WO2023196897A1 - Il-12 prodrugs - Google Patents

Il-12 prodrugs Download PDF

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Publication number
WO2023196897A1
WO2023196897A1 PCT/US2023/065429 US2023065429W WO2023196897A1 WO 2023196897 A1 WO2023196897 A1 WO 2023196897A1 US 2023065429 W US2023065429 W US 2023065429W WO 2023196897 A1 WO2023196897 A1 WO 2023196897A1
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Prior art keywords
compound
inducible
prodrug
tumor
fold
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PCT/US2023/065429
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French (fr)
Inventor
William Winston
Cynthia Seidel-Dugan
Daniel Hicklin
Heather BRODKIN
Jose Andres SALMERON-GARCIA
Philipp Steiner
Christopher NIRSCHL
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Werewolf Therapeutics Inc.
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Publication of WO2023196897A1 publication Critical patent/WO2023196897A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • Cancer immunotherapy has rapidly established itself as the fourth pillar of cancer treatment largely owing to the clinical success of checkpoint inhibitors. Despite the durable responses achieved by some patients using these new therapies, the proportion of responders is still relatively low and restricted to only some cancer types. Tumor mutational burden, the presence or absence of T cell infiltration in tumors, and the overall immunosuppressive microenvironment of tumors greatly influences the response to immunotherapies.
  • immune checkpoint blockade can prevent the physiological stop-signal that arises in response to immune activation
  • other approaches can be used to positively stimulate the anti-tumor immune response.
  • One approach involves the use of immune-activating cytokines. Numerous preclinical and clinical studies have demonstrated the promise of cytokine therapy to increase anti-tumor immunity. In fact, these were some of the first cancer immunotherapies approved for clinical use. However, systemic toxicity and poor pharmacokinetic profiles have limited their clinical application.
  • Interleukin-12 is a heterodimeric 70 kDa cytokine composed of two covalently linked glycosylated subunits (p35 and p40) (Lieschke et al., 1997; Jana et al., 2014). It is a potent immune agonist and has been considered a promising therapeutic agent for oncology. However, IL- 12 has shown to have a narrow therapeutic window because they are highly potent and have a short serum half-life. Consequently, therapeutic administration of IL-12 produces undesirable systemic effects and toxicities.
  • cytokines i.e., IL-12
  • IL-12 cytokine-like growth factor-12
  • cytokine action e.g., a tumor microenvironment
  • cytokines due to the biology of cytokine and the inability to effectively target and control their activity, cytokines have not achieved the hoped for clinical advantages in the treatment in tumors.
  • IL-12 prodrugs include a native IL- 12 molecule attached through a protease cleavable linker to a half-life extension domain (e g., anti-human serum albumin antibody binding fragment such as a VH domain) and an IL-12 blocking element (e.g., anti-IL-12 antibody binding fragment, such as a Fab or scFv) to block binding of IL-12 to IL-12Rpi or IL-12RP2 receptors on normal tissue in the periphery.
  • a protease cleavable linker to block binding of IL-12 to IL-12Rpi or IL-12RP2 receptors on normal tissue in the periphery.
  • This disclosure relates to compositions and methods for treating cancer using an inducible IL-12 prodrug.
  • the inducible IL-12 prodrugs contain an attenuated IL-12 and have a long half-life in comparison to naturally occurring IL-12.
  • the IL-12 can be a mutein.
  • the IL-12 mutein can be aglycosylated or partially aglycosylated.
  • the inducible IL-12 prodrugs disclosed herein comprise two or more polypeptide chains, and the inducible IL-12 prodrug includes IL-12 subunits p35 and p40, a half-life extension element, an IL-12 blocking element and a protease cleavable linker.
  • the inducible IL- 12 prodrug can comprise two different polypeptides.
  • the first polypeptide can comprise an IL-12 subunit, and optionally an IL-12 blocking element.
  • the IL-12 blocking element when present is operably linked to the IL-12 subunit through a first protease cleavable linker.
  • the second polypeptide chain can comprise an IL-12 subunit operably linked to a half-life extension element through a second protease cleavable linker, and optionally a IL- 12 blocking element.
  • the IL-12 blocking element when present can be operably linked to the IL-12 subunit through a protease cleavable linker or can be operably linked to the half-life extension element through a linker that is optionally protease cleavable. Only one of the first and second polypeptide contains the IL- 12 blocking element. When the IL- 12 subunit in the first polypeptide is p35, the IL-12 subunit in the second polypeptide is p40, and when the IL-12 subunit in the first polypeptide is p40, the IL- 12 subunit in the second polypeptide is p35.
  • a preferred blocking element of this inducible IL-12 prodrug is a single chain antibody that binds IL-12 or an antigen binding fragment thereof.
  • the cleavable linkers in this inducible IL- 12 prodrug can be the same or different.
  • the inducible IL- 12 prodrug can comprise three different polypeptides.
  • one polypeptide chain comprises either the p35 or p40 IL- 12 subunit, but not both, and a second polypeptide comprises the other IL-12 subunit and the third polypeptide comprises at least a portion (component) of the blocking element.
  • the first polypeptide can comprise an IL- 12 subunit, and optionally a half-life extension element.
  • the half-life extension element when present is operably linked to the IL- 12 subunit through a protease cleavable linker.
  • the second polypeptide can comprise a IL-12 subunit, at least an antigen binding portion of an antibody light chain or an antigen binding portion of an antibody heavy chain, and optionally a half-life extension element.
  • the half-life extension element is operably linked to the IL- 12 subunit through a protease cleavable linker and the antibody heavy chain or light chain is either a) operably linked to the IL- 12 subunit through a second protease cleavable linker, or b) operably linked to the half-life extension element through an optionally cleavable linker.
  • the third polypeptide can comprise can an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the second polypeptide, or an antibody light chain that is complementary to the heavy chain in the second polypeptide and together with said light chain forms an IL-12 binding site.
  • the IL-12 subunit in the first polypeptide is p35
  • the IL-12 subunit in the second polypeptide is p40
  • the IL-12 subunit in the first polypeptide is p40
  • the IL-12 subunit in the second polypeptide is p35.
  • the IL-12 blocking element is preferably an antigen binding fragment of an antibody.
  • the antigen binding fragment comprises as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain.
  • the protease cleavable linkers in this inducible IL- 12 prodrug can be the same or different.
  • the inducible IL- 12 prodrug can comprise two different polypeptides wherein p35 and p40 are located on the same polypeptide chain.
  • a first polypeptide chain can comprise p35, p40, a half-life extension element and at least an antigen binding portion of an antibody light chain.
  • p35 and p40 can be operably linked, and the half-life extension element can be operably linked to p40 through a first protease cleavable linker and the antigen binding portion of an antibody light chain can be operably linked to p35 through a protease cleavable linker.
  • the half-life extension element can be operably linked to p35 through a protease cleavable linker and the antigen binding portion of an antibody light chain is operably linked to p40 through a protease cleavable linker.
  • the second polypeptide comprises at least an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the second polypeptide and together with said light chain forms and IL-12 binding site.
  • the protease cleavable linkers in this inducible IL-12 prodrug can be the same or different.
  • a first polypeptide chain can comprise p35, p40, a half-life extension element and at least an antigen binding portion of an antibody heavy chain.
  • p35 and p40 can be operably linked, and the half-life extension element can be operably linked to p40 or through a protease cleavable linker and the antigen binding portion of an antibody heavy chain can be operably linked to p35 through a protease cleavable linker.
  • the half-life extension element can be operably linked to p35 through a protease cleavable linker and the antigen binding portion of an antibody heavy chain can be operably linked to p40 through a second protease cleavable linker.
  • a second polypeptide comprises at least an antigen binding portion of an antibody light chain that is complementary to the heavy chain in the second polypeptide and together with said light chain forms and IL- 12 binding site.
  • the protease cleavable linkers in this inducible IL- 12 prodrug can be the same or different.
  • the inducible IL-12 prodrug comprises a first polypeptide that does not comprise a blocking element and the second polypeptide that has the formula: [A]-[L 1 ]-[B]- [L3]-[D] or [D]-[L3]-[B]-[L1]-[A] or [B]-[L1]-[A]-[L2]-[D] or [D]-[L1]-[A]-[L2]-[B], wherein, A is an IL-12 subunit; LI is a first protease-cleavable linker; L2 is a second protease cleavable linker; L3 is an optionally cleavable linker; B is a half-life extension element; and D is a blocking element.
  • the first polypeptide comprises the formula: [A]-[L1]-[D] or [D]- [L1]-[A]; and the second polypeptide has the formula: [A’]-[L2]-[B] or [B]-[L2]-[A’], wherein A and A’ independently are either p35 or p40, wherein when A is p35, A’ is p40 and when A is p40, A’ is p35; LI is a first protease cleavable linker; L2 is a second protease cleavable linker; B is a half-life extension element; and D is a blocking element.
  • This disclosure relates to a method for inducing immunological memory to a tumor.
  • the method comprises administering to a subject in need thereof and effective amount of an inducible IL-12 prodrug as described herein, wherein the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations.
  • This disclosure relates to a method for selectively activating effector CD8+ T cells in the tumor microenvironment, and/or to a method for selectively activating tumor infiltrating lymphocytes.
  • These methods comprise administering to a subject in need thereof and effective amount of an inducible IL- 12 prodrug as described herein, wherein the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results a significantly higher frequency of CD8+ T cells that produce TNF, IFNgamma and/or Granzyme B within the tumor.
  • the inducible IL-12 prodrug can be administered about twice a week or less frequently, once a week or less frequently or about once every two weeks or less frequently. In certain embodiments, the inducible IL- 12 prodrug can be administered about once every two weeks.
  • Preferred, inducible IL-12 prodrugs for use in the methods of this disclosure are Chimeric Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, or an amino acid sequence variant of any of the foregoing Chimeric Compound 1 comprises a first polypeptide chain of SEQ ID NO: 1 and a second polypeptide chain of SEQ ID NO: 7, and the amino acid sequence variant of Chimeric Compound 1 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO: 1 and a second polypeptide chain can comprise at least about 80% identity to SEQ ID NO:7.
  • Compound 2 comprises a first polypeptide chain of SEQ ID NO:2 and a second polypeptide chain of SEQ ID NO:7, and the amino acid sequence variant of Compound 2 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO:2 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO:7.
  • Compound 3 comprises a first polypeptide chain of SEQ ID NO:3 and a second polypeptide chain of SEQ ID NO:8, and the amino acid sequence variant of Compound 3 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO: 3 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO: 8.
  • Compound 4 comprises a first polypeptide chain of SEQ ID NON and a second polypeptide chain of SEQ ID NO:8, and the amino acid sequence variant of Compound 4 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NON and a second polypeptide chain that has at least about 80% identity to SEQ ID NO:8.
  • Compound 5 comprises a first polypeptide chain of SEQ ID NON and a second polypeptide chain of SEQ ID NO: 8, and the amino acid sequence variant of Compound 5 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO: 5 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO:8.
  • Compound 5 comprises a first polypeptide chain of SEQ ID NO:5 and a second polypeptide chain of SEQ ID NO:8, and the amino acid sequence variant of Compound 5 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO: 5 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO: 8.
  • Compound 6 comprises a first polypeptide chain of SEQ ID NO:6 and a second polypeptide chain of SEQ ID NO:8, and the amino acid sequence variant of Compound 6 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO: 6 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO:8.
  • FIG. 1 is a graph showing the in vitro activity of Chimeric Compound 1 in the IL- 12 HEK-Blue reporter assay, comparing intact Chimeric Compound 1 (squares) and cleaved Chimeric Compound 1 (triangles) to chimeric IL-12 (circles).
  • FIGs. 2A-2B show that Chimeric Compound 1 is well tolerated and induces tumor regression in a cleavage dependent manner.
  • FIG. 2A is a graph showing anti-tumor activity of Chimeric Compound 1 at various doses in the murine model. Chimeric Compound 1 was dosed intraperitonially twice a week for two weeks at 7 pg/dose and 43 pg/dose and a NC (Non- cleavable) version of Chimeric Compound 1 was dosed at 43 pg/dose.
  • FIG. 2B is a graphic depiction of the calculated therapeutic window for chimeric IL- 12 and Chimeric Compound 1 on a per molar basis using the same tumor model (MC38), based on the identification of the active and toxic dose level for both treatments.
  • FIGs. 3A-3G are graphs showing that Chimeric Compound 1 generates anti-tumor immunity and protective memory in multiple syngeneic tumor models.
  • FIGs. 3A-3E show antitumor activity of Chimeric Compound 1 at various doses in various murine syngeneic tumor models, CT26 model (FIG. 3A), B16-F10 model (FIG. 3B), EMT-6 model (FIG. 3C), A20 model (FIG. 3D), and EG7.OVA model (FIG. 3E). Mice were dosed twice a week with the dose noted on the figure legends for a total of two weeks.
  • FIGs. 3F and 3G are graphs showing tumor volume in the EMT6 (FIG. 3F) and MC38 (FIG. 3G) models re-challenged with the same tumor on the opposite flank over time, demonstrating that treatment with Chimeric Compound 1 induces immunological memory against the same tumor type.
  • FIGs. 4A-4F show that Chimeric Compound Itreatment reshapes the tumor microenvironment and induces activation of intratumoral effector cells (NKs and CD8+ T cells) in the MC38 model.
  • FIG. 4A is a heatmap of transcripts with statistically significant differences between the two treatments derived from nanostring analysis of bulk RNA from tumor samples. Transcripts were excluded from the heat map if they had average normalized counts below 50. Each lane represents an individual animal.
  • FIG. 4B shows a volcano plot of transcripts differentially expressed between Chimeric Compound 1 and vehicle treated mice.
  • FIGs. 4C-4D show the frequency of tumor infiltrating NK cells producing IFNy or Granzyme B.
  • FIGs. 5A-5F show that Chimeric Compound 1 treatment reshapes the tumor microenvironment and activates B16-F10 tumor infiltrating NK cells and CD8+ T cells.
  • FIG. 5A shows a heatmap of transcripts with statistically significant differences in expression between the two treatments derived from nanostring analysis of bulk RNA from tumor samples. Transcripts were excluded from the heatmap if they had average normalized counts below 50.
  • FIG. 5B shows a volcano plot of transcripts differentially expressed between Chimeric Compound 1 and vehicle-treated mice.
  • FIG. 5C shows graphs of pathway scores for vehicle and Chimeric Compound 1 for antigen processing, interferon, MHC, and NK Cell Functions.
  • FIG. 5D are graphs showing normalized counts from individual transcripts for vehicle and Chimeric Compound E
  • FIG. 5E is a flow cytometry diagram showing the frequency of tetramer+ CD8+ T cells producing IFN gamma and/or Granzyme B.
  • FIG. 5F are pie graphs showing the frequency of polyfunctional tetramer positive CD8+ T cells measured by examining co-expression of IFN gamma, TNF, and Granzyme B by flow cytometry.
  • FIG. 6 are pie graphs showing that Chimeric Compound Itreatment induces a sustained polyfunctional CD8+ T cells response.
  • Mice were implanted with EMT6 cells and randomized into treatment groups. Mice were dosed twice weekly for two weeks, and tumors were harvested at the indicated timepoints. The frequency of polyfunctional tumor infiltrating CD8+ T cells was measured by examining co-expression of IFN gamma, TNF, and Granzyme B. All animals in the vehicle group were out of the study by day 21 due to tumor burden.
  • FIG. 7A-7D shows that systemic administration of Chimeric Compound 1 results in CD8+ T cell infdtration and activation in the tumors assessed by immunofluorescence staining and the increase of IL- 12 and IFN gamma signaling by tumor infdtrating CD8+ T cells.
  • Mice were implanted with EMT6 cells and randomized into treatment groups. Mice were dosed twice weekly for two weeks, and tumors were harvested on Day 11. Nanostring GeoMX analysis was performed on FFPE tumor tissues.
  • FIG. 7A are immunofluorescence images of tumor infdtrating CD8+ T cells in vehicle and Chimeric Compound 1.
  • FIG. 7B is a graph showing differential gene expression analysis of tumor infdtrating CD8+ T cells.
  • FIGs. 7C-7D are heat maps showing genes associated with IL-12 (FIG. 7C) and IFN gamma (FIG. 7D) signaling.
  • FIGs. 8A-8B are graphs showing anti-tumor activity of Chimeric Compound 1 in various studies of the murine syngeneic MC38 tumor model.
  • FIG. 8A is a graph showing tumor growth over time in MC38 tumor bearing mice treated with Chimeric Compound 1 (+/-) daily FTY720 treatment.
  • FIG. 8B is a graph showing tumor growth over time in MC38 tumor bearing mice dosed twice a week with CD4, CD8, and NK cell depleting antibodies in conjunction with Chimeric Compound 1.
  • FIGs. 9A-9C show that Chimeric Compound 1 is preferentially activated within the TME and expands the therapeutic window compared to chimeric IL- 12.
  • FIGs. 9A-9B are graphs showing the presence of total Chimeric Compound 1 or free chimeric IL- 12 over time from plasma (FIG. 9A) or tumor (FIG. 9B) from MC38 tumor-bearing mice treated with Chimeric Compound 1. The area under the curve was calculated, and the ratio of total Chimeric Compound 1 to free chimeric IL-12 was calculated.
  • FIG. 9A-9A are graphs showing the presence of total Chimeric Compound 1 or free chimeric IL- 12 over time from plasma (FIG. 9A) or tumor (FIG. 9B) from MC38 tumor-bearing mice treated with Chimeric Compound 1. The area under the curve was calculated, and the ratio of total Chimeric Compound 1 to free chimeric IL-12 was calculated.
  • FIG. 9A-9A are graphs showing the presence of total Chimeric Compound 1
  • FIGS. 9C are pie chart graphs showing the frequency of polyfunctional CD8+ T cells in the tumor, peripheral blood, tumor draining or nontumor draining lymph nodes in MC38 tumor bearing mice dosed twice with Chimeric Compound 1.
  • the frequency of polyfunctional CD8+ T cells was measured by examining co-expression of IFN gamma, TNF, and Granzyme B after PMA/ionomycin restimulation.
  • FIGs. 10A-10F show Chimeric Compound 1 activates tumor infdtrating immune cell populations in the MC38 syngeneic tumor model.
  • FIG. 10A are representative flow plots of CD1 lb+ and CD103+ tumor infdtrating dendritic cells.
  • FIG. 10B is a graph showing the ratio of CD1 lb+ and CD103+ tumor infdtrating dendritic cells.
  • FIG. 10C is a graph showing the frequency of CD4+ T conventional cells with a THI phenotype (Tbet+ IFN gamma + TFN+).
  • FIG. 10D are representative flow plots showing the frequency of tumor infiltrating FoxP3+ Tregs producing IFN gamma and TNF.
  • FIGs. 10A are representative flow plots of CD1 lb+ and CD103+ tumor infdtrating dendritic cells.
  • FIG. 10B is a graph showing the ratio of CD1 lb+ and CD103+ tumor infdtrating
  • 10E-10F are graphs depicting the frequency of tumor infiltrating FoxP3+ Tregs producing IFN gamma and TNF (FIG. 10E) and Tbet (FIG. 10F). Unless otherwise stated, data are represented as the mean + SD, and P values are derived from t tests (**, p ⁇ 0.01; ***, p ⁇ 0.001; ****, p ⁇ 0.00001).
  • FIGs. 11A-11E show that systemic treatment with Chimeric Compound 1 expands novel TCR clones and increases overall clonality of the TCR repertoire.
  • FIG. 11A is a heat map depicting intratumoral CD8+ T cells downstream TCR signaling following vehicle and Chimeric Compound 1 treatment.
  • FIGs. 11B-11C are graphs depicting the clone frequency of individual VDJ recombination on the TCR-beta chain in an EMT-6 tumor model treated with vehicle and Chimeric Compound 1. Live T cells were isolated from EMT-6 tumors on Day 11 and TCR sequencing was performed.
  • FIG. 11D is graph depicting the clonality index score for vehicle and Chimeric Compound 1.
  • FIG. 1 IE is a graph depicting the frequency of the top fifty TCR clones plotted for each animal.
  • FIGs. 12A-12V show that Chimeric Compound 1 treatment drives increased mitochondrial respiration and fitness.
  • FIG. 12A is a heatmap of tumor infiltrating CD8+ T cells depicting genes associated with glycolysis.
  • FIGs. 12B-12C are graphs showing intake of 2- NDBG in EMT-6 TILs from either vehicle or Chimeric Compound 1 treated animals.
  • FIGs. 12D-12F are heatmaps of tumor infiltrating CD8+ T cells depicting genes associated with the TCA cycle (FIG. 12D), mitochondrial biogenesis (FIG. 12E), and mitochondrial translation (FIG. 12F).
  • FIGs. 12D shows that Chimeric Compound 1 treatment drives increased mitochondrial respiration and fitness.
  • FIG. 12A is a heatmap of tumor infiltrating CD8+ T cells depicting genes associated with glycolysis.
  • FIGs. 12B-12C are graphs showing intake of 2- NDBG in EMT-6 TILs from either vehicle or Chimeric Compound 1 treated animals.
  • FIGs. 12G, 12H, 12K, 12L, 120, 12P are graphs depicting EMT-6 infiltrating CD8+ T cells from either vehicle or Chimeric Compound 1 treated animals stained with mitotracker red (FIGs. 12G and 12H), TMRM (FIGs. 12K, 12L), MitoSOX (FIGs. 12O-12P).
  • FIGs. 121, 12 J, 12M, 12N, 12Q, 12R are graphs depicting EMT-6 infiltrating NK cells from either vehicle or Chimeric Compound 1 treated animals stained with mitotracker red (FIGs. 121 and 12J), TMRM (FIGs. 12M-12N), MitoSOX (FIGs. 12Q-12R).
  • FIGs. 12S-12T show that Chimeric Compound 1 preferentially expands new clones rather than previously present clones.
  • FIGs 12S and 12T disclose SEQ ID NOS 450-457 and 458-465, respectively, from left to right, which are sequences from TCRs and define T cell clones.
  • FIGs. 17A-17B are graphs showing the percentage of TCR repertoire for the top 50 shared clones.
  • FIGs. 12U-12V shows that Chimeric Compound 1 treatment increases mitochondrial mass and fitness in tumor infiltrating immune cells.
  • FIGs. 13A-13B show that Compound 36 is inducible, stable in human serum, and selectively processed by dissociated primary human tumor samples.
  • FIGs. 14A-14B are graphs showing IFN gamma production by intracellular cytokine staining with or without ex vivo restimulation in TILs from mice treated with either vehicle or Chimeric Compound 1.
  • FIGs. 15A-15B show selective activation of tumor infdtrating immune cells by Chimeric Compound 1.
  • FIG. 15A is a graph showing the frequency of conventional CD4+ T conventional cells (FoxP3-) producing IFNy and TNF in the tumor tissue compared to peripheral tissue.
  • FIG. 15B is a graph showing the frequency of NK cells producing IFNv and TNF in the tumor tissue compared to peripheral tissue.
  • FIGs. 16A, 16C, 16E, 16G, 161, 16K, 16M, 160, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, 16ZO are graphs showing the activity of IL- 12 prodrugs in a HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA).
  • Squares depict activity of the intact inducible IL- 12 prodrug and triangles depict the activity of the in vitro protease activated (cleaved) inducible IL-12 prodrug.
  • Circles depict activity of the control chimeric IL-12.
  • FIGs. 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays with elastase.
  • FTGs. 17A, 17C, 17E, 17G, and 171 are graphs showing results of analyzing inducible IL- 12 prodrugs in a syngeneic MC38 mouse tumor model. They show average tumor volume over time in mice treated with 5 pg, 50 pg, and 500 pg of each inducible IL-12 prodrug dosed biweekly. Data show the tumor volume was inhibited over time in a dose-dependent manner.
  • FIGs. 17B, 17D, 17F, 17H, and 17J are graphs showing body weight average of the groups over time.
  • FIGs. 18A-18N are schematic illustrations depicting various inducible IL-12 prodrugs.
  • the disclosure relates to inducible IL-12 polypeptide prodrugs that contain an attenuated IL- 12 and that have a long half-life in comparison to naturally occurring IL- 12.
  • the IL- 12 polypeptide prodrugs disclosed herein contain at least one polypeptide chain, and can contain two or more polypeptides, if desired.
  • the two or more polypeptide chains disclosed herein are different, i.e., the complexes can be heterodimers, heterotrimers, and the like.
  • the inducible IL- 12 prodrugs comprises a p35 IL- 12 subunit, a p40 IL- 12 subunit, a half-life extension element, an IL- 12 blocking element, and a protease cleavable linker.
  • the p35 subunit and the p40 subunit associate to form the TL-12 heterodimer, which has intrinsic IL-12 receptor agonist activity.
  • the IL-12 receptor agonist activity is attenuated and the circulating half-life is extended.
  • the IL-12 receptor agonist activity is attenuated through the blocking element.
  • the half-life extension element can also contribute to attenuation, for example through steric effects.
  • the blocking element is capable of blocking the activity of all or some of the receptor agonist activity of IL-12 by sterically blocking and/or noncovalently binding to IL- 12 (e.g., to p35, p40, or the p35p40 complex).
  • IL- 12 e.g., to p35, p40, or the p35p40 complex.
  • a form of IL-12 is released from the inducible IL-12 prodrug that is active (e.g., more active than the inducible IL-12 prodrugs).
  • the released IL-12 is at least 10 x more active than the inducible IL-12 prodrug.
  • the released IL-12 is at least 20 x, at least 30 x, at least 50 x, at least 100 x, at least 200 x, at least 300 x, at least 500 x, at least 1000 x, at least about 10,000X or more active than the inducible IL- 12 prodrug.
  • the form of IL- 12 that is released upon cleavage of the inducible IL- 12 prodrug typically has a short half-life, which is often substantially similar to the half-life of naturally occurring IL- 12. Even though the half-life of the inducible IL-12 prodrug is extended, toxicity is reduced or eliminated because the circulating inducible TL-12 prodrug is attenuated and active TL-12 is targeted to the desired site (e.g., tumor microenvironment).
  • the inducible IL-12 prodrug comprises two different polypeptide chains.
  • the first polypeptide chain comprises p35 and the second polypeptide chain comprises p40.
  • the p35 and p40 subunits associate to form a biologically active heterodimer.
  • the p35p40 heterodimer complex can be covalently linked, for example through a disulfide bond.
  • either the first of the second polypeptide can comprise an IL- 12 blocking element (e.g., an scFV that binds IL-12) that is operably linked to the IL-12 subunit through a protease cleavable linker.
  • the other polypeptide chain can further comprise a half-life extension element that is operably linked to the IL-12 subunit through a protease cleavable linker.
  • the inducible IL- 12 prodrug includes one functional blocking element and one functional half-life extension element.
  • the first polypeptide chain comprises an IL-12 blocking element
  • the second polypeptide chain does not comprise an IL-12 blocking element.
  • one polypeptide chain includes either p35 or p40, and further includes a half-life extension element and a blocking element, each of which is operably linked to the p35 or p40 through a protease cleavable linker (e.g., one or more protease cleavable linker), and the other polypeptide include the complementary IL- 12 subunit (e.g., either p40 or p35).
  • the IL-12 blocking element on the second polypeptide can be operably linked to the IL-12 subunit through a protease cleavable linker.
  • the IL- 12 blocking element can be operably linked to the half-life extension element through an optional protease cleavable linker.
  • the protease cleavable linkers on the first and second polypeptide chains can be the same or can be different.
  • the protease cleavable linkers on the first and second polypeptide chains are the same.
  • the blocking element in this inducible IL-12 prodrug can be a single chain antibody. Any single chain antibody that has binding specificity for IL-12 can be a blocking element.
  • the blocking element is a scFv.
  • the inducible TL-12 prodrugs disclosed herein preferably contain one half-life extension element and one blocking element, such elements can contain two or more components that are present on the same polypeptide chain or on different polypeptide chains.
  • components of the blocking element can present on separate polypeptide chains.
  • a first polypeptide chain can include an antibody light chain (VL+CL) or light chain variable domain (VL) and a second polypeptide can include an antibody heavy chain Fab fragment (VH + CHI) or heavy chain variable domain (VH) that is complementary to the VL+ CL or VL on the first polypeptide.
  • these components can associate in the inducible IL- 12 prodrugs to form an antigen-binding site, such as a Fab that binds IL- 12 and attenuates IL- 12 activity.
  • the p35 and p40 subunit can be located on the same polypeptide chain, and linked through and optionally protease cleavable linker.
  • at least one of the half-life extension element, the blocking element, or a component of the half-life extension or blocking element is on a separate polypeptide.
  • a first polypeptide can include p35 and p40, linked through an optionally cleavable polypeptide chain, and other elements of the inducible IL- 12 prodrug are located on a second polypeptide chain.
  • the first polypeptide chain comprises the p35 subunit, the p40 subunit, the half-life extension element, and a portion of an antibody light chain.
  • the second polypeptide contains a portion of an antibody heavy chain that is complementary to the antibody light chain. The portion of the antibody light chain together with the complementary heavy chain associate in the inducible IL-12 prodrug to form a binding site for IL-12.
  • the first polypeptide comprises the p35 subunit, the p40 subunit, the half-life extension element, and a portion of an antibody heavy chain.
  • the second polypeptide contains a portion of an antibody light chain that is complementary to the antibody heavy chain.
