatants that had not been stimulated for 24 hours prior to the assay. In this assay, mock-transfected CD19+ T cells were included as "bystander cells" in co-culture with the modified T cells described above.

[0092] IL-12 sustained activation assay Following the co-culture described above, the co-culture was depleted of IFNg, and then 20 microliters (mL) of the supernatant was collected for stimulation with Transact (1:500). After 20 hours of incubation, bystander cells were harvested, and INFg secretion from these cells was determined by MSD.

[0093] B. Results and Discussion As shown in Figure 1, the IL-12 stability variant was designed to remove the disulfide bridge present between the two subunits of wild-type (WT) IL-12 and introduce a Hurin-cleavable linker that is not present in either WT or naturally occurring variants of IL-12. To remove the disulfide bridge present in WT IL-12, the IL-12 stability variant was designed to lack a cystine (C) residue at position 177 of the p35 subunit (also referred to herein as the first subunit) and position 74 of the p40 subunit (also referred to herein as the second subunit). Instead of the cysteine ​​residue, the preferred IL-12 stability variant of this disclosure includes a serine (S) residue at position 177 of the p35 subunit (also referred to herein as the first subunit) and position 74 of the p40 subunit (also referred to herein as the second subunit). The IL-12 stability variant of this disclosure may include further mutations disclosed herein.

[0094] T cells were transduced with one of several IL-12 variants containing a Huurin-cleavable linker and with nucleic acids encoding wild-type single-stranded IL-12 as a control. IL-12 was immunoprecipitated from culture and assayed by Western blotting to determine the stability of the IL-12 dimer structure. Figure 2 confirms that the IL-12 stable variant was cleaved with approximately 100% efficiency when produced by and secreted from T cells.

[0095] As shown in Figure 3, the presence of p40 indicated that the molecule was secreted from all constructs. The absence of measurable p70 indicated that the molecule was cleaved and dissociated in equilibrium. The presence of proximal bystander T cell activity immediately after secretion (measured by IFN-γ production) indicated that the secreted IL-12 stable variant maintained its ability to signal through IL-12R, while the absence of proximal bystander activity after the incubation period indicated that the secreted IL-12 stable variant became inactive over time. [Examples]

[0096] Determination of the dissociation rate of IL-12 stability variants A. Methods and Materials Octet (registered trademark) analysis For each IL-12 stability variant, the biotinylated p35 subunit (p35-biotin) was captured on a streptavidin-coated surface. The captured p35 was associated with its corresponding p40 subunit in solution. The dissociation of the two subunits was measured as a function of time (seconds) by transferring the surface to a solution that did not contain p40. Throughout the assay, protein subunit interactions (capture, association, and dissociation) in each phase of exemplary IL-12 stability variants were marked by p35-p40 binding as a function of time (seconds). Octet®-mediated analysis, using biolayer interference (BLI) technology, evaluated biomolecular interactions in microwell plates. The Octet® system is commercially available.

[0097] B. Results and Discussion As used in this disclosure and illustrated in Figure 4, the term “half-life” is a different form of the pharmacokinetic use of the term “half-life.” In this experiment, the term “half-life” means describing the half-life of each p70 form of the destabilized variant after the variant has been secreted and diluted to a concentration comparable to the physiological concentration of WT IL-12 observed from samples obtained from peripheral circulating blood. At this point, p35 and p40 begin to dissociate at the rates shown in Figure 9. The measured half-life of hscIL12-p40-C177S-p35-C74S (also referred to herein as the “DS” variant) is approximately 10 minutes. This half-life profile is in contrast to the “DS+R181A” variant (hscIL12-p40-C177S-p35-C74S-R181A), which has a very short measured half-life of approximately 1 minute. While not intended to be theoretically rigid, the preference for one form over another in this experiment may be based on the objective of achieving a dissociation rate slow enough to diffuse the active molecule from therapeutic cells modified to secrete variant IL-12 across one to two cell diameters. Therefore, a preferred time course for dissociation occurs within a few minutes. Specific selection of mutations is made to adjust the stability of the IL-12 variant and achieve a desired half-life. [Examples]

