IL-2 Prodrug

Abstract

The present disclosure relates to methods and compositions for treating cancer using inducible IL-2 prodrugs.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 253,964, filed October 8, 2021, U.S. Provisional Application No. 63 / 290,941, filed December 17, 2021, and U.S. Application No. 63 / 328,524, filed April 7, 2022, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Cancer immunotherapy has rapidly established itself as the fourth pillar of cancer treatment, mainly due to the clinical success of checkpoint inhibitors (1-3). Despite the fact that durable responses have been achieved in some patients using these new therapies, the percentage of responders remains relatively low and limited to some cancer types. The mutational burden of the tumor, the presence or absence of T cell infiltration in the tumor, and the overall immunosuppressive microenvironment of the tumor have a significant impact on the response to immunotherapy. Although immune checkpoint blockade can prevent the physiological stop signals that arise in response to immune activation, other approaches can be used to actively stimulate antitumor immune responses. One approach involves the use of immune-activating cytokines. Numerous preclinical and clinical studies have demonstrated the promise of cytokine therapies to enhance antitumor immunity. Indeed, these were some of the first cancer immunotherapies approved for clinical use. However, their clinical application has been limited by systemic toxicity and poor pharmacokinetic profiles (4).

[0003] Interleukin (IL)-2 is a key cytokine that promotes immune-mediated killing of cancer cells, and its mechanism of action includes the 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-2Rγ), CD122 (IL-2Rγ), and CD132 (IL-2Rγ). Signaling is mediated through the heterodimerization of CD122 and CD132. Together, these molecules form the IL-2 intermediate affinity receptor, which is expressed on natural killer (NK) cells, monocytes, macrophages, and resting CD4+ and CD8+ T cells. The trimeric IL-2 high affinity receptor (CD25 / CD122 / CD132) is present on activated T cells and NK cells and constitutively expressed on CD4+FoxP3+ regulatory T cells (Tregs). IL-2 increases the proliferation and activation of T and NK cells and induces the differentiation of CD8+ T cells into effector and memory cells (5, 6). Recombinant human IL-2 (Proleukin) has been approved for clinical use in metastatic melanoma and renal cell carcinoma as a high-dose therapy, but this treatment is associated with severe side effects, including vascular leak syndrome and hypotension, limiting its practical application (5, 7).

[0004] To address the limitations of IL-2 high-dose therapy, several approaches have been pursued to develop next-generation IL-2 molecules that bind only to intermediate affinity receptors (CD122 / CD132) in hopes of reducing toxicity and lowering Treg activation (7-10). However, many of these molecules still activate IL-2 receptors on non-tumor specific immune cells located in normal tissues and thus may not minimize toxicity associated with IL-2 signaling. Molecules that block IL-2 signaling in the periphery while delivering fully active native IL-2 in the tumor microenvironment may be a more appropriate approach to achieve the full potential of IL-2 antitumor activity with minimal systemic toxicity.

[0005] Inducible forms of IL-2 that are conditionally activated in the tumor microenvironment through protease cleavage and release fully active native IL-2 cytokine in the tumor to stimulate a strong anti-tumor immune response are described in WO2021097376. These IL-2 prodrugs contain a native IL-2 molecule linked to a half-life extension domain (e.g., an anti-human serum albumin antibody binding fragment such as a VH domain) via a protease-cleavable linker and an IL-2 blocking element (e.g., an anti-IL-2 antibody binding fragment such as a Fab) that blocks the binding of IL-2 to IL-2β / γ receptors on peripheral normal tissues. Upon cleavage of the protease-cleavable linker, fully active native IL-2 is released in the tumor to stimulate a strong anti-tumor immune response. Summary of the Invention

[0006] The present disclosure relates to compositions and methods for treating cancer using an inducible IL-2 prodrug. The methods generally include administering to a subject in need of treatment 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.

[0007] The inducible IL-2 prodrug is conditionally active. The inducible IL-2 prodrug comprises two polypeptide chains. The first polypeptide chain can comprise, from the amino terminus to the carboxy terminus, an IL-2 polypeptide-protease cleavable linker-anti-human serum albumin (HSA) binding single antibody variable domain-preferably a protease cleavable linker-VH and CH1 of an antibody that binds to IL-2. The first polypeptide chain can comprise, from the amino terminus to the carboxy terminus, an IL-2 polypeptide-protease cleavable linker-VH and CH1 of an antibody that binds to IL-2-preferably a protease cleavable linker-anti-human serum albumin (HSA) binding single antibody variable domain. The second polypeptide chain comprises the VL and CL of an antibody that binds to IL-2, which together with the VH and CH1 of the first polypeptide chain form a Fab that binds to the IL-2 polypeptide. When the inducible IL-2 prodrug is not present at the site of interest (e.g., the tumor microenvironment), the prodrug typically remains intact. The intact prodrug has attenuated IL-2 receptor agonist activity. When the inducible IL-2 prodrug is at the site of interest (such as the tumor microenvironment), the protease-cleavable linker is cleaved by proteases active at the site of interest, releasing a non-attenuated form of IL-2. This conditional activity maintains the immunostimulatory effects of IL-2 while limiting the systemic toxicity associated with non-inducible IL-2 therapy. The intact IL-2 prodrug contains elements that extend its half-life, but does not contain the non-attenuated form of IL-2 after cleavage. As a result, the short half-life of IL-2 effectively limits toxicity outside the site of interest.

[0008] As further described and exemplified herein, after systemic administration, the amount of inducible IL-2 prodrug in the circulation (plasma) may be at least about 5 times greater than the amount in the tumor, for example, the amount in the circulation may be at least about 5 times, at least about 10 times, at least about 15 times, at least about 18 times, at least about 20 times, or at least about 25 times greater than the amount of inducible IL-2 prodrug in the tumor. Although more prodrug is found in the circulation than in the tumor microenvironment, the prodrug is more processed (cleaved) in the tumor microenvironment to release active IL-2. After systemic administration, at least about 40 times more prodrug may be cleaved to release active IL-2 in the tumor microenvironment compared to the circulation. In embodiments, cleavage of the inducible IL-2 prodrug may be at least about 45 times, at least about 50 times, at least about 55 times, at least about 60 times, at least about 65 times, at least about 70 times, at least about 75 times, at least about 80 times, at least about 85 times, at least about 90 times, at least about 93 times, at least about 95 times, or at least about 100 times greater in the tumor microenvironment compared to the circulation.

[0009] The present disclosure relates to a method for treating cancer, comprising administering to a subject in need of treatment an effective amount of an inducible interleukin-2 (IL-2) prodrug, the inducible IL-2 prodrug being administered systemically and activated by cleavage by a protease having higher activity in the tumor microenvironment than at other sites, resulting in at least about 40-fold greater cleavage of the inducible IL-2 prodrug in the tumor microenvironment compared to the circulation. The method can significantly increase the tumor-reactive CD8+ / Treg ratio.

[0010] The present disclosure relates to a method of inducing immunological memory against a tumor. The method includes administering to a subject in need of treatment an effective amount of an inducible interleukin-2 (IL-2) prodrug, where the inducible IL-2 prodrug is administered systemically and activated by cleavage by a protease that has higher activity in the tumor microenvironment than at other sites. After systemic administration, the amount of the inducible IL-2 prodrug in the circulation (plasma) may be at least about 5 times higher than the amount in the tumor, for example, the amount in the circulation may be at least about 5 times, at least about 10 times, at least about 15 times, at least about 18 times, at least about 20 times, or at least about 25 times higher than the amount of the inducible IL-2 prodrug in the tumor. After systemic administration, at least about 40 times more prodrug may be cleaved to release active IL-2 in the tumor microenvironment compared to the circulation. In embodiments, cleavage of an inducible IL-2 prodrug may be at least about 45 times, at least about 50 times, at least about 55 times, at least about 60 times, at least about 65 times, at least about 70 times, at least about 75 times, at least about 80 times, at least about 85 times, at least about 90 times, at least about 93 times, at least about 95 times, or at least about 100 times greater in the tumor microenvironment compared to the circulation. Immunological memory can be characterized by the presence of tumor-reactive CD8+ cells with a memory phenotype (e.g., CD8+CD44hiCD62low), tumor-reactive CD8+ cells that produce TNF, IFNγ, and / or granzyme B upon restimulation, or tumor-reactive CD8+ cells with a memory phenotype that produce TNF, IFNγ, and / or granzyme B upon restimulation.

[0011] The present disclosure relates to methods for selectively activating effector CD8+ T cells in a tumor microenvironment and methods for selectively activating tumor infiltrating lymphocytes. These methods include administering to a subject in need of treatment an effective amount of an inducible interleukin-2 (IL-2) prodrug, which is administered systemically and activated by cleavage by a protease with higher activity in the tumor microenvironment than at other sites, resulting in a significantly higher frequency of TNF and / or IFNγ producing CD8+ T cells in the tumor compared to peripheral tissues. After systemic administration, the amount of inducible IL-2 prodrug in the circulation (plasma) may be at least about 5 times greater than the amount in the tumor, for example, the amount in the circulation may be at least about 5 times, at least about 10 times, at least about 15 times, at least about 18 times, at least about 20 times, or at least about 25 times greater than the amount of inducible IL-2 prodrug in the tumor. After systemic administration, at least about 40 times more prodrug may be cleaved to release active IL-2 in the tumor microenvironment compared to the circulation. In embodiments, cleavage of the inducible IL-2 prodrug may be at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 93-fold, at least about 95-fold, or at least about 100-fold greater in the tumor microenvironment compared to the circulation. These methods can substantially increase the tumor-reactive CD8+ / Treg ratio in the tumor microenvironment.

[0012] In embodiments of the disclosed methods, the inducible IL-2 prodrug can be administered no more than about twice a week, no more than once a week, or no more than about once every two weeks. In certain embodiments, the inducible IL-2 prodrug can be administered no more than about once every two weeks.

[0013] Preferred inducible IL-2 prodrugs for use in the methods of the present 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 the present 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 having at least about 80% identity to SEQ ID NO: 1, and the 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 having at least about 80% identity to SEQ ID NO: 2 and a second polypeptide chain having 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 an amino acid sequence variant of compound 3 can comprise a first polypeptide chain having at least about 80% identity to SEQ ID NO:3 and a second polypeptide chain having 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 an amino acid sequence variant of compound 4 can comprise a first polypeptide chain having at least about 80% identity to SEQ ID NO:4 and a second polypeptide chain having at least about 80% identity to SEQ ID NO:5. [Brief description of the drawings]

[0014] [Figure 1]AF shows the design and development of an inducible IL-2 prodrug represented by Compound 1. A shows a diagram of the components of Compound 1. B shows a non-reducing SDS-PAGE comparing intact Compound 1 with protease-cleaved Compound 1 (IL-2, anti-HSA half-life extending domain, and Fab inactivation domain). C shows the in vitro activity of Compound 1 in a HEK-Blue IL-2 reporter assay comparing intact Compound 1 (squares) and protease-activated (cleaved) Compound 1 (triangles) with rhIL-2 (circles). D shows the in vitro activity of intact Compound 1 (squares) and cleaved Compound 1 (triangles) in primary human Tblasts compared to rhIL-2 (circles). E shows the in vitro activity of intact Compound 1 (squares) and cleaved Compound 1 (triangles) in primary mouse Tblasts compared to rhIL-2 (circles). F shows the in vitro activity of intact Compound 1-NC (circles) and truncated Compound 1-NC (triangles) in primary human Tblasts compared to rhIL-2 (squares). Curves in C-F represent at least duplicate wells and show the mean ± SD of individual points. Data are representative of at least two experiments.

