WO2023060242A1 - Il-2 prodrug - Google Patents

Il-2 prodrug Download PDF

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Publication number
WO2023060242A1
WO2023060242A1 PCT/US2022/077772 US2022077772W WO2023060242A1 WO 2023060242 A1 WO2023060242 A1 WO 2023060242A1 US 2022077772 W US2022077772 W US 2022077772W WO 2023060242 A1 WO2023060242 A1 WO 2023060242A1
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fold
tumor
prodrug
inducible
compound
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English (en)
French (fr)
Inventor
Jose Andres Salmeron-Garcia
William Winston
Daniel Hicklin
Cynthia Seidel-Dugan
Heather BRODKIN
Christopher NIRSCHL
Tiziana CAFARELLI
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Werewolf Therapeutics Inc
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Werewolf Therapeutics Inc
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Priority to EP22797609.9A priority Critical patent/EP4412712A1/en
Priority to JP2024520927A priority patent/JP2024538707A/ja
Priority to CA3233893A priority patent/CA3233893A1/en
Priority to KR1020247014914A priority patent/KR20240082401A/ko
Publication of WO2023060242A1 publication Critical patent/WO2023060242A1/en
Priority to US18/626,430 priority patent/US20240374687A1/en
Anticipated expiration legal-status Critical
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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]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Immunode 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 (4).
  • Interleukin (IL)-2 is a critical cytokine driving the immune-mediated killing of cancer cells, and whose mechanism of action includes stimulation of both innate and adaptive immune cells.
  • the IL-2 receptor (IL-2R) is composed of three subunits: cluster of differentiation (CD)25 (IL-2R ⁇ ), CD122 (IL-2R ⁇ ), and CD132 (IL-2R ⁇ ). Signal transduction is mediated through a heterodimer of CD122 and CD132. Together, these molecules form the IL-2 medium-affinity receptor, which is expressed on natural killer (NK) cells, monocytes, macrophages, and resting CD4+ and CD8+ T cells.
  • NK natural killer
  • the trimeric IL-2 high-affinity receptor (CD25/CD122/CD132) is present on activated T and NK cells and constitutively expressed on CD4+FoxP3+ regulatory T cells (Tregs).
  • IL-2 increases the proliferation and activation of T cells and NK cells, and induces the differentiation of CD8+ T cells into effector and memory cells (5,6).
  • Recombinant human IL- 2 (proleukin) is approved for clinical use in metastatic melanoma and renal cell carcinoma as a high-dose therapy, but this treatment is associated with serious side effects, including vascular leakage syndrome and hypotension, limiting its practical use (5,7).
  • IL-2 high-dose therapy To address the limitations of IL-2 high-dose therapy, several approaches have been pursued to develop next-generation IL-2 molecules that only bind the medium-affinity receptor (CD122/CD132) in the hope of alleviating toxicities and reducing the activation of Tregs (7-10). However, many of these molecules still activate IL-2 receptors on non-tumor specific immune cells located in normal tissues and therefore, may not minimize toxicities associated with IL-2 signaling. Molecules that block IL-2 signaling in the periphery while delivering a fully active native IL-2 in the tumor microenvironment may be a more appropriate approach to achieve the full potential of IL-2 anti-tumor activity with minimal systemic toxicities.
  • CD122/CD132 medium-affinity receptor
  • IL-2 prodrugs include a native IL-2 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-2 blocking element (e.g., anti-IL-2 antibody binding fragment, such as a Fab) to block binding of IL-2 to IL-2 ⁇ / ⁇ receptors on normal tissue in the periphery.
  • 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-2 blocking element (e.g., anti-IL-2 antibody binding fragment, such as a Fab) to block binding of IL-2 to IL-2 ⁇ / ⁇ receptors on normal tissue in the periphery.
  • a half- life extension domain e.g., anti-human serum albumin antibody binding fragment such as a VH domain
  • an IL-2 blocking element
  • compositions and methods for treating cancer using an inducible IL-2 prodrug generally comprises administering to a subject in need thereof an effective amount of an inducible IL-2 prodrug.
  • the inducible IL-2 prodrug can be Compound 1, Compound 2, Compound 3, or Compound 4.
  • the inducible IL-2 prodrug can be any one of Compounds 5-29.
  • the inducible IL-2 prodrug is conditionally active.
  • the inducible IL-2 prodrug comprises two polypeptide chains.
  • the first polypeptide chain can comprise from amino to carboxy terminus: the IL-2 polypeptide – a protease cleavable linker – an anti-human serum albumin (HSA) binding single antibody variable domain – a linker that is preferably protease cleavable – VH and CH1 of an antibody that binds IL-2.
  • the first polypeptide chain can comprise from amino to carboxy terminus: the IL-2 polypeptide – a protease cleavable linker – VH and CH1 of an antibody that binds IL-2 – a linker that is preferably protease cleavable – an anti-human serum albumin (HSA) binding single antibody variable domain.
  • the second polypeptide chain comprises a VL and CL of an antibody that binds IL-2 and that together with the VH and CH1 of the first polypeptide chain form a Fab that binds the IL-2 polypeptide.
  • the prodrug typically remains intact.
  • the intact prodrug has attenuated IL-2 receptor agonist activity.
  • the protease cleavable linker is cleaved by a protease active in the site of interest, releasing an unattenuated form of IL-2.
  • This conditional activity preserves the immune stimulatory effects of IL-2 while limiting the systemic toxicity associated with non-inducible IL-2 therapy.
  • the intact IL-2 prodrug contains an element that extends its half-life, but the post-cleavage unattenuated form of IL-2 does not. As a result, the short half-life of IL-2 effectively limits toxicity outside of the site of interest.
  • the amount of inducible IL-2 prodrug in the circulation can be at least about 5-fold greater than the amount in the tumor, e.g., the amount in the circulation can be about 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-2 prodrug in the tumor. While more prodrug is found in the circulation than in the tumor microenvironment, the prodrug is processed (cleaved) to a greater extent in the tumor microenvironment to release active IL-2.
  • This disclosure relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of an inducible interleukin-2 (IL-2) prodrug, wherein the inducible IL-2 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-2 prodrug in the tumor microenvironment compared with the circulation.
  • the method can result in a significant increase in the tumor reactive CD8+/Treg ratio.
  • 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 interleukin-2 (IL-2) prodrug, wherein the inducible IL-2 prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations.
  • the amount of inducible IL-2 prodrug in the circulation can be at least about 5-fold greater that the amount in the tumor, e.g., the amount in the circulation can be about 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-2 prodrug in the tumor.
  • the immunological memory can be characterized by the presence of tumor reactive CD8+ cells with a memory phenotype (e.g., CD8+CD44hiCD62low), by tumor reactive CD8+ cells that produce TNF, IFNgamma and/or granzyme B upon restimulation, or tumor reactive CD8+ cells with a memory phenotype that produce TNF, IFNgamma and/or granzyme B upon restimulation.
  • This disclosure relates to a method for selectively activating effector CD8+ T cells in the tumor microenvironment, and to a method for selectively activating tumor infiltrating lymphocytes.
  • IL-2 inducible interleukin-2
  • IL-2 prodrug 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. cleavage by a protease that has higher activity in the tumor microenvironment than in other locations.
  • the amount of inducible IL-2 prodrug in the circulation can be at least about 5-fold greater that the amount in the tumor, e.g., the amount in the circulation can be about 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-2 prodrug in the tumor.
  • the inducible IL-2 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-2 prodrug can be administered about once every two weeks.
  • Preferred, inducible IL-2 prodrugs for use in the methods of this disclosure are Compound 1, Compound 2, Compound 3, Compound 4 or an amino acid sequence variant of any of the foregoing.
  • Other preferred inducible IL-2 prodrugs for use in the methods of this disclosure are Compounds 5-29.
  • Compound 1 comprises a first polypeptide chain of SEQ ID NO:1 and a second polypeptide chain of SEQ ID NO:5, and the amino acid sequence variant of 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:5.
  • Compound 2 comprises a first polypeptide chain of SEQ ID NO:2 and a second polypeptide chain of SEQ ID NO:5, 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:5.
  • Compound 3 comprises a first polypeptide chain of SEQ ID NO:3 and a second polypeptide chain of SEQ ID NO:5, 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:5.
  • Compound 4 comprises a first polypeptide chain of SEQ ID NO:1 and a second polypeptide chain of SEQ ID NO:4, 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 NO:4 and a second polypeptide chain that has at least about 80% identity to SEQ ID NO:5. 3.
  • FIGs.1A-1F show the design and development of inducible IL-2 prodrugs represented by Compound 1.
  • FIG.1A is a diagram of the components of Compound 1.
  • FIG.1B depicts a non-reduced SDS-PAGE comparing intact and protease-cleaved Compound 1 (IL-2, anti-HSA half-life extension domain, and the Fab inactivation domain).
  • FIG.1C shows the in vitro activity of Compound 1 in the HEK-Blue IL-2 reporter assay comparing intact (squares), and protease- activated (cleaved) Compound 1 (triangles) to rhIL-2 (circles).
  • FIG.1D shows the in vitro activity of intact (squares) and cleaved (triangles) Compound 1 in primary human Tblasts compared to rhIL-2 (circles).
  • FIG.1E shows the in vitro activity of intact (squares) and cleaved (triangles) Compound 1 in primary murine Tblasts compared to rhIL-2 (circles).
  • FIG.1F shows the in vitro activity of intact (circles) and cleaved (triangles) Compound 1-NC in primary human Tblasts compared with rhIL-2 squares).
  • FIGs.1C-1F curves are representative of at least duplicate wells and depict the mean ⁇ SD for individual points; data are representative of at least two experiments.
  • FIGs.2A-2J depict that Compound 1 induced tumor regression in a cleavage-dependent manner.