  • the portion of the antibody heavy chain together with the complementary light chain associate in the inducible IL- 12 prodrug to form a binding site for IL-12.
  • the p35 subunit and p40 subunit can be operably linked through an optional protease cleavable linker.
  • the p35 subunit and the p40 subunit are operably linked by a non-cleavable linker.
  • the half-life extension element is preferably operably linked to either the p35 subunit or the p40 subunit through a protease cleavable linker.
  • the inducible IL-12 prodrug can include a first polypeptide in which p35 or p40 is operably linked to a half-life extension element through a protease cleavable linker.
  • the inducible IL- 12 prodrug can include a first polypeptide in which p35 or p40 is operably linked to a half-life extension element through a protease cleavable linker, and the half-life extension element is further operably linked to a blocking element (or component of a blocking element) through an optionally protease cleavable linker.
  • the inducible IL-12 prodrug comprises at least one additional polypeptide that includes the IL-12 subunit (p40 or p35) that is not present on the first polypeptide. Additional arrangements of the elements of the inducible IL- 12 prodrug are envisioned and encompassed by this disclosure.
  • the blocking element can be operably linked to either the p35 subunit or the p40 subunit through a protease cleavable linker.
  • One of the half-life extension element or the blocking element can be operably linked to the p35 subunit, and the other of the half-life or extension element or the blocking element can be operably linked to the p40 subunit.
  • the blocking element can be operably linked to the p40 subunit.
  • the blocking element in this inducible IL-12 prodrug is preferably a Fab.
  • the inducible IL-12 prodrugs can comprise three polypeptide chains. Typically, one polypeptide chain comprises either the p35 or p40 IL- 12 subunit, but not both, and a second polypeptide comprises the other IL-12 subunit and the third polypeptide comprises at least a portion (component) of the blocking element.
  • the IL- 12 subunit on the first polypeptide is p35
  • the IL-12 subunit on the second polypeptide is p40.
  • the IL- 12 subunit on the second polypeptide is p35.
  • the p35 and p40 subunits can associate to form a biologically active heterodimer.
  • the p35p40 heterodimer complex can be covalently linked, for example through a disulfide bond.
  • the first polypeptide can additionally comprise a half-life extension element that when present is operably linked to the IL- 12 subunit through a protease cleavable linker.
  • the second polypeptide further comprises a portion of the blocking element, and the third polypeptide can comprise the remainder of the blocking element.
  • the IL-12 blocking element can be antigen binding fragment of an antibody that is formed by the interaction of polypeptide two and polypeptide three, e g. a Fab fragment.
  • the second polypeptide can comprise at least an antigen binding portion of an antibody light chain.
  • the second polypeptide can comprise at least an antigen binding portion of an antibody heavy chain.
  • the antigen binding portion of an antibody light chain or the antigen binding portion of the heavy chain can be operably linked to the IL- 12 subunit through a protease cleavable linker.
  • the second polypeptide can contain a half-life extension element. When the second polypeptide contains the half-life extension element, the first polypeptide does not contain the half-life extension element.
  • the half-life extension element can be operably linked to the IL- 12 subunit through a protease cleavable linker.
  • the half-life extension element can be operably linked to a portion of the blocking element (e.g., an antigen binding portion of an antibody light chain or the antigen binding portion of the heavy chain) through an optional protease cleavable linker.
  • the antibody heavy chain or light chain can be operably linked to the IL- 12 subunit through a protease cleavable linker.
  • the antibody heavy chain or light chain can be operably linked to the IL- 12 subunit through an optionally cleavable linker.
  • the protease cleavable linkers on the first, second, and/or polypeptide chains can be the same or can be different.
  • Chimeric Compound 1, and Compounds 2, 3, 4, 5, and 6 are specific examples of inducible IL-12 prodrugs that comprise two polypeptide chains for use according to this disclosure. Chimeric Compound 1, and Compounds 2, 3, 4, 5, and 6 and additional details regarding their activity is disclosed in International Application No.: PCT/US2021/33014. [047] Compounds 7, 8, 17, 18, 21-28, 34, and 35 are specific examples of inducible IL-12 prodrugs that comprise one polypeptide chain for use according to this disclosure. Compounds 9- 13, 15, 19, 20, 29-31, and 36 are specific examples of inducible IL-12 prodrugs that comprise two polypeptide chains for use according to this disclosure.
  • TL-12 prodrugs that comprise three polypeptide chains for use according to this disclosure.
  • Table 1 Exemplary Inducible TL-12 prodrugs
  • the TL-12 can be a mutein, if desired.
  • the TL-12 mutein retains TL-12 activity, for example intrinsic IL-12 receptor agonist activity.
  • IL-12 subunits, p35 and/or p40 can be muteins.
  • the invention also relates to certain single chain IL-12 inducible polypeptides.
  • the single chain IL-12 polypeptides disclosed herein comprise IL-12, a blocking element, a half-life extension element, and a protease cleavable linker.
  • IL-12 has receptor agonist activity for its cognate IL-12 receptor.
  • IL-12 receptor activating activity is attenuated when the blocking element binds to IL-12.
  • active IL-12 polypeptide is released.
  • Single chain inducible IL- 12 polypeptides have been disclosed in International Application No.: PCT/US2019/032320 and International Application No.: PCT/US2019/032322.
  • [050] Contemplated herein are domains which extend the half-life of the inducible IL- 12 prodrug. Increasing the in vivo half-life of therapeutic molecules with naturally short half-lives allows for a more acceptable and manageable dosing regimen without sacrificing effectiveness. [051]
  • the half-life extension element increases the in vivo half-life and provides altered pharmacodynamics and pharmacokinetics of the inducible IL- 12 prodrug. Without being bound by theory, the half-life extension element alters pharmacodynamics properties including alteration of tissue distribution, penetration, and diffusion of the inducible IL-12 prodrug.
  • the half-life extension element can improve tissue targeting, tissue penetration, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension element.
  • an exemplary way to improve the pharmacokinetics of a polypeptide is by expression of an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor.
  • HSA human IgGs
  • HSA transferrin
  • HSA may also be directly bound to the pharmaceutical compositions or bound via a short linker. Fragments of HSA may also be used. HSA and fragments thereof can function as both a blocking element and a half-life extension element. Human IgGs and Fc fragments can also carry out a similar function.
  • the serum half-life extension element can also be antigen-binding polypeptide that binds to a protein with a long serum half-life such as serum albumin, transferrin and the like.
  • polypeptides include antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like.
  • antigen-binding domain include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
  • non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
  • antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that binds to or associates with one or more target antigens.
  • the half-life extension element as provided herein is preferably a human serum albumin (HSA) binding domain, and antigen binding polypeptide that binds human serum albumin or an immunoglobulin Fc or fragment thereof.
  • HSA human serum albumin
  • the half-life extension element of a inducible IL-12 prodrug extends the half-life of inducible IL-12 prodrug or the by at least about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about 10 days or more. In some embodiments, the half-life extension element extends the half-life of a inducible IL-12 prodrug to at least 2-3 days, 3-4 days, 4-5 days, 5-6 days, 6-7 days, 7-8 days or more.
  • the blocking element can be any element that binds to IL- 12 and inhibits the ability of the inducible IL-12 prodrug to bind and activate its receptor.
  • the blocking element can inhibit the ability of the IL-12 to bind and/or activate its receptor e.g., by sterically blocking and/or by noncovalently binding to the IL-12 prodrug.
  • the blocking element disclosed herein can bind to pl9, p35, p40, the p35p40 heterodimeric complex, or the pl9p40 heterodimeric complex.
  • blocking elements include the full length or an IL-12-binding fragment or mutein of the cognate receptor of IL-12.
  • Antibodies and antigen-binding fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like that bind IL-12 can also be used.
  • Suitable antigen-binding domain that bind IL-12 can also be used, include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
  • suitable blocking polypeptides include polypeptides that sterically inhibit or block binding of IL- 12 to its cognate receptor.
  • moieties can also function as half-life extending elements.
  • a peptide that is modified by conjugation to a water-soluble polymer can sterically inhibit or prevent binding of the cytokine to its receptor.
  • a water-soluble polymer such as PEG
  • Polypeptides, or fragments thereof, that have long serum half-lives can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin and the like, as well as fragments and muteins of such polypeptides.
  • Preferred IL- 12 blocking elements are single chain variable fragments (scFv) or Fab fragments.
  • the scFv blocking elements comprise the amino acid sequence as set forth in SEQ ID NOs: 144-188.
  • the Fab blocking element comprises the amino acid sequence as set forth in SEQ ID NOs: 189-194.
  • the IL-12 antibody fragments encompassed by SEQ ID NOs: 144-194 have been optimized to enhance the developability of the inducible IL- 12 prodrug disclosed herein.
  • Preferred antibody light chain blocking elements comprise SEQ ID NOs: 192-193. These preferred components can be located on one polypeptide chain and the complementary antigen binding portion of the heavy chain can be located on a second polypeptide chain.
  • Preferred heavy chain blocking elements comprise SEQ ID NOs: 189-191 and 194. These preferred components can be located on one polypeptide chain and the complementary light chain is located on a second polypeptide chain. The antibody light chain and the antibody heavy chain together form a binding site for IL- 12.
  • the IL-12 blocking element comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to SEQ ID NOs: 144-194, e.g., over the full length of SEQ ID Nos: 144-194.
  • amino acid sequence of the CDRs in not altered, and amino acid substitutions are present in the framework regions.
  • the disclosure also relates to functional variants of IL-12 blocking elements comprising SEQ ID NOs: 144-194.
  • the functional variants of IL-12 blocking elements comprising SEQ ID NOs: 144-194 generally differ from SEQ ID NOs: 144-194 by one or a few amino acids (including substitutions, deletions, insertions, or any combination thereof), and substantially retain their ability to bind to the IL-12 polypeptide (e.g., the p35 subunit, the p40 subunit, or the p35p40 complex) and inhibit binding of IL- 12 to its cognate receptor.
  • the IL-12 polypeptide e.g., the p35 subunit, the p40 subunit, or the p35p40 complex
  • the functional variant can contain at least one or more amino acid substitutions, deletions, or insertions relative to the IL-12 blocking element comprising SEQ ID NOs: 144-194.
  • the functional variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations compared to the IL-12 blocking element comprising SEQ ID NOs: 144-194.
  • the functional variant differs from the IL- 12 blocking element comprising SEQ ID NOs: 144-194 by less than 10, less, than 8, less than 5, less than 4, less than 3, less than 2, or one amino acid alterations, e.g., amino acid substitutions or deletions.
  • the functional variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NOs: 144-194.
  • the amino acid substitution can be a conservative substitution or a non-conservative substitution, but preferably is a conservative substitution.
  • the functional variants of the IL-12 blocking element may comprise 1, 2, 3, 4, or 5 or more non-conservative amino acid substitutions compared the IL- 12 blocking elements comprising SEQ ID NOs: 144-194. Non-conservative amino acid substitutions could be recognized by one of skill in the art.
  • the functional variant of the separation moiety preferably contains no more than 1, 2, 3, 4, or 5 amino acid deletions.
  • an inducible IL-12 prodrug that contains a blocking element having specificity for IL- 12 and contains a half-life extension element.
  • the blocking element is an antibody or antigen binding fragment that has binding specificity for TL-12, specifically the IL- 12 subunit beta precursor (p40) as defined by SEQ ID NO: 421, disclosed herein.
  • the antibody or antigen binding fragment comprises an antigen binding domain that binds to the residues shown in Table 2 of SEQ ID NO: 421.
  • This disclosure relates to an antibody or antigenbinding fragment that binds the IL-12 epitope defined by the amino acid residues shown in Table 2, and to an inducible IL- 12 prodrug that contains such an antibody or antigen-binding fragment, and to the use of such an antibody or antigen-binding fragment for the preparation of an inducible IL-12 prodrug, or a medicament containing such an inducible IL-12 prodrug.
  • the inducible IL- 12 prodrug comprises one or more linker sequences.
  • a linker sequence serves to provide flexibility between the polypeptides, such that, for example, the blocking element is capable of inhibiting the activity of IL-12.
  • the linker can be located between the IL- 12 subunit, the half-life extension element, and/or the blocking element.
  • the inducible IL- 12 prodrug comprises a protease cleavable linker.
  • the protease cleavable linker can comprise one or more cleavage sites for one or more desired protease.
  • the desired protease is enriched or selectively expressed at the desired target site of IL-12 (e.g., the tumor microenvironment).
  • the inducible IL-12 prodrug is preferentially or selectively cleaved at the target site of desired IL- 12 activity.
  • Suitable linkers are typically less than about 100 amino acids. Such linkers can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 1 to 100 amino acids.
  • the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acids in length.
  • Preferred linkers are typically from about 5 amino acids to about 30 amino acids.
  • the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked domain.
  • the linker is cleavable by a cleaving agent, e.g., an enzyme.
  • the separation moiety comprises a protease cleavage site.
  • the separation moiety comprises one or more cleavage sites.
  • the separation moiety can comprise a single protease cleavage site.
  • the separation moiety can also comprise 2 or more protease cleavage sites. For example, 2 cleavage sites, 3 cleavage sites, 4, cleavage sites, 5 cleavage sites, or more.
  • the separation moiety comprises 2 or more protease cleavage sites
  • the cleavage sites can be cleaved by the same protease or different proteases.
  • a separation moiety comprising two or more cleavage sites is referred to as a “tandem linker.”
  • the two or more cleavage sites can be arranged in any desired orientation, including, but not limited tom one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, or one cleavage site following by another cleavage site with intervening amino acids between the two cleavage sites.
  • protease-cleavable linkers are disease specific protease-cleavable linkers. Also preferred are protease-cleavable linkers that are preferentially cleaved at a desired location in the body, such as the tumor microenvironment, relative to the peripheral circulation.
  • the rate at which the protease-cleavable linker is cleaved in the tumor microenvironment can be at least about 10 times, at least about 100 times, at least about 1000 times or at least about 10,000 times faster in the desired location in the body, e.g., the tumor microenvironment, in comparison to in the peripheral circulation (e.g., in plasma).
  • Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtili sin-like protease, actinidain, bromelain, calpain
  • Proteases capable of cleaving linker amino acid sequences can, for example, be selected from the group consisting of a prostate specific antigen (PSA), a matrix metalloproteinase (MMP), an A Disintigrin and a Metalloproteinase (ADAM), a plasminogen activator, a cathepsin, a caspase, a tumor cell surface protease, and an elastase.
  • the MMP can, for example, be matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 14 (MMP 14).
  • the linker can be cleaved by a cathepsin, such as, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K and/or Cathepsin L.
  • a cathepsin such as, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K and/or Cathepsin L.
  • the linker can be cleaved by MMP 14 or Cathepsin L.
  • Proteases useful for cleavage of linkers and for use in the inducible IL- 12 prodrug disclosed herein are presented in Table 3, and exemplary proteases and their cleavage site are presented in Table 4.
  • Exemplary protease cleavable linkers include, but are not limited to kallikrein cleavable linkers, thrombin cleavable linkers, chymase cleavable linkers, carboxypeptidase A cleavable linkers, cathepsin cleavable linkers, elastase cleavable linkers, FAP cleavable linkers, ADAM cleavable linkers, PR-3 cleavable linkers, granzyme M cleavable linkers, a calpain cleavable linkers, a matrix metalloproteinase (MMP) cleavable linkers, a plasminogen activator cleavable linkers, a caspase cleavable linkers, a tryptase cleavable linkers, or a tumor cell surface protease.
  • MMP matrix metalloproteinase
  • MMP9 cleavable linkers Specifically, MMP9 cleavable linkers, ADAM cleavable linkers, CTSL1 cleavable linkers, FAPa cleavable linkers, and cathepsin cleavable linkers.
  • Some preferred protease-cleavable linkers are cleaved by a MMP and/or a cathepsin.
  • the separation moieties disclosed herein are typically less than 100 amino acids.
  • Such separation moieties can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 1 to 100 amino acids.
  • the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acids in length.
  • Preferred linkers are typically from about 5 amino acids to about 30 amino acids.
  • the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked domains.
  • the separation moiety comprises the sequence GPAGLYAQ (SEQ ID NO: 195); GPAGMKGL (SEQ ID NO: 196); PGGPAGIG (SEQ ID NO: 197); ALFKSSFP (SEQ ID NO: 198); ALFFSSPP (SEQ ID NO: 199); LAQRLRSS (SEQ ID NO: 200);
  • LAQKLKSS (SEQ ID NO; 201); GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (SEQ ID NO: 202); RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (SEQ ID NO: 203);
  • KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO: 204); RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO: 205); KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO: 206); SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO: 207); RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (SEQ ID NO: 208); GGGALFKSSFPLAQKLKSSPGGPAGIGGGR (SEQ ID NO: 209); RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (SEQ ID NO: 210); RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (SEQ ID NO: 211); RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO: 212);
  • GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO: 213); GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO: 214); RGGALFKSSFPLAQKLKSSGPAGLYAQGGK (SEQ ID NO: 215); RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (SEQ ID NO: 216); SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (SEQ ID NO: 217); KGGPGGPAGTGPLAQRLRSSALFKSSFPGR (SEQ TD NO: 218);
  • Certain preferred separation moieties comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198).
  • the separation moieties disclosed herein can comprise one or more cleavage motif or functional variants that are the same or different.
  • the separation moieties can comprise 1, 2, 3, 4, 5, or more cleavage motifs or functional variants.
  • Separation moieties comprising 30 amino acids can contain 2 cleavage motifs or functional variants, 3 cleavage motifs or functional variants or more.
  • a “functional variant” of a separation moiety retains the ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease) and are not cleaved or cleaved with low efficiency in the periphery (e.g., serum).
  • a target site e.g., a tumor microenvironment that expresses high levels of the protease
  • the functional variants retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation moiety comprising any one of SEQ ID NOs: 195-220 or 447-448.
  • the separation moieties comprising more than one cleavage motif can be selected from SEQ ID NOs: 195-201 or 447-448 and combinations thereof.
  • Preferred separation moieties comprising more than one cleavage motif comprise the amino acids selected from SEQ ID NO: 202-220.
  • the separation moiety can comprise both ALFKSSFP (SEQ ID NO: 198) and GPAGLYAQ (SEQ ID NO: 195).
  • the separation moiety can comprise two cleavage motifs that each have the sequence GPAGLYAQ (SEQ ID NO: 195).
  • the separation moiety can comprise two cleavage motifs that each have the sequence ALFKSSFP (SEQ ID NO: 198).
  • the separation moiety can comprise a third cleavage motif that is the same or different.
  • the separation moiety comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NOs: 195 to SEQ ID NO: 220 or 447-448 over the full length of SEQ ID NO: 195-220 or SEQ ID NOS 447-448.
  • the disclosure also relates to functional variants of separation moieties comprising SEQ ID NOs: 195-220 or 447-448.
  • the functional variants of separation moieties comprising SEQ ID NOs: 195-220 or 447-448 generally differ from SEQ ID NOs: 195-220 or 447-448 by one or a few amino acids (including substitutions, deletions, insertions, or any combination thereof), and substantially retain their ability to be cleaved by a protease.
  • the functional variants can contain at least one or more amino acid substitutions, deletions, or insertions relative to the separation moi eties comprising SEQ ID NOs: 195-220 or 447-448.
  • the functional variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations comparted to the separation moieties comprising SEQ ID NOs: 195-220 or 447-448.
  • the functional variant differs from the separation moiety comprising SEQ ID NOs: 195-220 by less than 10, less, than 8, less than 5, less than 4, less than 3, less than 2, or one amino acid alterations, e.g., amino acid substitutions or deletions.
  • the functional variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NOs: 195-220 or 447-448.
  • the amino acid substitution can be a conservative substitution or a non-conservative substitution, but preferably is a conservative substitution.
  • the functional variants of the separation moieties may comprise 1, 2, 3, 4, or 5 or more non-conservative amino acid substitutions compared the separation moieties comprising SEQ ID NOs: 195-220 or 447-448.
  • Non-conservative amino acid substitutions could be recognized by one of skill in the art.
  • the functional variant of the separation moiety preferably contains no more than 1, 2, 3, 4, or 5 amino acid deletions.
  • separation moieties comprising 8 amino acid protease substrates (e.g., SEQ ID Nos: 195-201 or 447-448) contain amino acid at positions P4, P3, P2, Pl, Pl’, P2’, P3’, P4’, wherein the sissile bond is between Pl and Pl’.
  • amino acid positions for the separation moiety comprising the sequence GPAGLYAQ SEQ ID NO: 195
  • sequence GPAGLYAQ SEQ ID NO: 195
  • Amino acids positions for the separation moiety comprising the sequence ALFKSSFP (SEQ ID NO: 198) can be described as follows:
  • amino acids surrounding the cleavage site e.g., positions Pl and Pl’for SEQ ID NOs: 195-201 or 447-448) are not substituted.
  • the separation moiety comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) or a functional variant of SEQ ID NO: 195 or a function variant of SEQ ID NO: 198.
  • a functional variant of PAGLYAQ (SEQ ID NO: 447) or ALFKSSFP (SEQ ID NO: 198) can comprise one or more amino acid substitutions, and substantially retain their ability to be cleaved by a protease.
  • the functional variants of GPAGLYAQ (SEQ ID NO: 195) is cleaved by MMP14, and the functional variant of ALFKSSFP (SEQ ID NO: 198) is cleaved by Capthepsin L (CTSL1).
  • the functional variants also retain their ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease).
  • the functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation moiety comprising amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.
  • the functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) comprise no more than 1, 2, 3, 4, or 5 conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198).
  • the amino acids at position PI and Pl’ are not substituted.
  • the amino acids at positions Pl and Pl’ in SEQ ID NO: 195 are G and L
  • the amino acids at positions Pl and Pl’ in SEQ ID NO: 198 are K and S.
  • the functional variant of GPAGLYAQ can preferably comprise one or more of the following: a) an arginine amino acid substitution at position P4, b) a leucine, valine, asparagine, or proline amino acid substitution at position P3, c) a asparagine amino acid substitution at position P2, d) a histidine, asparagine, or glycine amino acid substitution at position Pl, e) a asparagine, isoleucine, or leucine amino acid substitution at position Pl’, f) a tyrosine or arginine amino acid substitution at position P2’, g) a glycine, arginine, or alanine amino acid substitution at position P3’, h) or a serine, glutamine, or lysine amino acid substitution at position P4’.
  • GPAGLYAQ The following amino acid substitutions are disfavored in functional variants of GPAGLYAQ (SEQ ID NO: 195): a) arginine or isoleucine at position P3, b) alanine at position P2, c) valine at position Pl , d) arginine, glycine, asparagine, or threonine at position Pl’, e) aspartic acid or glutamic acid at position P2’, f) isoleucine at position P3’, g) valine at position P4’.
  • the functional variant of GPAGLYAQ does not comprise an amino acid substitution at position Pl and/or Pl’.
  • the amino acid substitution of the functional variant of GPAGLYAQ preferably comprises an amino acid substitution at position P4 and/or P4’.
  • the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprise a leucine at position P4, or serine, glutamine, lysine, or phenylalanine at position P4.
  • the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprise a glycine, phenylalanine, or a proline at position P4’.
  • amino acid substitutions at position P2 or P2’ of GPAGLYAQ are not preferred.
  • the functional variant of GPAGLYAQ comprises the amino acid sequence selected from SEQ ID NOs: 221- 295.
  • Specific functional variants of GPAGLYAQ include GPLGLYAQ (SEQ ID NO: 259), and GPAGLKGA (SEQ ID NO: 249).
  • the functional variants of LFKSSFP preferably comprises hydrophobic amino acid substitutions.
  • the functional variant of LFKSSFP can preferably comprise one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, phenylalanine at position P3; (c) arginine, leucine, alanine, glutamine, or histatine at position P2; (d) phenylalanine, histidine, threonine, alanine, or glutamine at position Pl; has histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine at position Pl’, (f
  • aspartic acid and/or glutamic acid are generally disfavored and avoided.
  • the following amino acid substitutions are also disfavored in functional variants of LFKSSFP (SEQ ID NO: 448): (a) alanine, serine, or glutamic acid at position P3; (b) proline, threonine, glycine, or aspartic acid at position P2; (c) proline at position Pl ; (d) proline at position Pl ’; (e) glycine at position P2’; (f) lysine or glutamic acid at position P3’; (g) aspartic acid at position P4’.
  • amino acid substitution of the functional variant of LFKSSFP preferably comprises an amino acid substitution at position P4 and/or Pl. In some embodiments, an amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 448) at position P4’ is not preferred.
  • the functional variant of LFKSSFP comprises the amino acid sequence selected from SEQ ID NOs: 296- 374.
  • Specific functional variants of LFKSSFP include ALFFSSPP (SEQ ID NO: 199), ALFKSFPP (SEQ ID NO: 346), ALFKSLPP (SEQ ID NO: 347); ALFKHSPP (SEQ ID NO: 335); ALFKSIPP (SEQ ID NO: 348); ALFKSSLP (SEQ ID NO: 356); or SPFRSSRQ (SEQ ID NO: 297).
  • the separation moieties disclosed herein can form a stable complex under physiological conditions with the amino acid sequences (e.g. domains) that they link, while being capable of being cleaved by a protease.
  • the separation moiety is stable (e.g., not cleaved or cleaved with low efficiency) in the circulation and cleaved with higher efficiency at a target site (i.e. a tumor microenvironment).
  • fusion polypeptides that include the linkers disclosed herein can, if desired, have a prolonged circulation half-life and/or lower biological activity in the circulation in comparison to the components of the fusion polypeptide as separate molecular entities.
  • the linkers when in the desired location (e.g., tumor microenvironment) the linkers can be efficiently cleaved to release the components that are joined together by the linker and restoring or nearly restoring the half-life and biological activity of the components as separate molecular entities.
  • the separation moiety desirably remains stable in the circulation for at least 2 hours, at least 5, hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.
  • the separation moiety is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% in the circulation as compared to the target location.
  • the separation moiety is also stable in the absence of an enzyme capable of cleaving the linker. However, upon expose to a suitable enzyme (i.e., a protease), the separation moiety is cleaved resulting in separation of the linked domain.
  • a suitable enzyme i.e., a protease
  • compositions comprising an inducible IL-12 prodrug described herein, a vector comprising the polynucleotide encoding the inducible IL-12 prodrug or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier.
  • compositions comprising the inducible IL- 12 prodrugs as described herein are suitable for administration in vitro or in vivo.
  • pharmaceutically acceptable carrier includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the subject to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • compositions are sterile.
  • compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21 st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005).
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic, although the formulate can be hypertonic or hypotonic if desired.
  • the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer’s solution, and dextrose solution.
  • the pH of the solution is generally about 5 to about 8 or from about 7 to 7.5.
  • Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptides.
  • Matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. Certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • Carriers are those suitable for administration of the IL-12 or nucleic acid sequences encoding the inducible IL-12 prodrugs to humans or other subjects.
  • the inducible TL-12 prodrug described herein is encapsulated in nanoparticles.
  • the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods.
  • the inducible IL-12 prodrug is attached to liposomes.
  • the inducible IL-12 prodrug are conjugated to the surface of liposomes.
  • the inducible IL- 12 prodrugs are encapsulated within the shell of a liposome.
  • the liposome is a cationic liposome.
  • the inducible IL- 12 prodrug described herein are contemplated for use as a medicament.
  • Administration is effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition.
  • the dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient’s size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently.
  • An “effective dose” refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.
  • the inducible IL-12 prodrug or nucleic acid sequences encoding the inducible IL-12 prodrug are administered by a vector.
  • a vector There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and/or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein.
  • compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express and preferably secrete the encoded chimeric polypeptide or to therapeutically deliver nucleic acids to a subject.
  • the components of the IL-12 polypeptide disclosed herein are typically operably linked in frame to encode a fusion protein.
  • plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the nucleic acid molecule and/or polypeptide in the cells into which it is delivered.
  • Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general and methods of making them are described by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). The construction of replicationdefective adenoviruses has been described (Berkner et al., J. Virol. 61:1213-20 (1987); Massie et al., Mol. Cell. Biol.
  • Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites.
  • Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.
  • VLPs Virus like particles
  • Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).
  • the inducible IL-12 prodrugs disclosed herein can be delivered by subviral dense bodies (DBs).
  • DBs transport proteins into target cells by membrane fusion.
  • Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).
  • the provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006/110728.
  • Non-viral based delivery methods can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding polypeptides, wherein the nucleic acids are operably linked to an expression control sequence.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif), and Invitrogen/Life Technologies (Carlsbad, Calif). Vectors typically contain one or more regulatory regions.
  • Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
  • a suitable host cell such as CHO cells.
  • Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g., P-actin promoter or EFla promoter, or from hybrid or chimeric promoters (e g., CMV promoter fused to the P-actin promoter).
  • viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g., P-actin promoter or EFla promoter, or from hybrid or chimeric promoters (e g., CMV promoter fused to the
  • Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' or 3' to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 base pairs (bp) in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the promoter and/or the enhancer can be inducible (e.g., chemically or physically regulated).
  • a chemically regulated promoter and/or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal.
  • a physically regulated promoter and/or enhancer can, for example, be regulated by environmental factors, such as temperature and light.
  • the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize the expression of the region of the transcription unit to be transcribed Tn certain vectors, the promoter and/or enhancer region can be active in a cell type specific manner.