[0015] [Figure 2A] Figure 1 shows that compound 1 induces tumor regression in a cleavage-dependent manner. Figure 2 shows a series of graphs showing tumor volume over time in mice treated with various doses of compound 1, compound-NC (non-cleavable control), or vehicle. Spider plots of individual mice are shown (dashed lines), and the mean tumor volume of the group is shown in bold. [Figure 2B] Figure 1 shows that Compound 1 induced tumor regression in a cleavage-dependent manner. Figure 2 shows the weight and survival of individual mice over time treated with either Compound 1 or WW0177 (a Compound 1 variant lacking the inactivation domain). Figure 3 shows the weight and survival of individual mice over time. Treatment with WW0177 was discontinued after two doses due to excessive toxicity, while mice treated with Compound 1 received all four doses. [Figure 2C]Figure 1 shows that compound 1 induced tumor regression in a cleavage-dependent manner. Western blots of compound 1 diluted in mouse plasma from either wild-type or MC38 tumor-bearing mice and incubated at 37°C for 24, 48, or 72 hours prior to compound 1 processing are shown. Intact and cleaved controls were prepared in vitro. Data represents n=3 mice. [Figure 2D] Figure 1 shows that compound 1 induced tumor regression in a cleavage-dependent manner. Figure 2 shows a graph of tumor volume over time in mice treated with effective doses of either compound 1 (total 5.04 μM) or rhIL-2 (total 15.5 μM). Spider plots of individual mice are shown. [Figure 2E] Figure 1 shows that Compound 1 induced tumor regression in a cleavage-dependent manner. Figure 2 shows total IL-2 in plasma from tumor-bearing mice over time. Samples were taken at various time points and analyzed for the presence of total inducible IL-2 protein using an ELISA that detects both intact Compound 1 and free IL-2. Presented as mean ± SD, area under the curve measurements were calculated using GraphPad Prism software. [Figure 2F] Figure 1 shows that Compound 1 induced tumor regression in a cleavage-dependent manner. Figure 2 shows total IL-2 over time in tumor samples from tumor-bearing mice. Samples were taken at various time points and analyzed for the presence of total inducible IL-2 protein using an ELISA that detects both intact Compound 1 and free IL-2. Presented as mean ± SD, area under the curve measurements were calculated using GraphPad Prism software. [Figure 2G] Figure 1 shows that compound 1 induced tumor regression in a cleavage-dependent manner. Figure 2 shows total IL-2 in plasma from tumor-bearing mice over time. Samples were taken at various time points and analyzed for the presence of total inducible IL-2 protein using an ELISA that detects free human IL-2 using AlphaLISA specific for unblocked human IL-2. Mice were dosed only twice, and the timing of dosing is indicated by the arrows on the dependent (x) axis. Values ​​are presented as mean ± SD and area under the curve measurements were calculated using GraphPad Prism software. [Figure 2H] Figure 1 shows that compound 1 induced tumor regression in a cleavage-dependent manner. Figure 2 shows total IL-2 in tumor samples from tumor-bearing mice over time. Samples were taken at various time points and analyzed for the presence of total inducible IL-2 protein using an ELISA that detects free human IL-2 using AlphaLISA specific for unblocked human IL-2. Mice were dosed only twice, and the timing of dosing is indicated by the arrows on the dependent (x) axis. Values ​​are presented as mean ± SD and area under the curve measurements were calculated using GraphPad Prism software. [Figure 2I] Figure 2 shows that compound 1 induced tumor regression in a cleavage-dependent manner. Figure 3 shows the tumor volume (mm3) on day 18 with vehicle, 25 μg, 50 μg, 100 μg and 300 μg of compound 1, and 300 μg of compound 1-NC. [Figure 2J] Figure 1 shows that compound 1 induced tumor regression in a cleavage-dependent manner.Figure 2 shows that depletion of CD8+ T cells by twice-weekly anti-CD8 antibody treatment significantly reduced the anti-tumor activity of compound 1 in mice.

[0016] [Figure 3A] 1 demonstrates that Compound 1 induced an anti-tumor memory response. The frequency of tetramer-positive CD8+ T cells in splenocytes is shown. [Figure 3B] 1 demonstrates that Compound 1 induced an anti-tumor memory response. The frequency of tetramer-positive CD8+ T cells in splenocytes is shown. [Figure 3C] 1 demonstrates that Compound 1 induced an anti-tumor memory response. Expression of memory cell markers CD62L and CD44 in tetramer-positive CD8+ T cells in splenocytes is shown. [Figure 3D] 1 demonstrates that Compound 1 induced an anti-tumor memory response. Expression of memory cell markers CD62L and CD44 in tetramer-positive CD8+ T cells in splenocytes is shown. [Figure 3E] 1 demonstrates that Compound 1 induced an anti-tumor memory response. The frequency of tetramer-positive CD8+ T cells producing TNF or IFNγ is shown. [Figure 3F] 1 demonstrates that Compound 1 induced an anti-tumor memory response. The frequency of tetramer-positive CD8+ T cells producing TNF or IFNγ is shown. [Figure 3G] 1 is a pie chart showing the analysis of polyfunctional tetramer-positive CD8+ T cells co-expressing IFNγ and TNF, demonstrating that Compound 1 induced an anti-tumor memory response. [Figure 3H] Demonstration that Compound 1 induced anti-tumor memory response. Schematic diagram of tumor challenge and rechallenge study. Naive mice or mice that previously rejected MC38 tumor after IL-2 INDUKINE™ protein treatment were rechallenged with MC38 tumor cells 60 days after the initial implantation. During rechallenge, these mice were not treated. [Figure 3I] Demonstrating that Compound 1 induced antitumor memory responses. Graphs showing tumor volume measured over time from MC38 CR (n=15) or naive (n=33) mice. Data are presented as mean ± SD, and P values ​​are obtained from t-test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).

[0017] [Figure 4A] Figure 1 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. A heat map of transcripts with statistically significant differences in expression between the two treatments (Compound 1 and vehicle control) is shown. Transcripts with an average normalized number below 50 were excluded from the heat map. Each lane represents an individual animal. [Figure 4B] 1 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. FIG. 1 is a plot of differentially expressed transcripts between Compound 1 and vehicle-treated mice. [Figure 4C]Figure 1 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. Specific pathway scores of Compound 1-treated or vehicle-treated mice are shown. P values ​​are from two-way ANOVA with multiple comparisons (***, P<0.001; ****, P<0.0001). [Figure 4D] Figure 1 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. Normalized gene counts from selected immune checkpoint genes are shown. [Figure 4E] 1 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. Figures from flow cytometry analysis of TIL density of various immune populations are shown, including fold change information between vehicle and Compound 1 treatment groups. [Figure 4F] Figure 1 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. The ratio of total CD8+ T cells or tetramer-positive CD8+ T cells to Tregs within TILs is shown, with fold change information between vehicle and Compound 1 treatment groups. [Figure 4G] Figure 1 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. Figure 2 shows the frequency of tetramer-positive CD8+ T cells producing IFNγ after restimulation with PMA / ionomycin. [Figure 4H] Figure 1 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. Figure 2 shows the frequency of tetramer-positive CD8+ T cells producing IFNγ after restimulation with PMA / ionomycin. [Figure 4I] FIG. 1 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. FIG. 1 shows a pie chart of the analysis of polyfunctional tetramer-positive CD8+ T cells by examining the co-expression of IFNγ, TNF, and granzyme B after PMA / ionomycin restimulation. [Figure 4J] Figure 2 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors.The frequency of IFNγ-producing tumor-infiltrating FoxP3+ Tregs in the vehicle (control) and Compound 1 groups is shown. [Figure 4K] Figure 2 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors.The frequency of IFNγ-producing tumor-infiltrating FoxP3+ Tregs in the vehicle (control) and Compound 1 groups is shown. [Figure 4L] Figure 2 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. Figure 2 shows the frequency of tumor-infiltrating FoxP3+ Tregs producing TNF after PMA / ionomycin restimulation in the vehicle (control) and Compound 1 groups. [Figure 4M] Figure 1 shows that treatment with Compound 1 increased immune cell activation and infiltration of MC38 tumors. The frequency of tumor-infiltrating FoxP3+ Tregs producing TNF after PMA / ionomycin restimulation in the vehicle (control) and Compound 1 groups is shown. Data are expressed as mean ± SD, unless otherwise stated, and P values ​​are obtained from t-test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).

[0018] [Figure 5A] Figure 1 shows that systemic treatment with Compound 1 preferentially activated tumor-infiltrating T cells. Figure 2 shows the frequency of tetramer-negative CD8+ T cells in TILs, splenocytes, DLN, and peripheral blood in vehicle (control) and Compound 1 groups. Data are presented as mean ± SD, unless otherwise stated, and P values ​​are obtained from t-test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). [Figure 5B] Figure 1 shows that systemic treatment with Compound 1 preferentially activated tumor-infiltrating T cells. Figure 2 shows the frequency of IFNγ-producing CD4+ non-Tregs in TILs, splenocytes, DLN, and peripheral blood in vehicle (control) and Compound 1 groups after restimulation with PMA / ionomycin. Data are presented as mean ± SD, unless otherwise stated, and P values ​​are obtained from t-test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). [Figure 5C]Figure 1 shows that systemic treatment with compound 1 preferentially activated tumor-infiltrating T cells. Figure 2 shows tumor volume over time in mice treated with either vehicle (n=10), compound 1 alone (n=10), or compound 1 with daily FTY720 treatment (n=10). FTY720 administration was initiated 24 hours before starting compound 1 treatment (dose 25 μg) and maintained daily (dose 10 μg) throughout the experiment. Tumor volume (mean ± SEM) was measured over time. Data are presented as mean ± SD unless otherwise stated, and P values ​​are derived from t-test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).

[0019] [Figure 6A] Figure 1 shows that treatment with Compound 1 increased CD8+ T cell activation and Treg vulnerability in B16-F10. Shown are tumor volumes measured over time in mice treated with vehicle, Compound 1 at 100 μg / animal and 200 μg / animal, and Compound 1 in combination with an anti-PD1 inhibitor at 100 μg / animal and 200 μg / animal. Data from individual mice (dashed lines) are shown with group means shown in bold. [Figure 6B] Figure 1 shows that treatment with Compound 1 increased CD8+ T cell activation and Treg vulnerability in B16-F10. A heat map of transcripts with statistically significant differences in expression between the two treatments (vehicle control and Compound 1) is shown. Transcripts with an average normalized number below 50 were excluded from the heat map. Each lane represents an individual animal. [Figure 6C] Figure 1 shows that treatment with Compound 1 increased CD8+ T cell activation and Treg vulnerability in B16-F10. Figure 2 shows the results of restimulating TILs and examining effector cytokine and protein production and proliferation. Figure 3 shows representative flow plots of tetramer-positive CD8+ T cells. [Figure 6D]Figure 1 shows that treatment with Compound 1 increased CD8+ T cell activation and Treg vulnerability in B16-F10. Figure 2 shows the results of restimulating TILs and examining their effector cytokine and protein production and proliferation. Figure 3 shows quantitative analysis from individual mice. [Figure 6E] Figure 1 shows that treatment with Compound 1 increased CD8+ T cell activation and Treg vulnerability in B16-F10. Figure 2 shows the results of restimulating TILs and examining effector cytokine and protein production and proliferation. Figure 3 shows representative flow plots of NK cells. [Figure 6F] Figure 1 shows that treatment with Compound 1 increased CD8+ T cell activation and Treg vulnerability in B16-F10. Figure 2 shows the results of restimulating TILs and examining their effector cytokine and protein production and proliferation. Figure 3 shows quantitative analysis from individual mice. [Figure 6G] Figure 1 shows that treatment with Compound 1 increased CD8+ T cell activation and Treg vulnerability in B16-F10. Figure 2 shows the results of restimulating TILs and examining effector cytokine and protein production and proliferation. Figure 3 shows representative flow plots of FoxP3+ Tregs. [Figure 6H] Figure 1 shows the results of restimulating TILs for effector cytokine and protein production and proliferation. Graphs show quantitative analysis from individual mice. P values ​​are from t-test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).