[0098] Demonstration of localized IL-12 stability variant activity in vitro. A. Methods and Materials Bystander assay Transduced or mock-transduced modified T cells (modified cells) or bystander cells secreting an IL-12 stable variant were incubated in separate cultures for 6 days. Cellular marker (LNGFR) expression was normalized in each population (stimulated with 1:100 transact) incubated for 20 hours prior to the start of the bystander study. Modified T cells (concentration of 200,000 cells per well) and bystander cells (e.g., mock-transfected CD19-expressing T cells) (concentration of 100,000 cells / well) were stimulated with transact 1:100 (bystander cells) or 1:1000 (modified cells) for 8 hours and then co-cultured for 10 hours. In co-cultures established in flat-bottom wells, bystander cells were placed adjacent to the modified cells, while in co-cultures established in trans-wells, bystander cells were placed at a distance from the modified cells and separated by a membrane that allowed proteins (e.g., IL-12 stable variants) to diffuse through but not the cells. Each co-culture configuration was then exposed to a Protein Transport Inhibitor Cocktail for 2 hours before the collection of cells and supernatant for analysis. Bystander cells were collected for analysis of intracellular IFN-γ expression using ICS. Bystander cell supernatant was collected for p40 detection using MSD.

[0099] B. Results and Discussion As shown in Figure 11, the exemplary IL-12 stable variants of this disclosure exhibited local activity in vitro. Bystander cells proximal to modified T cells secreting the IL-12 stable variant demonstrated activation as indicated by intracellular expression of IFN-γ, while bystander cells distal to the IL-12 stable variant-secreting T cells did not. Proximal and distal bystander cells were exposed to similar levels of the IL-12 stable variant under either flat-bottom well or transwell conditions, respectively. These results demonstrate local activity, which is an important indicator of safety in vivo.

[0100] These results indicate that an inducible promoter (e.g., 4xNFAT-hBG) enabled minimal basal IL-12 production from T cells and substantially increased IL-12 production after T cell activation. The study also showed that the IL-12 stability variant dissociated into an inactive state after cleavage with an approximately 10-minute half-life, and that proximal bystander T cells were activated while distal bystander T cells were not, demonstrating that the function was limited to the site where expression was induced.

Example

[0101] Effect of IL-12 Stability Variant on T Cell-Mediated Cytolysis of Tumor Cells As demonstrated in the above example, single-chain IL-12 variants were designed, each variant being a fusion protein containing two IL-12 subunits linked by a furin-cleavable peptide linker and the sequences of two subunits modified to remove intermolecular disulfide bonds between them (C177S substitution of p40 and C74S substitution of p35). The dissociation rate of the cleaved IL-12 heterodimer was measured using the biolayer interferometry method. T cells were engineered with a lentiviral vector expressing either wild-type (wt) scIL-12 (scIL-12 in which the p40 and p35 subunits were not modified) or an engineered inducible promoter (e.g., 4xNFAT-hBG)-controlled scIL-12 variant; T cells were also engineered to constitutively express a TCR specific for the tumor antigen NY-ESO-1. IL-12 activity in proximal or distal bystander immune cells was measured by IFN-γ production in T cells co-cultured with engineered T cells producing IL-12, either directly or separated by a transwell membrane.

[0102] This example describes further functional testing of exemplary IL-12 stability variants. Engineered T cells were functionally evaluated in vitro by repeated loading with A375 tumor (melanoma) cells. Details of the test are described below. + A375 tumor (melanoma) cells were used for repeated loading to functionally evaluate in vitro. The details of the test are described below.