  • the promoter and/or enhancer region can be active in all eukaryotic cells, independent of cell type.
  • Preferred promoters of this type are the CMV promoter, the SV40 promoter, the P-actin promoter, the EF 1 a promoter, and the retroviral long terminal repeat (LTR).
  • the vectors also can include, for example, origins of replication and/or markers.
  • a marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell.
  • the marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed.
  • selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E.
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide.
  • Tag sequences such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAGTM tag (Kodak; New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GFP glutathione S-transferase
  • GST glutathione S-transferase
  • polyhistidine polyhistidine
  • c-myc hemagglutinin
  • FLAGTM tag FLAGTM tag
  • a disease, disorder or condition associated with a target antigen comprising administering to a subject in need thereof a inducible IL-12 prodrug as described herein.
  • Diseases, disorders, or conditions include, but are not limited to, cancer, inflammatory disease, an immunological disorder, autoimmune disease, infectious disease (i.e., bacterial, viral, or parasitic disease).
  • the disease, disorder, or condition is cancer.
  • any suitable cancer may be treated with the inducible IL-12 prodrugs provided herein.
  • suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease,
  • ALL acute lympho
  • provided herein is a method of enhancing an immune response in a subject in need thereof by administering an effective amount of an inducible IL- 12 prodrug provided herein to the subject.
  • the enhanced immune response may prevent, delay, or treat the onset of cancer, a tumor, or a viral disease.
  • the inducible IL- 12 prodrug enhances the immune response by activating the innate and adaptive immunities.
  • the methods described herein increase the activity of Natural Killer Cells and T lymphocytes.
  • the inducible IL- 12 prodrug provided herein can induce IFNy release from Natural Killer cells as well as CD4+ and CD8+ T cells.
  • This disclosure relates to a method for selectively activating tumor infiltrating lymphocytes, comprising administering to a subject in need thereof and effective amount of an inducible interleukin-2 (IL- 12) prodrug as described herein.
  • IL- 12 inducible interleukin-2
  • the inducible IL- 12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results a significantly higher frequency of CD8+ T cells that produce IFNgamma and/or Granzyme.
  • This disclosure relates to a method for inducing immunological memory to a tumor, comprising administering to a subject in need thereof an effective amount of an inducible interleukin- 12 (IL-12) prodrug as described herein.
  • IL-12 interleukin- 12
  • the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other areas.
  • This disclosure relates to a method for selectively activating effector CD8+ T cells in the tumor microenvironment, comprising administering to a subject in need thereof an effective amount of an inducible interleukin- 12 (IL-12) prodrug as described herein.
  • IL-12 interleukin- 12
  • the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results a significantly higher frequency of CD8+ T cells that produce TNF and/or IFNgamma within the tumor in comparison to peripheral tissue.
  • the method can further involve the administration of one or more additional agents to treat cancer, such as chemotherapeutic agents (e.g., Adriamycin, Cerubidine, Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), immuno-oncology agents (e.g., anti-PD-Ll, anti- CTLA4, anti-PD-1, anti-CD47, anti-GD2), cellular therapies (e.g., CAR-T, T-cell therapy), oncolytic viruses and the like.
  • chemotherapeutic agents e.g., Adriamycin, Cerubidine, Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine
  • immuno-oncology agents e
  • Non-limiting examples of anti-cancer agents include acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacytidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil
  • Immune checkpoint proteins include, for example, PD-1 which binds ligands PD-L1 (B7-H1 , CD274) and PD-L2 (B7-DC, CD273), CTLA-4 (GDI 52) which binds B7-1 (CD80) and B7-2 (CD86), LAG 3 (CD223) which binds Galectin3, LSECtin and FGL1; TIM3 (HAVCR2) which binds ligands Ceacaml and Galectin9; TIGIT (VSTM3, WUCAM) which binds CD112 and CD155; BTLA (CD272) which binds HVEM (TNFRSF14), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD160 (BY55) which bind HVEM
  • Therapeutic agents such as antibodies, that bind immune checkpoint proteins and inhibit their immunosuppressive activity include the anti-PDl antibodies pembrolizumab (KEYTRUDA), dostarlimab (JEMPERLI), cemiplimab-rwlc (LIBATYO), nivolumab (OPDIVO), camrelizumab, tislelizumab, toripalimab, and sintilimab (TYVYT); the anti-PD-Ll antibodies avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ); the anti-CTLA-4 antibody ipilimumab (YERVOY).
  • KEYTRUDA pembrolizumab
  • JEMPERLI dostarlimab
  • LIBATYO cemiplimab-rwlc
  • OPDIVO nivolumab
  • camrelizumab tislelizumab
  • the inducible IL-12 prodrug is administered in combination with an agent for the treatment of the particular disease, disorder, or condition.
  • Agents include, but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroidal, peptide, and the like), radiotherapies (y-rays, C- rays, and/or the directed delivery of radioisotopes, microwaves, UV radiation and the like), gene therapies (e.g., antisense, retroviral therapy and the like) and other immunotherapies.
  • the inducible IL-12 prodrug is administered in combination with anti-diarrheal agents, anti-emetic agents, analgesics and/or non-steroidal anti-inflammatory agents.
  • the terms “activatable,” “activate,” “induce,” and “inducible” refers to a polypeptide complex that has an attenuated activity form (e.g., attenuated receptor binding and/or agonist activity) and an activated form.
  • the polypeptide complex is activated by protease cleavage of the linker that causes the blocking element and half-life extension element to dissociate from the polypeptide complex.
  • the induced/activated polypeptide complex can bind with increased affinity/avidity to the IL-12 receptor.
  • an antibody or immunoglobulin is intended to refer to immunoglobulin molecules comprised of two heavy (H) chains.
  • H heavy chain
  • mammals e g., humans, rodents, and monkey
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multi specific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, or tetrameric antibodies comprising two heavy chain and two light chain molecules.
  • monospecific antibodies monospecific antibodies
  • multi specific antibodies including bispecific antibodies
  • human antibodies humanized antibodies
  • chimeric antibodies immunoglobulins
  • synthetic antibodies or tetrameric antibodies comprising two heavy chain and two light chain molecules.
  • tetrameric antibodies comprising two heavy chain and two light chain molecules.
  • the term “attenuated” as used herein is an IL-12 receptor agonist that has decreased receptor agonist activity as compared to the IL-12 receptor’s naturally occurring agonist.
  • An attenuated IL- 12 agonist can have at least about 10X, at least about 50X, at least about 100X, at least about 250X, at least about 500X, at least about 1000X or less agonist activity as compared to the receptor’s naturally occurring agonist.
  • a IL-12 polypeptide complex that contains IL-12 as described herein is described as “attenuated” or having “attenuated activity”, it is meant that the IL-12 polypeptide complex is an attenuated IL-12 receptor agonist.
  • cancer refers to the physiological condition in mammals in which a population of cells is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate and/or certain morphological features. Often cancers can be in the form of a tumor or mass, but may exist alone within the subject, or may circulate in the blood stream as independent cells, such a leukemic or lymphoma cells.
  • the term cancer includes all types of cancers and metastases, including hematological malignancy, solid tumors, sarcomas, carcinomas and other solid and non-solid tumors. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., triple negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancers.
  • Triple negative breast cancer refers to breast cancer that is negative for expression of the genes for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu.
  • a “conservative” amino acid substitution generally refers to substitution of one amino acid residue with another amino acid residue from within a recognized group which can change the structure of the peptide but biological activity of the peptide is substantially retained.
  • Conservative substitutions of amino acids are known to those skilled in the art. Conservative substitutions of amino acids can include, but not limited to, substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • half-life extension element in the context of the polypeptide complex disclosed herein, refers to a chemical element, preferable a polypeptide that increases the serum half-life and improve pK, for example, by altering its size (e.g., to be above the kidney filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.
  • a polypeptide comprising an IL-12 subunit and an IL- 12 blocking element are operably linked by a protease cleavable linker in a polypeptide complex when the IL-12 blocking element is capable of inhibiting the IL-12 receptor-activating activity of the IL-12 polypeptide, but upon cleavage of the protease cleavable linker the inhibition of the IL- 12 receptor-activating activity of the IL- 12 polypeptide by the IL- 12 blocking element is decreased or eliminated, for example because the TL-12 blocking element can diffuse away from the IL- 12.
  • peptide As used herein, the terms “peptide”, “polypeptide”, or “protein” are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.
  • subject herein to refers to any animal, such as any mammal, including but not limited to, humans, non-human primates, rodents, and the like.
  • the mammal is a mouse.
  • the mammal is a human.
  • the term “therapeutically effective amount” refers to an amount of a compound described herein (i.e., a IL-12 polypeptide complex) that is sufficient to achieve a desired pharmacological or physiological effect under the conditions of administration.
  • a “therapeutically effective amount” can be an amount that is sufficient to reduce the signs or symptoms of a disease or condition (e.g., a tumor).
  • a therapeutically effective amount of a pharmaceutical composition can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmaceutical composition to elicit a desired response in the individual. An ordinarily skilled clinician can determine appropriate amounts to administer to achieve the desired therapeutic benefit based on these and other considerations.
  • Chimeric Compound 1 an IL-12 prodrug that includes murine IL- 12 p35 and human IL- 12 p40 and thus is active in mouse models, was used as a surrogate for human IL-12 prodrugs that include human p35 and p40.
  • a chimeric IL-12 that included murine p35 and human p40 was used as a control.
  • Chimeric Compound 1 includes murine IL-12 p35 and is a surrogate for Compound 36 which includes human IL-12 p35 and is otherwise the same as Chimeric Compound 1.
  • HEK-Blue IL- 12 cells (InvivoGen) were plated in suspension at a density of 50,000 cells/well in culture media with or without 15 or 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL-12, chimeric IL-12 (mouse p35/human p40), activatable chimeric IL-12, or activatable hIL-12 for 20-24 hours at 37°C and 5% CO2. Activity of uncleaved and cleaved activatable hIL-12 was tested. Cleaved inducible hIL-12 was generated by incubation with active MMP9 or CTSL-1.
  • HSA human serum albumin
  • IL-12 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay. Results confirm that IL- 12 fusion proteins are active and inducible. Results are shown in FIG. 1.
  • SEAP Secreted Alkaline Phosphatase
  • mice in vivo work was performed in accordance with current regulations and standards and the NTH at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of an Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • Female, 6-8 week- old C57B1/6 mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation.
  • a total of 5 x 10 5 MC38 cells were injected subcutaneously and monitored for tumor growth.
  • Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was 100-150 mm 3 , and mice were randomized into treatment groups on Day 0.
  • mice receiving Chimeric Compound 1 were dosed twice a week for two weeks (Days 1, 4, 8, and 11) unless otherwise noted.
  • Inducible IL-12 prodrugs used in these studies included Chimeric Compound 1.
  • Mice receiving recombinant chimeric IL-12 (chimeric IL-12 or WW0295) were dosed twice a day for 5 days before receiving a 2-day break (5/2 regimen) and the cycle was repeated for a total of two weeks. All treatments were administered by intraperitoneal injection. Body weight and tumor volume were both measured twice weekly for the duration of the study.
  • mice in vivo work was performed in accordance with current regulations and standards and the NIH at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of an Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • Female, 6-8 week-old C57B1/6 mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation.
  • a total of 1 x 10 5 B16-F10 cells were injected subcutaneously and monitored for tumor growth.
  • Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was 50-100 mm 3 , and mice were randomized into treatment groups on Day 0.
  • mice receiving Chimeric Compound 1 were dosed on Days 1 and 4, and tumors were harvested 24 hours after the second dose (Day 5). Inducible TL-12 prodrugs used in these studies included Chimeric Compound 1 . All treatments were administered by intraperitoneal injection. See, FIGs. 3B and FIG. 5A-5F.
  • mice in vivo work was performed in accordance with current regulations and standards and the NIH at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of an Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • Female, 6-8 week- old C57B1/6 mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation.
  • a total of 1 x 10 5 EMT6 cells were injected subcutaneously and monitored for tumor growth.
  • Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was 50-100 mm 3 , and mice were randomized into treatment groups on Day 0.
  • mice receiving Chimeric Compound 1 were dosed twice a week for two weeks (Days 1, 4, 8, and 11) unless otherwise noted.
  • Inducible IL- 12 prodrugs used in these studies included Chimeric Compound 1. All treatments were administered by intraperitoneal injection. In some experiments, mice that rejected tumors previously were then rechallenged with 1 x 10 5 EMT6 cells on the opposite flank four months after the initial rejection. In those experiments, age matched, tumor naive animals were used as a control. In some experiments, tumor samples were harvested and incubated in 5- 10 mLs of 10% neutral buffered formalin for at least 72 hours before being embedded in paraffin and mounted on slides. Unstained slides were submitted to Nanostring for immunofluorescence staining and geospatial transcriptional analysis using a Nanostring GeoMX DSP system. See, FIGs. 3C, 3F, 6, and 7A-7D.
  • mice in vivo work was performed in accordance with current regulations and standards and the NIH at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of an Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • Female, 6-8 week- old Balb/C mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation.
  • a total of 3 x 10 5 CT26 cells were injected subcutaneously and monitored for tumor growth. Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was 30-60 mm 3 , and mice were randomized into treatment groups on Day 0.
  • mice receiving Chimeric Compound 1 were dosed twice a week for two weeks (Days 1, 4, 8, and 11) unless otherwise noted.
  • mice in vivo work was performed in accordance with current regulations and standards and the NTH at Covance (Ann Arbor, MI) with the approval of an Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • Female, 6-8 week-old C57B1/6 mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation. A total of 1 x 10 6 EG7.
  • OVA cells were injected subcutaneously and monitored for tumor growth. Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was ⁇ 93 mm 3 , and mice were randomized into treatment groups on Day 0.
  • mice receiving Chimeric Compound 1 were dosed twice a week for two weeks (Days 1, 4, 8, and 11) unless otherwise noted.
  • mice All mouse in vivo work was performed in accordance with current regulations and standards and the NIH at Covance (Ann Arbor, MI) with the approval of an Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • Female, 6-8 week-old Balb/C mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation.
  • a total of 5 x 10 5 A20 cells were injected subcutaneously and monitored for tumor growth. Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was -90-130 mm 3 , and mice were randomized into treatment groups on Day 0.
  • mice receiving Chimeric Compound 1 were dosed twice a week for two weeks (Days 1, 4, 8, and 11) unless otherwise noted.
  • MC38 and B16-F10 tumors were chopped into small pieces ( ⁇ 5 mm 3 ) in phenol -free RPMI-1640 (ThermoFisher) before being enzymatically digested with Collagenase IV (3 mg/mL, Gibco, 17104019) at 37°C for 35 minutes while shaking. After digestion, tumor samples were mechanically dissociated through a 70 pM cell strainer. EMT6 tumors were processed using gentleMACSTM C-Tubes from Miltenyi Biotech (130-093-237).
  • tumors were cut into small pieces ( ⁇ 5 mm 3 ) in HBSS containing 1.25 mg/mL Collagenase Type IV (Gibco, 17104019), 0.0025mg/mL Hyaluronidase (Sigma-Aldrich, H3506), and O.Olmg/mL DNASE I (Worthington, LS002004).
  • Samples were placed on a gentleMACSTM Octo Dissociator and processed using program 37C_m_TDK_l before samples were passed through a 70 pM cell strainer to remove any undigested tumor pieces. Single cell suspensions were then counted and analyzed by flow cytometry.
  • NanoString analysis 5 x 10 5 cells were frozen in 100 pL of RLT Lysis buffer (Qiagen, 1053393). RNA samples were shipped to LakePharma and analyzed using the nCounter Mouse PanCancer Immune Profiling Codeset Panel with the nCounter FLEX analysis system. NanoString analysis was performed using nSolverTM Software with the Advanced Analysis module installed.
  • MitoTracker Deep Red FM (ThermoFisher, # M46753), MitoTracker Green FM (ThermoFisher, M46750) MitoSOX Red (ThermoFisher, M36008), and TMRM (ThermoFisher, T668) staining was performed at 37°C/5% CO2 in RPMI 1640 media (Gibco, A10491-01) with 10% heat inactivated FBS (Gibco, 10082-147) and Penicillin/Streptomycin (Gibco, 15140-122) for 1 hour. Cells were then washed with FACs buffer and stained for extracellular markers. Specific antibody clones are detailed as follows.
  • Fluorescent dye-conjugated antibodies specific for the following proteins were purchased from BioLegend: CD8a APC, clone 53-67; CD4 BV650, clone RM4-5; CD3 AF700, clone 17A2; CD45 BV605, clone 30-F11; CD49b APC/Cy7, clone DX5; CD25 BV421, clone PC61; CD25 APC/Fire 750, clone PC61; Ki67 PeCy7, clone 16A8; Ki67 AF700, clone 16A8; granzyme B FITC, clone GB11; IfNy PE, clone XMG1.2; F4/80 Pe/Dazzle 594, clone BM8; CD3 Complex PeCy7, clone 17A2; FC Block, clone 93.
  • Fluorescent dye-conjugated antibodies specific for the following proteins were purchased from eBioscience: CD45 BUV395, clon30-Fl l; CD4 BUV496, clone GK1.5; CD8 BUV563, 53.6-7; TNF BV750, clone MP6-XT22; CD49B Pe-Cy5, clone DX5, FoxP3 AF488, clone FJK-16s; FoxP3 eFlour450, clone FJK-16s.
  • the fluorescent dye-conjugated tetramer against the MulV pl5E peptide KSPWFTTL (SEQ ID NO: 449) was purchased from ThermoFisher Scientific (50-168-9385).
  • the Live/Dead Blue Dye was also purchased from ThermoFisher Scientific (L23105). 11. Pharmacokinetic A nalysis
  • Plasma and tumor samples were collected at indicated time points by Charles River Laboratories and stored at -80°C.
  • MC38 tumor lysates were generated by homogenizing each tumor with a Qiagen TissueRuptor with disposable probes (Qiagen) in ice cold Lysis Buffer (IX Tris Buffered Saline (Sigma-Aldrich, T5912-1L), 1 mM EDTA (Sigma-Aldrich, 3690-100mL), 1 % Triton X-100 (Sigma-Aldrich, XIOO-lOOOmL), with protease inhibitors (Sigma-Aldrich, P8340-1L) in diH2O).
  • Qiagen TissueRuptor with disposable probes Qiagen
  • IX Tris Buffered Saline Sigma-Aldrich, T5912-1L
  • 1 mM EDTA Sigma-Aldrich, 3690-100mL
  • Plasma and tumor samples were analyzed using a sandwich ELISA on the MSD platform, which detects both intact Chimeric Compound 1 and free/released IL-12.
  • Free IL- 12 level was quantified using an in-house developed ECLIA assay on MSD MESOTM QuickPlex SQ 120 system. Data acquisition and analysis were performed using MSD Workbench 4.0.12, and pharmacokinetic parameters were calculated using Phoenix WinNonlin Version 8.1.
  • Compound 36 was incubated in human serum (BioIVT) from healthy donors in duplicate for each timepoint. Time zero (TO) samples were immediately frozen at -80°C. The remaining samples were incubated at 37°C for 24 (T24) or 72 (T72) hours before being stored at 80°C. Stability of Compound 36 was assessed by western blot analysis using the JESS system (Protein Simple, SM-W004) according to the manufacturer’s general protocol. Input controls (intact and protease cleaved) were also analyzed.
  • Human PBMCs were isolated from whole blood (BioIVT) using FicolLPaque Plus (GE Healthcare, GE17-1440-03) according to the manufacturer’ s protocol and frozen in Recovery Cell Culture Freezing Media (Gibco, 12648010) for later use.
  • Tblasts To generate activated T cells (Tblasts), PBMCs were thawed, counted, and stimulated with 5 pg/mL ofPHA (Sigma-Aldrich, L1668-5MG) for 72 hours before being frozen.
  • Tblasts were thawed, counted, and plated in a 96- well round bottom plate, and incubated with titrated amounts of intact or protease-activated (cleaved) INDUKINETM proteins or chimeric IL- 12. After 72 hours, IFNy production was measured using a Human IFNy specific AlphaLisa Kit (Perkin Elmer, AL217C) according to the manufacturer’s protocol with a Perkin Elmer Enspire Alpha Reader running Enspire Manager Software (V4.13.3005.1482).
  • Flow cytometry plots were generated with Flowlo Software (vl0.5.30) and are representative samples. All the quantitative plots were generated using GraphPad Prism 8 Software for Windows (64-Bit) (San Diego, CA). For in vitro activity assays, data were analyzed using a non-linear sigmoidal, 4PL curve fit model without constraints. Statistical analysis was also performed using GraphPad Prism software (San Diego, CA). Two sample comparisons used a student’s /-test while comparisons of more than two groups used an analysis of variance (ANOVA) test with multiple comparisons. Antitumor effects over time were analyzed by using a mixed-effects model.
  • ANOVA analysis of variance
  • NanoString dataset statistical analysis was performed using nSolverTM software with the Advanced Analysis Module installed. Pathway analysis was performed using Partek software (v 10.0.22.0428), based on transcripts that were significantly different following mWTX-330 with an FDR step-up of 0.05.
  • Chimeric Compound 1 is a Selectively Activated Inducible IL-12 Prodrug that Generates a Robust, Cleavage Dependent Anti-Tumor Immune Response in Multiple Models
  • a selectively inducible IL-12 prodrug was developed.
  • HEK-Blue IL- 12 reporter cells were incubated with either intact or protease activated Chimeric Compound 1, and IL- 12 signaling was assessed.
  • intact Chimeric Compound 1 had 175-fold less activity than either cleaved Chimeric Compound 1 or chimeric IL- 12. (FIG. 1).
  • non- cleavable (NC) variant of Chimeric Compound 1 had less activity than even the lowest dose of Chimeric Compound 1 demonstrating that the full potency of Chimeric Compound 1 is dependent on in vivo processing of the molecule.
  • Chimeric Compound 1 treatment also generated robust anti-tumor immunity in less immune cell infiltrated (“colder") syngeneic tumor models, including CT26 (FIG. 3A), B16-F10 (FIG. 3B), and EMT-6 (FIG. 3C), demonstrating the broad activity of this molecule in vivo. While the use of a non-cleavable control demonstrated the necessity of processing for full activity, these data did not directly indicate that processing was occurring in the TME.
  • FTY720 is a small molecule inhibitor of Sphingosine- 1 -phosphate receptor- 1 which prevents lymphocyte egress from secondary lymphoid tissues, and effectively isolates the TILs from the normal recirculating population of immune cells in vivo.
  • Chimeric Compound 1/FTY720 co-treated animals retained the potent early anti-tumor activity associated with Chimeric Compound 1 treatment, although tumor control was less complete after dosing with the INDUKINETM molecule had stopped (FIG. 8A) [0153]
  • chimeric IL- 12 had a half-life of only 4 hours, while Chimeric Compound 1 had a half-life of nearly 16 hours (FIG. 9A). Furthermore, only about 2% of the Chimeric Compound 1 found in the plasma was in the form of the cleaved molecule. Tn contrast, when the same analysis was performed on tumor samples (FIG. 9B), nearly 45% of the molecule was unmasked IL- 12 and inter-tumoral exposure was maintained far beyond what was achieved by treatment with chimeric IL-12.
  • Chimeric Compound 1 was compared to those same populations in the tumor draining and non-draining lymph nodes, as well as the peripheral blood following Chimeric Compound 1 treatment. While Chimeric Compound 1 treatment resulted in a significant increase in the frequency of polyfunctional CD8+ T cells within the MC38 tumors, no such increase was observed in the lymph nodes or in the peripheral blood (FIG. 9C). Likewise, among CD4+ T conventional cells (FoxP3-) (FIG. 15A) and NK Cells (FIG. 15B), Chimeric Compound 1 preferentially increased the frequency of cells producing effector cytokines in the tumor compared to the peripheral tissues.
  • Chimeric Compound 1 treatment resulted in significant enrichment of several immune related signaling pathways, including “PD-L1 Expression and PD-1 Checkpoint in Cancer”, “NK Cell Cytotoxicity”, and “TH1 and TH2 Differentiation”.
  • Chimeric Compound 1 treatment resulted in a significant increase in the frequency of tumor infiltrating NK cells producing IFNy, TNF, and Granzyme B (FIG. 4C-4D).
  • Chimeric Compound 1 treatment resulted in NK, NKT, CD4+ T conventional cells, and CD8+ T cells producing such elevated levels of ZFNy that it was measurable by intracellular cytokine staining without ex vivo restimulation (FIGs. 14A-14B).
  • Chimeric Compound 1 treatment results in rapid tumor rejection, making it technically challenging to fully investigate the kinetics of immune activation.
  • Chimeric Compound 1 treatment of the EMT-6 tumor model generates complete rejections over a longer period, which is favorable to a more thorough analysis of an ongoing CD8+ T cell response (FIG. 3C). Therefore, mice bearing established EMT-6 tumors were randomized into treatment groups and dosed twice a week for two weeks with either vehicle or Chimeric Compound 1. Tumors and plasma were then harvested at various timepoints.
  • the frequency of poly functional CD8+ T cells did expand over the course of the experiment, but eventually retracted in line with eventual tumor growth.
  • Chimeric Compound 1 treatment increased the frequency of polyfunctional CD8+ T cells over that of the control animals as soon as Day 5 after the start of treatment (FIG. 6), and this frequency continued to expand even after exposure to Chimeric Compound 1 was undetectable.
  • IL-12 (FIG. 7C) and IFNy signaling (FIG, 7D) were both significantly upregulated, confirming the local release of unmasked IL-12 and subsequent production of IFNy within the TME.
  • Chimeric Compound 1 treatment also increased expression of transcripts downstream of TCR signaling (FIG. 11A).
  • T cells were isolated from EMT-6 tumors following Chimeric Compound 1 or vehicle treatment and sent for TCR sequencing.
  • Chimeric Compound 1 Substantially Increases Mitochondrial Activity in Tumor Infdtrating CD8+ T cells and NK Cells
  • CD8+ T cells have substantial energy requirements and rely heavily on glucose uptake and glycolysis to quickly generate the energy necessary to perform their effector functions, before transitioning towards mitochondria dependent oxidative phosphorylation as they develop into long-lived memory cells.
  • recent publications have demonstrated that tumor infdtrating CD8+ T cells often fail to induce significant mitochondrial respiration compared to those activated in the spleen or lymph nodes, suggesting that the TME negatively impacts the metabolic health of effector cells.
  • Chimeric Compound 1 treatment resulted in a significant enrichment of transcripts associated with glycolysis (FIG. 12A).
  • Chimeric Compound 1 treatment may increase glucose uptake by tumor infiltrating CD8+ T cells, and thereby lead to increased glycolysis
  • 2-NDBG non-metabolizable fluorescent glucose analog
  • tumor infiltrating CD8+ T cells from Chimeric Compound 1 treated animals actually had slightly less glucose uptake than those from vehicle animals (FIGs. 12B-12C). Therefore, rather than simply increasing glucose uptake by tumor infiltrating CD8+ T cells, Chimeric Compound 1 treatment was instead reprogramming those cells to utilize glucose more efficiently than those from vehicle treated animals.
  • Chimeric Compound 1 treatment In addition to driving increased glycolysis, Chimeric Compound 1 treatment also enriched for transcripts associated with the TCA cycle, mitochondrial biogenesis, and mitochondrial translation, suggesting that Chimeric Compound 1 treatment may enhance the mitochondrial activity and health of tumor infiltrating effector cells (FIGs. 12D-12F).
  • TILs were isolated from vehicle or Chimeric Compound 1 treated animals and mitochondrial phenotyping was performed by flow cytometry.
  • Mitotracker Red is a dye that specifically stains actively respirating mitochondria due to its pH sensitivity. While tumor infiltrating CD8+ T cells from vehicle treated animals had limited evidence of ongoing active mitochondrial respiration, those from Chimeric Compound 1 treated animals had significantly increased levels of active respiration (FIGs. 12G-12H).
  • NK cells FIGs. 12I-12J
  • total CD4+ T cells FIG. 12U
  • This increase was primarily due to increased mitochondrial activity, rather than simply an increase in total mitochondrial mass, as Chimeric Compound 1 treatment only slightly increased the total mitochondrial mass of tumor infiltrating NK cells, CD8+ T cells, and total CD4+ T cells (FIG. 12V).
  • TMRM staining also revealed that Chimeric Compound 1 treatment significantly increased the mitochondrial membrane potential in both CD8+ T cells (FIGs. 12K-12L) as well as NK cells (FIGs. 12M-12N).
  • ROS Mitochondrial reactive oxygen species
  • Oxidative phosphorylation is the primary energy source for memory T cells, and increased dependence on this pathway has been associated with superior anti-tumor immunity and a “stem-cell like” phenotype.
  • Tumor infiltrating CD8+ T cells from Chimeric Compound 1 treated mice also significantly upregulated expression of genes associated with T cell sternness, including Tcf7, Cxcr3, and I12ry while significantly downregulating expression of several genes associated with CD8+ T cell exhaustion, including Pdcdl, Havcr2, and Lag3.
  • Compound 36 a fully human inducible IL-12 Prodrug, is Stable in Human Serum and Preferentially Activated by Primary Human Tumor Samples
  • Compound 36 is identical to Chimeric Compound 1 except that it contains fully human IL-12 as the payload.
  • intact Compound 36 had substantially less activity than either cleaved Compound 36 or recombinant human IL-12 in a HEK-Blue IL-12 reporter assay.