  • FIG.2A is a series of graphs that show tumor volume over time in mice treated with various doses of Compound 1, Compound-NC (non-cleavable control), or vehicle. Spider plots for individual mice are shown (dashed lines), and the average tumor volume for the group is shown as the bold line.
  • FIG.2B are graphs that show the body weight and survival from individual mice over time is shown treated with either Compound 1 or WW0177 (a Compound 1 variant lacking the inactivation domain). Body weight and survival from individual mice over time is shown.
  • FIG.2D are graphs showing the tumor volume over time in mice treated with efficacious amounts of either Compound 1 (5.04 ⁇ M total) or rhIL-2 (15.5 ⁇ M total). Spider plots for individual mice are shown.
  • FIG.2E is a graph showing total IL-2 over time in the plasma from tumor-bearing mice. Samples were taken at various timepoints and analyzed for either the presence of the total inducible IL-2 protein using an ELISA that detects both intact Compound 1 as well as free IL-2.
  • FIG.2F is a graph showing total IL-2 over time in tumor samples from tumor-bearing mice. Samples were taken at various timepoints and analyzed for either the presence of the total inducible IL-2 protein using an ELISA that detects both intact Compound 1 as well as free IL-2.
  • FIG.2G is a graph showing total IL-2 over time in the plasma from tumor-bearing mice.
  • FIG.2H a graph showing total IL-2 over time in tumor samples from tumor-bearing mice. Samples were taken at various timepoints and analyzed for either the presence of the total inducible IL-2 protein using an ELISA that detects free human IL-2 using an AlphaLISA specific for unblocked human IL-2.
  • FIG. 2E-2H are presented as the mean ⁇ SD, and area under the curve measurements were calculated using GraphPad Prism software.
  • FIG.2I is a graph showing tumor volume (mm 3 ) at day 18 with vehicle, Compound 1 at 25 ⁇ g, 50 ⁇ g, 100 ⁇ g and 300 ⁇ g, and Compound 1-NC at 300 ⁇ g.
  • FIG. 2J is a graph showing that the anti-tumor activity of Compound 1 was greatly reduced in mice when CD8+ T cells were depleted by anti-CD8 antibody treatment twice per week.
  • FIG.3A-3I demonstrate that Compound 1 induced anti-tumor memory response.
  • FIGs. 3A-3B show the frequency of tetramer-positive CD8+ T cells in splenocytes.
  • FIG.3C-3D show the expression of the memory cell markers CD62L and CD44 on tetramer-positive CD8+ T cells in splenocytes.
  • FIG.3E-3F show the frequency of tetramer-positive CD8+ T cells producing TNF or IFN ⁇ .
  • FIG.3G are pie graphs showing the analysis of polyfunctional tetramer-positive CD8+ T cells co-expressing IFN ⁇ and TNF.
  • FIG.3H is a schematic of a tumor challenge and rechallenge study.
  • FIG.4A depicts a heatmap of transcripts with statistically significant differences in expression between the two treatments (Compound 1 and Vehicle control). Transcripts were excluded from the heat map if they had average normalized counts below 50. Each lane represents an individual animal.
  • FIG.4B is a plot of transcripts differentially expressed between Compound 1 and vehicle-treated mice.
  • FIG.4C depict specific pathway scores for Compound 1 or vehicle-treated mice. P values are derived from a 2-way ANOVA with multiple comparisons (***, P ⁇ 0.001; ****, P ⁇ 0.0001).
  • FIG.4D shows normalized gene counts from selected immune checkpoint genes.
  • FIG.4E depicts diagrams from flow cytometry analysis of TIL density of various immune populations, including fold change information between the vehicle- and Compound 1-treated groups.
  • FIG.4F show the ratio of total CD8+ T cells or tetramer-positive CD8+ T cells to Tregs within the TILs, including fold change information between the vehicle- and Compound 1-treated groups.
  • FIGs.4G-4H show the frequency of tetramer-positive CD8+ T cells producing IFN ⁇ after re-stimulation with PMA/lonomycin.
  • FIG.4I shows pie graphs of the analysis of polyfunctional tetramer-positive CD8+ T cells by examining co-expression of IFN ⁇ , TNF, and granzyme B after PMA/lonomycin restimulation.
  • FIGs.4J-4K show the frequency of tumor-infiltrating FoxP3+ Tregs producing IFN ⁇ in the vehicle (control) and Compound 1 groups.
  • FIGs.4L-4M show the frequency of tumor-infiltrating FoxP3+ Tregs producing TNF after PMA/lonomycin restimulation in the vehicle (control) and Compound 1 groups. Unless otherwise stated, data are presented as the mean ⁇ SD, and P values are derived from t tests (*, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001). [018]
  • FIGs.5A-5C show that systemic treatment with Compound 1 preferentially activated tumor-infiltrating T cells.
  • FIG.5A are graphs showing the frequency of tetramer-negative CD8+ T cells in vehicle (control) and Compound 1 groups in the TILs, spleenocytes, DLN, and peripheral blood.
  • FIG.5B are graphs showing the frequency of CD4+ non-Tregs producing IFN ⁇ in vehicle (control) and Compound 1 groups in TILs, spleenocytes, DLN, and peripheral blood after re-stimulation with PMA/lonomycin.
  • FIGs.6A-6G show that treatment with Compound 1 increased CD8+ T cell activation and Treg fragility in B16-F10.
  • FIG.6A shows tumor volume measured over time in mice treated with vehicle, Compound 1 at 100 ⁇ g/animal and 200 ⁇ g/animal, and Compound 1 at 100 ⁇ g/animal and 200 ⁇ g/animal in combination with an anti-PD1 inhibitor. Data from individual mice (dashed lines) are depicted with the group average presented in the bold line.
  • FIG.6B depicts a heatmap of transcripts with statistically significant differences in expression between the two treatments (vehicle control and Compound 1). Transcripts were excluded from the heat map if they had average normalized counts below 50. Each lane represents an individual animal.
  • FIGs.6C–6H are a series of graphs showing the results of TILs that were re-stimulated and examined for production of effector cytokines and proteins and proliferation.
  • FIG.6C are representative flow plots of tetramer-positive CD8+ T cells.
  • FIG.6D are graphs showing the quantitative analysis from individual mice.
  • FIG.6E are representative flow plots of NK cells and
  • FIG.6F are graphs showing the quantitative analysis from individual mice.
  • FIG.6G are representative flow plots of FoxP3+ Tregs and FIG.6H are graphs showing the quantitative analysis from individual mice.
  • P values are derived from t tests (*, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001).
  • FIGs.7A-7B show that Compound 1 was stable in human serum and selectively processed by human tumor cells.
  • FIGs.8A-8D show activity of Compound 1 in additional human donors and mice.
  • FIGs.8A-8B show CD25 expression by Tblasts over time in response to PHA stimulation.
  • Data in FIG.8A are representative of an individual human donor.
  • FIGs.8C-8D are graphs showing in vitro activity of Compound 1 in primary human Tblasts (FIG.8C) and murine Tblasts (FIG.8D) derived from additional donors, comparing intact (circle) and protease-activated (cleaved) WTX-124 (downward triangle) with rhIL-2 (upward triangle).
  • FIGs.9A-9B shows that Compound 1 is well tolerated in mice.
  • FIG.9B is a graph showing a representative therapeutic window of rhIL-2 and Compound 1 in MC38 tumor-bearing mice. [023] FIGs.10A-10B shows that effector cytokine production of MC38 tumor-infiltrating tetramer-positive CD8+T cells.
  • FIGs.11 is a graph showing that PD-1 monotherapy does not have anti-tumor activity in B16-F10.
  • FIGs.12A-12B shows the results of a baseline tumor-infiltrating lymphocyte analysis. MC38 and B16-F10 tumors were implanted and allowed to grow to an average volume of 100 mm 3 before tumors were harvested for TIL analysis.
  • FIG.12A shows the gating strategy for the identification of various immune cell populations.
  • FIG.12B shows the frequency of various immune populations within the CD45+ population.
  • FIGs.13A-13G shows the results of Compound 1 treatment in mice.
  • MC38 tumor cells were implanted and allowed to grow to an average volume of 100–150 mm 3 before mice were randomized into treatment groups. Labels in the legend represent the dose per mouse per dosing day. Mice were dosed IP twice a week for a total of four doses.
  • FIG. 13B is a graph showing tumor volume measured over time and is depicted as the mean +/- SEM.
  • FIG.13C is a graph showing the results of MC38 tumor bearing mice randomized and dosed with either vehicle, Compound 1, or a IL-2 prodrug missing the half life extension element. Tumor volume was measured over time and is depicted as the mean +/- SEM.
  • FIG.13D shows the results of mice dosed with equimolar amounts of recombinant human IL-2 (5 total doses over three days), WW0177 (2 doses over three days), or Compound 1 (2 doses over three days), before mice were injected intravenously with Evan’s Blue solution. Evan’s Blue extravasation into the lungs was measured 30 minutes following intravenous administration of the dye.
  • FIG.13D shows the results of mice dosed with equimolar amounts of recombinant human IL-2 (5 total doses over three days), WW0177 (2 doses over three days), or Compound 1 (2 doses over three days), before mice were injected intravenously with Evan’s Blue solution. Evan’s Blue extravasation into the lungs was measured 30 minutes
  • FIG. 13E is a graph showing detection of either recombinant human IL-2 or recombinant mouse IL-2 by a human specific IL-2 ELISA.
  • FIG.13F is a graph showing detection of either Compound 1 or free IL-2 by a human specific IL-2 Alphalisa.
  • FIG.13G is a graph showing the Therapeutic Window representation of rhIL-2, WW0177, or Compound 1 in MC38 tumor-bearing mice. P values are derived from t tests (*, P ⁇ 0.05; ***, P ⁇ 0.001).
  • FIGs.14A-14C is a graph showing that Compound 1 is superior to equimolar amounts of recombinant human IL-2 at activating B16-F10 TILs.