[0103] A. Methods and Materials T cell production was performed over 7 days using TCM medium (CTS® OpTmizer® medium supplemented with L-glutamine, GlutaMAX®, OpTmizer® cell supplement, Immune Cell Serum Replacement, 200 IU / mL IL-2, 1,200 IU / mL IL-7, and 200 IU / mL IL-15). On day 0, CD4 + and CD8 + T cells were thawed in TMC medium and 2 × 10⁻⁶ cells were mixed. 6 The cells were mixed in a 1:1 ratio at a concentration of [number] cells / mL and activated with TransACT® at a 1:100 dilution for 24-28 hours.

[0104] On day 1, T cells were simultaneously transduced using two lentiviral vectors. The first vector encoded an antigen-specific TCR expressed by a constitutive promoter, and the second vector encoded a human LNGFR expressed by a constitutive promoter and an IL-12 mutant protein expressed by a T cell activation-inducing promoter in which its four NFAT domains were linked to an hBG minimal promoter domain.

[0105] On day 2, at least 24 hours after 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.

[0106] On day 7, cells were counted, viability was assessed by AOPI staining, then harvested, and evaluated phenotypically by 1) flow cytometry for activation markers, memory markers, and transduction efficiency of both TCR and IL-12 (by LNGFR markers), and functionally by repeated loading with target cell lines (sequential kill assay).

[0107] Sequential kill assays were performed in RPMI1640 + 10% FBS medium. On day 1 of stimulation, target cells (A375-NLR) were counted, seeded on appropriate plates, and incubated at 37°C for 2 hours to adhere. Effector T cells (fresh or frozen from the above production) were then co-cultured with target cells in an E:T ratio of 1:5. The E:T ratio was used to determine viable TCRs. + Based on T cell count, IL-12 expression was characterized by a constitutive LNGFR tag, and expression differences were maintained within 10% in all samples. These co-culture plates were then placed in IncuCyte®, read every 2 hours, to measure changes in target cells over time (by NLR); a decrease in NLR over time indicates target cell clearance. Small amounts of supernatant were collected 24 hours after seeding effector cells and frozen at -80°C for subsequent analysis of cytokine levels.

[0108] Three days after the start of co-culture, a new plate of target cells was seeded as described above, and 25% of the previous culture was transferred to this new plate to initiate repeated loading / second stimulation of effector cells for the sequential kill assay. Cells remaining on the first plate were then phenotypically evaluated by flow cytometry for cell expansion, activation markers, memory markers, and transduction efficiency (LNGFR) of both TCR and IL-12.

[0109] As previously described, the supernatant was collected again 24 hours after seeding effector cells and frozen as described above for subsequent analysis of cytokine levels. Cells continued to be replated every 3 days until repeated loading, providing the necessary solution to distinguish between the functions of various IL-12 constructs (determined by NLR reduction in IncuCyte®). After the completion of the sequential kill assay, the collected supernatant was analyzed by MSD at 1:5 and 1:20 dilutions (due to the concentration difference between IL-12 and IFN-γ) according to the manufacturer's instructions.

[0110] B. Results and Discussion Four single-stranded human IL-12 variants were created and tested: (1) "DS-furin variant": The variant contains a C177S mutation in the p40 sequence and a C74S mutation in the p35 sequence; (2) "DS-furin-I52C-A179C variant": The variant contains C177S and A179C mutations in the p40 sequence, as well as C74S and I52C mutations in the p35 sequence, in which the native disulfide bond between C177 of p40 and C74 of p35 is removed, and instead a new disulfide bond can be formed between 179C of p40 and 52C of p35; (3) "DS-furin-I52C-A179C-R181A variant": Same as (2) except that the p35 sequence further contains the R181A mutation; and (4) "p40-C177S-R181A-p35-C74S" variant: Same as (1) except that the p35 sequence further contains the R181A mutation.