  • intact Compound 36 was 61 -fold less active on average than the cleaved molecule.
  • IL- 12 has long been a cytokine of great interest for oncology due to its potential to induce innate and adaptive immune responses (9,11) and its promising anti-tumor preclinical data (12,18,20,34,35). Nevertheless, despite this interest, the poor pharmacokinetic properties of this cytokine and the unacceptable levels of toxicity associated with its systemic administration have prevented its use in clinical settings (9,10,24,36). To address these concerns, we developed an inducible IL-12 prodrug, Compound 36. Compound 36 a prodrug molecule, designed to be an infrequently administered, systemically delivered therapy with targeted intra-tumoral activation that releases native IL- 12 into the tumor microenvironment.
  • Chimeric Compound 1 demonstrated anti -turn or activity in the MC38 tumor model that was dependent on in vivo cleavage of the inducible IL- 12 prodrug by the tumor. Furthermore, Chimeric Compound 1 was a very potent monotherapy in several mouse tumor models with varying levels of baseline infdtration, including complete responses in a model refractory to anti-PD-1 treatment (EMT-6). These complete responses translated into robust immune memory against subsequent rechallenge with the same tumor cell line, highlighting the role of the immune system in tumor rejection.
  • EMT-6 anti-PD-1 treatment
  • Chimeric Compound 1 proved to be well-tolerated in mice compared with recombinant chimeric IL- 12 treatment, while maintaining the potential to induce complete tumor regressions, resulting in an almost 10-fold improvement of the therapeutic window compared to the unblocked cytokine. Improvement of the therapeutic window is a key feature of inducible IL-12 prodrugs and is necessary to facilitate clinical development of potent cytokines for oncology treatment. [0167] Chimeric Compound 1 treatment robustly activated various tumor infiltrating innate and adaptive effector cell populations, supporting a mechanism of action where infiltration and activation of multiple effector cells plays a fundamental role in initial tumor control.
  • IL-12 as a direct activator of effector cell populations and as a driver of cross presenting dendritic cell activation will likely set apart therapeutics based on this cytokine compared to other treatments when it comes to triggering efficacy in “cold” tumors. Indeed, we observed this effect using a model of a “cold” tumor, the poorly infiltrated EMT-6 model. NanoString Digital Spatial Profiling demonstrated that systemic treatment with Chimeric Compound 1 enhanced deep infiltration of EMT-6 tumors by CD8+ T cells and confirmed the intratumoral increase in IL-12 and IFNy signaling, as well as the significant upregulation of transcripts associated with robust CD8+ T cell activation.
  • Treatment also significantly increased the clonality of the TCR repertoire among tumor infiltrating T cells and drove the expansion of several novel clones, suggesting that systemic Chimeric Compound 1 treatment resulted in the activation of a de novo T cell response to unique tumor antigens, which may be key to the CD8+ T cell dependent tumor rejection observed earlier.
  • systemic Chimeric Compound 1 treatment had a substantial effect on the metabolism of the tumor infiltrating effector cells, transforming the metabolic status of not just the activated, tumor infiltrating CD8+ T cells but also that of the intratumoral NK cells.
  • the TME is known to have several distinct characteristics when compared to a typical cellular environment, including a lower pH, hypoxic conditions, and significant competition for extracellular glucose, all of which may impair effector cell activity.
  • we observed a slight reduction in extracellular glucose uptake following Chimeric Compound 1 treatment suggesting that Chimeric Compound 1 treatment is not simply driving greater glucose uptake by tumor infiltrating cells, but is instead facilitating an increase in metabolic efficiency and increasing oxidative phosphorylation.
  • Chimeric Compound 1 In addition to the metabolic reinvigoration of the effector cells, treatment with Chimeric Compound 1 likely increased the sternness of the tumor infiltrating CD8+ T cells, with upregulation of TCF7 (the mRNA transcript associated with TCF1, the protein) and decreased expression of PD-1 and Tim-3 by these cells. Together, these data suggest that Chimeric Compound 1 treatment results in a robust and all-encompassing re-programming of the tumor infiltrating CD8+ T cell response.
  • TCF7 the mRNA transcript associated with TCF1, the protein
  • the MC38 cell line a rapidly growing colon adenocarcinoma cell line, was used. Using this tumor model, the ability of IL-12 prodrugs to affect tumor growth and body weight was examined.
  • mice were anaesthetized with isoflurane for implantation of cells to reduce the ulcerations.
  • Female C57BL/6 mice were set up with 5xl0 5 MC38 tumor cells in 0% Matrigel sc in flank. Cell injection volume was 0.1 mL/mouse
  • Mouse age at start date was 8 to 12 weeks. Pair matches were performed when tumors reach an average size of 100 - 150 mm 3 and began treatment. This was Day 1 of the study. Body weights were taken at initiation and then biweekly to the end. Caliper measurements were taken biweekly to the end. Any adverse reactions were reported immediately. Any individual animal with a single observation of > than 25% body weight loss or three consecutive measurements of >20% body weight loss was euthanized.
  • Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy. Nat Commun. Nature Research; 2020; 11.
  • Mirlekar B Pylayeva-Gupta Y. IL-12 family cytokines in cancer and immunotherapy. Cancers (Basel). 2021.

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Abstract

This disclosure relates to methods and compositions for treating cancer using an inducible IL-12 prodrug.

Description

TL-12 PRODRUGS
[01] This application claims the benefit of U.S. Provisional Application No. 63/328,708, filed on April 7, 2022, and U.S. Application No. 63/382,694, field on November 7, 2022, the entire contents of each of which are incorporated herein by reference.
1. BACKGROUND
[02] Cancer immunotherapy has rapidly established itself as the fourth pillar of cancer treatment largely owing to the clinical success of checkpoint inhibitors. Despite the durable responses achieved by some patients using these new therapies, the proportion of responders is still relatively low and restricted to only some cancer types. Tumor mutational burden, the presence or absence of T cell infiltration in tumors, and the overall immunosuppressive microenvironment of tumors greatly influences the response to immunotherapies. Although immune checkpoint blockade can prevent the physiological stop-signal that arises in response to immune activation, other approaches can be used to positively stimulate the anti-tumor immune response. One approach involves the use of immune-activating cytokines. Numerous preclinical and clinical studies have demonstrated the promise of cytokine therapy to increase anti-tumor immunity. In fact, these were some of the first cancer immunotherapies approved for clinical use. However, systemic toxicity and poor pharmacokinetic profiles have limited their clinical application.
[03] Interleukin-12 (IL-12) is a heterodimeric 70 kDa cytokine composed of two covalently linked glycosylated subunits (p35 and p40) (Lieschke et al., 1997; Jana et al., 2014). It is a potent immune agonist and has been considered a promising therapeutic agent for oncology. However, IL- 12 has shown to have a narrow therapeutic window because they are highly potent and have a short serum half-life. Consequently, therapeutic administration of IL-12 produces undesirable systemic effects and toxicities. This is exacerbated by the need to administer large quantities of cytokines (i.e., IL-12) in order to achieve the desired levels of cytokine at the intended site of cytokine action (e.g., a tumor microenvironment). Unfortunately, due to the biology of cytokine and the inability to effectively target and control their activity, cytokines have not achieved the hoped for clinical advantages in the treatment in tumors.
[04] Inducible forms of IL- 12, that are conditionally activated in the tumor microenvironment through protease cleavage to release the fully active, native IL-12 cytokine within the tumor to stimulate a potent anti-tumor immune response, are described in International Application Nos. PCT/US2019/032320, PCT/US2019/032322, and PCT/US2021/033014. These IL-12 prodrugs include a native IL- 12 molecule attached through a protease cleavable linker to a half-life extension domain (e g., anti-human serum albumin antibody binding fragment such as a VH domain) and an IL-12 blocking element (e.g., anti-IL-12 antibody binding fragment, such as a Fab or scFv) to block binding of IL-12 to IL-12Rpi or IL-12RP2 receptors on normal tissue in the periphery. Upon cleavage of the protease cleavable linker, fully active native IL-12 is released within the tumor to stimulate a potent anti-tumor immune response.
2. SUMMARY
[05] This disclosure relates to compositions and methods for treating cancer using an inducible IL-12 prodrug. The inducible IL-12 prodrugs contain an attenuated IL-12 and have a long half-life in comparison to naturally occurring IL-12. If desired, the IL-12 can be a mutein. The IL-12 mutein can be aglycosylated or partially aglycosylated. The inducible IL-12 prodrugs disclosed herein comprise two or more polypeptide chains, and the inducible IL-12 prodrug includes IL-12 subunits p35 and p40, a half-life extension element, an IL-12 blocking element and a protease cleavable linker.
[06] The inducible IL- 12 prodrug can comprise two different polypeptides. The first polypeptide can comprise an IL-12 subunit, and optionally an IL-12 blocking element. The IL-12 blocking element when present is operably linked to the IL-12 subunit through a first protease cleavable linker. The second polypeptide chain can comprise an IL-12 subunit operably linked to a half-life extension element through a second protease cleavable linker, and optionally a IL- 12 blocking element. The IL-12 blocking element when present can be operably linked to the IL-12 subunit through a protease cleavable linker or can be operably linked to the half-life extension element through a linker that is optionally protease cleavable. Only one of the first and second polypeptide contains the IL- 12 blocking element. When the IL- 12 subunit in the first polypeptide is p35, the IL-12 subunit in the second polypeptide is p40, and when the IL-12 subunit in the first polypeptide is p40, the IL- 12 subunit in the second polypeptide is p35. A preferred blocking element of this inducible IL-12 prodrug is a single chain antibody that binds IL-12 or an antigen binding fragment thereof. The cleavable linkers in this inducible IL- 12 prodrug can be the same or different.
[07] The inducible IL- 12 prodrug can comprise three different polypeptides. Typically, one polypeptide chain comprises either the p35 or p40 IL- 12 subunit, but not both, and a second polypeptide comprises the other IL-12 subunit and the third polypeptide comprises at least a portion (component) of the blocking element. The first polypeptide can comprise an IL- 12 subunit, and optionally a half-life extension element. The half-life extension element when present is operably linked to the IL- 12 subunit through a protease cleavable linker.
[08] The second polypeptide can comprise a IL-12 subunit, at least an antigen binding portion of an antibody light chain or an antigen binding portion of an antibody heavy chain, and optionally a half-life extension element. When the half-life extension element is present, it is operably linked to the IL- 12 subunit through a protease cleavable linker and the antibody heavy chain or light chain is either a) operably linked to the IL- 12 subunit through a second protease cleavable linker, or b) operably linked to the half-life extension element through an optionally cleavable linker.
[09] The third polypeptide can comprise can an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the second polypeptide, or an antibody light chain that is complementary to the heavy chain in the second polypeptide and together with said light chain forms an IL-12 binding site. When the IL-12 subunit in the first polypeptide is p35, the IL-12 subunit in the second polypeptide is p40, and when the IL-12 subunit in the first polypeptide is p40, the IL-12 subunit in the second polypeptide is p35. In this inducible IL-12 prodrug, the IL-12 blocking element is preferably an antigen binding fragment of an antibody. The antigen binding fragment comprises as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain. The protease cleavable linkers in this inducible IL- 12 prodrug can be the same or different.
[010] The inducible IL- 12 prodrug can comprise two different polypeptides wherein p35 and p40 are located on the same polypeptide chain. A first polypeptide chain can comprise p35, p40, a half-life extension element and at least an antigen binding portion of an antibody light chain. p35 and p40 can be operably linked, and the half-life extension element can be operably linked to p40 through a first protease cleavable linker and the antigen binding portion of an antibody light chain can be operably linked to p35 through a protease cleavable linker. Alternatively, the half-life extension element can be operably linked to p35 through a protease cleavable linker and the antigen binding portion of an antibody light chain is operably linked to p40 through a protease cleavable linker. The second polypeptide comprises at least an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the second polypeptide and together with said light chain forms and IL-12 binding site. The protease cleavable linkers in this inducible IL-12 prodrug can be the same or different.
[OH] In an alternative format, a first polypeptide chain can comprise p35, p40, a half-life extension element and at least an antigen binding portion of an antibody heavy chain. p35 and p40 can be operably linked, and the half-life extension element can be operably linked to p40 or through a protease cleavable linker and the antigen binding portion of an antibody heavy chain can be operably linked to p35 through a protease cleavable linker. Alternatively, the half-life extension element can be operably linked to p35 through a protease cleavable linker and the antigen binding portion of an antibody heavy chain can be operably linked to p40 through a second protease cleavable linker. A second polypeptide comprises at least an antigen binding portion of an antibody light chain that is complementary to the heavy chain in the second polypeptide and together with said light chain forms and IL- 12 binding site. The protease cleavable linkers in this inducible IL- 12 prodrug can be the same or different.
[012] In one example, the inducible IL-12 prodrug comprises a first polypeptide that does not comprise a blocking element and the second polypeptide that has the formula: [A]-[L 1 ]-[B]- [L3]-[D] or [D]-[L3]-[B]-[L1]-[A] or [B]-[L1]-[A]-[L2]-[D] or [D]-[L1]-[A]-[L2]-[B], wherein, A is an IL-12 subunit; LI is a first protease-cleavable linker; L2 is a second protease cleavable linker; L3 is an optionally cleavable linker; B is a half-life extension element; and D is a blocking element.
[013] In another example, the first polypeptide comprises the formula: [A]-[L1]-[D] or [D]- [L1]-[A]; and the second polypeptide has the formula: [A’]-[L2]-[B] or [B]-[L2]-[A’], wherein A and A’ independently are either p35 or p40, wherein when A is p35, A’ is p40 and when A is p40, A’ is p35; LI is a first protease cleavable linker; L2 is a second protease cleavable linker; B is a half-life extension element; and D is a blocking element.
[014] This disclosure relates to a method for inducing immunological memory to a tumor. The method comprises administering to a subject in need thereof and effective amount of an inducible IL-12 prodrug as described herein, wherein the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations. This disclosure relates to a method for selectively activating effector CD8+ T cells in the tumor microenvironment, and/or to a method for selectively activating tumor infiltrating lymphocytes. These methods comprise administering to a subject in need thereof and effective amount of an inducible IL- 12 prodrug as described herein, wherein the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results a significantly higher frequency of CD8+ T cells that produce TNF, IFNgamma and/or Granzyme B within the tumor.
[015] In embodiments of the methods of this disclosure the inducible IL-12 prodrug can be administered about twice a week or less frequently, once a week or less frequently or about once every two weeks or less frequently. In certain embodiments, the inducible IL- 12 prodrug can be administered about once every two weeks.
[016] Preferred, inducible IL-12 prodrugs for use in the methods of this disclosure are Chimeric Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, or an amino acid sequence variant of any of the foregoing Chimeric Compound 1 comprises a first polypeptide chain of SEQ ID NO: 1 and a second polypeptide chain of SEQ ID NO: 7, and the amino acid sequence variant of Chimeric Compound 1 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO: 1 and a second polypeptide chain can comprise at least about 80% identity to SEQ ID NO:7. Compound 2 comprises a first polypeptide chain of SEQ ID NO:2 and a second polypeptide chain of SEQ ID NO:7, and the amino acid sequence variant of Compound 2 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO:2 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO:7. Compound 3 comprises a first polypeptide chain of SEQ ID NO:3 and a second polypeptide chain of SEQ ID NO:8, and the amino acid sequence variant of Compound 3 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO: 3 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO: 8. Compound 4 comprises a first polypeptide chain of SEQ ID NON and a second polypeptide chain of SEQ ID NO:8, and the amino acid sequence variant of Compound 4 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NON and a second polypeptide chain that has at least about 80% identity to SEQ ID NO:8. Compound 5 comprises a first polypeptide chain of SEQ ID NON and a second polypeptide chain of SEQ ID NO: 8, and the amino acid sequence variant of Compound 5 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO: 5 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO:8. Compound 5 comprises a first polypeptide chain of SEQ ID NO:5 and a second polypeptide chain of SEQ ID NO:8, and the amino acid sequence variant of Compound 5 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO: 5 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO: 8. Compound 6 comprises a first polypeptide chain of SEQ ID NO:6 and a second polypeptide chain of SEQ ID NO:8, and the amino acid sequence variant of Compound 6 can comprise a first polypeptide chain that has at least about 80% identity to SEQ ID NO: 6 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO:8.
3. BRIEF DESCRIPTION OF THE DRAWINGS
[017] The drawings are not necessarily to scale or exhaustive. Instead, the emphasis is generally placed upon illustrating the principles of the inventions described herein. The accompanying drawings, which constitute part of the specification, illustrate several embodiments consistent with the disclosure and, together with the description, serve to explain the principles of the disclosure. In the drawings:
[018] FIG. 1 is a graph showing the in vitro activity of Chimeric Compound 1 in the IL- 12 HEK-Blue reporter assay, comparing intact Chimeric Compound 1 (squares) and cleaved Chimeric Compound 1 (triangles) to chimeric IL-12 (circles).
[019] FIGs. 2A-2B show that Chimeric Compound 1 is well tolerated and induces tumor regression in a cleavage dependent manner. FIG. 2A is a graph showing anti-tumor activity of Chimeric Compound 1 at various doses in the murine model. Chimeric Compound 1 was dosed intraperitonially twice a week for two weeks at 7 pg/dose and 43 pg/dose and a NC (Non- cleavable) version of Chimeric Compound 1 was dosed at 43 pg/dose. FIG. 2B is a graphic depiction of the calculated therapeutic window for chimeric IL- 12 and Chimeric Compound 1 on a per molar basis using the same tumor model (MC38), based on the identification of the active and toxic dose level for both treatments.
[020] FIGs. 3A-3G are graphs showing that Chimeric Compound 1 generates anti-tumor immunity and protective memory in multiple syngeneic tumor models. FIGs. 3A-3E show antitumor activity of Chimeric Compound 1 at various doses in various murine syngeneic tumor models, CT26 model (FIG. 3A), B16-F10 model (FIG. 3B), EMT-6 model (FIG. 3C), A20 model (FIG. 3D), and EG7.OVA model (FIG. 3E). Mice were dosed twice a week with the dose noted on the figure legends for a total of two weeks. FIGs. 3F and 3G are graphs showing tumor volume in the EMT6 (FIG. 3F) and MC38 (FIG. 3G) models re-challenged with the same tumor on the opposite flank over time, demonstrating that treatment with Chimeric Compound 1 induces immunological memory against the same tumor type.
[021] FIGs. 4A-4F show that Chimeric Compound Itreatment reshapes the tumor microenvironment and induces activation of intratumoral effector cells (NKs and CD8+ T cells) in the MC38 model. FIG. 4A is a heatmap of transcripts with statistically significant differences between the two treatments derived from nanostring analysis of bulk RNA from tumor samples. Transcripts were excluded from the heat map if they had average normalized counts below 50. Each lane represents an individual animal. FIG. 4B shows a volcano plot of transcripts differentially expressed between Chimeric Compound 1 and vehicle treated mice. FIGs. 4C-4D show the frequency of tumor infiltrating NK cells producing IFNy or Granzyme B. FIGs. 4E are flow cytometry images showing the frequency of tetramer positive CD8+ T cells producing IFNy and/or TNF. FIG. 4F are pie chart graphs showing the frequency of polyfunctional tetramer positive CD8+ T cells measured by examining co-expression of IFNy, TNF, and Granzyme B. [022] FIGs. 5A-5F show that Chimeric Compound 1 treatment reshapes the tumor microenvironment and activates B16-F10 tumor infiltrating NK cells and CD8+ T cells. FIG. 5A shows a heatmap of transcripts with statistically significant differences in expression between the two treatments derived from nanostring analysis of bulk RNA from tumor samples. Transcripts were excluded from the heatmap if they had average normalized counts below 50. Each lane represents an individual animal. FIG. 5B shows a volcano plot of transcripts differentially expressed between Chimeric Compound 1 and vehicle-treated mice. FIG. 5C shows graphs of pathway scores for vehicle and Chimeric Compound 1 for antigen processing, interferon, MHC, and NK Cell Functions. FIG. 5D are graphs showing normalized counts from individual transcripts for vehicle and Chimeric Compound E FIG. 5E is a flow cytometry diagram showing the frequency of tetramer+ CD8+ T cells producing IFN gamma and/or Granzyme B. FIG. 5F are pie graphs showing the frequency of polyfunctional tetramer positive CD8+ T cells measured by examining co-expression of IFN gamma, TNF, and Granzyme B by flow cytometry.
[023] FIG. 6 are pie graphs showing that Chimeric Compound Itreatment induces a sustained polyfunctional CD8+ T cells response. Mice were implanted with EMT6 cells and randomized into treatment groups. Mice were dosed twice weekly for two weeks, and tumors were harvested at the indicated timepoints. The frequency of polyfunctional tumor infiltrating CD8+ T cells was measured by examining co-expression of IFN gamma, TNF, and Granzyme B. All animals in the vehicle group were out of the study by day 21 due to tumor burden.
[0241 FIG. 7A-7D shows that systemic administration of Chimeric Compound 1 results in CD8+ T cell infdtration and activation in the tumors assessed by immunofluorescence staining and the increase of IL- 12 and IFN gamma signaling by tumor infdtrating CD8+ T cells. Mice were implanted with EMT6 cells and randomized into treatment groups. Mice were dosed twice weekly for two weeks, and tumors were harvested on Day 11. Nanostring GeoMX analysis was performed on FFPE tumor tissues. FIG. 7A are immunofluorescence images of tumor infdtrating CD8+ T cells in vehicle and Chimeric Compound 1. FIG. 7B is a graph showing differential gene expression analysis of tumor infdtrating CD8+ T cells. FIGs. 7C-7D are heat maps showing genes associated with IL-12 (FIG. 7C) and IFN gamma (FIG. 7D) signaling.
[025] FIGs. 8A-8B are graphs showing anti-tumor activity of Chimeric Compound 1 in various studies of the murine syngeneic MC38 tumor model. FIG. 8A is a graph showing tumor growth over time in MC38 tumor bearing mice treated with Chimeric Compound 1 (+/-) daily FTY720 treatment. FIG. 8B is a graph showing tumor growth over time in MC38 tumor bearing mice dosed twice a week with CD4, CD8, and NK cell depleting antibodies in conjunction with Chimeric Compound 1.
[026] FIGs. 9A-9C show that Chimeric Compound 1 is preferentially activated within the TME and expands the therapeutic window compared to chimeric IL- 12. FIGs. 9A-9B are graphs showing the presence of total Chimeric Compound 1 or free chimeric IL- 12 over time from plasma (FIG. 9A) or tumor (FIG. 9B) from MC38 tumor-bearing mice treated with Chimeric Compound 1. The area under the curve was calculated, and the ratio of total Chimeric Compound 1 to free chimeric IL-12 was calculated. FIG. 9C are pie chart graphs showing the frequency of polyfunctional CD8+ T cells in the tumor, peripheral blood, tumor draining or nontumor draining lymph nodes in MC38 tumor bearing mice dosed twice with Chimeric Compound 1. The frequency of polyfunctional CD8+ T cells was measured by examining co-expression of IFN gamma, TNF, and Granzyme B after PMA/ionomycin restimulation.
[027] FIGs. 10A-10F show Chimeric Compound 1 activates tumor infdtrating immune cell populations in the MC38 syngeneic tumor model. FIG. 10A are representative flow plots of CD1 lb+ and CD103+ tumor infdtrating dendritic cells. FIG. 10B is a graph showing the ratio of CD1 lb+ and CD103+ tumor infdtrating dendritic cells. FIG. 10C is a graph showing the frequency of CD4+ T conventional cells with a THI phenotype (Tbet+ IFN gamma + TFN+). FIG. 10D are representative flow plots showing the frequency of tumor infiltrating FoxP3+ Tregs producing IFN gamma and TNF. FIGs. 10E-10F are graphs depicting the frequency of tumor infiltrating FoxP3+ Tregs producing IFN gamma and TNF (FIG. 10E) and Tbet (FIG. 10F). Unless otherwise stated, data are represented as the mean + SD, and P values are derived from t tests (**, p <0.01; ***, p < 0.001; ****, p <0.00001).
[028] FIGs. 11A-11E show that systemic treatment with Chimeric Compound 1 expands novel TCR clones and increases overall clonality of the TCR repertoire. FIG. 11A is a heat map depicting intratumoral CD8+ T cells downstream TCR signaling following vehicle and Chimeric Compound 1 treatment. FIGs. 11B-11C are graphs depicting the clone frequency of individual VDJ recombination on the TCR-beta chain in an EMT-6 tumor model treated with vehicle and Chimeric Compound 1. Live T cells were isolated from EMT-6 tumors on Day 11 and TCR sequencing was performed. FIG. 11D is graph depicting the clonality index score for vehicle and Chimeric Compound 1. FIG. 1 IE is a graph depicting the frequency of the top fifty TCR clones plotted for each animal.
[029] FIGs. 12A-12V show that Chimeric Compound 1 treatment drives increased mitochondrial respiration and fitness. FIG. 12A is a heatmap of tumor infiltrating CD8+ T cells depicting genes associated with glycolysis. FIGs. 12B-12C are graphs showing intake of 2- NDBG in EMT-6 TILs from either vehicle or Chimeric Compound 1 treated animals. FIGs. 12D-12F are heatmaps of tumor infiltrating CD8+ T cells depicting genes associated with the TCA cycle (FIG. 12D), mitochondrial biogenesis (FIG. 12E), and mitochondrial translation (FIG. 12F). FIGs. 12G, 12H, 12K, 12L, 120, 12P are graphs depicting EMT-6 infiltrating CD8+ T cells from either vehicle or Chimeric Compound 1 treated animals stained with mitotracker red (FIGs. 12G and 12H), TMRM (FIGs. 12K, 12L), MitoSOX (FIGs. 12O-12P). FIGs. 121, 12 J, 12M, 12N, 12Q, 12R are graphs depicting EMT-6 infiltrating NK cells from either vehicle or Chimeric Compound 1 treated animals stained with mitotracker red (FIGs. 121 and 12J), TMRM (FIGs. 12M-12N), MitoSOX (FIGs. 12Q-12R). Data are presented as mean + SD, and P values are derived from t tests (*, p<0.05, ** p<0.01; ***, p<0.001, pO.OOOl). FIGs. 12S-12T show that Chimeric Compound 1 preferentially expands new clones rather than previously present clones. FIGs 12S and 12T disclose SEQ ID NOS 450-457 and 458-465, respectively, from left to right, which are sequences from TCRs and define T cell clones. FIGs. 17A-17B are graphs showing the percentage of TCR repertoire for the top 50 shared clones. FIGs. 12U-12V shows that Chimeric Compound 1 treatment increases mitochondrial mass and fitness in tumor infiltrating immune cells.
[030] FIGs. 13A-13B show that Compound 36 is inducible, stable in human serum, and selectively processed by dissociated primary human tumor samples. FIG. 13A depicts a western blot analysis of Compound 36 that was diluted into healthy human serum from n = 6 donors and incubated at 37 °C for 24 or 72 hours before analysis. FIG. 13B is a graph of protein cleavage after incubation with primary human dissociated tumor samples (n = 88) or primary human healthy cells (n = 13) exposed to Compound 36 for 48 hours before protein cleavage was measured by activity in a human Tblast assay. Box plots represent the 25th and 75th percentile, while the line represents the median value for each indication. Whiskers represent the minimum and maximum values within a given indication.
[031] FIGs. 14A-14B are graphs showing IFN gamma production by intracellular cytokine staining with or without ex vivo restimulation in TILs from mice treated with either vehicle or Chimeric Compound 1.
[032] FIGs. 15A-15B show selective activation of tumor infdtrating immune cells by Chimeric Compound 1. FIG. 15A is a graph showing the frequency of conventional CD4+ T conventional cells (FoxP3-) producing IFNy and TNF in the tumor tissue compared to peripheral tissue. FIG. 15B is a graph showing the frequency of NK cells producing IFNv and TNF in the tumor tissue compared to peripheral tissue.
[033] FIGs. 16A, 16C, 16E, 16G, 161, 16K, 16M, 160, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, 16ZO are graphs showing the activity of IL- 12 prodrugs in a HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares depict activity of the intact inducible IL- 12 prodrug and triangles depict the activity of the in vitro protease activated (cleaved) inducible IL-12 prodrug. Circles depict activity of the control chimeric IL-12. EC50 values for each are shown in the tables (N.D. = not determined). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirm that inducible IL- 12 prodrugs are active and inducible. FIGs. 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays with elastase. [034] FTGs. 17A, 17C, 17E, 17G, and 171 are graphs showing results of analyzing inducible IL- 12 prodrugs in a syngeneic MC38 mouse tumor model. They show average tumor volume over time in mice treated with 5 pg, 50 pg, and 500 pg of each inducible IL-12 prodrug dosed biweekly. Data show the tumor volume was inhibited over time in a dose-dependent manner. FIGs. 17B, 17D, 17F, 17H, and 17J are graphs showing body weight average of the groups over time.
[035] FIGs. 18A-18N are schematic illustrations depicting various inducible IL-12 prodrugs.