  • B16-F1 tumors were implanted and allowed to grow to an average volume of 100 mm 3 before mice were randomized into treatment groups. Mice were dosed IP twice a week for two weeks with either vehicle (hollow circles; or anti-PD-1 (solid circles, 200 ⁇ g)).
  • FIGs.14B-14C show the results of tumors from mice treated with either the vehicle, Compound 1 (200 ⁇ g/dose) or equimolar amounts of recombinant human IL-2 that were harvested on Day 5.
  • FIG.14B shows quantitative analysis of CD25 expression and FIG.14C Ki67 expression by various immune cell subsets. P values are derived from one way ANOVA analysis with multiple comparisons (*, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001).
  • FIGs.15A-15C are graphs showing that a variant of Compound 1 containing a non-alpha IL-2 mutein (Compound 5) has no anti-tumor activity when compared to the same dose of Compound 1.
  • FIG.15A are graphs showing tumor volume measured overtime in MC38 tumor beating mice treated either with vehicle, Compound 1 (containing a native IL-2 payload, 100 ⁇ g/dose), or a variant of Compound 1 containing a non-alpha IL-2 mutein as a payload (100 ⁇ g/dose) (Compound 5).
  • FIG.15B are graphs depicting the frequency of tumor infiltrating tetramer +CD8+ T cells producing Granzyme B, IFN ⁇ , or TNF on day 5.
  • FIG.15C are graphs showing the frequency of tumor infiltrating NK cells producing Granzyme B or IFN ⁇ . 4.
  • This disclosure relates to compositions and methods for treating cancer using an inducible IL-2 prodrug.
  • the method generally comprises administering to a subject in need thereof an effective amount of an inducible IL-2 prodrug.
  • the inducible IL-2 prodrug can be Compound 1, Compound 2, Compound 3, or Compound 4.
  • the inducible IL-2 prodrug can be any one of Compounds 5-29.
  • the inducible IL-2 prodrugs can selectively activate IL-2 in the tumor microenvironment and decreases IL-2-related toxicity while improving anti-tumor effects in patients with cancer.
  • inducible IL-2 is preferentially activated in tumor tissue by tumor-associated proteases, releasing active IL-2 in the tumor microenvironment.
  • in vitro assays confirmed that the activity of an inducible IL-2 prodrug (Compound 1) is dependent on proteolytic activation, and an inducible IL-2 prodrug treatment results in complete rejection of established tumors in a cleavage-dependent manner.
  • Compound 1 an inducible IL-2 prodrug
  • the inventors show that treatment with inducible IL-2 prodrug triggers the activation of T cells and natural killer cells, and markedly shifts the immune activation profile of the tumor microenvironment, resulting in significant inhibition of tumor growth in syngeneic tumor models.
  • inducible IL-2 prodrug minimizes the toxicity of IL-2 treatment in the periphery while retaining the full pharmacology of IL-2 in the tumor microenvironment, supporting its further development as a novel cancer immunotherapy treatment.
  • A. IL-2 Prodrugs [031] The inducible IL-2 prodrug for use in the methods and compositions of this disclosure overcome the toxicity and short half-life problems that have severely limited the clinical use of cytokines in oncology.
  • the inducible IL-2 prodrug contains an IL-2 polypeptide that has receptor agonist activity of native IL-2, including binding to and activating signaling through IL-2R ⁇ / ⁇ / ⁇ and IL-2R ⁇ / ⁇ , but in the context of the inducible pro-drug, the cytokine receptor agonist activity is attenuated, and the circulating half-life is extended.
  • the prodrug includes protease cleavage sequences, which are cleaved by proteases that are associated with, and are typically enriched or selectively present in, the tumor microenvironment.
  • the inducible IL-2 prodrugs are preferentially (or selectively) and efficiently cleaved in the tumor microenvironment to release active IL-2, and to limit IL-2 activity substantially to the tumor microenvironment.
  • the IL-2 that is released upon cleavage has a short half-life, which is substantially similar to the half-life of naturally occurring IL-2, further restricting IL-2 activity to the tumor microenvironment. Even though the half-life of the inducible IL-2 prodrug is extended, toxicity is dramatically reduced or eliminated because the circulating prodrug has attenuated IL-2 activity, and active IL-2 is restricted to the tumor microenvironment.
  • the inducible IL-2 prodrug comprises two polypeptide chains.
  • the first polypeptide chain can comprise from amino to carboxy terminus: the IL-2 polypeptide – a protease cleavable linker – an anti-human serum albumin (HSA) binding single antibody variable domain – a linker that is preferably protease cleavable – VH and CH1 of an antibody that binds IL-2.
  • the first polypeptide chain can comprise from amino to carboxy terminus: the IL-2 polypeptide – a protease cleavable linker – VH and CH1 of an antibody that binds IL-2 – a linker that is preferably protease cleavable – an anti-human serum albumin (HSA) binding single antibody variable domain.
  • the second polypeptide chain comprises a VL and CL of an antibody that binds IL-2 and that together with the VH and CH1 of the first polypeptide chain form a Fab that binds the IL-2 polypeptide.
  • Compounds 1, 2, 3 and 4 are specific examples of inducible IL-2 prodrugs for use according to this disclosure. Compounds 1, 2, 3, and 4 and additional details regarding their activity is disclosed in WO2021/097376. Compounds 5-29 are additional examples of inducible IL-2 prodrugs for use according to this disclosure. Table 1. Inducible IL-2 prodrugs
  • Amino acid sequence variants of compounds 1, 2, 3 and 4 that retain attenuated IL-2 activity in the periphery and that release active IL-2 upon protease cleavage in the tumor microenvironment can also be used in accordance with this disclosure.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:1 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:2 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:3 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:4 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 1 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 8.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 1 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 9.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 1 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 10.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 1 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 11.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 1 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 12.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 13 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 14 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 15 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 16 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 17 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 18 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 19 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 20 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 21 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 22 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 23 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 24 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 25 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 26 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 27 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 28 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 29 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 30 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 31 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 32 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.
  • the protease cleavage site contain no amino acid replacements, or only conservative amino acid replacements, so that the sequence variant prodrug is cleaved in the tumor microenvironment and releases IL-2 to substantially the same degree as the corresponding parental prodrug.
  • the complementarity determining regions of the anti-HAS single variable domain and the anti-IL2 Fab contain no amino acid replacements, or only conservative amino acid replacements, so that a) the serum half-life of the sequence variant prodrug is substantially the same as the corresponding parental prodrug, and b) the attenuation of IL-2 agonist activity of the sequence variant prodrug is substantially the same as the corresponding parental prodrug.
  • Exemplary amino acid substitutions are provided in Table 2. Table 2. Exemplary amino acid substitutions
  • compositions for treating cancer using an inducible IL-2 prodrug optionally in combination with one or more additional therapeutic agents, such as a chemotherapeutic agents, cytokines, oncolytic viruses, immune-oncology agents, or a check point inhibitor s(e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody).
  • additional therapeutic agents such as a chemotherapeutic agents, cytokines, oncolytic viruses, immune-oncology agents, or a check point inhibitor s(e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody).
  • chemotherapeutic agents e.g., cyclophosphamide, mechlorethamine, melphalan, chlorambucil, ifosfamide, busulfan, N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mitomycin, diaziquone (AZQ), cisplatin, carboplatin, oxaliplatin, procarbazine, hexamethylmelamine, methotrexate, pemetrexed, fluorouracil (e.g.5- fluorouracil), capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, pen
  • Therapeutic agents such as antibodies, that bind immune checkpoint proteins and inhibit their immunosuppressive activity include the anti-PD1 antibodies pembrolizumab (KEYTRUDA), dostarlimab (JEMPERLI), cemiplimab-rwlc (LIBATYO), nivolumab (OPDIVO), camrelizumab, tislelizumab, toripalimab, and sintilimab (TYVYT); the anti-PD-L1 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-2 prodrug and any additional therapeutic agents is typically administered systemically, for example by intravenous injection or preferably intravenous infusion.
  • Other types of administration can be used, such as orally, parenterally, intravenous, intravenously, intra-articularly, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, intrahepatically, intracranially, nebulization/inhalation, by installation via bronchoscopy, or intratumorally.
  • the methods and compositions disclosed herein can be used to treat any suitable cancer, in particular solid tumors, such as sarcomas and carcinomas.
  • the methods and compositions disclosed herein can be used to treat 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, histio
  • the non-small cell lung cancer can be, for example, adenocarcinoma NSCLC, squamous cell NSCLC or large cell carcinoma NSCLC.
  • the methods and compositions disclosed herein can be used to treat 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, gest
  • the methods and compositions disclosed herein are used to treat melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell cancer (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability high or mismatch repair deficient cancer, microsatellite instability high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial carcinoma, tumor mutational burden high cancer, cutaneous squamous cell carcinoma (cSCC), triple negative breast cancer (TNBC), urothelial carcinoma, colorectal cancer or oesophageal carcinoma.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • HNSCC head and neck squamous cell
  • the methods and compositions disclosed herein are used to treat glioblastoma.
  • the methods and compositions disclosed herein are used to treat Merkel Cell Carcinoma (MCC), Urothelial Carcinoma (UC), Renal Cell Carcinoma (RCC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), triple negative breast cancer (TNBC), endometrial cancer, cutaneous squamous cell carcinoma (CSCC), basal cell carcinoma (BCC), melanoma, malignant pleural mesothelioma, classical Hodgkin lymphoma (cHL), squamous cell carcinoma of the head and neck (SCCHN), hepatocellular carcinoma (HCC), esophageal squamous cell carcinoma (ESCC), non-squamous non-small cell lung cancer, or nasopharyngeal carcinoma (NPC).
  • MCC Merkel Cell Carcinoma
  • UC Urothelial Carcinoma
  • RRCC Renal
  • the methods and compositions disclosed herein are used to treat colon cancer, lung cancer, melanoma, renal cell carcinoma, or breast cancer.