[0020] [Figure 7A] Compound 1 is stable in human serum and selectively processed by human tumor cells. Compound 1 was diluted in healthy human serum from n=3 donors and incubated at 37° C. for 24 or 72 hours, and then the processing of Compound 1 was measured by Western blot analysis of IL-2. [Figure 7B] Figure 1 shows that compound 1 was stable in human serum and selectively processed by human tumor cells. Graphs of total activation upon exposure of compound 1 to primary cells and measuring ex vivo inducible IL-2 protein cleavage in primary human tumor samples (n=97) and primary human healthy cells (n=13) are shown.

[0021] [Figure 8A] Figure 1 shows the activity of Compound 1 in additional human donors. CD25 expression by Tblasts over time in response to PHA stimulation. Data are representative of individual human donors. [Figure 8B] Figure 1 shows the activity of Compound 1 in additional human donors. CD25 expression by Tblasts over time in response to PHA stimulation. Data pooled from n=3 donors. [Figure 8C] Figure 1 shows the activity of Compound 1 in additional mice.Figure 2 shows the in vitro activity of Compound 1 in primary human Tblasts from additional donors, comparing intact (circles) and protease-activated (cleaved) WTX-124 (downward triangles) with rhIL-2 (upward triangles). [Figure 8D] Figure 1 shows the activity of Compound 1 in additional mice.Figure 2 shows the in vitro activity of Compound 1 in mouse Tblasts from additional donors, comparing intact (circles) and protease-activated (cleaved) WTX-124 (downward triangles) with rhIL-2 (upward triangles).

[0022] [Figure 9]A-B show that compound 1 is well tolerated in mice. A shows the body weight over time after randomization of mice implanted with MC38 tumor cells grown to an average volume of 100-150 mm3 into treatment groups. Labels in the legend indicate the dose per mouse per day of administration. Mice were intraperitoneally administered twice a week for a total of four times, and body weight was measured over time. The average body weight of n=12 mice per group is shown. B shows the typical therapeutic window of rhIL-2 and compound 1 in MC38 tumor-bearing mice.

[0023] [Figure 10] A-B show effector cytokine production of MC38 tumor-infiltrating tetramer-positive CD8+ T cells. After MC38 tumor cells were implanted and allowed to grow to a mean volume of 100 mm3, mice were randomly assigned to treatment groups. Mice were dosed twice weekly with compound (100 μg) or PBS vehicle. Tumors were harvested 24 h after the second dose and dissociated for further analysis. Frequency of tetramer-positive CD8+ T cells producing TNF (A) or granzyme B (B) after restimulation with PMA / ionomycin. P values ​​are derived from t-test (*, P<0.05).

[0024] [Figure 11] Figure 1 shows that PD-1 monotherapy has no anti-tumor activity in B16-F10. After B16-F10 tumors were implanted and allowed to grow to a mean volume of 100 mm3, mice were randomly assigned to treatment groups. Mice were administered either vehicle (open circles) or anti-PD-1 (filled circles, 200 μg) intraperitoneally twice a week for 2 weeks. Tumor volumes were measured over time. Data represent n=10 mice per group.

[0025] [Figure 12A] Results of baseline tumor infiltrating lymphocyte analysis are shown. MC38 and B16-F10 tumors were implanted and allowed to grow to a mean volume of 100 mm3 before tumor harvest for TIL analysis. Gating strategies for identifying different immune cell populations are shown. [Figure 12B] Results of baseline tumor infiltrating lymphocyte analysis are shown. MC38 and B16-F10 tumors were implanted and allowed to grow to a mean volume of 100 mm3 before tumor harvest for TIL analysis. The frequency of various immune populations within the CD45+ population is shown.

[0026] [Figure 13A] Results of compound 1 treatment in mice. MC38 tumor cells were implanted and allowed to grow to a mean volume of 100-150 mm3, after which mice were randomly assigned to treatment groups. Labels in the legend represent dose per mouse per day of treatment. Mice were dosed intraperitoneally twice weekly for a total of four doses. Graph showing body weights measured over time. Mean body weights are shown for n=12 mice per group. [Figure 13B] Figure 1 shows the results of compound 1 treatment in mice. MC38 tumor cells were implanted and allowed to grow to a mean volume of 100-150 mm3, after which mice were randomly assigned to treatment groups. Labels in the legend represent dose per mouse per day of treatment. Mice were dosed intraperitoneally twice weekly for a total of four doses. Graph showing tumor volume measured over time, shown as mean + / - SEM. [Figure 13C] Figure 1 shows the results of Compound 1 treatment in mice. After implanting MC38 tumor cells and allowing them to grow to a mean volume of 100-150 mm3, mice were randomly assigned to treatment groups. Labels in the legend represent doses per mouse per day of treatment. Mice were administered intraperitoneal doses twice weekly for a total of four doses. Figure 1 shows the results of MC38 tumor-bearing mice that were randomized to receive either vehicle, Compound 1, or an IL-2 prodrug lacking a half-life extension component. Tumor volumes were measured over time and are shown as mean + / - SEM. [Figure 13D]Results of compound 1 treatment in mice are shown. MC38 tumor cells were implanted and allowed to grow to a mean volume of 100-150 mm3, after which mice were randomly assigned to treatment groups. Labels in the legend represent doses per mouse per day of treatment. Mice were intraperitoneally dosed twice weekly for a total of four doses. Mice were dosed with equimolar amounts of recombinant human IL-2 (a total of five doses over three days), WW0177 (two doses over three days), or compound 1 (two doses over three days), followed by intravenous injection of Evans blue solution. Extravasation of Evans blue into the lungs was measured 30 min after intravenous administration of the dye. [Figure 13E] Figure 1 shows the results of compound 1 treatment in mice. MC38 tumor cells were implanted and allowed to grow to a mean volume of 100-150 mm3, after which mice were randomly assigned to treatment groups. Labels in the legend represent doses per mouse per day of treatment. Mice were dosed intraperitoneally twice weekly for a total of four doses. Figure 1 shows the detection of either recombinant human IL-2 or recombinant murine IL-2 by human-specific IL-2 ELISA. [Figure 13F] Figure 1 shows the results of Compound 1 treatment in mice. MC38 tumor cells were implanted and allowed to grow to a mean volume of 100-150 mm3, after which mice were randomly assigned to treatment groups. Labels in the legend represent doses per mouse per day of treatment. Mice were dosed intraperitoneally twice weekly for a total of four doses. Figure 1 shows the detection of either Compound 1 or free IL-2 by Human Specific IL-2 Alphalisa. [Figure 13G]Results of compound 1 treatment in mice are shown. MC38 tumor cells were implanted and allowed to grow to a mean volume of 100-150 mm3, after which mice were randomly assigned to treatment groups. Labels in the legend represent doses per mouse per day of treatment. Mice were administered intraperitoneally twice weekly for a total of four doses. Graphs depicting therapeutic window expression 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). ELISA, enzyme-linked immunosorbent assay; IL-2, interleukin-2; IP, intraperitoneal; MC, mouse colon; rhIL-2, recombinant human IL-2; SEM, standard error; TW, therapeutic window.

[0027] [Figure 14A] FIG. 1 shows that compound 1 is superior to equimolar amounts of recombinant human IL-2 in activating B16-F10 TILs. After B16-F1 tumors were implanted and allowed to grow to a mean volume of 100 mm3, mice were randomly assigned to treatment groups. Mice were administered either vehicle (open circles) or anti-PD-1 (closed circles, 200 μg) intraperitoneally twice weekly for 2 weeks. FIG. 1 shows tumor volume measured over time. Data represents n=10 mice per group and is shown as mean + / - SEM. [Figure 14B] Figure 1 shows that Compound 1 is superior to equimolar amounts of recombinant human IL-2 in activating B16-F10 TILs. After implanting B16-F1 tumors and allowing them to grow to an average volume of 100 mm3, mice were randomly assigned to treatment groups. Mice were administered either vehicle (open circles) or anti-PD-1 (closed circles, 200 μg) intraperitoneally twice weekly for 2 weeks. Results are shown for tumors harvested on day 5 from mice treated with either vehicle, Compound 1 (200 μg / dose), or equimolar amounts of recombinant human IL-2. Quantitative analysis of CD25 expression is shown. [Figure 14C]Figure 1 shows that Compound 1 is superior to equimolar amounts of recombinant human IL-2 in activating B16-F10 TILs. After B16-F1 tumors were implanted and allowed to grow to a mean volume of 100 mm3, mice were randomly assigned to treatment groups. Mice were administered either vehicle (open circles) or anti-PD-1 (closed circles, 200 μg) intraperitoneally twice weekly for 2 weeks. Results are shown for tumors harvested on day 5 from mice treated with either vehicle, Compound 1 (200 μg / dose), or equimolar amounts of recombinant human IL-2. Ki67 expression by various immune cell subsets is shown. P values ​​are from one-way ANOVA with multiple comparisons (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).

[0028] [Figure 15A] 1 is a graph showing that a variant of Compound 1 containing a non-α IL-2 mutein (Compound 5) has no antitumor activity when compared to the same dose of Compound 1. 2 is a graph showing tumor volume measured over time in MC38 tumor-bearing mice treated with either vehicle, Compound 1 (containing a native IL-2 payload, 100 μg / dose), or a variant of Compound 1 containing a non-α IL-2 mutein as the payload (100 μg / dose) (Compound 5). [Figure 15B] FIG. 1 is a graph showing that a variant of Compound 1 (Compound 5) containing a non-alpha IL-2 mutein has no antitumor activity when compared to the same dose of Compound 1. FIG. 2 is a graph showing the frequency of tumor-infiltrating tetramer+ CD8+ T cells producing granzyme B, IFNγ, or TNF on day 5. [Figure 15C] FIG. 1 is a graph showing that a variant of Compound 1 (Compound 5) containing a non-α IL-2 mutein has no antitumor activity when compared to the same dose of Compound 1. FIG. 2 is a graph showing the frequency of tumor-infiltrating NK cells producing granzyme B or IFNγ. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present disclosure relates to compositions and methods for treating cancer using an inducible IL-2 prodrug. The method generally includes administering an effective amount of an inducible IL-2 prodrug to a subject in need of treatment. 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 can selectively activate IL-2 in the tumor microenvironment and reduce IL-2-associated toxicity while improving antitumor efficacy in cancer patients. The inventors herein demonstrate and illustrate that inducible IL-2 is preferentially activated in tumor tissue by tumor-associated proteases and releases active IL-2 in the tumor microenvironment. In vitro assays confirmed that the activity of the inducible IL-2 prodrug (Compound 1) is dependent on proteolytic activation and that inducible IL-2 prodrug treatment completely rejects established tumors in a cleavage-dependent manner.