4. DETAILED DESCRIPTION
A. IL-12 Prodrugs
[036] The disclosure relates to inducible IL-12 polypeptide prodrugs that contain an attenuated IL- 12 and that have a long half-life in comparison to naturally occurring IL- 12. The IL- 12 polypeptide prodrugs disclosed herein contain at least one polypeptide chain, and can contain two or more polypeptides, if desired. The two or more polypeptide chains disclosed herein are different, i.e., the complexes can be heterodimers, heterotrimers, and the like. The inducible IL- 12 prodrugs comprises a p35 IL- 12 subunit, a p40 IL- 12 subunit, a half-life extension element, an IL- 12 blocking element, and a protease cleavable linker. The p35 subunit and the p40 subunit associate to form the TL-12 heterodimer, which has intrinsic IL-12 receptor agonist activity. Tn the context of the inducible IL-12 prodrug, the IL-12 receptor agonist activity is attenuated and the circulating half-life is extended. The IL-12 receptor agonist activity is attenuated through the blocking element. The half-life extension element can also contribute to attenuation, for example through steric effects. The blocking element is capable of blocking the activity of all or some of the receptor agonist activity of IL-12 by sterically blocking and/or noncovalently binding to IL- 12 (e.g., to p35, p40, or the p35p40 complex). Upon cleavage of the protease cleavable linker a form of IL-12 is released from the inducible IL-12 prodrug that is active (e.g., more active than the inducible IL-12 prodrugs). Typically, the released IL-12 is at least 10 x more active than the inducible IL-12 prodrug. Preferably, the released IL-12 is at least 20 x, at least 30 x, at least 50 x, at least 100 x, at least 200 x, at least 300 x, at least 500 x, at least 1000 x, at least about 10,000X or more active than the inducible IL- 12 prodrug.
[037] The form of IL- 12 that is released upon cleavage of the inducible IL- 12 prodrug typically has a short half-life, which is often substantially similar to the half-life of naturally occurring IL- 12. Even though the half-life of the inducible IL-12 prodrug is extended, toxicity is reduced or eliminated because the circulating inducible TL-12 prodrug is attenuated and active TL-12 is targeted to the desired site (e.g., tumor microenvironment).
[0381 It will be appreciated by those skilled in the art, that the number of polypeptide chains, and the location of the p35 and p40 subunits, the half-life extension element, the protease cleavable linker(s), and the blocking element (and components of such elements, such as a VH or VL domain) on the polypeptide chains can vary and is often a matter of design preference. All such variations are encompassed by this disclosure.
[039] In embodiments, the inducible IL-12 prodrug comprises two different polypeptide chains. Typically, the first polypeptide chain comprises p35 and the second polypeptide chain comprises p40. The p35 and p40 subunits associate to form a biologically active heterodimer. The p35p40 heterodimer complex can be covalently linked, for example through a disulfide bond.
[040] In embodiments, either the first of the second polypeptide can comprise an IL- 12 blocking element (e.g., an scFV that binds IL-12) that is operably linked to the IL-12 subunit through a protease cleavable linker. The other polypeptide chain can further comprise a half-life extension element that is operably linked to the IL-12 subunit through a protease cleavable linker. Preferably, the inducible IL- 12 prodrug includes one functional blocking element and one functional half-life extension element. For example, when the first polypeptide chain comprises an IL-12 blocking element, the second polypeptide chain does not comprise an IL-12 blocking element. In other embodiments, one polypeptide chain includes either p35 or p40, and further includes a half-life extension element and a blocking element, each of which is operably linked to the p35 or p40 through a protease cleavable linker (e.g., one or more protease cleavable linker), and the other polypeptide include the complementary IL- 12 subunit (e.g., either p40 or p35). The IL-12 blocking element on the second polypeptide can be operably linked to the IL-12 subunit through a protease cleavable linker. Alternatively, the IL- 12 blocking element can be operably linked to the half-life extension element through an optional protease cleavable linker. The protease cleavable linkers on the first and second polypeptide chains can be the same or can be different. Preferably, the protease cleavable linkers on the first and second polypeptide chains are the same. The blocking element in this inducible IL-12 prodrug can be a single chain antibody. Any single chain antibody that has binding specificity for IL-12 can be a blocking element. Preferably, the blocking element is a scFv. [041] While the inducible TL-12 prodrugs disclosed herein preferably contain one half-life extension element and one blocking element, such elements can contain two or more components that are present on the same polypeptide chain or on different polypeptide chains. Illustrative of this, and as disclosed and exemplified herein, components of the blocking element can present on separate polypeptide chains. For example, a first polypeptide chain can include an antibody light chain (VL+CL) or light chain variable domain (VL) and a second polypeptide can include an antibody heavy chain Fab fragment (VH + CHI) or heavy chain variable domain (VH) that is complementary to the VL+ CL or VL on the first polypeptide. In such situations, these components can associate in the inducible IL- 12 prodrugs to form an antigen-binding site, such as a Fab that binds IL- 12 and attenuates IL- 12 activity.
[042] In embodiments, the p35 and p40 subunit can be located on the same polypeptide chain, and linked through and optionally protease cleavable linker. In such embodiments of two or multichain prodrugs, at least one of the half-life extension element, the blocking element, or a component of the half-life extension or blocking element is on a separate polypeptide. For example, a first polypeptide can include p35 and p40, linked through an optionally cleavable polypeptide chain, and other elements of the inducible IL- 12 prodrug are located on a second polypeptide chain. In another example, the first polypeptide chain comprises the p35 subunit, the p40 subunit, the half-life extension element, and a portion of an antibody light chain. The second polypeptide contains a portion of an antibody heavy chain that is complementary to the antibody light chain. The portion of the antibody light chain together with the complementary heavy chain associate in the inducible IL-12 prodrug to form a binding site for IL-12. In another example, the first polypeptide comprises the p35 subunit, the p40 subunit, the half-life extension element, and a portion of an antibody heavy chain. In this example the second polypeptide contains a portion of an antibody light chain that is complementary to the antibody heavy chain. The portion of the antibody heavy chain together with the complementary light chain associate in the inducible IL- 12 prodrug to form a binding site for IL-12. In these inducible IL-12 prodrugs, the p35 subunit and p40 subunit can be operably linked through an optional protease cleavable linker. Preferably, the p35 subunit and the p40 subunit are operably linked by a non-cleavable linker.
[043] In the inducible IL-12 prodrugs disclosed herein, the half-life extension element is preferably operably linked to either the p35 subunit or the p40 subunit through a protease cleavable linker. For example, the inducible IL-12 prodrug can include a first polypeptide in which p35 or p40 is operably linked to a half-life extension element through a protease cleavable linker. In another example, the inducible IL- 12 prodrug can include a first polypeptide in which p35 or p40 is operably linked to a half-life extension element through a protease cleavable linker, and the half-life extension element is further operably linked to a blocking element (or component of a blocking element) through an optionally protease cleavable linker. In such exemplary embodiments, the inducible IL-12 prodrug comprises at least one additional polypeptide that includes the IL-12 subunit (p40 or p35) that is not present on the first polypeptide. Additional arrangements of the elements of the inducible IL- 12 prodrug are envisioned and encompassed by this disclosure. For example, the blocking element can be operably linked to either the p35 subunit or the p40 subunit through a protease cleavable linker. One of the half-life extension element or the blocking element can be operably linked to the p35 subunit, and the other of the half-life or extension element or the blocking element can be operably linked to the p40 subunit. When the half-life extension element is operably linked to the p35 subunit, the blocking element can be operably linked to the p40 subunit. When the half-life extension element is operably linked the p40 subunit, the blocking element can be operably linked to the p35 subunit. The blocking element in this inducible IL-12 prodrug is preferably a Fab.
[044] The inducible IL-12 prodrugs can comprise three polypeptide chains. Typically, one polypeptide chain comprises either the p35 or p40 IL- 12 subunit, but not both, and a second polypeptide comprises the other IL-12 subunit and the third polypeptide comprises at least a portion (component) of the blocking element. When the IL- 12 subunit on the first polypeptide is p35, the IL-12 subunit on the second polypeptide is p40. When the IL-12 subunit on the first polypeptide is p40, the IL- 12 subunit on the second polypeptide is p35. When the polypeptides are expressed and folded, the p35 and p40 subunits can associate to form a biologically active heterodimer. The p35p40 heterodimer complex can be covalently linked, for example through a disulfide bond.
[045] In some embodiments, the first polypeptide can additionally comprise a half-life extension element that when present is operably linked to the IL- 12 subunit through a protease cleavable linker. The second polypeptide further comprises a portion of the blocking element, and the third polypeptide can comprise the remainder of the blocking element. In such a inducible IL-12 prodrug, the IL-12 blocking element can be antigen binding fragment of an antibody that is formed by the interaction of polypeptide two and polypeptide three, e g. a Fab fragment. In embodiments, the second polypeptide can comprise at least an antigen binding portion of an antibody light chain. Alternatively, the second polypeptide can comprise at least an antigen binding portion of an antibody heavy chain. The antigen binding portion of an antibody light chain or the antigen binding portion of the heavy chain can be operably linked to the IL- 12 subunit through a protease cleavable linker. In some embodiments, the second polypeptide can contain a half-life extension element. When the second polypeptide contains the half-life extension element, the first polypeptide does not contain the half-life extension element. The half-life extension element can be operably linked to the IL- 12 subunit through a protease cleavable linker. Alternatively or in addition, the half-life extension element can be operably linked to a portion of the blocking element (e.g., an antigen binding portion of an antibody light chain or the antigen binding portion of the heavy chain) through an optional protease cleavable linker. When the half-life extension element is present and operably linked to the IL- 12 subunit, the antibody heavy chain or light chain can be operably linked to the IL- 12 subunit through a protease cleavable linker, Alternatively, when the half-life extension element is present and operably linked to the IL- 12 subunit, the antibody heavy chain or light chain can be operably linked to the IL- 12 subunit through an optionally cleavable linker. The protease cleavable linkers on the first, second, and/or polypeptide chains can be the same or can be different.
[046] Chimeric Compound 1, and Compounds 2, 3, 4, 5, and 6 are specific examples of inducible IL-12 prodrugs that comprise two polypeptide chains for use according to this disclosure. Chimeric Compound 1, and Compounds 2, 3, 4, 5, and 6 and additional details regarding their activity is disclosed in International Application No.: PCT/US2021/33014. [047] Compounds 7, 8, 17, 18, 21-28, 34, and 35 are specific examples of inducible IL-12 prodrugs that comprise one polypeptide chain for use according to this disclosure. Compounds 9- 13, 15, 19, 20, 29-31, and 36 are specific examples of inducible IL-12 prodrugs that comprise two polypeptide chains for use according to this disclosure. Compounds 14, 16, 32, and 33 are specific examples of inducible IL- 12 prodrugs that comprise three polypeptide chains for use according to this disclosure. Table 1 . Exemplary Inducible TL-12 prodrugs
Figure imgf000017_0001
[048] As described above, the TL-12 can be a mutein, if desired. The TL-12 mutein retains TL-12 activity, for example intrinsic IL-12 receptor agonist activity. IL-12 subunits, p35 and/or p40 can be muteins.
[049] The invention also relates to certain single chain IL-12 inducible polypeptides. The single chain IL-12 polypeptides disclosed herein comprise IL-12, a blocking element, a half-life extension element, and a protease cleavable linker. IL-12 has receptor agonist activity for its cognate IL-12 receptor. IL-12 receptor activating activity is attenuated when the blocking element binds to IL-12. Upon cleavage of the protease cleavable linkers, active IL-12 polypeptide is released. Single chain inducible IL- 12 polypeptides have been disclosed in International Application No.: PCT/US2019/032320 and International Application No.: PCT/US2019/032322.
B. Half-Life Extension Element
[050] Contemplated herein are domains which extend the half-life of the inducible IL- 12 prodrug. Increasing the in vivo half-life of therapeutic molecules with naturally short half-lives allows for a more acceptable and manageable dosing regimen without sacrificing effectiveness. [051] The half-life extension element, increases the in vivo half-life and provides altered pharmacodynamics and pharmacokinetics of the inducible IL- 12 prodrug. Without being bound by theory, the half-life extension element alters pharmacodynamics properties including alteration of tissue distribution, penetration, and diffusion of the inducible IL-12 prodrug. In some embodiments, the half-life extension element can improve tissue targeting, tissue penetration, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension element. Without being bound by theory, an exemplary way to improve the pharmacokinetics of a polypeptide is by expression of an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor. Three types of proteins, e.g., human IgGs, HSA (or fragments), and transferrin, persist for much longer in human serum than would be predicted just by their size, which is a function of their ability to bind to receptors that are recycled rather than degraded in the lysosome. These proteins, or fragments retain FcRn binding and are routinely linked to other polypeptides to extend their serum half-life. HSA may also be directly bound to the pharmaceutical compositions or bound via a short linker. Fragments of HSA may also be used. HSA and fragments thereof can function as both a blocking element and a half-life extension element. Human IgGs and Fc fragments can also carry out a similar function.
[052] The serum half-life extension element can also be antigen-binding polypeptide that binds to a protein with a long serum half-life such as serum albumin, transferrin and the like. Examples of such polypeptides include antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like. Other suitable antigen-binding domain include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that binds to or associates with one or more target antigens.
[053] The half-life extension element as provided herein is preferably a human serum albumin (HSA) binding domain, and antigen binding polypeptide that binds human serum albumin or an immunoglobulin Fc or fragment thereof.
[054] The half-life extension element of a inducible IL-12 prodrug extends the half-life of inducible IL-12 prodrug or the by at least about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about 10 days or more. In some embodiments, the half-life extension element extends the half-life of a inducible IL-12 prodrug to at least 2-3 days, 3-4 days, 4-5 days, 5-6 days, 6-7 days, 7-8 days or more.
C. Blocking Element
[055] The blocking element can be any element that binds to IL- 12 and inhibits the ability of the inducible IL-12 prodrug to bind and activate its receptor. The blocking element can inhibit the ability of the IL-12 to bind and/or activate its receptor e.g., by sterically blocking and/or by noncovalently binding to the IL-12 prodrug. The blocking element disclosed herein can bind to pl9, p35, p40, the p35p40 heterodimeric complex, or the pl9p40 heterodimeric complex.
[0561 Examples of suitable blocking elements include the full length or an IL-12-binding fragment or mutein of the cognate receptor of IL-12. Antibodies and antigen-binding fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like that bind IL-12 can also be used. Other suitable antigen-binding domain that bind IL-12 can also be used, include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block binding of IL- 12 to its cognate receptor. Advantageously, such moieties can also function as half-life extending elements. For example, a peptide that is modified by conjugation to a water-soluble polymer, such as PEG, can sterically inhibit or prevent binding of the cytokine to its receptor. Polypeptides, or fragments thereof, that have long serum half-lives can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin and the like, as well as fragments and muteins of such polypeptides.
[057] Preferred IL- 12 blocking elements are single chain variable fragments (scFv) or Fab fragments. The scFv blocking elements comprise the amino acid sequence as set forth in SEQ ID NOs: 144-188. Alternatively, the Fab blocking element comprises the amino acid sequence as set forth in SEQ ID NOs: 189-194. The IL-12 antibody fragments encompassed by SEQ ID NOs: 144-194 have been optimized to enhance the developability of the inducible IL- 12 prodrug disclosed herein.
[058] Preferred antibody light chain blocking elements comprise SEQ ID NOs: 192-193. These preferred components can be located on one polypeptide chain and the complementary antigen binding portion of the heavy chain can be located on a second polypeptide chain. Preferred heavy chain blocking elements comprise SEQ ID NOs: 189-191 and 194. These preferred components can be located on one polypeptide chain and the complementary light chain is located on a second polypeptide chain. The antibody light chain and the antibody heavy chain together form a binding site for IL- 12.
[059] In some embodiments, the IL-12 blocking element comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to SEQ ID NOs: 144-194, e.g., over the full length of SEQ ID Nos: 144-194. Typically, the amino acid sequence of the CDRs in not altered, and amino acid substitutions are present in the framework regions.
[060] The disclosure also relates to functional variants of IL-12 blocking elements comprising SEQ ID NOs: 144-194. The functional variants of IL-12 blocking elements comprising SEQ ID NOs: 144-194 generally differ from SEQ ID NOs: 144-194 by one or a few amino acids (including substitutions, deletions, insertions, or any combination thereof), and substantially retain their ability to bind to the IL-12 polypeptide (e.g., the p35 subunit, the p40 subunit, or the p35p40 complex) and inhibit binding of IL- 12 to its cognate receptor.
[061] The functional variant can contain at least one or more amino acid substitutions, deletions, or insertions relative to the IL-12 blocking element comprising SEQ ID NOs: 144-194. The functional variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations compared to the IL-12 blocking element comprising SEQ ID NOs: 144-194. In some preferred embodiments, the functional variant differs from the IL- 12 blocking element comprising SEQ ID NOs: 144-194 by less than 10, less, than 8, less than 5, less than 4, less than 3, less than 2, or one amino acid alterations, e.g., amino acid substitutions or deletions. In other embodiments, the functional variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NOs: 144-194. The amino acid substitution can be a conservative substitution or a non-conservative substitution, but preferably is a conservative substitution.
[062] In other embodiments, the functional variants of the IL-12 blocking element may comprise 1, 2, 3, 4, or 5 or more non-conservative amino acid substitutions compared the IL- 12 blocking elements comprising SEQ ID NOs: 144-194. Non-conservative amino acid substitutions could be recognized by one of skill in the art. The functional variant of the separation moiety preferably contains no more than 1, 2, 3, 4, or 5 amino acid deletions.
[063] Also disclosed herein is an inducible IL-12 prodrug that contains a blocking element having specificity for IL- 12 and contains a half-life extension element. The blocking element is an antibody or antigen binding fragment that has binding specificity for TL-12, specifically the IL- 12 subunit beta precursor (p40) as defined by SEQ ID NO: 421, disclosed herein. The antibody or antigen binding fragment comprises an antigen binding domain that binds to the residues shown in Table 2 of SEQ ID NO: 421. This disclosure relates to an antibody or antigenbinding fragment that binds the IL-12 epitope defined by the amino acid residues shown in Table 2, and to an inducible IL- 12 prodrug that contains such an antibody or antigen-binding fragment, and to the use of such an antibody or antigen-binding fragment for the preparation of an inducible IL-12 prodrug, or a medicament containing such an inducible IL-12 prodrug.
[064] Table 2. Epitope binding residues in the IL- 12 subunit beta precursor
Figure imgf000022_0001
D. Protease Cleavable Linker
[065] As disclosed herein, the inducible IL- 12 prodrug comprises one or more linker sequences. A linker sequence serves to provide flexibility between the polypeptides, such that, for example, the blocking element is capable of inhibiting the activity of IL-12. The linker can be located between the IL- 12 subunit, the half-life extension element, and/or the blocking element. As described herein the inducible IL- 12 prodrug comprises a protease cleavable linker. The protease cleavable linker can comprise one or more cleavage sites for one or more desired protease. Preferably, the desired protease is enriched or selectively expressed at the desired target site of IL-12 (e.g., the tumor microenvironment). Thus, the inducible IL-12 prodrug is preferentially or selectively cleaved at the target site of desired IL- 12 activity.
[066] Suitable linkers are typically less than about 100 amino acids. Such linkers can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 1 to 100 amino acids. In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acids in length. Preferred linkers are typically from about 5 amino acids to about 30 amino acids.
[067] Preferably the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked domain. In a preferred embodiment, the linker is cleavable by a cleaving agent, e.g., an enzyme. Preferably, the separation moiety comprises a protease cleavage site. In some cases, the separation moiety comprises one or more cleavage sites. The separation moiety can comprise a single protease cleavage site. The separation moiety can also comprise 2 or more protease cleavage sites. For example, 2 cleavage sites, 3 cleavage sites, 4, cleavage sites, 5 cleavage sites, or more. In cases the separation moiety comprises 2 or more protease cleavage sites, the cleavage sites can be cleaved by the same protease or different proteases. A separation moiety comprising two or more cleavage sites is referred to as a “tandem linker.” The two or more cleavage sites can be arranged in any desired orientation, including, but not limited tom one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, or one cleavage site following by another cleavage site with intervening amino acids between the two cleavage sites.
[068] Of particular interest in the present invention are disease specific protease-cleavable linkers. Also preferred are protease-cleavable linkers that are preferentially cleaved at a desired location in the body, such as the tumor microenvironment, relative to the peripheral circulation. For example, the rate at which the protease-cleavable linker is cleaved in the tumor microenvironment can be at least about 10 times, at least about 100 times, at least about 1000 times or at least about 10,000 times faster in the desired location in the body, e.g., the tumor microenvironment, in comparison to in the peripheral circulation (e.g., in plasma).
[069] Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtili sin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin- 1 converting enzyme, thrombin, FAP (FAPoc), dipeptidyl peptidase, meprins, granzymes and dipeptidyl peptidase IV (DPPIV/CD26). Proteases capable of cleaving linker amino acid sequences (which can be encoded by the chimeric nucleic acid sequences provided herein) can, for example, be selected from the group consisting of a prostate specific antigen (PSA), a matrix metalloproteinase (MMP), an A Disintigrin and a Metalloproteinase (ADAM), a plasminogen activator, a cathepsin, a caspase, a tumor cell surface protease, and an elastase. The MMP can, for example, be matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 14 (MMP 14). In addition, or alternatively, the linker can be cleaved by a cathepsin, such as, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K and/or Cathepsin L. Preferably, the linker can be cleaved by MMP 14 or Cathepsin L.
[070] Proteases useful for cleavage of linkers and for use in the inducible IL- 12 prodrug disclosed herein are presented in Table 3, and exemplary proteases and their cleavage site are presented in Table 4.
Table 3. Proteases relevant to inflammation and cancer
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Table 4. Exemplary Proteases and Protease Recognition Sequences
Figure imgf000026_0002
Figure imgf000027_0001
[071] Exemplary protease cleavable linkers include, but are not limited to kallikrein cleavable linkers, thrombin cleavable linkers, chymase cleavable linkers, carboxypeptidase A cleavable linkers, cathepsin cleavable linkers, elastase cleavable linkers, FAP cleavable linkers, ADAM cleavable linkers, PR-3 cleavable linkers, granzyme M cleavable linkers, a calpain cleavable linkers, a matrix metalloproteinase (MMP) cleavable linkers, a plasminogen activator cleavable linkers, a caspase cleavable linkers, a tryptase cleavable linkers, or a tumor cell surface protease. Specifically, MMP9 cleavable linkers, ADAM cleavable linkers, CTSL1 cleavable linkers, FAPa cleavable linkers, and cathepsin cleavable linkers. Some preferred protease-cleavable linkers are cleaved by a MMP and/or a cathepsin. [072] The separation moieties disclosed herein are typically less than 100 amino acids. Such separation moieties can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 1 to 100 amino acids. In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acids in length. Preferred linkers are typically from about 5 amino acids to about 30 amino acids.
[073] Preferably the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked domains.
[074] In some embodiments, the separation moiety comprises the sequence GPAGLYAQ (SEQ ID NO: 195); GPAGMKGL (SEQ ID NO: 196); PGGPAGIG (SEQ ID NO: 197); ALFKSSFP (SEQ ID NO: 198); ALFFSSPP (SEQ ID NO: 199); LAQRLRSS (SEQ ID NO: 200);
LAQKLKSS (SEQ ID NO; 201); GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (SEQ ID NO: 202); RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (SEQ ID NO: 203);
KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO: 204); RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO: 205); KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO: 206); SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO: 207); RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (SEQ ID NO: 208); GGGALFKSSFPLAQKLKSSPGGPAGIGGGR (SEQ ID NO: 209); RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (SEQ ID NO: 210); RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (SEQ ID NO: 211); RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO: 212);
GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO: 213); GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO: 214); RGGALFKSSFPLAQKLKSSGPAGLYAQGGK (SEQ ID NO: 215); RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (SEQ ID NO: 216); SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (SEQ ID NO: 217); KGGPGGPAGTGPLAQRLRSSALFKSSFPGR (SEQ TD NO: 218);
KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (SEQ ID NO: 219); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (SEQ ID NO: 220) [075] Certain preferred separation moieties comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). The separation moieties disclosed herein can comprise one or more cleavage motif or functional variants that are the same or different. The separation moieties can comprise 1, 2, 3, 4, 5, or more cleavage motifs or functional variants. Separation moieties comprising 30 amino acids can contain 2 cleavage motifs or functional variants, 3 cleavage motifs or functional variants or more. A “functional variant” of a separation moiety retains the ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease) and are not cleaved or cleaved with low efficiency in the periphery (e.g., serum). For example, the functional variants retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation moiety comprising any one of SEQ ID NOs: 195-220 or 447-448.
[076] The separation moieties comprising more than one cleavage motif can be selected from SEQ ID NOs: 195-201 or 447-448 and combinations thereof. Preferred separation moieties comprising more than one cleavage motif comprise the amino acids selected from SEQ ID NO: 202-220.
[077] The separation moiety can comprise both ALFKSSFP (SEQ ID NO: 198) and GPAGLYAQ (SEQ ID NO: 195). The separation moiety can comprise two cleavage motifs that each have the sequence GPAGLYAQ (SEQ ID NO: 195). Alternatively or additionally, the separation moiety can comprise two cleavage motifs that each have the sequence ALFKSSFP (SEQ ID NO: 198). The separation moiety can comprise a third cleavage motif that is the same or different.
[078] In some embodiments, the separation moiety comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NOs: 195 to SEQ ID NO: 220 or 447-448 over the full length of SEQ ID NO: 195-220 or SEQ ID NOS 447-448.
[079] The disclosure also relates to functional variants of separation moieties comprising SEQ ID NOs: 195-220 or 447-448. The functional variants of separation moieties comprising SEQ ID NOs: 195-220 or 447-448 generally differ from SEQ ID NOs: 195-220 or 447-448 by one or a few amino acids (including substitutions, deletions, insertions, or any combination thereof), and substantially retain their ability to be cleaved by a protease.
[080] The functional variants can contain at least one or more amino acid substitutions, deletions, or insertions relative to the separation moi eties comprising SEQ ID NOs: 195-220 or 447-448. The functional variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations comparted to the separation moieties comprising SEQ ID NOs: 195-220 or 447-448. In some preferred embodiments, the functional variant differs from the separation moiety comprising SEQ ID NOs: 195-220 by less than 10, less, than 8, less than 5, less than 4, less than 3, less than 2, or one amino acid alterations, e.g., amino acid substitutions or deletions. In other embodiments, the functional variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NOs: 195-220 or 447-448. The amino acid substitution can be a conservative substitution or a non-conservative substitution, but preferably is a conservative substitution.
[081] In other embodiments, the functional variants of the separation moieties may comprise 1, 2, 3, 4, or 5 or more non-conservative amino acid substitutions compared the separation moieties comprising SEQ ID NOs: 195-220 or 447-448. Non-conservative amino acid substitutions could be recognized by one of skill in the art. The functional variant of the separation moiety preferably contains no more than 1, 2, 3, 4, or 5 amino acid deletions.
[082] The amino acid sequences disclosed in the separation moieties can be described by the relative linear position in the separation moiety with respect to the sissile bond. As will be well- understood by persons skilled in the art, separation moieties comprising 8 amino acid protease substrates (e.g., SEQ ID Nos: 195-201 or 447-448) contain amino acid at positions P4, P3, P2, Pl, Pl’, P2’, P3’, P4’, wherein the sissile bond is between Pl and Pl’. For example, amino acid positions for the separation moiety comprising the sequence GPAGLYAQ (SEQ ID NO: 195 ) can be described as follows:
Figure imgf000030_0002
[083] Amino acids positions for the separation moiety comprising the sequence ALFKSSFP (SEQ ID NO: 198) can be described as follows:
Figure imgf000030_0001
Figure imgf000031_0001
[084] Preferably, the amino acids surrounding the cleavage site (e.g., positions Pl and Pl’for SEQ ID NOs: 195-201 or 447-448) are not substituted.
[085] In embodiments, the separation moiety comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) or a functional variant of SEQ ID NO: 195 or a function variant of SEQ ID NO: 198. As described herein, a functional variant of PAGLYAQ (SEQ ID NO: 447) or ALFKSSFP (SEQ ID NO: 198) can comprise one or more amino acid substitutions, and substantially retain their ability to be cleaved by a protease. Specifically, the functional variants of GPAGLYAQ (SEQ ID NO: 195) is cleaved by MMP14, and the functional variant of ALFKSSFP (SEQ ID NO: 198) is cleaved by Capthepsin L (CTSL1). The functional variants also retain their ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease). For example, the functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation moiety comprising amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.
[086] Preferably, the functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) comprise no more than 1, 2, 3, 4, or 5 conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). Preferably, the amino acids at position PI and Pl’ are not substituted. The amino acids at positions Pl and Pl’ in SEQ ID NO: 195 are G and L, and the amino acids at positions Pl and Pl’ in SEQ ID NO: 198 are K and S.
[087] The functional variant of GPAGLYAQ (SEQ ID NO: 195) can preferably comprise one or more of the following: a) an arginine amino acid substitution at position P4, b) a leucine, valine, asparagine, or proline amino acid substitution at position P3, c) a asparagine amino acid substitution at position P2, d) a histidine, asparagine, or glycine amino acid substitution at position Pl, e) a asparagine, isoleucine, or leucine amino acid substitution at position Pl’, f) a tyrosine or arginine amino acid substitution at position P2’, g) a glycine, arginine, or alanine amino acid substitution at position P3’, h) or a serine, glutamine, or lysine amino acid substitution at position P4’. The following amino acid substitutions are disfavored in functional variants of GPAGLYAQ (SEQ ID NO: 195): a) arginine or isoleucine at position P3, b) alanine at position P2, c) valine at position Pl , d) arginine, glycine, asparagine, or threonine at position Pl’, e) aspartic acid or glutamic acid at position P2’, f) isoleucine at position P3’, g) valine at position P4’. In some embodiments, the functional variant of GPAGLYAQ (SEQ ID NO: 195) does not comprise an amino acid substitution at position Pl and/or Pl’.