  • the methods and compositions disclosed herein are used to treat melanoma.
  • the methods and compositions disclosed herein can be used to treat melanoma in subjects with unresectable or metastatic melanoma.
  • the methods and compositions disclosed herein can be used for the adjuvant treatment of subjects with melanoma with involvement of lymph node(s) following complete resection.
  • the methods and compositions disclosed herein are used to treat non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the methods and compositions disclosed herein can be used to treat NSCLC in subjects with NSCLC expressing PD-L1 (e.g., Tumor Proportion Score (TPS) ⁇ 1%) as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is: stage III where subjects are not candidates for surgical resection or definitive chemoradiation, or metastatic.
  • PD-L1 Tumor Proportion Score (TPS) ⁇ 1%
  • TPS Tumor Proportion Score
  • the methods and compositions disclosed herein can be used to treat NSCLC in patients with metastatic NSCLC whose tumors express PD-L1 (TPS ⁇ 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy.
  • the methods and compositions disclosed herein can be used in combination with pemetrexed and platinum chemotherapy, as first-line treatment of patients with metastatic nonsquamous NSCLC, with no EGFR or ALK genomic tumor aberrations.
  • the methods and compositions disclosed herein can be used in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, as first-line treatment of patients with metastatic squamous NSCLC.
  • the methods and compositions disclosed herein are used to treat SCLC.
  • the methods and compositions disclosed herein can be used to treat SCLC in subjects with metastatic SCLC with disease progression on or after platinum- based chemotherapy and at least one other prior line of therapy.
  • the methods and compositions disclosed herein are used to treat HNSCC.
  • the methods and compositions disclosed herein can be used to treat HNSCC in subjects with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 (e.g., Combined Positive Score (CPS) ⁇ 1) as determined by an FDA- approved test.
  • the methods and compositions disclosed herein can be used to treat HNSCC in subjects with recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy.
  • the methods and compositions disclosed herein can be used in combination with platinum and fluorouracil for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC.
  • the methods and compositions disclosed herein are used to treat cHL.
  • the methods and compositions disclosed herein can be used to treat cHL in subjects with relapsed or refractory cHL.
  • the methods and compositions disclosed herein can be used to treat cHL in pediatric subjects with refractory cHL, or cHL that has relapsed after 2 or more lines of therapy.
  • the methods and compositions disclosed herein are used to treat PMBCL.
  • the methods and compositions disclosed herein can be used to treat PMBCL in subjects with refractory PMBCL, or in subjects who have relapsed after 2 or more prior lines of therapy.
  • the methods and compositions disclosed herein are used to treat urothelial carcinoma.
  • the methods and compositions disclosed herein can be used to treat urothelial carcinoma in subjects with locally advanced or metastatic urothelial carcinoma who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (e.g., Combined Positive Score (CPS) ⁇ 10) as determined by an FDA- approved test, or in subjects who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status.
  • PD-L1 e.g., Combined Positive Score (CPS) ⁇ 10
  • the methods and compositions disclosed herein can be used to treat urothelial carcinoma in subjects with locally advanced or metastatic urothelial carcinoma who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum- containing chemotherapy.
  • the methods and compositions disclosed herein can be used to treat urothelial carcinoma in subjects with Bacillus Calmette-Guerin (BCG)- unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.
  • BCG Bacillus Calmette-Guerin
  • NMIBC non-muscle invasive bladder cancer
  • CIS carcinoma in situ
  • the methods and compositions disclosed herein are used to treat Microsatellite Instability-High (MSI-H) or Mismatch Repair Deficient (dMMR) Cancer.
  • MSI-H Microsatellite Instability-High
  • dMMR Mismatch Repair Deficient
  • the methods and compositions disclosed herein can be used to treat MSI- H or dMMR cancer in subjects with unresectable or metastatic MSI-H or dMMR cancer wherein the solid tumors have progressed following prior treatment and the subject has no satisfactory alternative treatment options, or wherein the colorectal cancer has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.
  • the methods and compositions disclosed herein are used to treat Microsatellite Instability-High (MSI-H) or Mismatch Repair Deficient (dMMR) Colorectal Cancer.
  • MSI-H Microsatellite Instability-High
  • dMMR Mismatch Repair Deficient
  • the methods and compositions disclosed herein can be used to treat MSI-H or dMMR colorectal cancer in subjects with unresectable or metastatic MSI-H or dMMR colorectal cancer.
  • the methods and compositions disclosed herein are used to treat gastric cancer.
  • the methods and compositions disclosed herein can be used to treat gastric cancer in subjects with recurrent locally advanced or metastatic gastric or gastroesophageal junction adenocarcinoma whose tumors express PD-L1 (e.g., Combined Positive Score (CPS) ⁇ 1) as determined by an FDA-approved test, with disease progression on or after 2 or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2/neu-targeted therapy.
  • PD-L1 e.g., Combined Positive Score (CPS) ⁇ 1
  • CPS Combined Positive Score
  • the methods and compositions disclosed herein are used to treat esophageal cancer.
  • the methods and compositions disclosed herein can be used to treat esophageal cancer in subjects with locally advanced or metastatic esophageal or gastroesophageal junction (GEJ) (e.g., tumors with epicenter 1 to 5 centimeters above the GEJ) carcinoma that is not amenable to surgical resection or definitive chemoradiation, in combination with platinum- and fluoropyrimidine-based chemotherapy.
  • GEJ gastroesophageal junction
  • the methods and compositions disclosed herein can be used to treat esophageal cancer in subjects with locally advanced or metastatic esophageal or gastroesophageal junction (GEJ) (e.g., tumors with epicenter 1 to 5 centimeters above the GEJ) carcinoma that is not amenable to surgical resection or definitive chemoradiation, after one or more prior lines of systemic therapy for patients with tumors of squamous cell histology that express PD-L1 (CPS ⁇ 10) as determined by an FDA-approved test.
  • GEJ gastroesophageal junction
  • the methods and compositions disclosed herein are used to treat cervical cancer.
  • the methods and compositions disclosed herein can be used to treat cervical cancer in subjects with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (e.g., Combined Positive Score (CPS) ⁇ 1) as determined by an FDA-approved test.
  • PD-L1 e.g., Combined Positive Score (CPS) ⁇ 1
  • CPS Combined Positive Score
  • the methods and compositions disclosed herein are used to treat HCC.
  • the methods and compositions disclosed herein can be used to treat HCC in subjects who have been previously treated with sorafenib.
  • the methods and compositions disclosed herein are used to treat MCC.
  • the methods and compositions disclosed herein can be used to treat MCC in subjects with recurrent locally advanced or metastatic MCC.
  • the methods and compositions disclosed herein are used to treat RCC.
  • the methods and compositions disclosed herein can be used in combination with axitinib, for the first-line treatment of patients with advanced RCC.
  • the methods and compositions disclosed herein are used to treat endometrial carcinoma.
  • the methods and compositions disclosed herein can be used in combination with lenvatinib, for the treatment of subjects with advanced endometrial carcinoma that is not MSI-H or dMMR, who have disease progression following prior systemic therapy and are not candidates for curative surgery or radiation.
  • the methods and compositions disclosed herein are used to treat Tumor Mutational Burden-High (TMB-H) Cancer.
  • TMB-H Tumor Mutational Burden-High
  • the methods and compositions disclosed herein can be used to treat TMB-H cancer in subjects with unresectable or metastatic tumor mutational burden-high (e.g., ⁇ 10 mutations/megabase (mut/Mb)) solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options.
  • the methods and compositions disclosed herein are used to treat Cutaneous Squamous Cell Carcinoma (cSCC).
  • the methods and compositions disclosed herein can be used to treat cSCC in subjects with recurrent or metastatic cutaneous squamous cell carcinoma that is not curable by surgery or radiation.
  • the methods and compositions disclosed herein are used to treat Triple-Negative Breast Cancer (TNBC).
  • TNBC Triple-Negative Breast Cancer
  • the methods and compositions disclosed herein can be used in combination with chemotherapy, for the treatment of subjects with locally recurrent unresectable or metastatic TNBC whose tumors express PD-L1 (e.g., Combined Positive Score (CPS) ⁇ 10) as determined by an FDA approved test.
  • PD-L1 e.g., Combined Positive Score (CPS) ⁇ 10
  • CPS Combined Positive Score
  • the methods and compositions disclosed herein can be used to treat Merkel cell carcinoma (MCC).
  • a combination comprising Avelumab can be used to treat MCC in subjects with metastatic MCC.
  • the methods and compositions disclosed herein can be used to treat Urothelial Carcinoma (UC).
  • UC Urothelial Carcinoma
  • a combination comprising avelumab can be used to treat UC in subjects with locally advanced or metastatic UC who have disease progression during or following platinum-containing chemotherapy.
  • a combination comprising avelumab can be used to treat UC in subjects with locally advanced or metastatic UC who have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.
  • the methods and compositions disclosed herein can be used to treat Renal Cell Carcinoma (RCC).
  • RCC Renal Cell Carcinoma
  • a combination comprising avelumab and axitinib can be used in a subject with advanced RCC.
  • the methods and compositions disclosed herein can be used to treat urothelial carcinoma (UC).
  • UC urothelial carcinoma
  • Durvalumab can be used to treat UC in subjects with locally advanced or metastatic urothelial carcinoma who have disease progression during or following platinum-containing chemotherapy.
  • a combination comprising Durvalumab can be used to treat UC in subjects with locally advanced or metastatic urothelial carcinoma who have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.
  • the methods and compositions disclosed herein can be used to treat non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • a combination comprising Durvalumab can be used to treat NSCLC in subjects with unresectable, Stage III non-small cell lung cancer (NSCLC) whose disease has not progressed following concurrent platinum-based chemotherapy and radiation therapy.
  • the methods and compositions disclosed herein can be used to treat small cell lung cancer (SCLC).