[0111] T cells were transduced by lentiviral vectors to express variants and wt scIL-12 under the control of an engineered inducible promoter (e.g., 4xNFAT-hBG). The T cells were also transduced to constitutively express a NY-ESO-1-specific TCR. Transduced T cells were subsequently co-cultured with A375 target cells, and the effects of these IL-12 variants on T cell-mediated cytolysis of A375 tumor cells were compared (see Materials and Methods above). All TCR-expressing T cells underwent enhanced tumor cell lysis in the presence of IL-12 (representative variants and wild-type single-stranded IL-12, Figure 13A-C). Analysis of the final repeated stimulation showed that T cells expressing the DS-furin variant, DS-furin-I52C-A179C variant, DS-furin-I52C-A179C-R181A variant, or wild-type scIL-12 all performed significantly better than T cells expressing TCR alone (p<0.05, Figure 13D), but the p40-C177S-R181A-p35-C74S variant showed a tendency toward significant difference (p=0.07). At the same time, no significant difference in cytotoxicity was observed between wild-type scIL-12 and any IL-12 variant.

[0112] These results demonstrate that cell activation-induced expression of IL-12 stable variants exhibits cytotoxic effects on proximal cells similar to those of wild-type IL-12. [Examples]

[0113] Effect of IL-12 stability variants on T cell-mediated efficacy in vivo This example describes the functional testing of an IL-12 stability variant in vivo. In vivo T cell efficacy and systemic IL-12 exposure were evaluated using mice with A375 tumors that were intravenously treated with engineered T cells.

[0114] A. Methods and Materials T cell production was carried out as described in Example 4. After production, IL-12-expressing T cells were subjected to magnetic selection of LNGFR according to the manufacturer's (StemCell) instructions before freezing. A alicot of cells from each condition was then thawed and evaluated for cytotoxicity (IncuCyte®), phenotype (flow cytometry), and viability (AOPI) to confirm functionality before in vivo testing. Functional evaluation of T cell efficacy in vivo was then performed using an A375 xenograft model with NSG-MHC I / II DKO mice (Jackson Labs).

[0115] A375 cell production was carried out in RPMI1640 medium supplemented with 10% FBS. Cells were passaged / expanded every 2-3 days and harvested when the required number of cells was reached. Cells were first washed with PBS and then treated with TrypLE at 37°C for 5 minutes. The detached cells were then washed with RPMI1640, counted, and resuspended in HBSS at an appropriate concentration. 1 × 10⁶ cells were resuspended in 100 μL. 6 Either 10⁴ or 2.5 × 10⁶ cells were then mixed 1:1 with Matrigel® before subcutaneous injection into the flank of the mouse. The tumors were then expanded to the desired volume (average 100 mm²). 3Measurements were taken three times a week until the target was reached. Mice were then randomized to various treatment conditions and simultaneously transduced with various IL-12 variants (1 × 10⁶). 6 pc or 2.5 × 10 6 One of several antigen-specific TCR-expressing T cells was injected.

[0116] Readout of T cell and IL-12 efficacy included tumor volume, T cell persistence by blood PK, and measurement of plasma cytokine levels of IL-12 and IFN-γ. Tumor measurements were performed twice weekly by caliper, and blood was collected for measurement of blood PK and plasma cytokine levels on days 1, 7, 14, 21, 28, and 42 after T cell infusion. 30 μL of blood was diluted 1:5 in PBS + 0.1% BSA solution, then centrifuged at 2000 × g for 15 minutes at 4°C to separate plasma from erythrocytes. Plasma was collected from the supernatant and used in an MSD assay performed at a 1:10 dilution according to the manufacturer's instructions. 50 μL of blood was then treated three times with ACK lysis buffer, washing with cell staining buffer (BioLegend) between treatments to reduce erythrocyte contamination. The remaining cells were then blocked with 20% normal mouse serum in cell staining buffer, stained with antibodies against constitutively expressed TCR, human CD3, human CD4, human CD8, human CD271 (LNGFR), and mouse CD45 (excluding mouse immune cells), and subsequently analyzed by flow cytometry.