[088] The amino acid substitution of the functional variant of GPAGLYAQ (SEQ ID NO: 195) preferably comprises an amino acid substitution at position P4 and/or P4’. For example, the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprise a leucine at position P4, or serine, glutamine, lysine, or phenylalanine at position P4. Alternatively or additionally, the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprise a glycine, phenylalanine, or a proline at position P4’.
[089] In some embodiments, the amino acid substitutions at position P2 or P2’ of GPAGLYAQ (SEQ ID NO: 195) are not preferred.
[090] In some embodiments, the functional variant of GPAGLYAQ (SEQ ID NO: 195) comprises the amino acid sequence selected from SEQ ID NOs: 221- 295. Specific functional variants of GPAGLYAQ (SEQ ID NO: 195) include GPLGLYAQ (SEQ ID NO: 259), and GPAGLKGA (SEQ ID NO: 249).
[091] The functional variants of LFKSSFP (SEQ ID NO: 448) preferably comprises hydrophobic amino acid substitutions. The functional variant of LFKSSFP (SEQ ID NO: 448) can preferably comprise one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, phenylalanine at position P3; (c) arginine, leucine, alanine, glutamine, or histatine at position P2; (d) phenylalanine, histidine, threonine, alanine, or glutamine at position Pl; has histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine at position Pl’, (f) phenylalanine, leucine, isoleucine, lysine, alanine, glutamine, or proline at position P2’; (g) phenylalanine, leucine, glycine, serine, valine, histidine, alanine, or asparagine at position P3’; and phenylalanine, histidine, glycine, alanine, serine, valine, glutamine, lysine, or leucine.
[092] The inclusion of aspartic acid and/or glutamic acid in functional variants of SEQ ID NO: 448 are generally disfavored and avoided. The following amino acid substitutions are also disfavored in functional variants of LFKSSFP (SEQ ID NO: 448): (a) alanine, serine, or glutamic acid at position P3; (b) proline, threonine, glycine, or aspartic acid at position P2; (c) proline at position Pl ; (d) proline at position Pl ’; (e) glycine at position P2’; (f) lysine or glutamic acid at position P3’; (g) aspartic acid at position P4’.
[0931 The amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 448) preferably comprises an amino acid substitution at position P4 and/or Pl. In some embodiments, an amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 448) at position P4’ is not preferred.
[094] In some embodiments, the functional variant of LFKSSFP (SEQ ID NO: 448) comprises the amino acid sequence selected from SEQ ID NOs: 296- 374. Specific functional variants of LFKSSFP (SEQ ID NO: 448) include ALFFSSPP (SEQ ID NO: 199), ALFKSFPP (SEQ ID NO: 346), ALFKSLPP (SEQ ID NO: 347); ALFKHSPP (SEQ ID NO: 335); ALFKSIPP (SEQ ID NO: 348); ALFKSSLP (SEQ ID NO: 356); or SPFRSSRQ (SEQ ID NO: 297).
[095] The separation moieties disclosed herein can form a stable complex under physiological conditions with the amino acid sequences (e.g. domains) that they link, while being capable of being cleaved by a protease. For example, the separation moiety is stable (e.g., not cleaved or cleaved with low efficiency) in the circulation and cleaved with higher efficiency at a target site (i.e. a tumor microenvironment). Accordingly, fusion polypeptides that include the linkers disclosed herein can, if desired, have a prolonged circulation half-life and/or lower biological activity in the circulation in comparison to the components of the fusion polypeptide as separate molecular entities. Yet, when in the desired location (e.g., tumor microenvironment) the linkers can be efficiently cleaved to release the components that are joined together by the linker and restoring or nearly restoring the half-life and biological activity of the components as separate molecular entities.
[096] The separation moiety desirably remains stable in the circulation for at least 2 hours, at least 5, hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.
[097] In some embodiments, the separation moiety is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% in the circulation as compared to the target location. The separation moiety is also stable in the absence of an enzyme capable of cleaving the linker. However, upon expose to a suitable enzyme (i.e., a protease), the separation moiety is cleaved resulting in separation of the linked domain. E. Pharmaceutical Compositions
[098] Also provided herein, are pharmaceutical compositions comprising an inducible IL-12 prodrug described herein, a vector comprising the polynucleotide encoding the inducible IL-12 prodrug or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier.
[099] Provided herein are pharmaceutical formulations or compositions containing the inducible IL-12 prodrugs as described herein and a pharmaceutically acceptable carrier. Compositions comprising the inducible IL- 12 prodrugs as described herein are suitable for administration in vitro or in vivo. The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the subject to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.
[0100] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic, although the formulate can be hypertonic or hypotonic if desired. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer’s solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or from about 7 to 7.5. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptides. Matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. Certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
Carriers are those suitable for administration of the IL-12 or nucleic acid sequences encoding the inducible IL-12 prodrugs to humans or other subjects. [0101] Tn some embodiments of the pharmaceutical compositions, the inducible TL-12 prodrug described herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical compositions, the inducible IL-12 prodrug is attached to liposomes. In some instances, the inducible IL-12 prodrug are conjugated to the surface of liposomes. In some instances, the inducible IL- 12 prodrugs are encapsulated within the shell of a liposome. In some instances, the liposome is a cationic liposome.
[0102] The inducible IL- 12 prodrug described herein are contemplated for use as a medicament. Administration is effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient’s size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently. An “effective dose” refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.
[0103] Optionally, the inducible IL-12 prodrug or nucleic acid sequences encoding the inducible IL-12 prodrug are administered by a vector. There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and/or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express and preferably secrete the encoded chimeric polypeptide or to therapeutically deliver nucleic acids to a subject. The components of the IL-12 polypeptide disclosed herein are typically operably linked in frame to encode a fusion protein. [0104] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the nucleic acid molecule and/or polypeptide in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general and methods of making them are described by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). The construction of replicationdefective adenoviruses has been described (Berkner et al., J. Virol. 61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virol. 57:267-74 (1986); Davidson et al., J. Virol. 61: 1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)). The benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.
[0105] The provided inducible IL- 12 prodrugs and/or nucleic acid molecules can be delivered via virus like particles. Virus like particles (VLPs) consist of viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).
[0106] The inducible IL-12 prodrugs disclosed herein can be delivered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003). The provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006/110728.
[0107] Non-viral based delivery methods, can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding polypeptides, wherein the nucleic acids are operably linked to an expression control sequence. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif), and Invitrogen/Life Technologies (Carlsbad, Calif). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to make the inducible IL-12 prodrugs by expression in a suitable host cell, such as CHO cells.
[0108] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g., P-actin promoter or EFla promoter, or from hybrid or chimeric promoters (e g., CMV promoter fused to the P-actin promoter). Of course, promoters from the host cell or related species are also useful herein. [0109] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' or 3' to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 base pairs (bp) in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
[0110] The promoter and/or the enhancer can be inducible (e.g., chemically or physically regulated). A chemically regulated promoter and/or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. A physically regulated promoter and/or enhancer can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize the expression of the region of the transcription unit to be transcribed Tn certain vectors, the promoter and/or enhancer region can be active in a cell type specific manner. Optionally, in certain vectors, the promoter and/or enhancer region can be active in all eukaryotic cells, independent of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the P-actin promoter, the EF 1 a promoter, and the retroviral long terminal repeat (LTR).
[0111] The vectors also can include, for example, origins of replication and/or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.
F. Therapeutic Applications
[0112] Also provided herein, are methods and uses for the treatment of a disease, disorder or condition associated with a target antigen comprising administering to a subject in need thereof a inducible IL-12 prodrug as described herein. Diseases, disorders, or conditions include, but are not limited to, cancer, inflammatory disease, an immunological disorder, autoimmune disease, infectious disease (i.e., bacterial, viral, or parasitic disease). Preferably, the disease, disorder, or condition is cancer.
[0113] Any suitable cancer may be treated with the inducible IL-12 prodrugs provided herein. Illustrative suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor. In embodiments, the cancer is melanoma or breast cancer.
[0114] In some embodiments, provided herein is a method of enhancing an immune response in a subject in need thereof by administering an effective amount of an inducible IL- 12 prodrug provided herein to the subject. The enhanced immune response may prevent, delay, or treat the onset of cancer, a tumor, or a viral disease. Without being bound by theory, the inducible IL- 12 prodrug enhances the immune response by activating the innate and adaptive immunities. In some embodiments, the methods described herein increase the activity of Natural Killer Cells and T lymphocytes. In some embodiments, the inducible IL- 12 prodrug provided herein, can induce IFNy release from Natural Killer cells as well as CD4+ and CD8+ T cells. [0115] This disclosure relates to a method for selectively activating tumor infiltrating lymphocytes, comprising administering to a subject in need thereof and effective amount of an inducible interleukin-2 (IL- 12) prodrug as described herein. Typically, the inducible IL- 12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results a significantly higher frequency of CD8+ T cells that produce IFNgamma and/or Granzyme.
[0116] This disclosure relates to a method for inducing immunological memory to a tumor, comprising administering to a subject in need thereof an effective amount of an inducible interleukin- 12 (IL-12) prodrug as described herein. Typically, the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other areas.
[0117] This disclosure relates to a method for selectively activating effector CD8+ T cells in the tumor microenvironment, comprising administering to a subject in need thereof an effective amount of an inducible interleukin- 12 (IL-12) prodrug as described herein. Typically, the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results a significantly higher frequency of CD8+ T cells that produce TNF and/or IFNgamma within the tumor in comparison to peripheral tissue.
[0118] The method can further involve the administration of one or more additional agents to treat cancer, such as chemotherapeutic agents (e.g., Adriamycin, Cerubidine, Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), immuno-oncology agents (e.g., anti-PD-Ll, anti- CTLA4, anti-PD-1, anti-CD47, anti-GD2), cellular therapies (e.g., CAR-T, T-cell therapy), oncolytic viruses and the like. Non-limiting examples of anti-cancer agents that can be used include acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacytidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-nl interferon alpha-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinzolidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. [0119] The IL-12 prodrug can be administered to a subject in need thereof in combination with an immune checkpoint inhibitor. Immune checkpoint proteins include, for example, PD-1 which binds ligands PD-L1 (B7-H1 , CD274) and PD-L2 (B7-DC, CD273), CTLA-4 (GDI 52) which binds B7-1 (CD80) and B7-2 (CD86), LAG 3 (CD223) which binds Galectin3, LSECtin and FGL1; TIM3 (HAVCR2) which binds ligands Ceacaml and Galectin9; TIGIT (VSTM3, WUCAM) which binds CD112 and CD155; BTLA (CD272) which binds HVEM (TNFRSF14), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD160 (BY55) which bind HVEM; CD 134 (TNRFSR4, 0X40) which binds CD252 (OX-40L). Therapeutic agents, such as antibodies, that bind immune checkpoint proteins and inhibit their immunosuppressive activity include the anti-PDl antibodies pembrolizumab (KEYTRUDA), dostarlimab (JEMPERLI), cemiplimab-rwlc (LIBATYO), nivolumab (OPDIVO), camrelizumab, tislelizumab, toripalimab, and sintilimab (TYVYT); the anti-PD-Ll antibodies avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ); the anti-CTLA-4 antibody ipilimumab (YERVOY).
[0120] In some embodiments of the methods described herein, the inducible IL-12 prodrug is administered in combination with an agent for the treatment of the particular disease, disorder, or condition. Agents include, but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroidal, peptide, and the like), radiotherapies (y-rays, C- rays, and/or the directed delivery of radioisotopes, microwaves, UV radiation and the like), gene therapies (e.g., antisense, retroviral therapy and the like) and other immunotherapies. In some embodiments, the inducible IL-12 prodrug is administered in combination with anti-diarrheal agents, anti-emetic agents, analgesics and/or non-steroidal anti-inflammatory agents.
G. Definitions
[0121] All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present disclosure. When a range of values is expressed, it includes embodiments using any particular value within the range. Further, reference to values stated in ranges includes each and every value within that range. All ranges are inclusive of their endpoints and combinable. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The use of “or” will mean “and/or” unless the specific context of its use dictates otherwise.
[0122] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.
[0123] As used herein, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.
[0124] Unless otherwise indicated, the terms “at least,” “less than,” and “about,” or similar terms preceding a series of elements or a range are to be understood to refer to every element in the series or range. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
[0125] As used herein, the terms “activatable,” “activate,” “induce,” and “inducible” refers to a polypeptide complex that has an attenuated activity form (e.g., attenuated receptor binding and/or agonist activity) and an activated form. The polypeptide complex is activated by protease cleavage of the linker that causes the blocking element and half-life extension element to dissociate from the polypeptide complex. The induced/activated polypeptide complex can bind with increased affinity/avidity to the IL-12 receptor.
[0126] The terms “antibody” and “immunoglobulin” are used interchangeably herein. An antibody or immunoglobulin, as used herein, is intended to refer to immunoglobulin molecules comprised of two heavy (H) chains. Typically, antibodies in mammals (e g., humans, rodents, and monkey’s) comprise four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multi specific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, or tetrameric antibodies comprising two heavy chain and two light chain molecules. One of skill in the art would recognize that other forms of antibodies exist (e.g. camelid and shark antibodies).
[0127] The term “attenuated” as used herein is an IL-12 receptor agonist that has decreased receptor agonist activity as compared to the IL-12 receptor’s naturally occurring agonist. An attenuated IL- 12 agonist can have at least about 10X, at least about 50X, at least about 100X, at least about 250X, at least about 500X, at least about 1000X or less agonist activity as compared to the receptor’s naturally occurring agonist. When a IL-12 polypeptide complex that contains IL-12 as described herein is described as “attenuated” or having “attenuated activity”, it is meant that the IL-12 polypeptide complex is an attenuated IL-12 receptor agonist.
[0128] The term “cancer” refers to the physiological condition in mammals in which a population of cells is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate and/or certain morphological features. Often cancers can be in the form of a tumor or mass, but may exist alone within the subject, or may circulate in the blood stream as independent cells, such a leukemic or lymphoma cells. The term cancer includes all types of cancers and metastases, including hematological malignancy, solid tumors, sarcomas, carcinomas and other solid and non-solid tumors. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., triple negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancers. Triple negative breast cancer refers to breast cancer that is negative for expression of the genes for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu.
[0129] A “conservative” amino acid substitution, as used herein, generally refers to substitution of one amino acid residue with another amino acid residue from within a recognized group which can change the structure of the peptide but biological activity of the peptide is substantially retained. Conservative substitutions of amino acids are known to those skilled in the art. Conservative substitutions of amino acids can include, but not limited to, substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. For instance, a person of ordinary skill in the art reasonably expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological activity of the resulting molecule.
[0130] As used herein, the term “half-life extension element” in the context of the polypeptide complex disclosed herein, refers to a chemical element, preferable a polypeptide that increases the serum half-life and improve pK, for example, by altering its size (e.g., to be above the kidney filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.
[0131] As used herein, the term “operably linked” in the context of a polypeptide complex refers to the orientation of the components of a polypeptide complex that permits the components to function in their intended manner. For example, a polypeptide comprising an IL-12 subunit and an IL- 12 blocking element are operably linked by a protease cleavable linker in a polypeptide complex when the IL-12 blocking element is capable of inhibiting the IL-12 receptor-activating activity of the IL-12 polypeptide, but upon cleavage of the protease cleavable linker the inhibition of the IL- 12 receptor-activating activity of the IL- 12 polypeptide by the IL- 12 blocking element is decreased or eliminated, for example because the TL-12 blocking element can diffuse away from the IL- 12.
[0132] As used herein, the terms “peptide”, “polypeptide”, or “protein” are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.
[0133] The term “subject” herein to refers to any animal, such as any mammal, including but not limited to, humans, non-human primates, rodents, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.
[0134] As used herein, the term “therapeutically effective amount” refers to an amount of a compound described herein (i.e., a IL-12 polypeptide complex) that is sufficient to achieve a desired pharmacological or physiological effect under the conditions of administration. For example, a “therapeutically effective amount” can be an amount that is sufficient to reduce the signs or symptoms of a disease or condition (e.g., a tumor). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. A therapeutically effective amount of a pharmaceutical composition can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmaceutical composition to elicit a desired response in the individual. An ordinarily skilled clinician can determine appropriate amounts to administer to achieve the desired therapeutic benefit based on these and other considerations.
5. EQUVALENTS
[0135] It will be readily apparent to those skilled in the art that other suitable modifications and adaptions of the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments. Having now described certain compounds and methods in detail, the same will be more clearly understood by reference to the following examples, which are introduced for illustration only and not intended to be limiting. 6. EXAMPLES
[0136] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided herein.
[0137] In the mouse studies described herein Chimeric Compound 1, an IL-12 prodrug that includes murine IL- 12 p35 and human IL- 12 p40 and thus is active in mouse models, was used as a surrogate for human IL-12 prodrugs that include human p35 and p40. A chimeric IL-12 that included murine p35 and human p40 was used as a control. Chimeric Compound 1 includes murine IL-12 p35 and is a surrogate for Compound 36 which includes human IL-12 p35 and is otherwise the same as Chimeric Compound 1.
Example 1.
1.1 Materials and Methods
1. HEK-Blue IL-12 Reporter Assay
[0138] HEK-Blue IL- 12 cells (InvivoGen) were plated in suspension at a density of 50,000 cells/well in culture media with or without 15 or 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL-12, chimeric IL-12 (mouse p35/human p40), activatable chimeric IL-12, or activatable hIL-12 for 20-24 hours at 37°C and 5% CO2. Activity of uncleaved and cleaved activatable hIL-12 was tested. Cleaved inducible hIL-12 was generated by incubation with active MMP9 or CTSL-1. IL-12 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay. Results confirm that IL- 12 fusion proteins are active and inducible. Results are shown in FIG. 1.
2. Cell Lines
[0139] All cell lines were grown and maintained by Charles River Laboratories (Morrisville, NC and Worcester, MA) according to ATCC guidelines and kept in culture for no longer than 2 weeks. Frozen cells were thawed and maintained for 1-3 passages before implantation. B16-F10, CT26, and EMT-6 cell lines were cultured in RPML1640 with L-Glutamine (Gibco, 11875-085) with 10% heat-inactivated fetal calf serum (Gibco, 35-015-CV) while MC38 was cultured in Dulbecco's Modified Eagle Medium (Gibco, 1966-025) supplemented with 10% heat-inactivated fetal calf serum (Gibco, 16000-044). Prior to tumor implantation, cells were washed twice with PBS and counted. 3. MC38 Model Tumor Implantation
[0140] All mouse in vivo work was performed in accordance with current regulations and standards and the NTH at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of an Institutional Animal Care and Use Committee (IACUC). Female, 6-8 week- old C57B1/6 mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation. A total of 5 x 105 MC38 cells were injected subcutaneously and monitored for tumor growth. Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was 100-150 mm3, and mice were randomized into treatment groups on Day 0. Mice receiving Chimeric Compound 1 were dosed twice a week for two weeks (Days 1, 4, 8, and 11) unless otherwise noted. Inducible IL-12 prodrugs used in these studies included Chimeric Compound 1. Mice receiving recombinant chimeric IL-12 (chimeric IL-12 or WW0295) were dosed twice a day for 5 days before receiving a 2-day break (5/2 regimen) and the cycle was repeated for a total of two weeks. All treatments were administered by intraperitoneal injection. Body weight and tumor volume were both measured twice weekly for the duration of the study. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula: Tumor volume (mm3) = [(w2 x 1) / 2] where w = width and 1 = length, in mm, of a tumor. Mice were kept on study until tumors reached 1500 mm3, or the study reached the termination point at Day 45. See, FIGs. 2A-28, 3G, and 4A-4F.
4. Bl 6-F10 Model
All mouse in vivo work was performed in accordance with current regulations and standards and the NIH at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of an Institutional Animal Care and Use Committee (IACUC). Female, 6-8 week-old C57B1/6 mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation. A total of 1 x 105 B16-F10 cells were injected subcutaneously and monitored for tumor growth. Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was 50-100 mm3, and mice were randomized into treatment groups on Day 0. Mice receiving Chimeric Compound 1 were dosed on Days 1 and 4, and tumors were harvested 24 hours after the second dose (Day 5). Inducible TL-12 prodrugs used in these studies included Chimeric Compound 1 . All treatments were administered by intraperitoneal injection. See, FIGs. 3B and FIG. 5A-5F.
5. EMT- 6 Model
[0141] All mouse in vivo work was performed in accordance with current regulations and standards and the NIH at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of an Institutional Animal Care and Use Committee (IACUC). Female, 6-8 week- old C57B1/6 mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation. A total of 1 x 105 EMT6 cells were injected subcutaneously and monitored for tumor growth. Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was 50-100 mm3, and mice were randomized into treatment groups on Day 0. Mice receiving Chimeric Compound 1 were dosed twice a week for two weeks (Days 1, 4, 8, and 11) unless otherwise noted. Inducible IL- 12 prodrugs used in these studies included Chimeric Compound 1. All treatments were administered by intraperitoneal injection. In some experiments, mice that rejected tumors previously were then rechallenged with 1 x 105 EMT6 cells on the opposite flank four months after the initial rejection. In those experiments, age matched, tumor naive animals were used as a control. In some experiments, tumor samples were harvested and incubated in 5- 10 mLs of 10% neutral buffered formalin for at least 72 hours before being embedded in paraffin and mounted on slides. Unstained slides were submitted to Nanostring for immunofluorescence staining and geospatial transcriptional analysis using a Nanostring GeoMX DSP system. See, FIGs. 3C, 3F, 6, and 7A-7D.
6. CT26 Model
[0142] All mouse in vivo work was performed in accordance with current regulations and standards and the NIH at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of an Institutional Animal Care and Use Committee (IACUC). Female, 6-8 week- old Balb/C mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation. A total of 3 x 105 CT26 cells were injected subcutaneously and monitored for tumor growth. Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was 30-60 mm3, and mice were randomized into treatment groups on Day 0. Mice receiving Chimeric Compound 1 were dosed twice a week for two weeks (Days 1, 4, 8, and 11) unless otherwise noted. Inducible IL-12 prodrugs used in these studies included Chimeric Compound 1. All treatments were administered by intraperitoneal injection. Body weight and tumor volume were both measured twice weekly for the duration of the study. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula: Tumor volume (mm3) = [(w2x I) / 2] where w = width and / = length, in mm, of a tumor. Mice were kept on study until tumors reached 1500 mm3, or the study reached the termination point at Day 45. See, FIG. 3 A.
7. EG7.OVA Model
[0143] All mouse in vivo work was performed in accordance with current regulations and standards and the NTH at Covance (Ann Arbor, MI) with the approval of an Institutional Animal Care and Use Committee (IACUC). Female, 6-8 week-old C57B1/6 mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation. A total of 1 x 106 EG7. OVA cells were injected subcutaneously and monitored for tumor growth. Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was ~93 mm3, and mice were randomized into treatment groups on Day 0. Mice receiving Chimeric Compound 1 were dosed twice a week for two weeks (Days 1, 4, 8, and 11) unless otherwise noted. Inducible IL-12 prodrugs used in these studies included Chimeric Compound 1. All treatments were administered by intraperitoneal injection. Body weight and tumor volume were both measured twice weekly for the duration of the study. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula: Tumor volume (mm3) = [(w2x I) / 2] where w = width and I = length, in mm, of a tumor. Mice were kept on study until tumors reached 1500 mm3, or the study reached the termination point at Day 45. See, FIG. 3E.
8. A20 Model
[0144] All mouse in vivo work was performed in accordance with current regulations and standards and the NIH at Covance (Ann Arbor, MI) with the approval of an Institutional Animal Care and Use Committee (IACUC). Female, 6-8 week-old Balb/C mice from Charles River Laboratories were shaved on their flank 1 day prior to tumor cell implantation. A total of 5 x 105 A20 cells were injected subcutaneously and monitored for tumor growth. Extra mice were implanted in order to have sufficiently sized tumors for randomization. Tumor volume was monitored until the group average was -90-130 mm3, and mice were randomized into treatment groups on Day 0. Mice receiving Chimeric Compound 1 were dosed twice a week for two weeks (Days 1, 4, 8, and 11) unless otherwise noted. Inducible IL-12 prodrugs used in these studies included Chimeric Compound 1. All treatments were administered by intraperitoneal injection. Body weight and tumor volume were both measured twice weekly for the duration of the study. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula: Tumor volume (mm3) = [(w2x I) / 2] where w = width and I = length, in mm, of a tumor. Mice were kept on study until tumors reached 1500 mm3, or the study reached the termination point at Day 45. See, FIG. 3D.
9. Tumor Digestions and NanoString Analysis
[0145] MC38 and B16-F10 tumors were chopped into small pieces (< 5 mm3) in phenol -free RPMI-1640 (ThermoFisher) before being enzymatically digested with Collagenase IV (3 mg/mL, Gibco, 17104019) at 37°C for 35 minutes while shaking. After digestion, tumor samples were mechanically dissociated through a 70 pM cell strainer. EMT6 tumors were processed using gentleMACS™ C-Tubes from Miltenyi Biotech (130-093-237). Briefly, tumors were cut into small pieces (< 5 mm3) in HBSS containing 1.25 mg/mL Collagenase Type IV (Gibco, 17104019), 0.0025mg/mL Hyaluronidase (Sigma-Aldrich, H3506), and O.Olmg/mL DNASE I (Worthington, LS002004). Samples were placed on a gentleMACS™ Octo Dissociator and processed using program 37C_m_TDK_l before samples were passed through a 70 pM cell strainer to remove any undigested tumor pieces. Single cell suspensions were then counted and analyzed by flow cytometry. For NanoString analysis, 5 x 105 cells were frozen in 100 pL of RLT Lysis buffer (Qiagen, 1053393). RNA samples were shipped to LakePharma and analyzed using the nCounter Mouse PanCancer Immune Profiling Codeset Panel with the nCounter FLEX analysis system. NanoString analysis was performed using nSolver™ Software with the Advanced Analysis module installed.
10. Flow cytometry
[0146] All cell staining was performed in 96-well round bottom plates using FACs Buffer (PBS + 0.5 % BSA) or lx Permeabilization Buffer (eBioscience, 00-5223-56) where appropriate. Cells were first treated with FC block (BioLegend, 101320) at room temperature before tetramer staining was performed for 20 minutes. After tetramer staining, cells were then stained with a master mix of extracellular antibodies for 20 minutes at 4°C before being fixed/permeabilized overnight using the eBioscience™ Foxp3 Transcription Factor Staining Buffer Set according to the manufacturer’s protocol. The next day, samples were washed with Perm Buffer and stained with intracellular markers for 20 minutes at 4°C. Cells were analyzed on a Cytek Aurora system. Fluorescence minus one (FMO) and single stain controls (cells or OneComp ebeadsTM (Thermofisher, 01-1111-42)) were stained alongside cells. Unless otherwise noted, when flow cytometry was used to assess effector cytokine production, cells were restimulated with PMA (50ng/mL, Sigma-Aldrich, P1585) and lonomycin (Ipg/mL, Sigma-Aldrich, IO634-1MG) in the presence of lx Brefeldin A (Thermofisher Scientific, 00-4506-51) for 4 hours in complete media at 37°C prior to staining. Cells used for 2-NDBG assay were starved in glucose free RPML1640 Media (Gibco, 11879-020) for 1 hour, then incubated with 2-NDBG (Cayman Chemical, # 186689-07-6) for 1 hour at 37 °C/5% CO2 before being stained for extracellular markers. MitoTracker Deep Red FM (ThermoFisher, # M46753), MitoTracker Green FM (ThermoFisher, M46750) MitoSOX Red (ThermoFisher, M36008), and TMRM (ThermoFisher, T668) staining was performed at 37°C/5% CO2 in RPMI 1640 media (Gibco, A10491-01) with 10% heat inactivated FBS (Gibco, 10082-147) and Penicillin/Streptomycin (Gibco, 15140-122) for 1 hour. Cells were then washed with FACs buffer and stained for extracellular markers. Specific antibody clones are detailed as follows. Fluorescent dye-conjugated antibodies specific for the following proteins were purchased from BioLegend: CD8a APC, clone 53-67; CD4 BV650, clone RM4-5; CD3 AF700, clone 17A2; CD45 BV605, clone 30-F11; CD49b APC/Cy7, clone DX5; CD25 BV421, clone PC61; CD25 APC/Fire 750, clone PC61; Ki67 PeCy7, clone 16A8; Ki67 AF700, clone 16A8; granzyme B FITC, clone GB11; IfNy PE, clone XMG1.2; F4/80 Pe/Dazzle 594, clone BM8; CD3 Complex PeCy7, clone 17A2; FC Block, clone 93. Fluorescent dye-conjugated antibodies specific for the following proteins were purchased from eBioscience: CD45 BUV395, clon30-Fl l; CD4 BUV496, clone GK1.5; CD8 BUV563, 53.6-7; TNF BV750, clone MP6-XT22; CD49B Pe-Cy5, clone DX5, FoxP3 AF488, clone FJK-16s; FoxP3 eFlour450, clone FJK-16s. The fluorescent dye-conjugated tetramer against the MulV pl5E peptide KSPWFTTL (SEQ ID NO: 449) was purchased from ThermoFisher Scientific (50-168-9385). The Live/Dead Blue Dye was also purchased from ThermoFisher Scientific (L23105). 11. Pharmacokinetic A nalysis
[0147] Plasma and tumor samples were collected at indicated time points by Charles River Laboratories and stored at -80°C. MC38 tumor lysates were generated by homogenizing each tumor with a Qiagen TissueRuptor with disposable probes (Qiagen) in ice cold Lysis Buffer (IX Tris Buffered Saline (Sigma-Aldrich, T5912-1L), 1 mM EDTA (Sigma-Aldrich, 3690-100mL), 1 % Triton X-100 (Sigma-Aldrich, XIOO-lOOOmL), with protease inhibitors (Sigma-Aldrich, P8340-1L) in diH2O). Plasma and tumor samples were analyzed using a sandwich ELISA on the MSD platform, which detects both intact Chimeric Compound 1 and free/released IL-12. Free IL- 12 level was quantified using an in-house developed ECLIA assay on MSD MESOTM QuickPlex SQ 120 system. Data acquisition and analysis were performed using MSD Workbench 4.0.12, and pharmacokinetic parameters were calculated using Phoenix WinNonlin Version 8.1.