  • a combination comprising Durvalumab can be used in combination with etoposide and either carboplatin or cisplatin, as first-line treatment of adult subjects with extensive-stage small cell lung cancer (ES-SCLC).
  • ES-SCLC extensive-stage small cell lung cancer
  • the methods and compositions disclosed herein can be used to treat urothelial carcinoma (UC).
  • a combination comprising Atezolizumab can be used to treat UC in adult subjects with locally advanced or metastatic urothelial carcinoma who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (e.g., PD-L1 stained tumor-infiltrating immune cells [IC] covering ⁇ 5% of the tumor area), as determined by an FDA-approved test, or are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status, or have disease progression during or following any platinum-containing chemotherapy, or within 12 months of neoadjuvant or adjuvant chemotherapy.
  • the methods and compositions disclosed herein can be used to treat NSCLC.
  • a combination comprising Atezolizumab can be used to treat NSCLC in adult subjects with metastatic NSCLC whose tumors have high PD-L1 expression (e.g., PD-L1 stained ⁇ 50% of tumor cells [TC ⁇ 50%] or PD-L1 stained tumor- infiltrating immune cells [IC] covering ⁇ 10% of the tumor area [IC ⁇ 10%]), as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations.
  • PD-L1 expression e.g., PD-L1 stained ⁇ 50% of tumor cells [TC ⁇ 50%] or PD-L1 stained tumor- infiltrating immune cells [IC] covering ⁇ 10% of the tumor area [IC ⁇ 10%]
  • a combination comprising Atezolizumab can be used in combination with bevacizumab, paclitaxel, and carboplatin, for the first-line treatment of adult subjects with metastatic non- squamous NSCLC with no EGFR or ALK genomic tumor aberrations.
  • a combination comprising Atezolizumab can be used in combination with paclitaxel protein-bound and carboplatin for the first-line treatment of adult subjects with metastatic non-squamous NSCLC with no EGFR or ALK genomic tumor aberrations.
  • a combination comprising Atezolizumab can be used to treat NSCLC in adult subjects with metastatic NSCLC who have disease progression during or following platinum-containing chemotherapy.
  • the methods and compositions disclosed herein can be used to treat triple negative breast cancer (TNBC).
  • TNBC triple negative breast cancer
  • a combination comprising Atezolizumab can be used in combination with paclitaxel protein-bound for the treatment of adult subjects with unresectable locally advanced or metastatic TNBC whose tumors express PD- L1 (e.g., PD-L1 stained tumor-infiltrating immune cells [IC] of any intensity covering ⁇ 1% of the tumor area), as determined by an FDA approved test.
  • PD- L1 e.g., PD-L1 stained tumor-infiltrating immune cells [IC] of any intensity covering ⁇ 1% of the tumor area
  • SCLC Small cell lung cancer
  • a combination comprising Atezolizumab can be used in combination with carboplatin and etoposide, for the first-line treatment of adult subjects with extensive-stage small cell lung cancer (ES-SCLC).
  • the methods and compositions disclosed herein can be used to treat endometrial cancer.
  • a combination comprising Dostarlimab can be used to treat endometrial cancer in adult subjects with mismatch repair deficient (dMMR) recurrent or advanced endometrial cancer, as determined by an FDA-approved test, that has progressed on or following prior treatment with a platinum-containing regimen.
  • dMMR mismatch repair deficient
  • the methods and compositions disclosed herein can be used to treat cutaneous squamous cell carcinoma (CSCC).
  • CSCC cutaneous squamous cell carcinoma
  • a combination comprising Cemiplimab-rwlc can be used to treat CSCC in subjects with metastatic cutaneous squamous cell carcinoma (mCSCC) or locally advanced CSCC (laCSCC) who are not candidates for curative surgery or curative radiation.
  • mCSCC metastatic cutaneous squamous cell carcinoma
  • laCSCC locally advanced CSCC
  • the methods and compositions disclosed herein can be used to treat basal cell carcinoma (BCC).
  • a combination comprising Cemiplimab-rwlc can be used to treat BCC in subjects with locally advanced BCC (laBCC) previously treated with a hedgehog pathway inhibitor or for whom a hedgehog pathway inhibitor is not appropriate.
  • laBCC locally advanced BCC
  • the methods and compositions disclosed herein can be used to treat NSCLC.
  • a combination comprising Cemiplimab-rwlc can be used to treat NSCLC in subjects whose tumors have high PD-L1 expression (e.g., Tumor Proportion Score (TPS) ⁇ 50%) as determined by an FDA-approved test, with no EGFR, ALK or ROS1 aberrations, and is locally advanced where subjects are not candidates for surgical resection or definitive chemoradiation, or metastatic.
  • TPS Tumor Proportion Score
  • the methods and compositions disclosed herein can be used to treat melanoma.
  • a combination comprising Nivolumab can be used to treat melanoma in subjects with unresectable or metastatic melanoma, as a single agent or in combination with ipilimumab.
  • a combination comprising Nivolumab can be used to treat melanoma in subjects with melanoma with lymph node involvement or metastatic disease who have undergone complete resection, in the adjuvant setting.
  • the methods and compositions disclosed herein can be used to treat NSCLC.
  • a combination comprising Nivolumab can be used to treat NSCLC in adult subjects with metastatic non-small cell lung cancer expressing PD-L1 ( ⁇ 1%) as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, as first-line treatment in combination with ipilimumab.
  • a combination comprising NSCLC can be used to treat melanoma in adult subjects with metastatic or recurrent non-small cell lung cancer with no EGFR or ALK genomic tumor aberrations as first-line treatment, in combination with ipilimumab and 2 cycles of platinum-doublet chemotherapy.
  • a combination comprising NSCLC can be used to treat melanoma in subjects with metastatic non-small cell lung cancer and progression on or after platinum-based chemotherapy.
  • the methods and compositions disclosed herein can be used to treat malignant pleural mesothelioma.
  • a combination comprising Nivolumab can be used to treat malignant pleural mesothelioma in adult subjects with unresectable malignant pleural mesothelioma, as first-line treatment in combination with ipilimumab.
  • the methods and compositions disclosed herein can be used to treat RCC.
  • a combination comprising Nivolumab can be used to treat RCC in subjects with intermediate or poor risk advanced renal cell carcinoma, as a first-line treatment in combination with ipilimumab.
  • a combination comprising Nivolumab can be used to treat RCC in subjects with advanced renal cell carcinoma, as a first- line treatment in combination with cabozantinib.
  • a combination comprising Nivolumab can be used to treat RCC in subjects with advanced renal cell carcinoma who have received prior anti-angiogenic therapy.
  • the methods and compositions disclosed herein can be used to treat classical Hodgkin lymphoma (cHL).
  • a combination comprising Nivolumab can be used to treat cHL in adult subjects with cHL that has relapsed or progressed after autologous hematopoietic stem cell transplantation (HSCT) and brentuximab vedotin, or 3 or more lines of systemic therapy that includes autologous HSCT.
  • the methods and compositions disclosed herein can be used to treat squamous cell carcinoma of the head and neck (SCCHN).
  • a combination comprising Nivolumab can be used to treat SCCHN in subjects with recurrent or metastatic squamous cell carcinoma of the head and neck with disease progression on or after a platinum-based therapy.
  • the methods and compositions disclosed herein can be used to treat urothelial carcinoma (UC).
  • UC urothelial carcinoma
  • a combination comprising Nivolumab can be used to treat UC in subjects with locally advanced or metastatic urothelial carcinoma who have disease progression during or following platinum-containing chemotherapy or have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.
  • the methods and compositions disclosed herein can be used to treat colorectal cancer.
  • a combination comprising Nivolumab can be used to treat colorectal cancer in subjects with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer that has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan, as a single agent or in combination with ipilimumab.
  • MSI-H microsatellite instability-high
  • dMMR mismatch repair deficient metastatic colorectal cancer that has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan, as a single agent or in combination with ipilimumab.
  • the methods and compositions disclosed herein can be used to treat hepatocellular carcinoma (HCC).
  • a combination comprising Nivolumab can be used to treat HCC in subjects with HCC who have been previously treated with sorafenib, as a single agent or in combination with ipilimumab.
  • the methods and compositions disclosed herein can be used to treat esophageal squamous cell carcinoma (ESCC).
  • ESCC esophageal squamous cell carcinoma
  • a combination comprising Nivolumab can be used to treat ESCC in subjects with unresectable advanced, recurrent or metastatic esophageal squamous cell carcinoma after prior fluoropyrimidine- and platinum-based chemotherapy.
  • a combination comprising Camrelizumab can be used to treat cHL.
  • a combination comprising Tislelizumab can be used to treat non-squamous non-small cell lung cancer. In certain preferred embodiments, a combination comprising Tislelizumab can be used to treat hepatocellular carcinoma (HCC). [0117] In certain preferred embodiments, a combination comprising Toripalimab can be used to treat urothelial carcinoma. In certain preferred embodiments, a combination comprising Toripalimab can be used to treat melanoma. In certain preferred embodiments, a combination comprising Toripalimab can be used to treat nasopharyngeal carcinoma (NPC).
  • NPC nasopharyngeal carcinoma
  • a combination comprising Sintilimab can be used to treat non-squamous non-small cell lung cancer.
  • a combination comprising Sintilimab can be used to treat cHL.
  • the cancer to be treated using the methods and compositions of this disclosure can be metastatic cancer.
  • the methods and compositions disclosed herein can be used to treat metastatic renal clear cell carcinoma or metastatic cutaneous malignant melanoma.
  • additional therapeutic agents can be administered to the subject.