[0117] B. Results and Discussion TCR T cells producing + / - IL-12 (and IL-12 variants) were injected into NSG-MHC I / II DKO mice carrying A375 xenografts as described in the Materials and Methods section above. Tumor volume was measured twice weekly after injection to assess the efficacy of the treatment. Mice treated with TCR T cells expressing the p40-C177S-p35-C74S variant (i.e., DS-furin or stability-reduced variant) demonstrated a deeper antitumor response and improved survival compared to mice treated with T cells expressing wt scIL-12 or TCR alone (Figure 14A-C). In one study (Figure 14A), proliferation measured in IL-12-treated mice between days 10 and 30 was determined not to be true tumor progression due to significant T cell expansion at the tumor site. The data show that the DS-furin variant enhanced T cell cytotoxicity in vitro (Example 4) and similarly demonstrated potent and comparable antitumor effects in vivo. [Examples]

[0118] Effects of IL-12 on T cell proliferation and cytokine secretion in vivo; safety evaluation. This example describes further testing of the biological activity of IL-12 stability variants in vivo.

[0119] In this study, blood was collected from mice weekly on days 1, 7, 14, 21, 28, and 42 after infusion with NY-ESO-1 TCR-T cells ("TCR-T cells"), and analyzed by flow cytometry of blood PKs and cytokine levels by MSD as described above. Promoting TCR-T cell proliferation was observed in mice treated with TCR-T cells expressing IL-12 (wild-type or stable variant) compared with mice treated with TCR-T cells alone (Figure 15A). The proliferation profile of TCR-T cells expressing wt scIL12 was similar to that of TCR-T cells expressing the p40-C177S-p35-C74S ("Furin-DS") variant (Figures 15A and 15C). Mice treated with TCR-T cells expressing IL-12 (wt or the "Furin-DS mutant" stability-reduced variant) also showed enhanced levels of IFN-γ secretion compared to TCR-T cells alone (Figure 15B). Animals treated with TCR-T cells expressing the p40-C177S-p35-C74S mutant ("Furin-DS mutant") (Figures 15B and 15D) showed similar or higher levels of IFN-γ.

[0120] Furthermore, to investigate whether the expressed IL-12 stability variant showed an improvement in the safety profile, we evaluated the levels of IL-12p70 (active, dimeric form) and IL-12p40 (subunit of active single-chain IL-12 or inactive free subunit after cleavage of the p40-C177S-p35-C74S mutant). IL-12p40 levels were readily detected in the plasma of mice treated with TCR-T cells expressing IL-12 (wt or stability variant, Figure 15E). However, IL-12p70 levels were detected only in mice treated with TCR-T cells expressing wt scIL12 (Figure 15F), empirically demonstrating the autoinactivating function of the p40-C177S-p35-C74S mutant. This trend was reproduced in the second mouse test, confirming (not demonstrating) the reproducibility of the self-inactivation function of the p40-C177S-p35-C74S stable mutant.

[0121] To further demonstrate the safety benefits of the IL-12 stability variants of this disclosure, the worst-case scenario for the equilibrium concentration of circulating active p70 was calculated for the human DS variant using the measured circulating p40 concentration and the p35 / p40 dissociation rate measured from Octet® in an exemplary donor, resulting in a dissociation constant (KD) of approximately 60 nM (6E-08M) (Figure 9). As shown in Figure 12, the mean p70 measured at day 42 for WT IL-12 was 5951 pg / mL, and the maximum p70 measured for WT IL-12 was 26,562 pg / mL. The mean p70 calculated at day 42 for the human DS variant was 0.03 pg / mL, and the maximum p70 calculated for the human DS variant was 0.17 pg / mL. These calculated maximum levels of circulating p70 were well below the levels considered dangerous in vivo.