[0030] The present inventors show that treatment with an inducible IL-2 prodrug induces activation of T cells and natural killer cells, significantly altering the immune activation profile of the tumor microenvironment and resulting in significant inhibition of tumor growth in a syngeneic tumor model. The present inventors further show that the 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.

[0031] A. IL-2 Prodrugs Inducible IL-2 prodrugs for use in the disclosed methods and compositions overcome the toxicity and short half-life issues that have severely limited the clinical use of cytokines in oncology. Inducible IL-2 prodrugs include IL-2 polypeptides that have the receptor agonist activity of native IL-2, including binding to and activating signaling through IL-2Rα / β / γ and IL-2Rβ / γ, but in the context of an inducible prodrug, the cytokine receptor agonist activity is attenuated and the circulating half-life is extended. The prodrugs include protease cleavage sequences that are cleaved by proteases that are associated with, and typically abundantly or selectively present in, the tumor microenvironment. Thus, the inducible IL-2 prodrugs are preferentially (or selectively) and efficiently cleaved in the tumor microenvironment to release active IL-2, substantially restricting IL-2 activity to the tumor microenvironment. The half-life of the IL-2 released upon cleavage is short and substantially similar to the half-life of naturally occurring IL-2, further restricting IL-2 activity to the tumor microenvironment. Even if the half-life of an inducible IL-2 prodrug is extended, toxicity is dramatically reduced or eliminated because the circulating prodrug attenuates IL-2 activity and active IL-2 is restricted to the tumor microenvironment.

[0032] The inducible IL-2 prodrug comprises two polypeptide chains. The first polypeptide chain can comprise, from the amino terminus to the carboxy terminus, an IL-2 polypeptide-protease cleavable linker-anti-human serum albumin (HSA) binding single antibody variable domain-preferably a protease cleavable linker-VH and CH1 of an antibody that binds IL-2. The first polypeptide chain can comprise, from the amino terminus to the carboxy terminus, an IL-2 polypeptide-protease cleavable linker-VH and CH1 of an antibody that binds IL-2-preferably a protease cleavable linker-anti-human serum albumin (HSA) binding single antibody variable domain. The second polypeptide chain comprises the VL and CL of an antibody that binds IL-2, which together with the VH and CH1 of the first polypeptide chain form a Fab that binds an IL-2 polypeptide. Compounds 1, 2, 3 and 4 are specific examples of inducible IL-2 prodrugs for use according to the present disclosure. Further details regarding compounds 1, 2, 3, and 4 and their activities are disclosed in WO2021 / 097376. Compounds 5-29 are further examples of inducible IL-2 prodrugs for use according to the present disclosure. [Table 1]

[0033] Amino acid sequence variants of compounds 1, 2, 3, and 4 that retain attenuated IL-2 activity in the periphery and release active IL-2 upon protease cleavage in the tumor microenvironment can also be used in accordance with the present disclosure. For example, the prodrug can include a first polypeptide having 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 to SEQ ID NO:1, and a second polypeptide having 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 to SEQ ID NO:5.

[0034] The prodrug can include a first polypeptide having 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 to SEQ ID NO:2, and a second polypeptide having 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 to SEQ ID NO:5.

[0035] The prodrug can include a first polypeptide having 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 to SEQ ID NO:3, and a second polypeptide having 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 to SEQ ID NO:5.

[0036] The prodrug can include a first polypeptide having 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 to SEQ ID NO:4, and a second polypeptide having 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 to SEQ ID NO:5.

[0037] Amino acid sequence variants of compounds 5-29 that retain attenuated IL-2 activity in the periphery and release active IL-2 upon protease cleavage in the tumor microenvironment can also be used in accordance with the present disclosure.

[0038] The prodrug can include a first polypeptide having 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 to SEQ ID NO:1, and a second polypeptide having 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 to SEQ ID NO:8.

[0039] The prodrug can include a first polypeptide having 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 to SEQ ID NO:1, and a second polypeptide having 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 to SEQ ID NO:9.

[0040] The prodrug can include a first polypeptide having 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 to SEQ ID NO:10, and a second polypeptide having 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 to SEQ ID NO:10.

[0041] The prodrug can include a first polypeptide having 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 to SEQ ID NO:11, and a second polypeptide having 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 to SEQ ID NO:11.

[0042] The prodrug can include a first polypeptide having 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 to SEQ ID NO:1, and a second polypeptide having 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 to SEQ ID NO:12.

[0043] The prodrug can include a first polypeptide having 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 to SEQ ID NO:13, and a second polypeptide having 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 to SEQ ID NO:5.

[0044] The prodrug can include a first polypeptide having 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 to SEQ ID NO:14, and a second polypeptide having 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 to SEQ ID NO:5.

[0045] The prodrug can include a first polypeptide having 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 to SEQ ID NO:15, and a second polypeptide having 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 to SEQ ID NO:5.

[0046] The prodrug can include a first polypeptide having 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 to SEQ ID NO:16, and a second polypeptide having 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 to SEQ ID NO:5.

[0047] The prodrug can include a first polypeptide having 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 to SEQ ID NO:17, and a second polypeptide having 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 to SEQ ID NO:5.

[0048] The prodrug can include a first polypeptide having 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 to SEQ ID NO:18, and a second polypeptide having 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 to SEQ ID NO:5.

[0049] The prodrug can include a first polypeptide having 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 to SEQ ID NO:19, and a second polypeptide having 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 to SEQ ID NO:5.

[0050] The prodrug can include a first polypeptide having 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 to SEQ ID NO:20, and a second polypeptide having 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 to SEQ ID NO:5.

[0051] The prodrug can include a first polypeptide having 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 to SEQ ID NO:21, and a second polypeptide having 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 to SEQ ID NO:5.

[0052] The prodrug can include a first polypeptide having 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 to SEQ ID NO:22, and a second polypeptide having 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 to SEQ ID NO:5.

[0053] The prodrug can include a first polypeptide having 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 to SEQ ID NO:23, and a second polypeptide having 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 to SEQ ID NO:5.

[0054] The prodrug can include a first polypeptide having 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 to SEQ ID NO:24, and a second polypeptide having 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 to SEQ ID NO:5.

[0055] The prodrug can include a first polypeptide having 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 to SEQ ID NO:25, and a second polypeptide having 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 to SEQ ID NO:5.

[0056] The prodrug can include a first polypeptide having 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 to SEQ ID NO:26, and a second polypeptide having 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 to SEQ ID NO:5.

[0057] The prodrug can include a first polypeptide having 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 to SEQ ID NO:27, and a second polypeptide having 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 to SEQ ID NO:5.

[0058] The prodrug can include a first polypeptide having 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 to SEQ ID NO:28, and a second polypeptide having 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 to SEQ ID NO:5.

[0059] The prodrug can include a first polypeptide having 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 to SEQ ID NO:29, and a second polypeptide having 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 to SEQ ID NO:5.

[0060] The prodrug can include a first polypeptide having 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 to SEQ ID NO:30, and a second polypeptide having 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 to SEQ ID NO:5.

[0061] The prodrug can include a first polypeptide having 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 to SEQ ID NO:31, and a second polypeptide having 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 to SEQ ID NO:5.

[0062] The prodrug can include a first polypeptide having 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 to SEQ ID NO:32, and a second polypeptide having 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 to SEQ ID NO:5.

[0063] For all amino acid sequence variant prodrugs, the protease cleavage site preferably contains no amino acid substitutions or only conservative amino acid substitutions, such that the sequence variant prodrugs are cleaved in the tumor microenvironment and release IL-2 to substantially the same extent as the corresponding parent prodrug. Similarly, the complementarity determining regions of the anti-HAS single variable domain and anti-IL2 Fab preferably contain no amino acid substitutions or only conservative amino acid substitutions, such that a) the serum half-life of the sequence variant prodrugs is substantially the same as the corresponding parent prodrug, and b) the attenuation of the IL-2 agonist activity of the sequence variant prodrugs is substantially the same as the corresponding parent prodrug.

[0064] Exemplary amino acid substitutions are shown in Table 2. [Table 2]

[0065] B. Therapeutic Uses and Pharmaceutical Compositions The disclosure further relates to methods and compositions for treating cancer using an inducible IL-2 prodrug, optionally in combination with one or more additional therapeutic agents, such as a chemotherapeutic agent, a cytokine, an oncolytic virus, an immuno-oncology agent, or a checkpoint inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody).Appropriate 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, hexamethylcyclohexanediaminetetraacetate, cyclosalicylic acid ... Melamine, methotrexate, pemetrexed, fluorouracil (e.g., 5-fluorouracil), capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thioguanine, mercaptopurine, vincristine, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, etoposide, teniposide, doxorubicin, daunorubicin, epirubicin, idarubicin Examples of immune checkpoint proteins include PD-1, which binds to ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), CTLA-4 (CD152), which binds to B7-1 (CD80) and B7-2 (CD86), LAG3 (CD223), which binds to galectin 3, LSECtin, and FGL1, and ligand IL-1 (IL-1, CD274). These include TIM3 (HAVCR2), which binds to the cancer cells Ceacam1 and galectin-9; TIGIT (VSTM3, WUCAM), which binds to CD112 and CD155; BTLA (CD272), which binds to HVEM (TNFRSF14); B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), and CD160 (BY55), which bind to HVEM; and CD134 (TNRFSR4, OX40), which binds to CD252 (OX-40L).Therapeutic agents such as antibodies that bind to immune checkpoint proteins and inhibit their immunosuppressive activity include the anti-PD1 antibodies pembrolizumab (KEYTRUDA), dostallimab (JEMPERLI), cemiplimab-rwlc (LIBATYO), nivolumab (OPDIVO), camrelizumab, tislelizumab, toripalimab, and sintilimab (TYVYT); anti-PD-L1 antibodies avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ); and the anti-CTLA-4 antibody ipilimumab (YERVOY).

[0066] The inducible IL-2 prodrug and any additional therapeutic agent are typically administered systemically, for example, by intravenous injection or, preferably, by intravenous infusion. Other types of administration can be used, such as oral, parenteral, intravenous, intravenously, intraarticular, intraperitoneal, intramuscular, subcutaneous, intracavity, transdermal, intrahepatic, intracranial, aerosol / inhalation, bronchoscopic placement, or intratumor.

[0067] The methods and compositions disclosed herein can be used to treat any suitable cancer, particularly solid tumors such as sarcomas and carcinomas. For example, the methods and compositions disclosed herein can be used to treat acute lymphocytic 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, cancer of unknown primary, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, breast ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, noninvasive lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary site, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple In one embodiment, the present invention can be used to treat myeloma, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, glioblastoma, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumors, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoma, testicular cancer, throat cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor. The non-small cell lung cancer (NSCLC) can be, for example, adenocarcinoma NSCLC, squamous cell NSCLC, or large cell carcinoma NSCLC.

[0068] In certain embodiments, the methods and compositions disclosed herein are useful in treating adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, cancer of unknown primary, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, noninvasive lobular carcinoma, lung cancer, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary site, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer It can be used to treat cancers including thymoma, T-cell lymphoma, teratoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, non-Hodgkin's lymphoma, head and neck squamous cell carcinoma, malignant pleural mesothelioma, and Wilms' tumor.