  • anti-cancer agents such as chemotherapeutic agents immunocheck point inhibitors, other cytokines (such as IL-12, inducible IL-12 prodrugs, inducible IFN, inducible IFN prodrugs, IL-2 or IL-2 prodrugs), angiogenesis inhibitors, antibody-drug conjugates (e.g., trastuzumab emtansine (KADCYLA), trastuzumab deruxtecan (ENHERTU), enfortumab vedotin (PADCEV), sacituzumab govitecan (TRODELVY), cellular therapies (e.g., CAR-T, TCT-T, T-cell therapy, such as tumor infiltrating lymphocyte (TIL) therapy), oncolytic viruses, radiation therapy and/or small molecules, as describride further herein.
  • chemotherapeutic agents immunocheck point inhibitors such as IL-12, inducible IL-12 prodrugs, inducible IFN, inducible IFN pro
  • the pharmaceutical compositions can take a variety of forms, e.g., liquid, lyophilized, and typically contain a suitable pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are the non-active ingredient components of the pharmaceutical composition and are not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical formulation or composition in which it is contained. Carriers are frequently selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.
  • 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).
  • Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution.
  • 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 chimeric polypeptides or nucleic acid sequences encoding the chimeric polypeptides to humans or other subjects.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic, although the formulation can be hypertonic or hypotonic if desired. The pH of the solution is generally about 5 to about 8 or from about 7 to 7.5. [0124] Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders.
  • compositions for oral administration include powders or granules, suspension or solutions in water or non-aqueous media, capsules, sachets, or tables. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders are optionally desirable.
  • kits that includes a pharmaceutical composition that contains an a) inducible IL-2 prodrug composition, for example as a liquid composition or a lyophilized composition, in a suitable container (e.g., a vial, bag or the like), and b) a pembrolizumab composition, for example as a liquid composition or a lyophilized composition, in a suitable container (e.g., a vial, bag or the like).
  • the kit can further include other components, such as sterile water or saline for reconstitution of lyophilized compositions.
  • Cytokine is a well-known term of art that refers to any of a class of immunoregulatory proteins (such as interleukin or interferon) that are secreted by cells especially of the immune system and that are modulators of the immune system.
  • immunoregulatory proteins such as interleukin or interferon
  • Cytokine polypeptides that can be used in the fusion proteins disclosed herein include, but are not limited to transforming growth factors, such as TGF- ⁇ and TGF- ⁇ (e.g., TGFbeta1, TGFbeta2, TGFbeta3); interferons, such as interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , interferon-kappa and interferon-omega; interleukins, such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21 and IL-25; tumor necrosis factors, such as tumor necrosis factor alpha and lymphotoxin; chemokines (e.g., C-X-C motif chemokine 10 (CXCL10), CCL19,
  • “Chemokine” is a term of art that refers to any of a family of small cytokines with the ⁇ ability to induce directed ⁇ chemotaxis ⁇ in nearby responsive cells.
  • the terms “inducible” refer to the ability of a protein, i.e. IL-2, IL-12, or IFN, that is part of a prodrug, to bind its receptor and effectuate activity upon cleavage of the prodrug in the tumor microenvironment.
  • the inducible cytokine prodrugs disclosed herein have attenuated or no cytokine agonist activity, but upon cleavage in the tumor microenvironment release active cytokine.
  • “Attenuated” activity means that biological activity and typically cytokine (i.e., IL-2, IL- 12 or IFN) agonist activity is decreased as compared to the activity of the natural cytokine (i.e., IL-2, IL-12 or IFN).
  • the inducible cytokine prodrugs disclosed herein have attenuated cytokine receptor agonists activity, that is 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 natural cytokine (i.e., IL-2, IL-12 or IFN).
  • cytokine Upon cleavage in the tumor microenvironment, cytokine is released that is active. Typically, the cytokine that is released has cytokine receptor agonist activity that is at least about 10X, at least about 50X, at least about 100X, at least about 250X, at least about 500X, or at least about 1000x greater than the IL-2 receptor activating activity of the prodrug.
  • cytokine receptor agonist activity that is at least about 10X, at least about 50X, at least about 100X, at least about 250X, at least about 500X, or at least about 1000x greater than the IL-2 receptor activating activity of the prodrug.
  • 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” can be a vertebrate, more specifically a mammal (e.g. a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
  • patient or “subject” may be used interchangeably and can refer to a subject with a disease or disorder (e.g. cancer).
  • a disease or disorder e.g. cancer
  • patient or subject includes human and veterinary subjects.
  • treatment refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition.
  • treatment can refer to at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially complete reduction in the severity of an established disease or condition or symptom of the disease or condition, such as reduction in tumor volume, reduction in tumor burden, reduction in death.
  • a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control.
  • the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels.
  • treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
  • the terms “prevent”, “preventing”, and “prevention” of a disease or disorder refers to an action, for example, administration of the chimeric polypeptide or nucleic acid sequence encoding the chimeric polypeptide, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder.
  • references to “decreasing”, “reducing”, or “inhibiting” include a change of at least about 10%, of at least about 20%, of at least about 30%, of at least about 40%, of at least about 50%, of at least about 60%, of at least about 70%, of at least about 80%, of at least about 90% or greater as compared to a suitable control level.
  • Such terms can include but do not necessarily include complete elimination of a function or property, such as agonist activity.
  • sequence variant refers to an amino acid sequence of a polypeptide that has substantially similar biological activity as a reference polypeptide but differs in amino acid sequence or to the nucleotide sequence of a nucleic acid that has substantially similar biological activity (e.g., encodes a protein with substantially similar activity) as a reference sequence but differs in nucleotide sequence.
  • amino acid or nucleotide sequence of a “sequence variant” is highly similar (e.g. at least about 80% similar) to that of a reference sequence.
  • the identity can be calculated after aligning the two sequences so that the identity is at its highest level over a defined number of nucleotides or amino acids.
  • Optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman Adv. Appl. Math.2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.
  • conservative amino acid substitution is a term of art that refers to the replacement of an amino acid in a polypeptide with another amino acid that has similar biochemical properties, such as size, charge and hydrophobicity as a reference amino acid. It is well-known that conservative amino acid replacements in the amino acid sequence of a polypeptide frequently do not significantly alter the overall structure or function of the polypeptide. 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.
  • 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.
  • the term “effective amount,” as used herein, refers to the amount of agent (e.g., inducible IL-2 prodrug) that is administered to achieve the desired effect under the conditions of administration, such an amount that reduces tumor size, reduces tumor burden, extends progression free survival or extends overall survival.
  • agent e.g., inducible IL-2 prodrug
  • the actual effective amount selected will depend on the particular cancer being treated and its stage and other factors, such as the subject’s age, gender, weight, ethnicity, prior treatments and response to those treatments and other factors. Suitable amounts of inducible cytokine prodrug and any additional agents to be administered, and dosage schedules for a particular patient can be determined by a clinician of ordinary skill based on these and other considerations.
  • the methods and compositions disclosed herein are used to treat colon cancer, lung cancer, melanoma, renal cell carcinoma, breast cancer, squamous carcinoma of the head and neck.
  • the methods and compositions disclosed herein are used to treat melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell cancer (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability high or mismatch repair deficient cancer, microsatellite instability high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial carcinoma, tumor mutational burden high cancer, cutaneous squamous cell carcinoma (cSCC), triple negative breast
  • mice Prior to tumor implantation, cells were washed twice with PBS and counted. Cells were inoculated in PBS (efficacy studies) or 50% Matrigel (TIL harvests, Corning). Mice, Tumor Implantations, and in vivo dosing [0144] All mouse in vivo work was performed in accordance with current regulations and standards of the U.S. Department of Agriculture and the NIH at Charles River Laboratories (Morrisville, NC and Worcester, MA). Female, 6–8 week-old C57Bl/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 or 1 x 10 5 B16-F10 cells were injected subcutaneously and monitored for tumor growth.
  • 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 inducible IL-2 prodrugs were dosed twice a week. Mice receiving rhIL-2 were dosed twice a day for 5 days before receiving a 2-day break (5/2 regimen). In studies where PD-1 blockade was used, mice were dosed with anti-PD-1 (200 ⁇ g, clone RMP1-14, BioXCell) on a twice-weekly schedule. In studies using FTY720, mice were initially dosed with 25 ⁇ g on the first dose, then treated daily with 10 ⁇ g per dose throughout the course of the experiment.
  • anti-PD-1 200 ⁇ g, clone RMP1-14, BioXCell
  • mice In some studies antitumor activity was assessed in mice in which CD8+ cells were depleted. In those studies mice were dosed with anti-CD8 antibody (200 ⁇ g/dose, clone 2.43 from Bio X Cell) twice a week via intraperitoneal injection. The average tumor volume for each group is displayed as the mean +/- the SEM (FIG.2I). The results showed that repletion of CD8+ cells reduced the anti-tumor effect of Compound 1.
  • Compound 5 comprises a first polypeptide having SEQ ID NO: 6 and a second polypeptide comprising SEQ ID NO: 5 (FIGs.15A-15C). The results demonstrated, that the non-alpha mutein did not have significant anti-tumor activity in the model.
  • HEK-Blue IL-2 Reporter Assay [0150] The HEK-Blue IL-2 reporter cell assay was performed according to the manufacturer’s protocol (Invivogen). On assay Day 1, the cells were rinsed, resuspended in media containing 1.5% human serum albumin and plated at a concentration of 5 x 10 4 cells per well in a 96-well flat bottom plate. Titrated amounts of intact and protease-activated (cleaved) inducible IL-2 proteins or rhIL-2 were added to the cells to generate a full dose-response curve.
  • Human and murine primary cell assays [0151] Human PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare) according to the manufacturer’s protocol and frozen in Recovery Cell Culture Freezing Media (Gibco) for later use. To generate activated T cells (Tblasts), PBMCs were thawed, counted, and stimulated with 5 ⁇ g/mL of PHA (Sigma-Aldrich) for 72 hours before being frozen for later use.