[0122] These results demonstrate that only wt scIL-12 was detectable in the active state of peripheral blood, while the DS-furin variant was not, thus demonstrating the local activity of the variant that may improve the safety profile.

[0123] In conclusion, the results of the above examples demonstrate that IL-12 variants can deliver potent IL-12 stimulation to tumor sites while avoiding systemic exposure, thereby improving the efficacy of T-cell therapy while maintaining a favorable safety profile that ultimately enables effective administration of IL-12. [Examples]

[0124] Design of a mouse surrogate IL-12 stability variant with dissociation-like dynamics. A. Methods and Materials For characterization and the Octet® method, see Examples 1 and 2, respectively.

[0125] B. Results and Discussion As shown in Figure 5, mouse IL-12 stable variants (variants that signal through the mouse IL-12 receptor) respond to p35 / p40 interface mutations differently from human IL-12 stable variants (variants that signal through the human IL-12 receptor). Mouse p70 formation is particularly eliminated when the p35-p40 subunit disulfide is mutated (see plot on the right, see asterisk). Human conversion mutations S177R and T178I from certain mice appear to increase mouse p70 formation (see plot on the right, see bar on the right) (see also Figure 7).

[0126] Alignment of mouse (top) and human (bottom) sequences of the p35 subunit of WT IL-12 and scIL-12 containing the WT sequence suggests that several positions are important for either IL-12Rb2 or p35 / p40 interface dimerization. As shown in Figure 6, the tyrosine (Y) residues in alignment columns 2 and 4 are IL-12Rb2 interaction residues conserved in both humans and mice. On the other hand, the boxed positions in alignment column 3 are IL-12Rb2 interaction residues that are not conserved in both humans and mice. In alignment column 4, the double-boxed positions (ST in mouse and RI in human) are important p35 / p40 interface residues that are not conserved. On the other hand, the boxed valine (V) and arginine (R) residues in alignment column 4 are important p35 / p40 interface residues that are conserved.

[0127] Figure 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 the S177R mutation to mouse DS slows down dissociation. The Octet® data reflecting the activity of the IL12 stable variant that can signal through human IL-12R (the “human variant”) is shown in the upper row, while the Octet® data reflecting the activity of the IL12 stable variant that can signal through mouse IL-12R (the “mouse variant”) is shown in the lower row. The quantitative analysis of the data shown in Figure 8 is presented in Figures 9 and 10, demonstrating that mouse DS+S177R has a p35 / p40 dissociation rate comparable to human DS, providing a tool variant for in vivo testing of the same strain. [Examples]

[0128] Mouse genetic testing This example describes the functional testing of a mouse IL-12 (mIL-12) stability variant in vivo.

[0129] B16F10 cells (ATCC) were cultured in DMEM + 10% FBS + 1% Pen / Strep. B16F10 cells were seeded in 6-well dishes (XM cells / well) and transduced with lentiviruses expressing either sc-mIL-12 under the control of the constitutive UBC promoter, sc-mIL-12-p40-C175S-furin-p35-C70S-S177R (mutations at the positions of SEQ ID NO: 10 in mouse p40 and SEQ ID NO: 11 in mouse p35) under the control of the constitutive mPGK promoter, or sc-mIL-12-p35-C70S under the control of the constitutive UBC promoter to generate B16F10 cells capable of secreting mRNA. The cell lines were then subjected to puromycin selection (2 μg / mL) for 48 hours, and maintained in 1 μg / mL puromycin for 2 weeks, with cell passage as necessary, to generate a pure mIL-12 expressing cell line.

[0130] B16F10 cells expressing mIL-12 were then removed from puromycin selection and passaged for two weeks to demonstrate stable mIL-12 expression in the absence of selection. At each passage (approximately every three days), 50,000 cells were plated in 1 mL of medium in a 24-well plate. After 24 hours, the supernatant was collected, and IL-12 p40 levels were analyzed by MSD to determine the level of cytokine production (Figure 16). IL-12 secretion remained stable in culture for at least 10 days.