[0069] In certain preferred embodiments, 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 carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, high-frequency microsatellite instability or mismatch repair deficient cancer, high-frequency microsatellite instability or mismatch repair deficient colon cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial cancer, high tumor mutation burden cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), urothelial carcinoma, colon cancer, or oesophageal carcinoma. In certain preferred embodiments, the methods and compositions disclosed herein are used to treat glioblastoma.

[0070] In certain preferred embodiments, 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 carcinoma, cutaneous squamous cell carcinoma (CSCC), basal cell carcinoma (BCC), melanoma, malignant pleural mesothelioma, classical Hodgkin's 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).

[0071] Preferably, the methods and compositions disclosed herein are used to treat colon cancer, lung cancer, melanoma, renal cell carcinoma, or breast cancer.

[0072] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat melanoma. As an example, the methods and compositions disclosed herein can be used to treat melanoma in subjects with unresectable or metastatic melanoma. As another example, the methods and compositions disclosed herein can be used for adjuvant treatment of subjects with melanoma with lymph node(s) involvement after complete resection.

[0073] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat non-small cell lung cancer (NSCLC). As an example, the methods and compositions disclosed herein can be used to treat NSCLC in subjects with stage III or metastatic NSCLC whose tumors express PD-L1 (e.g., tumor proportion score (TPS) ≧1%) (as determined by an FDA-approved test), have no EGFR or ALK genomic tumor abnormalities, and the subject is not a candidate for surgical resection or definitive chemoradiotherapy. As another example, 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) and whose disease has progressed during or after platinum-containing chemotherapy. As another example, the methods and compositions disclosed herein can be used in combination with pemetrexed and platinum chemotherapy as a first-line treatment for patients with metastatic non-squamous NSCLC who have no EGFR or ALK genomic tumor abnormalities. As another example, the methods and compositions disclosed herein can be used in combination with carboplatin and either paclitaxel or protein-bound paclitaxel as a first line treatment for patients with metastatic squamous NSCLC.

[0074] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat SCLC. By way of example, the methods and compositions disclosed herein can be used to treat SCLC in subjects suffering from metastatic SCLC whose disease has progressed during or after platinum-based chemotherapy and at least one other prior line of treatment.

[0075] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat HNSCC. As an example, the methods and compositions disclosed herein can be used to treat HNSCC in subjects with metastatic or unresectable recurrent HNSCC whose tumors express PD-L1 (e.g., combined positive score (CPS) > 1) (as determined by an FDA-approved test). As another example, the methods and compositions disclosed herein can be used to treat HNSCC in subjects with recurrent or metastatic HNSCC whose disease has progressed during or after platinum-containing chemotherapy. As another example, 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 unresectable recurrent HNSCC.

[0076] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat cHL. As an example, the methods and compositions disclosed herein can be used to treat cHL in subjects with relapsed or refractory cHL. As another example, the methods and compositions disclosed herein can be used to treat cHL in pediatric subjects with refractory cHL or cHL that has relapsed after two or more lines of therapy.

[0077] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat PMBCL. By way of example, the methods and compositions disclosed herein can be used to treat PMBCL in subjects with refractory PMBCL or in subjects who have relapsed after two or more prior lines of therapy.

[0078] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat urothelial carcinoma. As an example, the methods and compositions disclosed herein can be used to treat urothelial carcinoma in subjects with locally advanced or metastatic urothelial carcinoma who are ineligible 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 who are ineligible for any platinum-containing chemotherapy regardless of PD-L1 status. As another example, the methods and compositions disclosed herein can be used to treat urothelial carcinoma in subjects with locally advanced or metastatic urothelial carcinoma who have progressed during or after platinum-containing chemotherapy, or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy. As another example, the methods and compositions disclosed herein can be used to treat urothelial carcinoma in subjects with bacillus Calmette-Guerin (BCG)-non-responsive high-risk non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary neoplasia who are ineligible for or choose not to undergo cystectomy.

[0079] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) cancers. By way of example, 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 whose solid tumors have progressed after previous treatment and have no satisfactory alternative treatment options, or whose colon cancer has progressed after treatment with fluoropyrimidines, oxaliplatin, and irinotecan.

[0080] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) colon cancer. By way of example, the methods and compositions disclosed herein can be used to treat MSI-H or dMMR colon cancer in subjects suffering from unresectable or metastatic MSI-H or dMMR colon cancer.

[0081] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat gastric cancer. By way of example, the methods and compositions disclosed herein can be used to treat gastric cancer in subjects with recurrent locally advanced or metastatic gastric cancer or gastroesophageal junction adenocarcinoma whose tumors express PD-L1 (e.g., a combined positive score (CPS) of ≧1) (as determined by an FDA-approved test) and whose disease has progressed during or after two or more lines of prior therapy, including fluoropyrimidine and platinum-containing chemotherapy, and, where appropriate, HER2 / neu targeted therapy.

[0082] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat esophageal cancer. As an example, the methods and compositions disclosed herein can be used in combination with platinum and fluoropyrimidine-based chemotherapy to treat esophageal cancer in subjects with locally advanced or metastatic esophageal or gastroesophageal junction (GEJ) (e.g., tumors with a center point 1-5 centimeters above the GEJ) cancer that are not suitable for surgical resection or definitive chemoradiotherapy. As another example, 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 a center point 1-5 centimeters above the GEJ) cancer that are not suitable for surgical resection or definitive chemoradiotherapy after one or more lines of prior systemic therapy for patients with squamous cell tumors that express PD-L1 (CPS≧10) (as determined by an FDA-approved test).

[0083] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat cervical cancer. By way of example, the methods and compositions disclosed herein can be used to treat cervical cancer in subjects with recurrent or metastatic cervical cancer whose tumors express PD-L1 (e.g., a combined positive score (CPS) of >= 1) (as determined by an FDA-approved test) and whose disease has progressed during or after chemotherapy.

[0084] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat HCC. In one example, the methods and compositions disclosed herein can be used to treat HCC in subjects who have previously been treated with sorafenib.

[0085] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat MCC. By way of example, the methods and compositions disclosed herein can be used to treat MCC in subjects suffering from recurrent locally advanced or metastatic MCC.

[0086] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat RCC.As an example, the methods and compositions disclosed herein can be used in combination with axitinib for the first-line treatment of patients with advanced RCC.

[0087] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat endometrial cancer.As an example, the methods and compositions disclosed herein can be used in combination with lenvatinib to treat subjects with non-MSI-H or non-dMMR advanced endometrial cancer whose disease has progressed after previous systemic therapy and who are not candidates for curative surgery or radiation therapy.

[0088] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat high tumor mutation burden (TMB-H) cancer. By way of example, the methods and compositions disclosed herein can be used to treat TMB-H cancer in subjects suffering from unresectable or metastatic high tumor mutation burden (e.g., 10 or more mutations per megabase (mut / Mb)) solid tumors (as determined by an FDA-approved test) that have progressed after previous treatment and have no satisfactory alternative treatment options.

[0089] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat cutaneous squamous cell carcinoma (cSCC). By way of example, 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 therapy.

[0090] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat triple-negative breast cancer (TNBC). By way of example, the methods and compositions disclosed herein can be used in combination with chemotherapy to treat 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).

[0091] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat Merkel cell carcinoma (MCC). As an example, a combination comprising avelumab can be used to treat MCC in subjects suffering from metastatic MCC.

[0092] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat urothelial carcinoma (UC). As one example, avelumab-containing combinations can be used to treat UC in subjects with locally advanced or metastatic UC whose disease has progressed during or after platinum-containing chemotherapy. As another example, avelumab-containing combinations can be used to treat UC in subjects with locally advanced or metastatic UC whose disease has progressed within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

[0093] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat renal cell carcinoma (RCC). As an example, a combination comprising avelumab and axitinib can be used in subjects with advanced RCC.

[0094] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat urothelial carcinoma (UC). As one example, a combination comprising durvalumab can be used to treat UC in a subject with locally advanced or metastatic urothelial carcinoma whose disease has progressed during or after platinum-containing chemotherapy. As another example, a combination comprising durvalumab can be used to treat UC in a subject with locally advanced or metastatic urothelial carcinoma whose disease has progressed within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

[0095] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat non-small cell lung cancer (NSCLC). As an example, a combination comprising durvalumab can be used to treat non-small cell lung cancer (NSCLC) in subjects with unresectable stage III NSCLC who have not progressed after combined platinum-based chemotherapy and radiation therapy.

[0096] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat small cell lung cancer (SCLC). As an example, a combination including durvalumab can be used in combination with etoposide and either carboplatin or cisplatin as a first-line treatment for adult subjects with advanced stage small cell lung cancer (ES-SCLC).

[0097] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat urothelial carcinoma (UC). As an example, a combination comprising atezolizumab can be used to treat UC in adult subjects with locally advanced or metastatic urothelial carcinoma who are ineligible for cisplatin-containing chemotherapy, whose tumors express PD-L1 (e.g., PD-L1 stained tumor-infiltrating immune cells [IC] cover 5% or more of the tumor area) (as determined by an FDA-approved test), or who are ineligible for any platinum-containing chemotherapy, regardless of PD-L1 status, or who have progressed during or after platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment.

[0098] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat NSCLC. As an example, 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., 50% or more of tumor cells stained with PD-L1 [TC≧50%] or PD-L1 stained tumor infiltrating immune cells [IC] covering 10% or more of the tumor area [IC≧10%]) (as determined by an FDA-approved test) and no EGFR or ALK genomic tumor abnormalities. As another example, 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 who do not have EGFR or ALK genomic tumor abnormalities. As another example, a combination comprising atezolizumab can be used in combination with protein-bound paclitaxel and carboplatin for the first-line treatment of adult subjects with metastatic non-squamous NSCLC who lack EGFR or ALK genomic tumor aberrations. As another example, a combination comprising atezolizumab can be used to treat NSCLC in adult subjects with metastatic NSCLC who have progressed on or after platinum-containing chemotherapy.

[0099] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat triple-negative breast cancer (TNBC). As an example, a combination comprising atezolizumab can be used in combination with protein-bound paclitaxel 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% or more of the tumor area) (as determined by an FDA-approved test).

[0100] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat small cell lung cancer (SCLC). As an example, a combination comprising atezolizumab can be used in combination with carboplatin and etoposide for the first-line treatment of adult subjects with advanced stage small cell lung cancer (ES-SCLC).

[0101] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat endometrial cancer. As an example, a combination comprising dostallimab can be used to treat endometrial cancer in adult subjects with mismatch repair deficient (dMMR) recurrent or progressive endometrial cancer (as determined by an FDA approved test) that has progressed during or after previous treatment with a platinum-containing regimen.

[0102] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat cutaneous squamous cell carcinoma (CSCC). As an example, 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 definitive radiation therapy.

[0103] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat basal cell carcinoma (BCC). As an example, a combination comprising cemiplimab-rwlc can be used to treat BCC in subjects with locally advanced BCC (laBCC) who have previously been treated with a hedgehog pathway inhibitor or for whom a hedgehog pathway inhibitor is not appropriate.

[0104] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat NSCLC. As an example, a combination comprising cemiplimab-rwlc can be used to treat locally advanced or metastatic NSCLC subjects whose tumors have high PD-L1 expression (e.g., tumor proportion score (TPS) ≧ 50%) (as determined by an FDA-approved test), no EGFR, ALK or ROS1 abnormalities, and who are not candidates for surgical resection or definitive chemoradiotherapy.