  • PHA Sigma-Aldrich
  • Tblasts were plated in a 96- well round bottom plate, and titrated amounts of intact or protease-activated (cleaved) inducible IL-2 proteins or rhIL-2 were added to the cells to generate a full dose-response curve. After 72 hours, proliferation was measured using Cell Titer glow reagent (Promega) according to the manufacturer’s protocol.
  • splenocytes were thawed, washed, and stimulated with 2 ⁇ g/mL of Concanavalin A (Sigma-Aldrich) for 72 hours before being frozen in Recovery Cell Culture Freezing Media (Gibco). T cell activation was performed in complete media (RPMI- 1640 media supplemented with 10 % FBS, 100 units/mL of penicillin, 100 ⁇ g/mL streptomycin and 0.1 % 2-mercaptoethanol). To measure inducible IL-2 protein activity, murine Tblasts were plated in a 96-well round bottom plate.
  • inducible IL-2 Stability in Murine Plasma and Human Serum [0153] Whole blood from 6–8 week-old female C57Bl/6 mice was used to generate plasma. Human serum was purchased from BioIVT. On Day 1 of the assay, inducible IL-2 was added to either the murine plasma or human serum before the samples were mixed and divided into three aliquots, which were incubated at 37°C for the indicated times before being frozen for later analysis.
  • inducible IL-2 To assess the enzymatic processing of inducible IL-2, samples were thawed, and inducible IL-2 cleavage was assessed using western blot analysis against human IL-2. Intact and protease-activated inducible IL-2 were included as positive and negative controls.
  • Western blot analysis was performed using the JESS system (Protein Simple) according to the manufacturer’s protocol. The primary anti-human IL-2 antibody was purchased from R&D Systems (AF-202-NA) and the anti-goat secondary antibody was purchased from Jackson Labs (AB_2338513). Samples and antibodies were loaded into a 12–230 kDA Jess separation module and run using a Jess system set to the standard settings for chemiluminescence.
  • Plasma and tumor samples were collected at indicated time points by Charles River Laboratories (Morrisville, North Carolina) and shipped on dry ice where they were stored at – 80 o C.
  • MC38 tumor lysates were generated by homogenizing each tumor with a Qiagen TissueRuptor homogenizer with disposable probes (Qiagen) in ice cold Lysis Buffer (1X Tris Buffered Saline, 1 mM EDTA, 1 % Triton X-100, with protease inhibitors in diH2O).
  • Plasma and tumor lysates were analyzed using the BioLegend IL-2 ELISA (431804), which detects both intact inducible IL-2 as well as free IL-2, as per manufacturer’s instructions. Intact inducible IL- 2 was used to generate a 12-point standard curve. To specifically analyze the level of free IL-2, samples were measured using an IL-2 AlphaLISA (PerkinElmer, AL221C), which detects free human IL-2 but not intact inducible IL-2 due to competition with the inactivation domain. All AlphaLISAs were performed according to manufacturer’s instructions and analyzed on a Perkin Elmer Enspire reader and software.
  • 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) at 37°C for 35 minutes while shaking. After digestion, tumor samples were mechanically dissociated through a 70 ⁇ M cell strainer.
  • 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.
  • Flow cytometry fluorescent dye–conjugated antibodies to the following proteins were purchased from Biolegend: CD8 ⁇ 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; IFN ⁇ PE, clone XMG1.2; F4/80 Pe/Dazzle 594, clone BM8; CD3 Complex PeCy7, clone 17A2; FC Block, clone 93.
  • Flow cytometry fluorescent dye–conjugated antibodies to the following proteins were purchased from eBioscience: CD45 BUV395, clon30-F11; 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 p15E peptide KSPWFTTL was purchased from ThermoFisher Scientific (50-168-9385).
  • the Live/Dead Blue Dye was also purchased from ThermoFisher Scientific (L23105).
  • Ex vivo inducible IL-2 prodrug processing assay [0158] Primary human healthy cells were purchased from either ATCC, Lonza, or Zen-Bio, and cultured according to the manufacturer’s protocol. Dissociated human tumor samples were purchased from Discovery Life Sciences. These samples are generated from primary human tumor samples that were surgically removed and enzymatically digested on site prior to being frozen. All purchased samples were shipped on dry ice and were stored in a liquid nitrogen freezer. [0159] To examine inducible IL-2 prodrug processing, samples were thawed, washed, and counted.
  • IL-2 Bioassay which utilizes thaw-and-use IL-2 reporter cells, was used to assess the IL-2 activity in the cell culture supernatants (Catalog # JA2201/JA2205). This bioassay was used according to the manufacturer’s protocol.
  • RLU Relative luminescence unit
  • Compound 1 an inducible IL-2 prodrug, was designed to enhance the clinical profile of recombinant human IL-2 treatment by facilitating less frequent systemic delivery, increasing the tumor exposure of the molecule, and decreasing the toxicity associated with high-dose IL-2 (FIG.1A).
  • Compound 1 includes native human IL-2, a Fab antibody fragment that prevents IL-2 from binding to the medium affinity IL-2 receptor (IL-2R ⁇ / ⁇ ), thereby acting as an inactivation domain, and an anti-human serum albumin ( ⁇ HSA) single domain antibody acting as a half-life extension domain.
  • IL-2R ⁇ / ⁇ medium affinity IL-2 receptor
  • ⁇ HSA anti-human serum albumin
  • human PBMCs were stimulated with PHA to form Tblasts, which express the high affinity IL-2 receptor (CD25/CD122/CD132) (FIGs.8A-8B) and respond to IL-2 signaling by proliferating.
  • Human Tblasts from multiple donors were exposed to rhIL-2, intact Compound 1, or cleaved Compound 1 for 72 hours and then Tblast proliferation was measured. In this system, intact Compound 1had less activity than either cleaved Compound 1or rhIL-2 across multiple donors (FIG.1D, FIG. 8C).
  • intact Compound 1 had approximately 23-fold less activity in terms of an increased EC 50 and plateaued at only 60% of the maximum activity seen with either cleaved Compound 1or rhIL-2.
  • the activity of intact and cleaved Compound 1 was also characterized in a mouse primary T blast assay. While cleaved Compound 1and rhIL-2 induced similar proliferation by murine Tblasts, intact Compound 1had almost no measurable activity in cells isolated from multiple mice (FIG.1E, FIG.8D).
  • Compound 1-NC a non-cleavable variant of Compound 1, named Compound 1-NC, was generated by replacing the linker sequence with a non-cleavable glycine/serine sequence.
  • Compound 1-NC was treated to the same enzymatic digestion as Compound 1before being tested in human Tblasts.
  • Compound 1-NC also acts as a control for the level of the in vivo activity derived specifically from intact Compound 1 (FIGs.1C-1E).
  • FOGs.1C-1E the level of the in vivo activity derived specifically from intact Compound 1
  • FIGs.2A, 2I and 13B tumor growth inhibition
  • doses of 100 ⁇ g, 150 ⁇ g, or 300 ⁇ g were all highly efficacious.
  • 24 mice in those dosing groups 23 had complete responses, with no measurable tumor remaining at the end of the experiment (FIG.2A). All of these dose levels were well tolerated by the mice, with no signs of body weight loss (FIG.9A).
  • WW0177 differs from Compound 1 in that it contains a non-cleavable linker sequence between the half-life extension domain and the fully active IL-2, and it does not have an inactivation domain, thereby representing the level of toxicity that should be expected if the inactivation domain was not functioning properly.
  • MC38 tumor-bearing mice were dosed with either WW0177 or Compound 1, and their weight was monitored over time (FIG.2B).
  • VLS Vascular leak syndrome
  • VLS is induced by high doses of recombinant IL-2 and can be measured by examining the amount of Evan’s Blue dye that leaks into the lungs following i.v. injection.
  • recombinant human IL-2, WW0177, or Compound 1 were administered in equimolar amounts, only recombinant human IL-2 and WW0177 resulted on detectable levels of Evans Blue leaking into the lungs, while the Compound 1 did not (FIG.13D).
  • Compound 1 was designed to facilitate less frequent, systemic delivery of the treatment without sacrificing potency and anti-tumor activity of high dose IL-2. Therefore, it was important to directly compare the activity of Compound 1 to native IL-2.
  • MC38 tumor-bearing mice were treated with titrated amounts of either Compound 1 as before (twice weekly for two weeks), or rhIL-2 dosed twice a day for two weeks (dosing regimen: 5 days dosing, 2 days rest schedule for 2 weeks).
  • Compound 1 dosing resulted in extended exposure in the plasma, with a half-life of approximately 20 hours, and exposure maintained over the course of 4 days (FIG.2E). Additionally, intraperitoneal dosing of Compound 1resulted in prolonged drug exposure within the tumor itself, demonstrating tissue penetrance by Compound 1 (FIG.2F). Total Compound 1levels reached a C max at 6 hours post-dosing in the plasma and peaked at 12 hours post-dosing in the tumor. [0171] Compound 1 was designed to restrict the systemic activity of IL-2 while delivering fully active IL-2 locally to the tumor via the use of cleavable linkers.
  • the therapeutic window (TW) of a therapy is defined as the ratio of the maximum tolerated dose and the lowest efficacious dose, thereby identifying the difference between activity and serious adverse events.
  • TW for proleukin is relatively small.
  • TW of rhIL-2 in MC8 tumor bearing mice was calculated to be less than 4-fold in our model (FIG.9B). Since the half-life extension element of WW0177 makes it a more active version of IL-2, less WW0177 is required to reach full efficacy compared to recombinant hIL-2. However, WW0177 also has a lower maximum tolerated dose, resulting in a TW of less than 2.
  • mice were examined for the presence of tumor-specific memory CD8+ T cells 6 months after the initial MC38 implantation (MC38 CR mice) (FIG. 3A).
  • KSPWFTTL Murine Leukemia Virus protein gp70
  • T cells specific for this antigen can be identified using fluorescently labeled MHC peptide complexes known as tetramers (18).