[0131] Fresh alicots of B16F10 cells expressing mIL-12 were then thawed and expanded. WT B16F10 cells expressing sc-mIL-12 or sc-mIL-12-p40-C175S-furin-p35-C70S-S177R were then mixed with parental B16F10 cells at varying frequencies (10%, 50%, and 100% IL-12 expressing cells). 1 × 10 6 These B16F10 cell mixtures were transplanted into C57BL / 6 mice and expanded in vivo.

[0132] Mice transplanted with parental B16F10 tumors showed rapid tumor growth, and all mice were removed from the study within 15 days. Mice transplanted with B16F10 cells expressing more than 10% IL-12 did not show tumor growth. Mice transplanted with tumors in which 10% of B16F10 cells expressed either WT sc-mIL-12 or sc-mIL-12-p40-C175S-furin-p35-C70S-S177R showed a significant delay in tumor growth with tumors expressing sc-mIL-12-p40-C175S-furin-p35-C70S-S177R, exhibiting a lower mean tumor volume (Figure 17A) and a reduced frequency of tumor growth (Figure 17B). Of the 10 mice transplanted with B16F10 expressing sc-mIL-12-p40-C175S-furin-p35-C70S-S177R, 4 remained tumor-free. However, of the 10 mice transplanted with B16F10 expressing WT sc-mIL-12, 2 remained tumor-free.

[0133] Furthermore, compared to mice expressing sc-mIL-12-p40-C175S-furin-p35-C70S-S177R, the inventors detected high levels of mIL-12p40, mIL-12-70, and IFN-γ in the plasma of B16F10 mice transplanted with wt sc-mIL-12, indicating that sc-mIL-12-p40-C175S-furin-p35-C70S-S177R has improved safety compared to wild-type IL-12 (Figures 18A-18C). Importantly, the inventors did not detect mIL-12p70 in the plasma of tumor-bearing mice expressing sc-mIL-12-p40-C175S-furin-p35-C70S-S177R.

[0134] Combined, these data demonstrated similar levels of efficacy between sc-mIL-12 and our stable mutant at the local level, but also showed the ability of our stable mutant to reduce systemic IL-12 activity.

[0135] array The following table provides a list of sequences disclosed herein (SEQ: sequence number).

[0136] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

Claims

1. It is an IL-12 variant, A p40 subunit sequence having an amino acid sequence that is at least 90% identical to Sequence ID No. 1, and It includes a p35 subunit sequence having an amino acid sequence that is at least 90% identical to Sequence ID No. 2, It lacks a natural disulfide bond between the p40 subunit sequence and the p35 subunit sequence. IL-12 variant.

2. The p40 subunit sequence contains a mutation that removes a cysteine ​​residue at the position corresponding to C177 in SEQ ID NO: 1, and in some cases the mutation is a C-to-S mutation; and / or The p35 subunit sequence contains a mutation that removes a cysteine ​​residue at the position corresponding to C74 in SEQ ID NO: 2, and in some cases, the mutation is a C-to-S mutation. The IL-12 variant according to claim 1.

3. The p35 subunit arrangement is, A mutation at the position corresponding to R181 in SEQ ID NO: 2, and in some cases, a mutation from R to A, or a mutation at the position corresponding to R183 in SEQ ID NO:

2. A mutation at the position corresponding to V185 in Sequence ID No. 2, and, in some cases, a mutation from V to A, A mutation at the position corresponding to R189 in sequence number 2, and in some cases, a mutation from R to K. An IL-12 variant according to claim 1 or 2, comprising one or more of the following.

4. The p40 subunit sequence includes a substitution of C at the position corresponding to A179 in sequence number 1. The p35 subunit sequence includes a substitution of C at the position corresponding to I52 in sequence number 2. The IL-12 variant according to claim 2 or 3.