[0105] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat melanoma.As an example, a combination comprising nivolumab can be used as a single agent or in combination with ipilimumab to treat melanoma in subjects with unresectable or metastatic melanoma.As another example, a combination comprising nivolumab can be used in an adjuvant setting to treat melanoma in subjects with melanoma with lymph node metastasis or metastatic disease who have undergone complete resection.

[0106] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat NSCLC. As an example, a combination comprising nivolumab can be used as a first-line treatment in combination with ipilimumab to treat NSCLC in adult subjects with metastatic non-small cell lung cancer that expresses PD-L1 (1% or more) (as determined by an FDA-approved test) and has no EGFR or ALK genomic tumor abnormalities. As another example, a combination comprising NSCLC can be used as a first-line treatment in combination with ipilimumab and two cycles of platinum combination chemotherapy to treat melanoma in adult subjects with metastatic or recurrent non-small cell lung cancer that has no EGFR or ALK genomic tumor abnormalities. As another example, a combination comprising NSCLC can be used to treat melanoma in subjects with metastatic non-small cell lung cancer that has progressed during or after platinum-based chemotherapy.

[0107] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat malignant pleural mesothelioma.As an example, a combination comprising nivolumab can be used to treat malignant pleural mesothelioma in adult subjects with unresectable malignant pleural mesothelioma as a first-line treatment in combination with ipilimumab.

[0108] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat RCC.As an example, a combination comprising nivolumab can be used as a first-line treatment in combination with ipilimumab to treat RCC in subjects with intermediate-risk or low-risk progressive renal cell carcinoma.As another example, a combination comprising nivolumab can be used as a first-line treatment in combination with cabozantinib to treat RCC in subjects with progressive renal cell carcinoma.As another example, a combination comprising nivolumab can be used to treat RCC in subjects with progressive renal cell carcinoma who have previously received antiangiogenic therapy.

[0109] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat classical Hodgkin's lymphoma (cHL). As an example, a combination comprising nivolumab can be used to treat cHL in adult subjects with cHL that has relapsed or progressed after 3 or more lines of systemic therapy, including autologous hematopoietic stem cell transplantation (HSCT) and brentuximab vedotin, or autologous HSCT.

[0110] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat squamous cell carcinoma of the head and neck (SCCHN). As an example, a combination comprising nivolumab can be used to treat SCCHN in subjects with recurrent or metastatic squamous cell carcinoma of the head and neck whose disease has progressed during or after platinum-based therapy.

[0111] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat urothelial carcinoma (UC). As an example, a combination comprising nivolumab can be used to treat UC in subjects with locally advanced or metastatic urothelial carcinoma whose disease has progressed during or after platinum-containing chemotherapy, or whose disease has progressed within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

[0112] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat colon cancer. As an example, a combination comprising nivolumab can be used as a single agent or in combination with ipilimumab to treat colon cancer in subjects with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colon cancer that has progressed after treatment with fluoropyrimidine, oxaliplatin and irinotecan.

[0113] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat hepatocellular carcinoma (HCC).As an example, a combination comprising nivolumab can be used as a single agent or in combination with ipilimumab to treat HCC in subjects with HCC who have previously been treated with sorafenib.

[0114] In certain preferred embodiments, the methods and compositions disclosed herein can be used to treat esophageal squamous cell carcinoma (ESCC).As an example, a combination comprising nivolumab can be used to treat ESCC in subjects with unresectable progressive, recurrent or metastatic esophageal squamous cell carcinoma after previous fluoropyrimidine and platinum-based chemotherapy.

[0115] In certain preferred embodiments, combinations including camrelizumab can be used to treat cHL.

[0116] In certain preferred embodiments, the combinations including tislelizumab can be used to treat non-squamous non-small cell lung cancer. In certain preferred embodiments, the combinations including tislelizumab can be used to treat hepatocellular carcinoma (HCC).

[0117] In certain preferred embodiments, the combination comprising toripalimab can be used to treat urothelial carcinoma. In certain preferred embodiments, the combination comprising toripalimab can be used to treat melanoma. In certain preferred embodiments, the combination comprising toripalimab can be used to treat nasopharyngeal carcinoma (NPC).

[0118] In certain preferred embodiments, the combinations comprising sintilimab can be used to treat non-squamous non-small cell lung cancer. In certain preferred embodiments, the combinations comprising sintilimab can be used to treat cHL.

[0119] The cancer treated using the methods and compositions of the present disclosure can be a metastatic cancer. The methods and compositions disclosed herein can be used to treat metastatic clear cell renal carcinoma or metastatic cutaneous malignant melanoma.

[0120] If necessary, an additional therapeutic agent can be administered to the subject. Typically, such additional therapeutic agents are anti-cancer agents such as chemotherapeutic agents, immune checkpoint 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)), cell therapy (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 further described herein.

[0121] Pharmaceutical compositions can take various forms, such as liquid, lyophilized, and typically contain a suitable pharma- ceutically acceptable carrier. A pharma-ceutically acceptable carrier (or excipient) is an inactive component of a pharmaceutical composition that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a detrimental manner with other components of the pharmaceutical formulation or composition in which it is contained. Carriers are often selected to minimize the degradation of the active component and to minimize adverse side effects in a subject.

[0122] 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 pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffer solutions such as Ringer's solution, and glucose solution. Other carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptide. The matrices are in the form of shaped articles, such as films, liposomes, or microparticles. Depending, for example, on the route of administration and the concentration of the composition to be administered, certain carriers may be more preferred. The carrier is one suitable for administering the chimeric polypeptide or the nucleic acid sequence encoding the chimeric polypeptide to humans or other subjects.

[0123] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include 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, dextrose Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on dextrose Ringer's solution), and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, are optionally present. Typically, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to render the formulation isotonic, but the formulation may be hypertonic or hypotonic as appropriate. The pH of the solution is generally about 5 to about 8 or about 7 to 7.5.

[0124] Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable as needed.

[0125] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. In some cases, thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders are desirable.

[0126] The present disclosure also relates to a kit comprising a pharmaceutical composition containing a) an inducible IL-2 prodrug composition, e.g., as a liquid composition or a lyophilized composition in a suitable container (e.g., vial, bag, etc.), and b) a pembrolizumab composition, e.g., as a liquid composition or a lyophilized composition in a suitable container (e.g., vial, bag, etc.). The kit may further comprise other components, such as sterile water or saline for reconstitution of the lyophilized composition.

[0127] C.Definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those of ordinary skill in the art to which the present invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference from that commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly used using conventional methods by those of ordinary skill in the art, such as, for example, the widely used molecular cloning methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Procedures involving the use of commercially available kits and reagents, where appropriate, are generally performed according to manufacturer-defined protocols and conditions unless otherwise noted.

[0128] "Cytokine" is a well-known term of art that refers specifically to any one of the immunoregulatory proteins (such as interleukins or interferons) secreted by cells of the immune system and that are modulators of the immune system. Cytokine polypeptides that may be used in the fusion proteins disclosed herein include, but are not limited to, transforming growth factors such as TGF-α and TGF-β (e.g., TGFβ1, TGFβ2, TGFβ3); interferons such as interferon-α, interferon-β, interferon-γ, interferon-κ, and interferon-ω; IL-1, IL-1α, 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-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60 interleukins, such as 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, CCL20, CCL21) and granulocyte-macrophage colony-stimulating factor (GM-CS), as well as fragments of such polypeptides (i.e., functional fragments of the foregoing) that activate the cognate receptor for the cytokine. "Chemokine" is a term of art referring to any of a family of small molecule cytokines that have the ability to induce directed chemotaxis in nearby responsive cells.

[0129] As used herein, the term "inducible" refers to the ability of the protein that is part of the prodrug, i.e., IL-2, IL-12, or IFN, to bind to its receptor and exert activity upon cleavage of the prodrug in the tumor microenvironment. The inducible cytokine prodrugs disclosed herein have reduced or no cytokine agonist activity but release active cytokines upon cleavage in the tumor microenvironment.

[0130] "Attenuated" activity means that the biological activity and typically the cytokine (i.e., IL-2, IL-12, or IFN) agonist activity is decreased compared to the activity of the native cytokine (i.e., IL-2, IL-12, or IFN). The inducible cytokine prodrugs disclosed herein have attenuated cytokine receptor agonist activity, i.e., at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 1000-fold or less agonist activity compared to the native cytokine (i.e., IL-2, IL-12, or IFN). Upon cleavage in the tumor microenvironment, the active cytokine is released. Typically, the released cytokine has a cytokine receptor agonist activity that is at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold greater than the IL-2 receptor activation activity of the prodrug.

[0131] As used herein, the terms "peptide", "polypeptide" or "protein" are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used interchangeably herein to refer to an amino acid sequence. It should be recognized that the term polypeptide is not used herein to imply a particular size or number of amino acids that make up the molecule, and that the peptides of the present invention can include up to a few or more amino acid residues.

[0132] As used throughout this specification, a "subject" may be a vertebrate, more specifically a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. The term does not denote a particular age or sex. Thus, both adult and neonatal subjects, whether male or female, are intended to be subjects. As used herein, "patient" or "subject" may be used interchangeably and may refer to a subject having a disease or disorder (e.g., cancer). The term patient or subject includes human and veterinary subjects.

[0133] As used herein, the terms "treatment", "treat" or "treating" refer to a method of reducing the effects of a disease or condition, or a symptom of the disease or condition. Thus, in the disclosed methods, 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 a substantially complete reduction (such as reduction in tumor volume, reduction in tumor burden, reduction in death, etc.) of the severity of an established disease or condition, or a symptom of the disease or condition. For example, a method for treating a disease is considered to be therapeutic if one or more symptoms of the disease are reduced by 10% in a subject compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete elimination of the disease, illness, or symptoms of the disease or illness.

[0134] As used herein, the terms "prevent," "preventing," and "prevention" of a disease or disorder refer to the act of administering a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, e.g., before or at about the same time that a subject begins to exhibit one or more symptoms of a disease or disorder, which inhibits or delays the onset or worsening of one or more symptoms of the disease or disorder.

[0135] As used herein, references to "decrease," "reduce," or "inhibit" include an alteration of 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 more, as compared to an appropriate control level. Such terms may, but do not necessarily, include the complete elimination of a function or property, such as agonist activity.

[0136] The term "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 a nucleotide sequence of a nucleic acid that has substantially similar biological activity as a reference sequence (e.g., encodes a protein with substantially similar activity) but differs in nucleotide sequence. Typically, the amino acid or nucleotide sequence of a "sequence variant" is highly similar (e.g., at least about 80% similar) to the amino acid or nucleotide sequence of the reference sequence. Those skilled in the art will readily understand how to determine the identity of two polypeptides or two nucleic acids. For example, the identity can be calculated after aligning the two sequences such that the identity is at its highest level over a defined number of nucleotides or amino acids. Optimal alignment of sequences for comparison can be performed 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 computer 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.

[0137] The term "conservative amino acid substitution" is a technical term 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 the reference amino acid. It is well known that conservative amino acid substitutions in the amino acid sequence of a polypeptide often do not significantly change the overall structure or function of the polypeptide. Conservative substitutions of amino acids are well known to those skilled in the art. Conservative substitutions of amino acids can include, but are not limited to, substitutions made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. For example, those skilled in the art would reasonably expect that the substitution of leucine alone with isoleucine or valine, the substitution of aspartic acid alone with glutamic acid, the substitution of threonine alone with serine, or the similar substitution of amino acids with structurally related amino acids will not significantly affect the biological activity of the resulting molecule.