  • Spleens from MC38 CR mice had a higher overall frequency of tetramer-positive CD8+ T cells than age matched tumor-na ⁇ ve mice (FIGs.3A-3B). Furthermore, although the tetramer-positive cells from tumor-na ⁇ ve mice largely maintained a na ⁇ ve phenotype, cells from MC38 CR mice were predominantly of an effector memory phenotype (CD44 hi CD62L low ) (FIGs.3C-3D). Furthermore, upon re-stimulation, tetramer- positive cells from MC38 CR mice secreted more of the effector cytokines TNF and IFN ⁇ (FIGs. 3E-3F).
  • MC38 tumor-bearing mice were randomized into treatment groups on Day 0 and treated with either vehicle or Compound 1on Day 1 and Day 4. Tumors were harvested 24 hours after their second dose. Total RNA was extracted from the single-cell suspensions and analyzed using the NanoString nCounter® PanCancer Mouse Immune Profiling Panel.
  • Compound 1 treatment resulted in a clear shift in the transcriptional profile, with 437/770 genes in the panel having statistically significant differences in expression compared with the control group (FIG. 4A-4B).
  • NanoString nSolverTM pathway analysis of this dataset revealed a series of immune activation-related pathways that were upregulated by Compound 1 treatment, including both broad immune activation signatures such as adaptive immunity and inflammation, as well as more specific signatures such as leukocyte function, NK cell function, and T cell function (FIG. 4C).
  • the expression of several transcripts associated with immune checkpoint proteins also increased following Compound 1 treatment, including PD-1, TIGIT, and CTLA-4 (FIG.4D).
  • TILs tumor- infiltrating lymphocytes
  • checkpoint proteins are upregulated during a typical immune response.
  • immune cell profiling by flow cytometry was also performed. As soon as 5 days after the initial dose, Compound 1 treatment resulted in a large increase in the density of infiltrating immune cells, including tumor-specific tetramer-positive CD8+ T cells ( ⁇ 19.8-fold increase) and to a lesser extent Tregs ( ⁇ 2.5-fold increase) (FIG.4E).
  • IL-2-based therapies on Tregs is a major topic of discussion in the scientific community, as there is some concern that IL-2 treatment will result in counterproductive Treg expansion, which may hinder immunotherapy in the clinic. This has led to the development of several IL-2 variant molecules that are engineered to avoid Treg engagement. In contrast, Compound 1 is not specifically engineered to avoid Tregs, as we hypothesize that the activity of fully active IL-2 on the cytolytic cells will overcome any possible Treg activation associated with the therapy.
  • Compound 1 treatment significantly increased the frequency of tetramer-positive CD8 T cells producing IFN ⁇ (FIGs.4G-4H), TNF (FIG.10A), and granzyme B (FIG.10B).
  • Compound 1 treatment also significantly increased the polyfunctionality of the tetramer-positive CD8+ T cells, with a greater frequency of tetramer-positive CD8+ T cells producing either two or all three of these effector cytokines compared with the control group (FIGs.4I).
  • Tregs can also produce effector cytokines such as TNF and IFN ⁇ , in a phenomenon known as “Treg Fragility” (20).
  • Tregs effector cytokines
  • Tumor specific activation of immune cells by Compound 1 to generate tumor rejection [0181] To confirm the effects of systemic Compound 1 treatment are selective for the tumor microenvironment, effector cytokine production by T cells derived from the tumor, spleen, peripheral blood, and draining lymph node were compared after Compound 1 treatment, using the same treatment schedule as previously described. Since the tetramer+ population is selectively enriched among CD8+ T cells within the tumor, the inclusion of these cells in the analysis could bias the comparison across different sites. Therefore, tetramer-negative CD8+ T cells were specifically examined across the various tissues.
  • mice were implanted with MC38 tumors that grew to around 100–150 mm3 before some mice were treated with Fingolimod, or FTY720.
  • FTY720 is a small molecule that blocks the sphingosine-1-phosphate receptors, thereby preventing lymphocyte egress from the thymus and secondary lymphoid tissues (21). Therefore, any anti- tumor activity seen in FTY720-treated mice is derived from the immune cells that have already infiltrated the tumor at the start of treatment, and not from the activation and subsequent trafficking of additional lymphocytes from secondary immune tissues.
  • Murine syngeneic tumor models vary in their baseline immune infiltration as well as their responses to immunotherapy. For example, in MC38 tumors, approximately 20% of the TILs are CD8+ T cells compared with only 2.5% in B16-F10 tumors (FIGs.12A-12B).
  • mice were injected subcutaneously with B16-F10 melanoma cells. Tumors were allowed to grow to an average volume of 100 mm 3 before mice were randomized to receive either PBS or various doses of WTX-124, using the same dosing schedule as before.
  • Compound 1 was more effective than an equimolar amount of recombinant hIL-2 at inducing CD25 expression and proliferation by tumor infiltrating NK cells, CD4+ NonTregs, total CD8+ T cells and tetramer+ CD8+ T cells, likely due to the enhanced PK profile of the IL-2 prodrug compared to the free cytokine.
  • an ex vivo processing assay was developed. Briefly, dissociated human tumor samples were incubated with Compound 1 or control proteins for 48 hours before the resultant IL-2 activity was measured. Using Compound 1-NC as a proxy for the baseline activity of the intact Compound 1 prodrug, and pre-cut Compound 1as representative of a fully activated molecule, the activity induced by incubation of Compound 1 with the dissociated human tumor samples was normalized to a range of 0–100% activation. In some instances, the primary human tumor samples also contained viable TILs, which had the capacity to consume some of the free IL-2 in the pre-cut Compound 1 positive control group.
  • IL-2 Before the advent of the modern field of immuno-oncology, high-dose IL-2 stood out as a treatment associated with complete responses, albeit in a minority of patients. However, the anti-tumor potential of proinflammatory cytokines, like IL-2, has been hindered by the serious toxicities linked to their systemic delivery and the engagement of target cells outside the tumor microenvironment (4). In the particular case of IL-2, several pharmaceutical and biotechnology companies have tried to minimize this problem by creating less-active forms of IL-2 (known as non-alpha molecules) that avoid activation of the IL-2 high-affinity receptor (7-10).
  • CD122/CD132 subunits which is responsible for the signal transduction of the cytokine
  • CD122/CD132 subunits which is responsible for the signal transduction of the cytokine
  • the non- ⁇ approach to IL-2 therapy may end up simply shifting the therapeutic window rather than improving it.
  • newly activated CD8+ T cells upregulate CD25 to form the high-affinity receptor, which is required for their sustained expansion in the presence of antigens.
  • CD8+ T cells lacking CD25 failed to expand in infected tissues, despite expression of the medium-affinity receptor (22).
  • inducible IL-2 addresses the challenges associated with rhIL-2 therapy.
  • Inducible IL-2 contains a native IL-2 to realize the full pharmacological potential of this cytokine in driving anti-tumor immunity.
  • the molecule is engineered as a prodrug to minimize the systemic toxicity and is conditionally activated to release IL-2 selectively in the tumor microenvironment.
  • the activity of Compound 1 was highly inducible in vitro in human reporter cell assay systems as well as in human and mouse primary cells. Likewise, Compound 1 was efficacious in mouse syngeneic models and this efficacy was dependent on the tumor-specific processing.
  • the half-life extension domain provides the opportunity for better drug exposure with less frequent dosing compared with the traditional dosing schedule for high-dose IL-2 therapy (proleukin).
  • IL-2 therapy proleukin
  • complete responses could be reliably generated in the MC38 mouse model by dosing twice a week
  • complete responses in 100% of the mice could also be achieved with doses as infrequent as once every 2 weeks, with slightly higher amounts of the prodrug.
  • the peripheral inactivation provided by the IL-2 inactivation domain allowed for the safe administration of this IL-2 prodrug to mice at doses > 20-fold higher than the dose required for potent efficacy but without obvious toxicity.
  • Compound 1 has a significantly wider therapeutic window than previously described for high-dose IL-2.
  • IFN- ⁇ is a fundamental effector cytokine that drives anti-tumor efficacy by amplifying the cellular immune component of the response and skewing CD4+ T cells towards a TH1 phenotype. Also, more recently, it has been shown that IFN- ⁇ directs the mechanistic fragility of Tregs (20).
  • Compound 1 An important feature of Compound 1 is the selective processing of the prodrug in the tumor, allowing for systemic delivery, good exposure, and activation of the prodrug to release fully active IL-2 in the tumor microenvironment. Indeed, Compound 1 was highly stable while in circulation as shown in mice and in non-human primates (data not shown), as well as when WTX-124 was exposed to healthy primary human cells or plasma.
  • Compound 1 was reliably processed by primary human dissociated tumor samples from a wide variety of different cancer types, demonstrating the potential for systemically administered Compound 1 to selectively deliver IL-2 to the site of the disease and positively contribute to the development of an effective immune response.
  • the clinical benefits and safety of Compound 1treatment will be examined in the upcoming Phase I trial, subject to FDA clearance, testing Compound 1either alone or in combination with the anti-PD-1 therapy pembrolizumab.
  • this work presents the design features and mechanistic characteristics of Compound 1, a novel, conditionally activated IL-2 prodrug that provides tumor-selective delivery of full potency IL-2 to activate tumor-specific immune cell populations.
  • CD4(+) T cells support polyfunctionality of cytotoxic CD8(+) T cells with memory potential in immunological control of tumor. Cancer Sci 2020;111:1958-68. 20. Overacre-Delgoffe AE, Chikina M, Dadey RE, Yano H, Brunazzi EA, Shayan G, et al. Interferon- ⁇ drives Treg fragility to promote anti-tumor immunity. Cell 2017;169:1130-41. e11. 21. 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 S1P receptor 1. Nature 2004;427:355-60. 22. D’Souza WN, Lefrandialis L. IL-2 is not required for the initiation of CD8 T cell cycling but sustains expansion. J Immunol 2003;171:5727-35.

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