12. Compound 36 Stability in Human Serum
[0148] Compound 36 was incubated in human serum (BioIVT) from healthy donors in duplicate for each timepoint. Time zero (TO) samples were immediately frozen at -80°C. The remaining samples were incubated at 37°C for 24 (T24) or 72 (T72) hours before being stored at 80°C. Stability of Compound 36 was assessed by western blot analysis using the JESS system (Protein Simple, SM-W004) according to the manufacturer’s general protocol. Input controls (intact and protease cleaved) were also analyzed. Samples and antibodies (anti-IL-12 primary (R&D Systems, AB-219-NA)) and secondary (Jackson Labs, 805-035-180) were loaded into a 12-230 kDA Jess separation module and run using a Jess system set to the standard settings for chemiluminescence, with modifications to the standard protocol for non-reducing conditions. Analysis of the resulting western blot was performed using Compass for Simple Western Software (v4.1.0).
13. Human Primary Cell Assays
[0149] Human PBMCs were isolated from whole blood (BioIVT) using FicolLPaque Plus (GE Healthcare, GE17-1440-03) according to the manufacturer’ s protocol and frozen in Recovery Cell Culture Freezing Media (Gibco, 12648010) for later use. To generate activated T cells (Tblasts), PBMCs were thawed, counted, and stimulated with 5 pg/mL ofPHA (Sigma-Aldrich, L1668-5MG) for 72 hours before being frozen. Tblasts were thawed, counted, and plated in a 96- well round bottom plate, and incubated with titrated amounts of intact or protease-activated (cleaved) INDUKINE™ proteins or chimeric IL- 12. After 72 hours, IFNy production was measured using a Human IFNy specific AlphaLisa Kit (Perkin Elmer, AL217C) according to the manufacturer’s protocol with a Perkin Elmer Enspire Alpha Reader running Enspire Manager Software (V4.13.3005.1482).
14. Ex Vivo Inducible IL-12 Pro-drug Protein Processing Assay
[0150] Primary human healthy cells were purchased from either ATCC, Lonza, or Zen-Bio, and cultured according to the manufacturers’ protocols. Dissociated human tumor samples were purchased from Discovery Life Sciences. To examine inducible IL-12 prodrug processing, samples were thawed, washed, and counted. Cells were then resuspended in media containing either intact Compound 36, a non-cleavable variant of Compound 36, or pre-cut Compound 36 for 48 hours before cell culture supernatants were collected and frozen for later analysis. Cell culture supernatants were then used to stimulate previously activated human Tblasts using the assay detailed above. IFNy production was normalized against the controls using the following equation to assess the level of processing.
Percent of Full Activity= (1 -((Sample -Uncleavable Ctrl))/ ((Cleaved Ctrl-Uncleavable Ctrl))) *100
15. Data Representation, Bioinformatic Analysis, and Statistics
[0151] Flow cytometry plots were generated with Flowlo Software (vl0.5.30) and are representative samples. All the quantitative plots were generated using GraphPad Prism 8 Software for Windows (64-Bit) (San Diego, CA). For in vitro activity assays, data were analyzed using a non-linear sigmoidal, 4PL curve fit model without constraints. Statistical analysis was also performed using GraphPad Prism software (San Diego, CA). Two sample comparisons used a student’s /-test while comparisons of more than two groups used an analysis of variance (ANOVA) test with multiple comparisons. Antitumor effects over time were analyzed by using a mixed-effects model. For the NanoString dataset, statistical analysis was performed using nSolverTM software with the Advanced Analysis Module installed. Pathway analysis was performed using Partek software (v 10.0.22.0428), based on transcripts that were significantly different following mWTX-330 with an FDR step-up of 0.05.
1.2 Results
1. Chimeric Compound 1 is a Selectively Activated Inducible IL-12 Prodrug that Generates a Robust, Cleavage Dependent Anti-Tumor Immune Response in Multiple Models
[0152] In order to address the clinical shortcomings of free cytokine therapy, a selectively inducible IL-12 prodrug was developed. To measure the difference in activity between intact and protease activated (cleaved) Chimeric Compound 1, HEK-Blue IL- 12 reporter cells were incubated with either intact or protease activated Chimeric Compound 1, and IL- 12 signaling was assessed. In this assay, intact Chimeric Compound 1 had 175-fold less activity than either cleaved Chimeric Compound 1 or chimeric IL- 12. (FIG. 1). To test whether Chimeric Compound 1 could generate anti -turn or immunity in vivo, MC38 tumor bearing animals were treated with titrated amounts of Chimeric Compound 1, and tumor growth was monitored over time. In this study, a variant of Chimeric Compound 1 with a non-cleavable linker (FIG. 1) was also included at the highest dose as a control. In this model, even the lowest tested dose of Chimeric Compound 1 (7 pg/dose) generated statistically significant tumor growth inhibition, with 43 pg/dose being sufficient to generate complete tumor rejections (FIG. 2A). In contrast, the non- cleavable (NC) variant of Chimeric Compound 1 had less activity than even the lowest dose of Chimeric Compound 1 demonstrating that the full potency of Chimeric Compound 1 is dependent on in vivo processing of the molecule. Chimeric Compound 1 treatment also generated robust anti-tumor immunity in less immune cell infiltrated (“colder") syngeneic tumor models, including CT26 (FIG. 3A), B16-F10 (FIG. 3B), and EMT-6 (FIG. 3C), demonstrating the broad activity of this molecule in vivo. While the use of a non-cleavable control demonstrated the necessity of processing for full activity, these data did not directly indicate that processing was occurring in the TME. FTY720 is a small molecule inhibitor of Sphingosine- 1 -phosphate receptor- 1 which prevents lymphocyte egress from secondary lymphoid tissues, and effectively isolates the TILs from the normal recirculating population of immune cells in vivo. Chimeric Compound 1/FTY720 co-treated animals retained the potent early anti-tumor activity associated with Chimeric Compound 1 treatment, although tumor control was less complete after dosing with the INDUKINE™ molecule had stopped (FIG. 8A) [0153] These data demonstrate that systemically administered Chimeric Compound 1 is processed in the TME, and that this local release of IL-12 is sufficient to generate early tumor growth inhibition.
[0154] In order to identify the effector cell populations responsible for the Chimeric Compound 1 induced anti -tumor immunity, MC38 tumor bearing mice were treated with Chimeric Compound 1 in conjunction with antibody-based depletion of individual effector cell populations.
[0155] Interestingly, while depletion of the CD8+ T cell population did not inhibit early tumor control, chimeric Compound treated mice without CD8+ T cells were ultimately unable to control tumor growth (FIG. 8B). In contrast, depletion of either NK cells or total CD4+ T cells alone did not inhibit Chimeric Compound 1 induced anti-tumor activity (FIG. 8B). However, in mice where all three populations were depleted, Chimeric Compound 1 treatment had little antitumor activity, suggesting that these cell types work jointly to reject MC38 tumors in response to treatment. Furthermore, when Chimeric Compound 1 treated mice that previously rejected either MC38 tumors (FIG. 3F) or EMT-6 tumors (FIG. 3G) were rechallenged, 100% of the animals were protected against tumor growth. Together, these data suggest that Chimeric Compound 1 treatment generates a robust and durable anti-tumor immune response that is dependent on in vivo cleavage/ activation of the molecule.
2. The Inducible IL-12 Prodrug Design of Chimeric Compound 1 Enhances its Pharmacokinetic Profile and Expands the Therapeutic Window of Chimeric IL-12
[0156] In the clinic, free cytokines have poor pharmacokinetic profiles with short half-lives, resulting in rapid clearance and poor exposure in patients, and leading to unreasonable dosing schedules. In order to examine whether the design of Chimeric Compound 1 enhanced the halflife and exposure of the molecule, MC38 tumor bearing mice were dosed with a single dose of either chimeric TL-12 or Chimeric Compound 1 , and peripheral blood and tumor samples were collected over time. Using a unique set of detection reagents, it was possible to separately measure the amount of total IL- 12 (blocked + unblocked) or to selectively measure the amount of unblocked IL- 12 present. In the plasma of tumor bearing mice, chimeric IL- 12 had a half-life of only 4 hours, while Chimeric Compound 1 had a half-life of nearly 16 hours (FIG. 9A). Furthermore, only about 2% of the Chimeric Compound 1 found in the plasma was in the form of the cleaved molecule. Tn contrast, when the same analysis was performed on tumor samples (FIG. 9B), nearly 45% of the molecule was unmasked IL- 12 and inter-tumoral exposure was maintained far beyond what was achieved by treatment with chimeric IL-12. To further corroborate the selective processing of Chimeric Compound 1 in the tumor, the activation status of tumor infdtrating CD8+ T cells, CD4+ T conventional cells, and NK cells was compared to those same populations in the tumor draining and non-draining lymph nodes, as well as the peripheral blood following Chimeric Compound 1 treatment. While Chimeric Compound 1 treatment resulted in a significant increase in the frequency of polyfunctional CD8+ T cells within the MC38 tumors, no such increase was observed in the lymph nodes or in the peripheral blood (FIG. 9C). Likewise, among CD4+ T conventional cells (FoxP3-) (FIG. 15A) and NK Cells (FIG. 15B), Chimeric Compound 1 preferentially increased the frequency of cells producing effector cytokines in the tumor compared to the peripheral tissues.
3. Chimeric Compound I Treatment Activates Various TIL Populations in the MC38 Model
[0157] To better understand the mechanism by which Chimeric Compound 1 treatment generates anti -turn or immunity, MC38 tumor-bearing mice were randomized into treatment groups on Day 0 and treated with either vehicle or Chimeric Compound 1 on Day 1 and Day 4. Tumors were harvested 24 hours after the second dose and analyzed by flow cytometry or NanoString analysis using the PanCancer Mouse Immune Profiling Panel. Systemic treatment with Chimeric Compound 1 had a striking effect on the transcriptional profile of the TME, with 364 of the 770 investigated transcripts having statistically significant differences in expression after treatment (FIG. 4A). Chimeric Compound 1 treatment resulted in significant enrichment of several immune related signaling pathways, including “PD-L1 Expression and PD-1 Checkpoint in Cancer”, “NK Cell Cytotoxicity”, and “TH1 and TH2 Differentiation”. In agreement with this analysis, Chimeric Compound 1 treatment resulted in a significant increase in the frequency of tumor infiltrating NK cells producing IFNy, TNF, and Granzyme B (FIG. 4C-4D). Interestingly, Chimeric Compound 1 treatment resulted in NK, NKT, CD4+ T conventional cells, and CD8+ T cells producing such elevated levels of ZFNy that it was measurable by intracellular cytokine staining without ex vivo restimulation (FIGs. 14A-14B). However, the signaling pathway with the highest enrichment score following treatment with Chimeric Compound 1 was “Antigen Processing and Presentation.” Gene set enrichment analysis revealed that Chimeric Compound Itreatment significantly enriched several gene sets associated with antigen presentation of exogenous peptides and/or antigens in either MHC class I or MHC class II proteins. Presentation of exogenously derived antigens in MHC class I proteins is a phenomenon known as cross presentation, and it is exclusively mediated by a unique population of dendritic cells, identified by the expression of CD 103(25). In agreement with the bioinformatic analysis, flow cytometric analysis demonstrated that Chimeric Compound 1 treatment significantly increased the frequency of the cross presenting CD103+ DC population among total DCs (FIGs. 10A-10B). [0158] Given the role of CD8+ T cells in Chimeric Compound 1 mediated tumor rejections (FIG. 8A) and the finding that Chimeric Compound 1 increased tumor infiltration by cross presenting DCs, it seemed likely that Chimeric Compound 1 treatment also enhanced CD8+ T cell activation. Indeed, differential expression analysis of the total RNA demonstrated that Chimeric Compound 1 treatment significantly increased the expression of many transcripts associated with activation of cytotoxic CD8+ T cells, including IFNy, Granzyme B, perforin, and TNF, in addition to several chemoattractant molecules (FIG. 4B). While Chimeric Compound 1 did not increase the frequency of tumor specific CD8+ T cells at this early time point, treatment did result in robust activation of the tumor specific CD8+ T cell population, as demonstrated by an increase in the frequency of tetramer+ polyfunctional CD8+ T cells (FIG. 4F), with nearly 100% of the tumor specific T cells producing IFNy. Comparable results were seen when considering the entire tumor infiltrating CD8+ T cell population, and not just the tetramer positive ones. Additionally, among CD4+ T cells, Chimeric Compound 1 treatment significantly increased the frequency of CD4+ T conventional cells with a TH1 phenotype (Tbet+ IFNy+ TNF+) (FIG. 10C). Finally, recent publications have highlighted the role of IFNy in driving Tregs away from regulatory activity and towards an effector phenotype, in a phenomenon known as Treg fragility. Chimeric Compound I treatment resulted in a significant subset of the FoxP3+ Treg population co-producing the effector cytokines TNF and IFNy (FIGs. 10D-10F) and expressing Tbet (FIG. 1 OF), demonstrating that systemic treatment with Chimeric Compound 1 can induce Treg instability in the TME. Altogether, these data demonstrate that systemic administration of Chimeric Compound 1 results in the transcriptional reprogramming of the TME, and the subsequent activation of various tumor infiltrating effector cell populations. 4. Chimeric Compound 1 Treatment Expands Unique TCR Clones and Increases TCR Clonality in the TME
[0159] In the MC38 tumor model, Chimeric Compound 1 treatment results in rapid tumor rejection, making it technically challenging to fully investigate the kinetics of immune activation. In contrast, Chimeric Compound 1 treatment of the EMT-6 tumor model generates complete rejections over a longer period, which is favorable to a more thorough analysis of an ongoing CD8+ T cell response (FIG. 3C). Therefore, mice bearing established EMT-6 tumors were randomized into treatment groups and dosed twice a week for two weeks with either vehicle or Chimeric Compound 1. Tumors and plasma were then harvested at various timepoints. Interestingly, in the control animals, the frequency of poly functional CD8+ T cells did expand over the course of the experiment, but eventually retracted in line with eventual tumor growth. In contrast, Chimeric Compound 1 treatment increased the frequency of polyfunctional CD8+ T cells over that of the control animals as soon as Day 5 after the start of treatment (FIG. 6), and this frequency continued to expand even after exposure to Chimeric Compound 1 was undetectable.
[0160] To better understand the transcriptional effects of Chimeric Compound 1 treatment specifically on the tumor infiltrating CD8+ T cell population, Geospatial NanoString analysis was performed on tumor samples from Day 11. This technology merges immunofluorescence with whole transcriptome analysis of specific cells, allowing for transcriptional analysis of a specific cell population while maintaining spatial information that would otherwise be lost during tissue dissociation. In the control group, CD8+ T cells were largely confined to the outer margins of the tumor (FIG. 7 A). In contrast, Chimeric Compound 1 treatment induced significant infiltration of the EMT-6 tumors, with CD8+ T cells penetrating deeply into the tumor tissue (FIG. 7A). Whole transcriptome analysis of the tumor infiltrating CD8+ T cells demonstrated that Chimeric Compound 1 treatment resulted in substantial transcriptional reprogramming of these cells, including upregulation of many genes associated with T cell activation such as Tbet, IFNy, Cd25, and chemoattractants known to be responsible for the recruitment of additional immune cells (FIG. 7B).
[0161] Among the pathways activated in tumor infiltrating CD8+ T cells by Chimeric Compound 1 treatment, IL-12 (FIG. 7C) and IFNy signaling (FIG, 7D) were both significantly upregulated, confirming the local release of unmasked IL-12 and subsequent production of IFNy within the TME. Chimeric Compound 1 treatment also increased expression of transcripts downstream of TCR signaling (FIG. 11A). Given the effects on cross presenting DCs, we hypothesized that Chimeric Compound 1 treatment may result in the activation of new T cell clones to generate anti-tumor immunity. In order to test this, T cells were isolated from EMT-6 tumors following Chimeric Compound 1 or vehicle treatment and sent for TCR sequencing. While the tumor infiltrating TCR repertoire of control animals was dominated by many low frequency clones, Chimeric Compound 1 treatment drove a robust expansion of several TCR clones (FIG. 1 IB), resulting in a significant increase in the overall clonality of the tumor infiltrating TCR repertoire (FIG. 11C). Indeed, when examining the overall frequency of the top fifty clones in each group, Chimeric Compound 1 treatment resulted in a substantial increase in the number of clones making up more than 1% of the total repertoire across multiple animals (FIG. 1 ID). Further analysis of this subset revealed that only 1 of the 8 clones common to both treatment groups expanded at least 10-fold with Chimeric Compound 1 treatment (FIG. 12S). In contrast, of the clones unique to the Chimeric Compound 1 treated group, all of them expanded over 10-fold compared to the control group (FIG. 12T), suggesting that Chimeric Compound 1 treatment is increasing the clonality of T cell populations primarily by expanding clones that were previously underrepresented, rather than increasing the frequency of already dominant clones.
5. Chimeric Compound 1 Substantially Increases Mitochondrial Activity in Tumor Infdtrating CD8+ T cells and NK Cells
[0162] Recently activated CD8+ T cells have substantial energy requirements and rely heavily on glucose uptake and glycolysis to quickly generate the energy necessary to perform their effector functions, before transitioning towards mitochondria dependent oxidative phosphorylation as they develop into long-lived memory cells. However, recent publications have demonstrated that tumor infdtrating CD8+ T cells often fail to induce significant mitochondrial respiration compared to those activated in the spleen or lymph nodes, suggesting that the TME negatively impacts the metabolic health of effector cells. Among tumor infiltrating CD8+ T cells, Chimeric Compound 1 treatment resulted in a significant enrichment of transcripts associated with glycolysis (FIG. 12A). Therefore, we hypothesized Chimeric Compound 1 treatment may increase glucose uptake by tumor infiltrating CD8+ T cells, and thereby lead to increased glycolysis However, when these cells were incubated with a non-metabolizable fluorescent glucose analog (2-NDBG), tumor infiltrating CD8+ T cells from Chimeric Compound 1 treated animals actually had slightly less glucose uptake than those from vehicle animals (FIGs. 12B-12C). Therefore, rather than simply increasing glucose uptake by tumor infiltrating CD8+ T cells, Chimeric Compound 1 treatment was instead reprogramming those cells to utilize glucose more efficiently than those from vehicle treated animals.
[0163] In addition to driving increased glycolysis, Chimeric Compound 1 treatment also enriched for transcripts associated with the TCA cycle, mitochondrial biogenesis, and mitochondrial translation, suggesting that Chimeric Compound 1 treatment may enhance the mitochondrial activity and health of tumor infiltrating effector cells (FIGs. 12D-12F). To test this, TILs were isolated from vehicle or Chimeric Compound 1 treated animals and mitochondrial phenotyping was performed by flow cytometry. Mitotracker Red is a dye that specifically stains actively respirating mitochondria due to its pH sensitivity. While tumor infiltrating CD8+ T cells from vehicle treated animals had limited evidence of ongoing active mitochondrial respiration, those from Chimeric Compound 1 treated animals had significantly increased levels of active respiration (FIGs. 12G-12H). Interestingly, this finding also extended to NK cells (FIGs. 12I-12J) and total CD4+ T cells (FIG. 12U). This increase was primarily due to increased mitochondrial activity, rather than simply an increase in total mitochondrial mass, as Chimeric Compound 1 treatment only slightly increased the total mitochondrial mass of tumor infiltrating NK cells, CD8+ T cells, and total CD4+ T cells (FIG. 12V). Furthermore, TMRM staining also revealed that Chimeric Compound 1 treatment significantly increased the mitochondrial membrane potential in both CD8+ T cells (FIGs. 12K-12L) as well as NK cells (FIGs. 12M-12N). Mitochondrial reactive oxygen species (ROS) have previously been linked both to NF AT signaling and subsequent production of IL -2 (31), as well as IFNy production by memory CD4+ T cells, and can be detected using the dye MitoSOX Red. Chimeric Compound 1 treatment increased the production of mitochondrial ROS species in both CD8+ T cells (FIGs. 12O-12P) as well as NK cells (FIGs. 12Q-12R).
[0164] Oxidative phosphorylation is the primary energy source for memory T cells, and increased dependence on this pathway has been associated with superior anti-tumor immunity and a “stem-cell like” phenotype. Tumor infiltrating CD8+ T cells from Chimeric Compound 1 treated mice also significantly upregulated expression of genes associated with T cell sternness, including Tcf7, Cxcr3, and I12ry while significantly downregulating expression of several genes associated with CD8+ T cell exhaustion, including Pdcdl, Havcr2, and Lag3. Together, these data demonstrate that systemic administration of Chimeric Compound 1 is sufficient to restore the mitochondrial respiration of tumor infiltrating CD8+ T and NK cells and pushes the CD8+ T cell population to adopt a more “stem-cell like” phenotype, which may translate into superior anti-tumor immunity.
6. Compound 36, a fully human inducible IL-12 Prodrug, is Stable in Human Serum and Preferentially Activated by Primary Human Tumor Samples
[0165] For preclinical murine studies, it was important to use a surrogate molecule that was active in mice. However, for clinical development, a fully human IL-12 payload will be used. Compound 36 is identical to Chimeric Compound 1 except that it contains fully human IL-12 as the payload. As with the murine surrogate molecule, intact Compound 36 had substantially less activity than either cleaved Compound 36 or recombinant human IL-12 in a HEK-Blue IL-12 reporter assay. Likewise, when exposed to stimulated primary human Tblasts from multiple donors, intact Compound 36 was 61 -fold less active on average than the cleaved molecule. In both of these in vitro assays, cleaved Compound 36 had activity similar to recombinant human IL-12. Furthermore, when Compound 36 incubated in serum from healthy human donors (n=6), no free IL-12 was detected after 72 hours at 37°C (FIG. 13A), confirming the stability of the molecule. In order to examine whether Compound 36 would be selectively processed by primary human tumor samples, an in vitro cleavage assay was developed. Briefly, primary human dissociated tumor samples from various indications, or primary human cells from healthy tissues were incubated for 48 hours with either Compound 36, pre-cut Compound 36, or an uncleavable variant of Compound 36 before the cell culture supernatants containing the processed inducible IL- 12 prodrug were collected. Since human Tblasts can differentiate between intact and cleaved Compound 36, primary human Tblasts were exposed to the cell culture supernatants and the production of IFNy was used as a surrogate marker for the processing of Compound 36. The results of this assay were then normalized to the uncleavable negative control (0% processed) and the positive pre-cleaved control (100% processed). Among the n=88 primary human tumor samples evaluated; Compound 36 was efficiently processed across all tested indications (FIG. 13B). In contrast, incubation of Compound 36 with primary human cells from various healthy tissues (n=13) resulted in no evidence of processing. These data suggest that Compound 36 is efficiently and selectively processed by primary human tumor samples and support the continued clinical development of this molecule.
1.3 Discussion
[0166] IL- 12 has long been a cytokine of great interest for oncology due to its potential to induce innate and adaptive immune responses (9,11) and its promising anti-tumor preclinical data (12,18,20,34,35). Nevertheless, despite this interest, the poor pharmacokinetic properties of this cytokine and the unacceptable levels of toxicity associated with its systemic administration have prevented its use in clinical settings (9,10,24,36). To address these concerns, we developed an inducible IL-12 prodrug, Compound 36. Compound 36 a prodrug molecule, designed to be an infrequently administered, systemically delivered therapy with targeted intra-tumoral activation that releases native IL- 12 into the tumor microenvironment. Our data with Chimeric Compound 1 demonstrated anti -turn or activity in the MC38 tumor model that was dependent on in vivo cleavage of the inducible IL- 12 prodrug by the tumor. Furthermore, Chimeric Compound 1 was a very potent monotherapy in several mouse tumor models with varying levels of baseline infdtration, including complete responses in a model refractory to anti-PD-1 treatment (EMT-6). These complete responses translated into robust immune memory against subsequent rechallenge with the same tumor cell line, highlighting the role of the immune system in tumor rejection. In the MC38 model, long term efficacy was dependent on the presence of CD8+ T cells, but overall tumor growth inhibition was driven by contributions from three main effector cell types, CD8+ T cells, CD4+ T conventional cells, and NK cells. The inducible IL-12 prodrug design also resulted in increased exposure and a favorable ratio of active IL-12 versus the blocked pro-drug molecule in the tumor tissue compared to the plasma, which correlated with selectively localized pharmacodynamic changes (effector cell polyfunctionality) observed in the tumor versus peripheral tissues. Importantly, Chimeric Compound 1 proved to be well-tolerated in mice compared with recombinant chimeric IL- 12 treatment, while maintaining the potential to induce complete tumor regressions, resulting in an almost 10-fold improvement of the therapeutic window compared to the unblocked cytokine. Improvement of the therapeutic window is a key feature of inducible IL-12 prodrugs and is necessary to facilitate clinical development of potent cytokines for oncology treatment. [0167] Chimeric Compound 1 treatment robustly activated various tumor infiltrating innate and adaptive effector cell populations, supporting a mechanism of action where infiltration and activation of multiple effector cells plays a fundamental role in initial tumor control. The tumor specific delivery of active IL-12 and subsequent induction of intratumoral IFNy also induced Treg fragility, which likely contributes to the potent efficacy delivered by Chimeric Compound 1 treatment. However, of equal importance, is the effect that Chimeric Compound 1 treatment had on antigen processing and presentation and the observed increase in tumor infiltration by crosspresenting dendritic cells. These cells are responsible for the de novo generation of new T cell responses to novel tumor antigens, and several publications have identified their importance in the generation of pre-clinical anti-tumor immunity(37,38). The dual role of IL-12 as a direct activator of effector cell populations and as a driver of cross presenting dendritic cell activation will likely set apart therapeutics based on this cytokine compared to other treatments when it comes to triggering efficacy in “cold” tumors. Indeed, we observed this effect using a model of a “cold” tumor, the poorly infiltrated EMT-6 model. NanoString Digital Spatial Profiling demonstrated that systemic treatment with Chimeric Compound 1 enhanced deep infiltration of EMT-6 tumors by CD8+ T cells and confirmed the intratumoral increase in IL-12 and IFNy signaling, as well as the significant upregulation of transcripts associated with robust CD8+ T cell activation. Treatment also significantly increased the clonality of the TCR repertoire among tumor infiltrating T cells and drove the expansion of several novel clones, suggesting that systemic Chimeric Compound 1 treatment resulted in the activation of a de novo T cell response to unique tumor antigens, which may be key to the CD8+ T cell dependent tumor rejection observed earlier.
[0168] Finally, systemic Chimeric Compound 1 treatment had a substantial effect on the metabolism of the tumor infiltrating effector cells, transforming the metabolic status of not just the activated, tumor infiltrating CD8+ T cells but also that of the intratumoral NK cells. The TME is known to have several distinct characteristics when compared to a typical cellular environment, including a lower pH, hypoxic conditions, and significant competition for extracellular glucose, all of which may impair effector cell activity. Despite the evidence for increased glycolysis, we observed a slight reduction in extracellular glucose uptake following Chimeric Compound 1 treatment, suggesting that Chimeric Compound 1 treatment is not simply driving greater glucose uptake by tumor infiltrating cells, but is instead facilitating an increase in metabolic efficiency and increasing oxidative phosphorylation. Recent studies have demonstrated that tumor infiltrating T cells often fail to robustly activate mitochondrial respiration compared to those that have been activated in the spleen or lymph nodes, suggesting that the TME impairs metabolic health of effector cells(27,28,30). In agreement with these studies, EMT-6 tumor infiltrating CD8+ T cells and NK cells from vehicle treated animals had very little evidence of ongoing mitochondrial respiration, despite efficiently taking up 2-NDBG in vitro. In contrast, effector cells from Compound 36 treated animals robustly upregulated active mitochondrial respiration, mitochondrial membrane potential, and mitochondrial reactive oxygen species. The transition of these cells strongly towards oxidative phosphorylation could be important in the context of the highly dysregulated metabolic environment within the tumor, where there is stiff competition for glucose, and every molecule must be used to its fullest extent to support effector cell activation.