5. The p40 subunit sequence includes sequence number 12, The p35 subunit sequence includes sequence number 13. The IL-12 variant according to claim 1.

6. The p40 subunit sequence includes sequence number 12, The p35 subunit sequence contains an amino acid sequence identical to that of sequence number 13, except for the R181A mutation. The IL-12 variant according to claim 1.

7. The p40 subunit sequence contains an amino acid sequence identical to that of sequence number 12, except for the A179C mutation. The p35 subunit sequence contains an amino acid sequence identical to sequence number 13, except for the I52C mutation. The IL-12 variant according to claim 1.

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

9. The p40 subunit sequence includes sequence number 10, The p35 subunit sequence contains an amino acid sequence that is identical to sequence number 11 except for a mutation at the C70 position, and in some cases, a C70S mutation. The IL-12 variant according to claim 1.

10. The p40 subunit sequence includes sequence number 10, The p35 subunit sequence contains an amino acid sequence identical to that of SEQ ID NO: 11, except for a mutation at position C70, possibly a C70S mutation, and possibly a mutation at position S177, possibly a S177R mutation. The IL-12 variant according to claim 1.

11. The p40 subunit sequence includes sequence number 10, The p35 subunit sequence contains an amino acid sequence that is identical to sequence number 11 except for a mutation at position N184, and in some cases the N184A mutation. The IL-12 variant according to claim 1.

12. An IL-12 variant according to any one of claims 1 to 11, wherein the p40 subunit sequence and the p35 subunit sequence are linked by a cleavable peptide linker.

13. The IL-12 variant according to claim 12, wherein the cleavable peptide linker includes a Hurin cleavage site, and optionally the cleavable linker includes SEQ ID NO:

5.

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

6.

15. An isolated nucleic acid molecule encoding an IL-12 variant according to any one of claims 1 to 14, wherein the nucleic acid molecule is mRNA or DNA, in some cases.

16. An expression vector comprising an isolated nucleic acid molecule as described in claim 15.

17. An expression vector according to claim 16, selected from a lentiviral vector, an adenovirus vector, and an adeno-associated virus (AAV) vector.

18. The expression vector according to claim 16 or 17, further comprising a tissue-specific or inducible promoter.

19. The expression vector according to claim 18, wherein the inducible promoter comprises a combination of an activated T cell nuclear factor (NFAT) domain and a human beta-globin (hBG) minimal promoter domain, and optionally the inducible promoter comprises Sequence ID No.

14.

20. A mammalian cell comprising the nucleic acid molecule described in claim 15 or the expression vector described in any one of claims 16 to 19.

21. The mammalian cell according to claim 20, which is a human immune cell.

22. The mammalian cells according to claim 21, wherein the human immune cells are T cells, NK cells, or TILs, which are optionally engineered to express a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), or a chimeric costimulatory receptor (CCR), and which optionally target tumor antigens.

23. A pharmaceutical composition comprising an IL-12 variant, a nucleic acid molecule, an expression vector, or a mammalian cell, and a pharmaceutically acceptable carrier, as described in any one of claims 1 to 22.

24. A method for stimulating the immune system or treating cancer in a human subject requiring such treatment, comprising the step of administering the pharmaceutical composition according to claim 23 to a human subject.

25. Use of an IL-12 variant, nucleic acid molecule, expression vector, or mammalian cell according to any one of claims 1 to 22 for the manufacture of a pharmaceutical product that stimulates the immune system or treats cancer in a human subject that requires it.

26. An IL-12 variant, nucleic acid molecule, expression vector, mammalian cell, or pharmaceutical composition according to any one of claims 1 to 23, for use in a process of stimulating the immune system or treating cancer in a human subject that requires it.

27. A step of culturing mammalian cells according to any one of claims 20 to 22 under conditions that enable the expression of an IL-12 variant; and Steps to isolate IL-12 variants from cultures A method for producing an IL-12 variant, including [the specified substance].

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