[0138] The term "effective amount" as used herein refers to the amount of agent (e.g., an inducible IL-2 prodrug) administered to achieve the desired effect under the conditions of administration, i.e., to reduce tumor size, reduce tumor burden, extend progression-free survival, or extend overall survival. The actual effective amount selected will depend on the particular cancer being treated and its stage, as well as other factors such as the subject's age, sex, weight, ethnicity, previous treatments and response to those treatments. The appropriate amount of inducible cytokine prodrug and any additional agents administered, as well as the dosing schedule for a particular patient, can be determined by a clinician skilled in the art based on these and other considerations.

[0139] Preferably, the methods and compositions disclosed herein are used to treat colon cancer, lung cancer, melanoma, renal cell carcinoma, breast cancer, and squamous cell carcinoma of the head and neck.

[0140] In certain preferred embodiments, 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 carcinoma (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 cancer, high tumor mutation burden cancer, cutaneous squamous cell carcinoma (cSCC), triple negative breast cancer (TNBC), urothelial carcinoma, colorectal cancer, or oesophageal carcinoma.

[0141] equivalent Other suitable modifications and variations of the methods of the present invention described herein will be apparent and may be made using appropriate equivalents without departing from the scope of the present disclosure or embodiments, as will be readily apparent to those skilled in the art. Although certain compounds and methods have been described in detail above, they will be more clearly understood by reference to the following examples, which are presented for illustrative purposes only and are not intended to be limiting. [Table 3-1-1] [Table 3-1-2] [Table 3-1-3] [Table 3-1-4] [Table 3-1-5] [Table 3-1-6] [Table 3-1-7] [Table 3-1-8] [Table 3-1-9] [Table 3-1-10] EXAMPLES

[0142] The following are examples of the methods and compositions of the present invention. It is understood that other various embodiments can be practiced given the general description provided herein.

[0143] 1.1 Materials and Methods cell line MC38 and B16-F10 cell lines were obtained from ATCC and were regularly checked for pathogen contamination. All cell lines were grown and maintained according to ATCC guidelines and kept in culture for no longer than 2 weeks. Frozen MC38 or B16-F10 cells were thawed and maintained for 1–3 passages in DMEM (ThermoFisher Scientific) supplemented with 10% heat-inactivated FCS (Gibco) and 1X penicillin / streptomycin (Gibco). Prior to tumor implantation, cells were washed twice with PBS and counted. Cells were seeded in PBS (efficacy studies) or 50% Matrigel (TIL harvest, Corning).

[0144] Mice, tumor implantation, and in vivo administration All mouse in vivo studies were performed at Charles River Laboratories (Morrisville, NC and Worcester, MA) in accordance with current USDA and NIH regulations and standards. Female C57Bl / 6 mice, 6-8 weeks old, from Charles River Laboratories were shaved on their flanks 1 day prior to tumor cell implantation. A total of 5 × 10 5 1 x 10 MC38 cells or 1 x 105 B16-F10 cells were injected subcutaneously and monitored for tumor growth. Additional mice were implanted to obtain tumors large enough for randomization. Group averages ranged from 100 to 150 mm. 3 Tumor volumes were monitored until tumor volume reached 100% and mice were randomly assigned to treatment groups on day 0. Mice receiving an inducible IL-2 prodrug were dosed twice weekly. Mice receiving rhIL-2 were dosed twice daily for 5 days followed by a 2-day rest period (5 / 2 regimen). In studies using PD-1 blockade, mice were dosed with anti-PD-1 (200 μg, clone RMP1-14, BioXCell) on a twice-weekly schedule. In studies using FTY720, mice were initially primed with 25 μg and then treated with 10 μg per dose daily throughout the duration of the experiment.

[0145] In some studies, antitumor activity was evaluated in mice depleted of CD8+ cells. In these studies, mice were administered anti-CD8 antibody (200 μg / dose, Bio X Cell clone 2.43) by intraperitoneal injection twice weekly. The average tumor volume for each group is shown as mean + / - SEM (Figure 2I). The results showed that repletion of CD8+ cells reduced the antitumor effect of compound 1.

[0146] In some studies, MC38 tumor-bearing mice were treated with either vehicle, compound 1 (containing the native IL-2 payload at 100 μg / dose), or a derived form of an IL-2 mutein (compound 5) that upon cleavage does not bind to the IL-2 receptor α but binds to the IL-2 receptor β-γ receptor at 100 μg / dose. Compound 5 contains a first polypeptide having SEQ ID NO:6 and a second polypeptide containing SEQ ID NO:5 (Figures 15A-C). Results showed that the non-α mutein had no significant antitumor activity in the model.

[0147] All treatments were administered by intraperitoneal injection and mice were administered for 2 weeks unless otherwise noted. Both body weight and tumor volume were measured twice weekly during the study. Tumors were measured in two dimensions using calipers and volumes were calculated using the following formula: tumor volume (mm3) = [(w2xl) / 2], where w = tumor width and l = tumor length in mm. The study continued until tumors reached 1500 mm3 or the study reached its endpoint on day 45. In some cases, mice that had fully regressed were kept for later memory experiments.

[0148] Mouse vascular leak syndrome model Mouse VLS experiments were performed at Biomodels LLC (Waltham, MA) in accordance with current regulations and standards of the US Department of Agriculture and NIH. Eight to ten week old female C57Bl / 6 were administered equimolar amounts of either recombinant human IL-2 (100 μg / dose, seven doses over four days), WW0177 (two doses on D0 and D3), or compound 1 (two doses on D0 and D3) by intraperitoneal injection. On day 3, animals were intravenously injected with Evans Blue dye and 30 minutes later animals were perfused with 50 mL of heparinized saline at a rate of 10 mL / min. Lungs were harvested and placed in formamide at 37°C for 24 hours. After 24 hours, extravasation of Evans Blue dye into the lungs was measured by measuring absorbance at 620 nm and 650 nm using a spectrometer and comparing the absorbance values ​​to a freshly prepared standard curve of Evans Blue dye.

[0149] Inducible IL-2 prodrug production, protease activation, and SDS page analysis Compound 1, recombinant human IL-2, and Compound 1-NC were produced. Proteins were expressed using Life Technologies' Expi293 Expression System according to the manufacturer's protocol. Four days after transfection, the cultures were spun down, filtered through 0.2 μm bottle-top filters, and rotated overnight in the presence of MabSelect resin. The following day, the culture / resin mixture was applied to a gravity column and the resin was washed with PBS (TEKNOVA, endotoxin tested). Proteins were eluted with 200 mM acetic acid (pH 3.5) and 50 mM NaCl and neutralized with 1 M Tris (pH 8). The eluates were pooled, dialyzed, concentrated, aliquoted, and stored at -80°C for future use. WTX-124 was dialyzed into 20 mM histidine (pH 6) and 150 mM NaCl, while recombinant human IL-2 and WW0177 were dialyzed against 1xPBS. The extinction coefficient of each protein was theoretically determined using SnapGene (v5.0.7), and protein concentrations were measured by A280. For SDS page gels, 3 μg of protein was loaded onto a 16% Tris-glycine gel (ThermoFisher) under non-reducing conditions.

[0150] HEK-Blue IL-2 reporter assay The HEK-Blue IL-2 reporter cell assay was performed according to the manufacturer's protocol (Invivogen). On assay day 1, cells were rinsed, resuspended in medium containing 1.5% human serum albumin, and plated at 5 × 10 cells per well in 96-well flat-bottom plates. 4 Cells were plated at a concentration of 100x100 cells. Titrated amounts of intact and protease-activated (cleaved) inducible IL-2 proteins or rhIL-2 were added to the cells to generate full dose-response curves. On day 2, SEAP levels were measured according to the manufacturer's protocol.

[0151] Human and mouse primary cell assays 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 μg / mL PHA (Sigma-Aldrich) for 72 h before being frozen for later use. To measure intact or protease-activated (cleaved) inducible IL-2 protein activity, Tblasts were plated in 96-well round-bottom plates and titrated amounts of intact or protease-activated (cleaved) inducible IL-2 protein or rhIL-2 were added to the cells to generate a full dose-response curve. After 72 h, proliferation was measured using Cell Titer glow reagent (Promega) according to the manufacturer's protocol.

[0152] For mouse Tblast experiments, splenocytes were thawed, washed, and stimulated with 2 μg / mL concanavalin A (Sigma-Aldrich) for 72 h before freezing in Recovery Cell Culture Freezing Media (Gibco). T cell activation was performed in complete medium (RPMI-1640 medium supplemented with 10% FBS, 100 units / mL penicillin, 100 μg / mL streptomycin, and 0.1% 2-mercaptoethanol). To measure inducible IL-2 protein activity, mouse Tblasts were plated in 96-well round-bottom plates. Titrated amounts of intact or protease-activated (cleaved) inducible IL-2 protein or rhIL-2 were added to the cells to generate a full dose-response curve. After 72 h, proliferation was measured using Cell Titer glow reagent (Promega) according to the manufacturer's protocol.

[0153] Stability of inducible IL-2 in mouse plasma and human serum 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 mouse plasma or human serum, after which the samples were mixed and divided into three aliquots, which were incubated at 37°C for the indicated times and then frozen for later analysis. To assess 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.

[0154] Western blot analysis was performed using the JESS system (Protein Simple) according to the manufacturer's protocol. Primary anti-human IL-2 antibody was purchased from R&D Systems (AF-202-NA) and 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 the Jess system set to standard settings for chemiluminescence. Analysis of the resulting Western blots was performed using Compass for Simple Western Software (v4.1.0).

[0155] Pharmacokinetic analysis Plasma and tumor samples were collected at the time points specified by Charles River Laboratories (Morrisville, North Carolina), shipped on dry ice, and stored at -80°C. MC38 tumor lysates were generated by homogenizing each tumor using a Qiagen TissueRuptor homogenizer equipped with a disposable probe (Qiagen) in ice-cold lysis buffer (1x Tris-buffered saline, 1 mM EDTA, 1% Triton® X-100, protease inhibitors in diH2O). Plasma and tumor lysates were analyzed using BioLegend IL-2 ELISA (431804), which detects both intact and free IL-2, according to the manufacturer's instructions. A 12-point standard curve was generated using intact and inducible IL-2. To specifically analyze the levels of free IL-2, samples were measured using an IL-2 AlphaLISA (PerkinElmer, AL221C). This IL-2 AlphaLISA detects free human IL-2 but not intact, inducible IL-2 due to competition with the inactivation domain. All AlphaLISAs were performed according to the manufacturer's instructions and analyzed on a Perkin Elmer Enspire reader and software.

[0156] Tumor digestion and NanoString analysis MC38 and B16-F10 tumors were minced into small pieces (<5 mm3) in phenol-free RPMI-1640 (Thermofisher) and then enzymatically digested with collagenase IV (3 mg / mL, Gibco) for 35 min at 37 °C with shaking. After digestion, tumor samples were mechanically dissociated through a 70 μM cell strainer. For flow cytometry analysis including effector cytokines, samples were restimulated for 4 h at 37 °C in complete medium containing phorbol 12-myristate 13-acetate (50 ng / mL, Sigma-Aldrich), ionomycin (1 μg / mL, Sigma-Aldrich), and 1XBrefeldin A (eBioscience). For NanoString analysis, 5 × 10 5Cells were frozen in 100 μL of RLT lysis buffer (Qiagen). 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.