[0169] In conjunction with the metabolic effects of the TME, effector cells also have to contend with another mechanism of immune regulation, exhaustion. This state is characterized by high co-expression of checkpoint proteins (such as PD-1, LAG-3, TIGIT, and TIM-3)(39), the loss of effector cytokine production, and the failure to proliferate following restimulation. However, recent publications have demonstrated that some tumor specific T cells maintain a more stemcell like phenotype, termed increased “sternness,” and have identified a crucial role for the transcription factor TCF1 in maintaining that phenotype (40). Furthermore, increased “sternness” has been associated with greater anti-tumor immunity. In addition to the metabolic reinvigoration of the effector cells, treatment with Chimeric Compound 1 likely increased the sternness of the tumor infiltrating CD8+ T cells, with upregulation of TCF7 (the mRNA transcript associated with TCF1, the protein) and decreased expression of PD-1 and Tim-3 by these cells. Together, these data suggest that Chimeric Compound 1 treatment results in a robust and all-encompassing re-programming of the tumor infiltrating CD8+ T cell response.
[0170] The mechanistic studies described herein not only identified the cell types responsible for the anti-tumor efficacy of Chimeric Compound 1 treatment, but also provide insight on how these effector cells are able to overcome the suppressive microenvironment within the tumor. Furthermore, these data demonstrate that the inducible IL- 12 prodrug design of Chimeric Compound 1 significantly increased the half-life of the molecule compared to recombinant IL- 12 and allowed for selective activation in the TME following systemic administration, resulting in a significant expansion of the therapeutic window. Additional studies with fully human Compound 36 demonstrated that the inducible IL- 12 prodrug was highly inducible and was cleaved by a majority of human tumor samples in vitro while demonstrating stability when incubated with normal primary cells and serum. Together, these data provide clear evidence for the continued preclinical development of this therapeutic molecule and support moving Compound 36 towards human clinical testing.
Example 2. MC38 Experiments (Study MC38-e52)
[0171] The MC38 cell line, a rapidly growing colon adenocarcinoma cell line, was used. Using this tumor model, the ability of IL-12 prodrugs to affect tumor growth and body weight was examined.
Table 5. Agents and treatment regimen
Figure imgf000066_0001
[0172] Mice were anaesthetized with isoflurane for implantation of cells to reduce the ulcerations. Female C57BL/6 mice were set up with 5xl05 MC38 tumor cells in 0% Matrigel sc in flank. Cell injection volume was 0.1 mL/mouse Mouse age at start date was 8 to 12 weeks. Pair matches were performed when tumors reach an average size of 100 - 150 mm3 and began treatment. This was Day 1 of the study. Body weights were taken at initiation and then biweekly to the end. Caliper measurements were taken biweekly to the end. Any adverse reactions were reported immediately. Any individual animal with a single observation of > than 25% body weight loss or three consecutive measurements of >20% body weight loss was euthanized. Any group with a mean body weight loss of >20 % or >10% mortality stopped dosing; the group was not euthanized, and recovery was allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint were euthanized. If the group treatment related body weight loss was recovered to within 10% of the original weights, dosing resumed at a lower dose or less frequent dosing schedule. Exceptions to non-treatment body weight % recovery were allowed on a case-by-case basis. Endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1500 mm3 or 40 days, whichever came first. When the endpoint was reached, the animals were euthanized. Results are shown in FIGs. 17A-17J.
References
1. Propper DJ, Balkwill FR. Harnessing cytokines and chemokines for cancer therapy. Nat. Rev. Clin. Oncol. 2022. page 237-53.
2. Berraondo P, Sanmamed MF, Ochoa MC, Etxeberria I, Aznar MA, Perez-Gracia JL, et al. Cytokines in clinical cancer immunotherapy. Br. J. Cancer. Nature Publishing Group; 2019. page 6-15.
3. Conlon KC, Miljkovic MD, Waldmann TA. Cytokines in the Treatment of Cancer. J Interf Cytokine Res. Mary Ann Liebert, Inc.; 2019;39:6-21.
4. Nirschl CJ, Brodkin HR, Hicklin DJ, Ismail N, Morris K, Seidel-Dugan C, et al. Discovery of a Conditionally Activated IL-2 that Promotes Antitumor Immunity and Induces Tumor Regression. Cancer Immunol Res. American Association for Cancer Research (AACR); 2022;10:581-96.
5. Chary ch DH, Hoch U, Langowski JL, Lee SR, Addepalli MK, Kirk PB, et al. NKTR-214, an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models. Clin Cancer Res. American Association for Cancer Research Inc ; 2016;22:680-90.
6. Klein C, Waldhauer I, Nicolini VG, Freimoser-Grundschober A, Nayak T, Vugts DJ, et al. Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines. Oncoimmunology. Taylor and Francis Inc.; 2017;6.
7. Lopes JE, Fisher JL, Flick HL, Wang C, Sun L, Emstoff MS, et al. ALKS 4230: A novel engineered IL-2 fusion protein with an improved cellular selectivity profile for cancer immunotherapy. J Immunother Cancer. BMJ Publishing Group; 2020;8:673. 8. Sharma M, Khong H, Fa’ak F, Bentebibel SE, Janssen LME, Chesson BC, et al.
Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy. Nat Commun. Nature Research; 2020; 11.
9. Waldmann TA. Cytokines in cancer immunotherapy. Cold Spring Harb Perspect Biol. Cold Spring Harbor Laboratory Press; 2018; 10.
10. Nguyen KG, Vrabel MR, Mantooth SM, Hopkins J J, Wagner ES, Gabaldon TA, et al. Localized Interleukin- 12 for Cancer Immunotherapy. Front. Immunol. 2020.
11. Del Vecchio M, Bajetta E, Canova S, Lotze MT, Wesa A, Parmiani G, et al. Interleukin-12: biological properties and clinical application. Clin Cancer Res. 2007;13:4677-85.
12. Lasek W, Zagozdzon R, Jakobisiak M. Interleukin 12: Still a promising candidate for tumor immunotherapy? Cancer Immunol. Immunother. 2014.
13. Tait Wojno ED, Hunter CA, Stumhofer JS. The Immunobiology of the Interleukin- 12 Family: Room for Discovery. Immunity. 2019.
14. Glassman CR, Mathiharan YK, Jude KM, Su L, Panova O, Lupardus PJ, et al. Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells. Cell. 2021;
15. Wang P, Li X, Wang J, Gao D, Li Y, Li H, et al. Re-designing Interleukin- 12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent. Nat Commun. Nature Publishing Group; 2017;8: 1395.
16. Wittrup KD, Kaufman HL, Schmidt MM, Irvine DJ. Intratumorally anchored cytokine therapy, https://doi.org/101080/1742524720222084070. Taylor & Francis; 2022;19:1-8.
17. Mirlekar B, Pylayeva-Gupta Y. IL-12 family cytokines in cancer and immunotherapy. Cancers (Basel). 2021.
18. Hewitt SL, Bailey D, Zielinski J, Apte A, Musenge F, Karp R, et al. Intratumoral IL 12 mRNA therapy promotes TH1 transformation of the tumor microenvironment. Clin Cancer Res. American Association for Cancer Research (AACR); 2020;26:6284-98.
19. Strauss J, Heery CR, Kim JW, Jochems C, Donahue RN, Montgomery AS, et al. First-inhuman phase I trial of a tumor-targeted cytokine (NHS-IL12) in subjects with metastatic solid tumors. Clin Cancer Res. American Association for Cancer Research Inc.; 2019;25:99-109.
20. Kueberuwa G, Kalaitsidou M, Cheadle E, Hawkins RE, Gilham DE. CD 19 CAR T Cells Expressing IL- 12 Eradicate Lymphoma in Fully Lymphoreplete Mice through Induction of Host Immunity. Mol Ther - Oncolytics. 2018;
21. Schoenhaut DS, Chua a O, Wolitzky a G, Quinn PM, Dwyer CM, McComas W, et al. Cloning and expression of murine IL-12. J Immunol. 1992;
22. Chang CH, Qiu J, O’ Sullivan D, Buck MD, Noguchi T, Curtis JD, et al. Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression. Cell. 2015;
23. Matloubian M, Lo CG, Cinamon G, Lesneski MJ, Xu Y, Brinkmann V, et al. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on SIP receptor 1. Nature. Nature; 2004;427:355-60.
24. Cohen J. IL-12 Deaths: Explanation and a Puzzle. Science (80- ). 1995;
25. Bedoui S, Whitney PG, Waithman J, Eidsmo L, Wakim L, Caminschi I, et al. Crosspresentation of viral and self antigens by skin-derived CD103+ dendritic cells. Nat Immunol. 2009;10:488-95.
26. Overacre-Delgoffe AE, Chikina M, Dadey RE, Yano H, Brunazzi EA, Shay an G, et al. Interferon-v Drives T reg Fragility to Promote Anti-tumor Immunity. Cell. 2017;169: 1130- 1141. el l 27. Pearce EL, Poffenberger MC, Chang CH, Jones RG. Fueling immunity: Insights into metabolism and lymphocyte function. Science (80-. ). 2013.
28. Scharping NE, Menk A V, Moreci RS, Whetstone RD, Dadey RE, Watkins SC, et al. The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction. Immunity. Elsevier; 2016;45:374-88.
29. McLane LM, Abdel -Hakeem MS, Wherry EJ. CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu. Rev. Immunol. 2019.
30. Siska PJ, Beckermann KE, Mason FM, Andrejeva G, Greenplate AR, Sender AB, et al. Mitochondrial dysregulation and glycolytic insufficiency functionally impair CD8 T cells infiltrating human renal cell carcinoma. JCI Insight. 2017;
31. Sena LA, Li S, Jairaman A, Prakriya M, Ezponda T, Hildeman DA, et al. Mitochondria Are Required for Antigen-Specific T Cell Activation through Reactive Oxygen Species Signaling. Immunity. 2013;
32. Rackov G, Tavakoli Zaniani P, Colomo del Pino S, Shokri R, Monserrat J, Alvarez-Mon M, et al. Mitochondrial reactive oxygen is critical for IL-12/IL-18-induced IFN-y production by CD4+ T cells and is regulated by Fas/FasL signaling. Cell Death Dis 2022 136. Nature Publishing Group; 2022;13:1-14.
33. Mo F, Yu Z, Li P, Oh J, Spolski R, Zhao L, et al. An engineered IL-2 partial agonist promotes CD8+ T cell sternness. Nature. 2021;
34. Hong Y, Robbins Y, Yang X, Mydlarz WK, Sowers A, Mitchell JB, et al. Cure of syngeneic carcinomas with targeted IL- 12 through obligate reprogramming of lymphoid and myeloid immunity. JCI Insight. 2022;
35. Xue D, Moon B, Liao J, Guo J, Zou Z, Han Y, et al. A tumor-specific pro-IL-12 activates preexisting cytotoxic T cells to control established tumors. Sci Immunol. American Association for the Advancement of Science; 2022;7.
36. Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB, et al. Effects of single-dose interleukin- 12 exposure on interleukin- 12 associated toxicity and interferon-y production. Blood. 1997;
37. Hildner K, Edelson BT, Purtha WE, Diamond M, Matsushita H, Kohyama M, et al. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science. 2008;322: 1097-100.
38. Broz ML, Binnewies M, Boldajipour B, Nelson AE, Pollack JL, Erie DJ, et al. Dissecting the Tumor Myeloid Compartment Reveals Rare Activating Antigen-Presenting Cells Critical for T Cell Immunity. Cancer Cell. Elsevier; 2014;26:638-52.
39. Nirschl CJ, Drake CG. Molecular pathways: coexpression of immune checkpoint molecules: signaling pathways and implications for cancer immunotherapy. Clin Cancer Res.
2013;19:4917-24.
40. Siddiqui I, Schaeuble K, Chennupati V, Fuertes Marraco SA, Calderon-Copete S, Pais Ferreira D, et al. Intratumoral Tcfl + PD-1 + CD8 + T Cells with Stem-like Properties Promote Tumor Control in Response to Vaccination and Checkpoint Blockade Immunotherapy.
Immunity. 2019.
7. SEQUENCE DISCLOSURE
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Claims

1. Use of an inducible interleukin- 12 (IL-12) prodrug for the manufacture of a medicament for selectively activating tumor infiltrating lymphocytes in a subject, comprising administering to the subject in need thereof and effective amount of the IL- 12 prodrug, wherein the inducible IL- 12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results a significantly higher frequency of CD8+ T cells that produce IFNgamma and/or Granzyme.
2. Use of an inducible interleukin- 12 (IL-12) prodrug for the manufacture of a medicament for inducing immunological memory to a tumor in a subject, comprising administering to the subject in need thereof an effective amount of the IL- 12 prodrug, wherein the inducible IL- 12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other areas.
3. Use of an inducible interleukin- 12 (IL-12) prodrug for the manufacture of a medicament for selectively activating effector CD8+ T cells in the tumor microenvironment in a subject, comprising administering to the subject in need thereof and effective amount of the IL-12 prodrug, wherein the inducible IL- 12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results a significantly higher frequency of CD8+ T cells that produce TNF and/or IFNgamma within the tumor in comparison to peripheral tissue.
4. The use of any one of the preceding claims, wherein the inducible IL-12 prodrug results in tumor reactive CD8+ T cells that produce IL-12 and IFNgamma.
5. Use of an inducible interleukin-2 (IL-12) prodrug for the manufacture of a medicament for treating cancer, comprising administering to a subject in need thereof and effective amount of the prodrug, wherein the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results in at least about 40-fold more cleavage of the inducible TL-12 prodrug in the tumor microenvironment compared with the circulation.
6. The use of claim 5, wherein at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75- fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 93-fold, at least about 95-fold, or at least about 100-fold more cleavage of the inducible IL-12 prodrug in the tumor microenvironment compared with the circulation.
7. The use of claim 5 or 6, wherein the administration results in an amount of inducible IL- 12 prodrug in the plasma that is at least about 5-fold greater than the amount of inducible IL-12 prodrug in the tumor.
8. The use of claim 7, wherein the among of inducible IL- 12 prodrug in the plasma is at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 18-fold, at least about 20-fold, or at least about 25-fold greater than the amount of inducible IL-12 prodrug in the tumor.
9. The use of any one of the preceding claims, wherein the inducible IL-12 prodrug is Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, or an amino acid sequence variant of any of the foregoing.
10. The use of anyone of the preceding claims, wherein the inducible IL-12 prodrug is Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20, Compound 21, Compound 22, Compound 23, Compound 24, Compound 25, Compound 26, Compound 27, Compound 28, Compound 29, Compound 30, Compound 31, Compound 32, Compound 33, Compound 34, Compound 35, Compound 36 or an amino acid sequence variant of any of the foregoing.
1 1 . The use of any one of the preceding claims, wherein the inducible TL-12 prodrug is administered about twice a week or less frequently.
12. The use of any one of the preceding claims, wherein the inducible IL-12 prodrug is administered about once a week or less frequently.
13. The use of any one of the preceding claims, wherein the inducible IL-12 is administered about once every 2 weeks.
14. The use of any one of the preceding claims, further comprising administering one or more additional agents.
15. The use of claim 14, wherein the one or more additional agents is an adoptive cell therapy.
16. The use of claim 15, wherein the adoptive cell therapy is a CAR-T cell therapy or a T-cell therapy.
The use of claim 15, wherein the one or more additional agents is a checkpoint inhibitor.
17. A method for selectively activating tumor infdtrating lymphocytes, comprising administering to a subject in need thereof and effective amount of an inducible interleukin-2 (IL- 12) prodrug, wherein the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results a significantly higher frequency of CD8+ T cells that produce IFNgamma and/or Granzyme.
18. A method for inducing immunological memory to a tumor, comprising administering to a subject in need thereof an effective amount of an inducible interleukin- 12 (11-12) prodrug, wherein the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other areas.
19. A method for selectively activating effector CD8+ T cells in the tumor microenvironment, comprising administering to a subject in need thereof and effective amount of an inducible interleukin- 12 (IL-12) prodrug, wherein the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results a significantly higher frequency of CD8+ T cells that produce TNF and/or IFNgamma within the tumor in comparison to peripheral tissue.
20. The method of any one of the preceding claims, wherein the inducible IL- 12 prodrug results in tumor reactive CD 8+ T cells that produce IL- 12 and IFNgamma.
21. A method for treating cancer, comprising administering to a subject in need thereof and effective amount of an inducible interleukin- 12 (IL-12) prodrug, wherein the inducible IL-12 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results in at least about 40- fold more cleavage of the inducible IL- 12 prodrug in the tumor microenvironment compared with the circulation.
22. The method of claim 21, wherein at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 93- fold, at least about 95-fold, or at least about 100-fold more cleavage of the inducible IL-12 prodrug in the tumor microenvironment compared with the circulation.
23. The method of claim 21 or 22, wherein the administration results in an amount of inducible IL-12 prodrug in the plasma that is at least about 5-fold greater than the amount of inducible IL-12 prodrug in the tumor.
24. The method of claim 23, wherein the among of inducible IL-12 prodrug in the plasma is at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 18-fold, at least about 20-fold, or at least about 25-fold greater than the amount of inducible TL-12 prodrug in the tumor.
25. The method of any one of the preceding claims, wherein the inducible IL-12 prodrug is Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 or an amino acid sequence variant of the foregoing.
26. The method of anyone of the preceding claims, wherein the inducible IL-12 prodrug is Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20, Compound 21, Compound 22, Compound 23, Compound 24, Compound 25, Compound 26, Compound 27, Compound 28, Compound 29, Compound 30, Compound 31, Compound 32, Compound 33, Compound 34, Compound 35, Compound 36 or an amino acid sequence variant of any of the foregoing.
27. The method of any one of the preceding claims, wherein the inducible IL- 12 prodrug is administered about twice a week or less frequently.
28. The method of any one of the preceding claims, wherein the inducible IL-12 prodrug is administered about once a week or less frequently.
29. The method of any one of the preceding claims, wherein the inducible IL- 12 is administered about once every 2 weeks.
30. The method of any one of the preceding claims, further comprising administering one or more additional agents.
31. The method of claim 30, wherein the one or more additional agents is an adoptive cell therapy.
32. The method of claim 31 , wherein the adoptive cell therapy is a CAR-T cell therapy or a T-cell therapy.
33. The method of claim 30, wherein the one or more additional agents is a checkpoint inhibitor.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006110728A2 (en) 2005-04-12 2006-10-19 The Uab Research Foundation Immunogenic cmv tegument aggregates
WO2019222296A1 (en) * 2018-05-14 2019-11-21 Werewolf Therapeutics, Inc. Activatable interleukin 12 polypeptides and methods of use thereof
WO2021236676A1 (en) * 2020-05-19 2021-11-25 Werewolf Therapeutics, Inc. Activatable il-12 polypeptides and methods of use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006110728A2 (en) 2005-04-12 2006-10-19 The Uab Research Foundation Immunogenic cmv tegument aggregates
WO2019222296A1 (en) * 2018-05-14 2019-11-21 Werewolf Therapeutics, Inc. Activatable interleukin 12 polypeptides and methods of use thereof
WO2021236676A1 (en) * 2020-05-19 2021-11-25 Werewolf Therapeutics, Inc. Activatable il-12 polypeptides and methods of use thereof

Non-Patent Citations (46)

* Cited by examiner, † Cited by third party
Title
"Remington: The Science and Practice of Pharmacy", 2005, LIPPICOTT WILLIAMS & WILKINS
BEDOUI SWHITNEY PGWAITHMAN JEIDSMO LWAKIM LCAMINSCHI I ET AL.: "Cross-presentation of viral and self antigens by skin-derived CD103+ dendritic cells", NAT IMMUNOL, vol. 10, 2009, pages 488 - 95
BERRAONDO PSANMAMED MFOCHOA MCETXEBERRIA IAZNAR MAPEREZ-GRACIA JL ET AL.: "Cytokines in clinical cancer immunotherapy", BR. J. CANCER. NATURE PUBLISHING GROUP, 2019, pages 6 - 15
BROZ MLBINNEWIES MBOLDAJIPOUR BNELSON AEPOLLACK JLERLE DJ ET AL.: "Cancer Cell", vol. 26, 2014, ELSEVIER, article "Dissecting the Tumor Myeloid Compartment Reveals Rare Activating Antigen-Presenting Cells Critical for T Cell Immunity", pages: 638 - 52
CHANG CHQIU JO' SULLIVAN DBUCK MDNOGUCHI TCURTIS JD ET AL.: "Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression", CELL, 2015
CHARY CH DHHOCH ULANGOWSKI JLLEE SRADDEPALLI MKKIRK PB ET AL.: "Clin Cancer Res. American Association for Cancer Research Inc.", vol. 22, 2016, article "NKTR-214, an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models", pages: 680 - 90
COHEN J.: "IL-12 Deaths: Explanation and a Puzzle", SCIENCE, vol. 80, 1995
CONLON KCMILJKOVIC MDWALDMANN TA: "J Interf Cytokine Res", vol. 39, 2019, MARY ANN LIEBERT, INC., article "Cytokines in the Treatment of Cancer", pages: 6 - 21
DAVIDSON ET AL., J. VIROL., vol. 61, 1987, pages 1226 - 39
DEL VECCHIO M, BAJETTA E, CANOVA S, LOTZE MT, WESA A, PARMIANI G: "Interleukin-12: biological properties and clinical application.", CLIN CANCER RES, vol. 13, 2007, pages 4677 - 85, XP055903839, DOI: 10.1158/1078-0432.CCR-07-0776
GARCEAGISSMANN, CURRENT OPINION IN BIOTECHNOLOGY, vol. 15, 2004, pages 513 - 7
GLASSMAN CRMATHIHARAN YKJUDE KMSU LPANOVA OLUPARDUS PJ ET AL.: "Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells", CELL, 2021
HAJ-AHMAD ET AL., J. VIROL., vol. 57, 1986, pages 267 - 74
HILDNER KEDELSON BTPURTHA WEDIAMOND MMATSUSHITA HKOHYAMA M ET AL.: "Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity", SCIENCE, vol. 322, 2008, pages 1097 - 100, XP055422537, DOI: 10.1126/science.1164206
HONG YROBBINS YYANG XMYDLARZ WKSOWERS AMITCHELL JB ET AL.: "Cure of syngeneic carcinomas with targeted IL-12 through obligate reprogramming of lymphoid and myeloid immunity", JCI INSIGHT, 2022
KLEIN CWALDHAUER INICOLINI VGFREIMOSER-GRUNDSCHOBER ANAYAK TVUGTS DJ ET AL.: "Oncoimmunology", vol. 6, 2017, TAYLOR AND FRANCIS INC., article "Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines"
KUEBERUWA GKALAITSIDOU MCHEADLE EHAWKINS REGILHAM DE: "CD19 CAR T Cells Expressing IL-12 Eradicate Lymphoma in Fully Lymphoreplete Mice through Induction of Host Immunity. Mol Ther", ONCOLYTICS, 2018
LASEK WZAGOZDZON RJAKOBISIAK M.: "Interleukin 12: Still a promising candidate for tumor immunotherapy?", CANCER IMMUNOL. IMMUNOTHER, 2014
LEONARD JPSHERMAN MLFISHER GLBUCHANAN LJLARSEN GATKINS MB ET AL.: "Effects of single-dose interleukin-12 exposure on interleukin-12 associated toxicity and interferon-γ production", BLOOD, 1997
LOPES JEFISHER JLFLICK HLWANG CSUN LERNSTOFF MS ET AL.: "J Immunother Cancer", vol. 8, 2020, BMJ PUBLISHING GROUP, article "ALKS 4230: A novel engineered IL-2 fusion protein with an improved cellular selectivity profile for cancer immunotherapy", pages: 673
MASSIE ET AL., MOL. CELL. BIOL., vol. 6, 1986, pages 2872 - 83
MATLOUBIAN MLO CGCINAMON GLESNESKI MJXU YBRINKMANN V ET AL.: "Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on SIP receptor 1. Nature", NATURE, vol. 427, 2004, pages 355 - 60
MCLANE LMABDEL-HAKEEM MSWHERRY EJ: "CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer", ANNU. REV. IMMUNOL, 2019
MIRLEKAR BPYLAYEVA-GUPTA Y: "IL-12 family cytokines in cancer and immunotherapy", CANCERS, 2021
MO FYU ZLI POH JSPOLSKI RZHAO L ET AL.: "An engineered IL-2 partial agonist promotes CD8+ T cell sternness", NATURE, 2021
NGUYEN KGVRABEL MRMANTOOTH SMHOPKINS JJWAGNER ESGABALDON TA ET AL.: "Localized Interleukin-12 for Cancer Immunotherapy", FRONT. IMMUNOL, 2020
NIRSCHL CJ, DRAKE CG: "Molecular pathways: coexpression of immune checkpoint molecules: signaling pathways and implications for cancer immunotherapy", CLIN CANCER RES, pages 4917 - 24
NIRSCHL CJBRODKIN HRHICKLIN DJISMAIL NMORRIS KSEIDEL-DUGAN C ET AL.: "Cancer Immunol Res", vol. 10, 2022, AMERICAN ASSOCIATION FOR CANCER RESEARCH (AACR, article "Discovery of a Conditionally Activated IL-2 that Promotes Antitumor Immunity and Induces Tumor Regression", pages: 581 - 96
OVERACRE-DELGOFFE AECHIKINA MDADEY REYANO HBRUNAZZI EASHAYAN G ET AL.: "Interferon-γ Drives T reg Fragility to Promote Anti-tumor Immunity", CELL, vol. 169, 2017, pages 1130 - 1141
PEARCE ELPOFFENBERGER MCCHANG CHJONES RG: "Fueling immunity: Insights into metabolism and lymphocyte function", SCIENCE, vol. 80, 2013
PEPPERL-KLINDWORTH ET AL., GENE THERAPY, vol. 10, 2003, pages 278 - 84
PROPPER DJBALKWILL FR: "Harnessing cytokines and chemokines for cancer therapy", NAT. REV. CLIN. ONCOL, 2022, pages 237 - 53, XP037766705, DOI: 10.1038/s41571-021-00588-9
RACKOV GTAVAKOLI ZANIANI PCOLOMO DEL PINO SSHOKRI RMONSERRAT JALVAREZ-MON M ET AL.: "Intratumorally anchored cytokine therapy", vol. 13, 2022, NATURE PUBLISHING GROUP, article "Mitochondrial reactive oxygen is critical for IL-12/IL-18-induced IFN-γ production by CD4+ T cells and is regulated by Fas/FasL signaling", pages: 1 - 14
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
SCHARPING NEMENK A VMORECI RSWHETSTONE RDDADEY REWATKINS SC ET AL.: "Immunity", vol. 45, 2016, ELSEVIER, article "The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction", pages: 374 - 88
SCHOENHAUT DSCHUA A OWOLITZKY A GQUINN PMDWYER CMMCCOMAS W ET AL.: "Cloning and expression of murine IL-12", J IMMUNOL, 1992
SENA LALI SJAIRAMAN APRAKRIYA MEZPONDA THILDEMAN DA ET AL.: "Mitochondria Are Required for Antigen-Specific T Cell Activation through Reactive Oxygen Species Signaling", IMMUNITY, 2013
SHARMA MKHONG HFA'AK FBENTEBIBEL SEJANSSEN LMECHESSON BC ET AL.: "Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy", NAT COMMUN. NATURE RESEARCH, vol. 11, 2020, XP055815480, DOI: 10.1038/s41467-020-14471-1
SIDDIQUI ISCHAEUBLE KCHENNUPATI VFUERTES MARRACO SACALDERON-COPETE SPAIS FERREIRA D ET AL.: "Intratumoral Tcfl + PD-1 + CD8 + T Cells with Stem-like Properties Promote Tumor Control in Response to Vaccination and Checkpoint Blockade Immunotherapy", IMMUNITY, 2019
SISKA PJBECKERMANN KEMASON FMANDREJEVA GGREENPLATE ARSENDER AB ET AL.: "Mitochondrial dysregulation and glycolytic insufficiency functionally impair CD8 T cells infiltrating human renal cell carcinoma", JCI INSIGHT, 2017
STRAUSS JHEERY CRKIM JWJOCHEMS CDONAHUE RNMONTGOMERY AS ET AL.: "Clin Cancer Res", vol. 25, 2019, AMERICAN ASSOCIATION FOR CANCER RESEARCH (AACR, article "First-inhuman phase I trial of a tumor-targeted cytokine (NHS-IL12) in subjects with metastatic solid tumors", pages: 6284 - 109
TAIT WOJNO EDHUNTER CASTUMHOFER JS: "The Immunobiology of the Interleukin-12 Family: Room for Discovery", IMMUNITY, 2019
WALDMANN TA.: "Cold Spring Harb Perspect Biol", 2018, COLD SPRING HARBOR LABORATORY PRESS, article "Cytokines in cancer immunotherapy", pages: 10
WANG PLI XWANG JGAO DLI YLI H ET AL.: "Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent", NAT COMMUN. NATURE PUBLISHING GROUP, vol. 8, 2017, pages 1395
XUE DMOON BLIAO JGUO JZOU ZHAN Y ET AL.: "Sci Immunol", vol. 7, 2022, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, article "A tumor-specific pro-IL-12 activates preexisting cytotoxic T cells to control established tumors"
ZHANG ET AL., BIOTECHNIQUES, vol. 15, 1993, pages 868 - 72

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