[0157] Flow cytometry All cell staining was performed in 96-well round bottom plates using FAC buffer (PBS + 0.5% BSA) or 1x permeabilization buffer (eBioscience) as appropriate. Cells were first treated with FC block (BioLegend) at room temperature, followed by tetramer staining for 20 min at room temperature. After tetramer staining, cells were washed and stained with a master mix of extracellular antibodies for 20 min at 4°C. Cells were then washed and fixed / permeabilized overnight using eBioscience™ Foxp3 Transcription Factor Staining Buffer Set according to the manufacturer's protocol. The next day, samples were washed with Perm buffer and stained with intracellular markers for 20 min at 4°C. Cells were then washed and analyzed on a Cytek Aurora system. Fluorescence minus one (FMO) and single stain controls were included in all stainings. In some cases, OneComp ebeads™ (Thermofisher) were stained in parallel with the cells to serve as single stain controls. Individual cell populations were defined as described by the gating strategy in Figures 12A-B. When effector cytokine production was assessed using flow cytometry, cells were restimulated with PMA (50 ng / mL, Sigma Aldrich) and ionomycin (1 μg / mL, Sigma Aldrich) in the presence of 1x Breldin A (Thermofisher Scientific) for 4 hours in complete medium at 37°C before staining. Flow cytometry fluorochrome-conjugated antibodies against the following proteins were purchased from Biolegend: CD8α 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; IFNγ PE, clone XMG1.2; F4 / 80 Pe / Dazzle 594, clone BM8; CD3 complex PeCy7, clone 17A2; FC block, clone 93.Flow cytometry fluorochrome-conjugated antibodies against the following proteins were purchased from eBioscience: CD45 BUV395, clone 30-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. Fluorochrome-conjugated tetramer against MulV p15E peptide KSPWFTTL (SEQ ID NO: 7) was purchased from ThermoFisher Scientific (50-168-9385). Live / Dead Blue Dye was also purchased from ThermoFisher Scientific (L23105).

[0158] Ex vivo inducible IL-2 prodrug processing assay 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 were generated from primary human tumor samples that were surgically removed, enzymatically digested in situ, and then frozen. All purchased samples were shipped on dry ice and stored in liquid nitrogen freezers.

[0159] To examine inducible IL-2 prodrug processing, samples were thawed, washed, and counted. Cells were then resuspended in X-Vivo 15 medium containing either Compound 1, Compound 1-NC, or precut Compound 1. After incubating the inducible IL-2 prodrug with the cells for 48 hours, cell culture supernatants were collected and frozen for later analysis. IL-2 activity in cell culture supernatants was assessed using an IL-2 bioassay (Promega) that utilizes thawed and used IL-2 reporter cells (catalog no. JA2201 / JA2205). This bioassay was used according to the manufacturer's protocol. Relative light unit (RLU) values ​​were converted to percent full activity using the following formula:

number

[0160] Data Representation and Statistics For mouse tumor experiments, mice were implanted with the respective tumor cell lines such that each group had at least n=8 mice per group at randomization and dosing initiation. The total number of implanted mice was calculated by adding 30% to the total number of animals required for the study to provide enough animals for proper randomization based on tumor size. Sample size was determined by previous experience with this model, and tumor measurements were performed in an unblinded fashion. Flow cytometry plots were generated using FlowJo software and are representative samples. All quantitative plots were generated using GraphPad Prism 8 software for Windows (64-bit) (San Diego, CA). For in vitro activity assays, data were analyzed using an unconstrained nonlinear sigmoidal 4PL curve-fitting model. Statistical analysis was also performed using GraphPad Prism software (San Diego, CA). Student's t-test was used for comparisons of two samples, and analysis of variance (ANOVA) test with multiple comparisons was used for comparisons of three or more groups. Antitumor effects over time were analyzed using mixed-effects models, whereas antitumor effects at specific time points were analyzed using unpaired t-tests. For the NanoString dataset, statistical analysis was performed using nSolverTM software with the Advanced Analysis Module installed.

[0161] 1.2 Results Inducible IL-2 signaling and activity is dependent on proteolytic activation Compound 1, an inducible IL-2 prodrug, was designed to enhance the clinical profile of recombinant human IL-2 therapy by facilitating more infrequent systemic delivery, increasing tumor exposure of the molecule, and reducing the toxicity associated with high-dose IL-2 (Figure 1A). Compound 1 contains native human IL-2, a Fab antibody fragment that prevents IL-2 from binding to the intermediate affinity IL-2 receptor (IL-2Rβ / γ), thereby acting as an inactivation domain, and an anti-human serum albumin (αHSA) single domain antibody that acts as a half-life extension domain. These two domains are linked to the IL-2 payload via a protease-cleavable linker sequence. Compound 1 in the prodrug state has an extended half-life and inhibits the activity of IL-2 by blocking the binding of the molecule to the IL-2 receptor. However, when the linker is enzymatically cleaved in the tumor tissue, the half-life extension domain and the inactivation domain are removed, releasing native IL-2 (Figure 1B).

[0162] To measure the difference in activity between intact and protease-cleaved compound 1, HEK-Blue IL-2 reporter cells were incubated with either recombinant human IL-2 (rhIL-2), intact compound 1, or protease-activated compound 1 (cleaved), and then IL-2 signaling was measured. In this assay, intact compound 1 was approximately 100-fold less active than rhIL-2 or cleaved compound 1 (Figure 1C). Furthermore, human PBMCs were stimulated with PHA to form Tblasts, which express the high-affinity IL-2 receptor (CD25 / CD122 / CD132) (Figures 8A-B) 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 1 was less active than either truncated Compound 1 or rhIL-2 across multiple donors (Figure 1D, Figure 8C). More specifically, intact Compound 1 inhibited the EC 50The activity was approximately 23-fold lower with increasing concentrations of IL-1 and plateaued at only 60% of the maximal activity seen with either truncated compound 1 or rhIL-2.

[0163] The activity of intact and truncated Compound 1 was also characterized in mouse primary T blast assays. Although truncated Compound 1 and rhIL-2 induced similar proliferation by mouse Tblasts, intact Compound 1 showed little measurable activity in cells isolated from multiple mice (Figure 1E, Figure 8D). To confirm that the activity of truncated Compound 1 was dependent on linker cleavage and not on an unknown processing event, a non-cleavable mutant of Compound 1 (designated Compound 1-NC) was generated by replacing the linker sequence with a non-cleavable glycine / serine sequence. As a control, Compound 1-NC was treated with the same enzymatic digestion as Compound 1 and then tested in human Tblasts. As expected, no difference in activity was observed between the intact and "truncated" forms of Compound 1-NC, indicating that linker cleavage is required to restore the full activity of IL-2 released from Compound 1 prodrug (Figure 1F).

[0164] Compound 1 treatment suppressed tumor growth in a cleavage-dependent manner To test whether compound 1 treatment could inhibit tumor growth, mice were implanted with MC38 tumor cells until tumors reached 100–150 mm 3Mice were randomized into treatment groups at 0.5-1.5°C. Mice were then treated twice weekly with vehicle (PBS) or titrated amounts of either Compound 1 or Compound 1-NC (noncleavable control) for a total of four doses. Given the residual activity observed with intact Compound 1 when tested at high concentrations in vitro, Compound 1-NC also serves as a control for the level of in vivo activity specifically derived from intact Compound 1 (Figures 1C-1E). In this model, even the lowest dose of Compound 1 (25 μg) resulted in statistically significant tumor growth inhibition (Figures 2A, 2I, and 13B). Furthermore, doses of 100 μg, 150 μg, or 300 μg were all highly effective. Of the 24 mice in these treatment groups, 23 showed complete responses, with no measurable tumors remaining at the end of the experiment (Figure 2A). Both of these dose levels were well tolerated by the mice, with no signs of weight loss (Figure 9A). In contrast, compound 1-NC had negligible effects on tumor growth even when administered at the highest tested dose (300 μg), demonstrating that the antitumor activity of compound 1 was dependent on the in vivo enzymatic cleavage of its linker. Furthermore, depletion of CD8+ T cells reduced the antitumor activity of compound 1 (Figure 2I), demonstrating that compound 1 supports the antitumor response of host effector cells.

[0165] A major obstacle to widespread clinical use of recombinant human IL-2 is the toxicity observed when this cytokine is administered systemically. Because compound 1 was designed to enhance the PK profile of IL-2 treatment, it was possible that the use of IL-2 with an extended half-life may actually result in even greater toxicity than the original free cytokine. Therefore, we tested whether the half-life extension element of compound 1 is necessary for anti-tumor activity. Indeed, when an inducible IL-2 prodrug lacking the half-life extension element (WW0057) was tested in vivo, this molecule failed to generate anti-tumor immunity even when administered at 10 times the fully effective dose of compound 1 (Figure 13C), demonstrating the necessity of including a half-life extension element to generate anti-tumor immunity with IL-2 prodrugs. We investigated whether the blocking element of compound 1 could prevent the expected increase in toxicity due to exposure to the half-life extended IL-2.

[0166] To better understand the effectiveness of the inactivation domain in limiting toxicity, a mutant of compound 1 without the blocking domain was created (WW0177). 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 does not have an inactivation domain, thereby representing the level of toxicity expected if the inactivation domain is not functioning properly. MC38 tumor-bearing mice were administered either WW0177 or compound 1 and their weight was monitored over time (Figure 2B). After only two doses of WW0177, treatment had to be discontinued due to weight loss, and only two of seven mice survived. In contrast, mice treated with four doses of compound 1 showed no signs of weight loss, despite receiving approximately 26 times the molar amount of IL-2 administered to the WW0177-treated group.

[0167] Although we lost a useful surrogate for monitoring overall toxicity with respect to immunotherapy, it was also important to examine the effect of compound 1 on organ-specific toxicity. Vascular leak syndrome (VLS) is the primary dose-limiting toxicity associated with high-dose IL-2 treatment in the clinic, limiting not only the clinical utility of high-dose IL-2 but also preventing IL-2 with extended half-life from being a viable clinical strategy. In mice, VLS is induced by high doses of recombinant IL-2 and can be measured by examining the amount of Evans Blue dye leaking into the lungs after intravenous injection. Consistent with the overall toxicity data, when recombinant human IL-2, WW0177, or compound 1 were administered in equimolar amounts, only recombinant human IL-2 and WW0177, but not compound 1, resulted in detectable levels of Evans Blue leaking into the lungs (Figure 13D). These data demonstrate that the blocking domain of compound 1 effectively blocks the activity of IL-2 in peripheral tissues and prevents the induction of VLS, compared to the peripherally active IL-2 molecule.

[0168] Although the inactivation domain of Compound 1 is highly effective, activity of this domain depends on the blocker remaining tethered to the IL-2 molecule (Figure 1). To examine the stability of Compound 1 in the periphery, Compound 1 was incubated in mouse plasma from either naive or MC38 tumor-bearing mice for 24, 48, or 72 hours, after which levels of intact Compound 1 were measured by Western blot. Consistent with the tolerability of this molecule, there was no evidence of Compound 1 cleavage across all time points tested (Figure 2C).

[0169] In addition to managing peripheral toxicity, compound 1 was designed to facilitate less frequent systemic delivery of treatment without sacrificing the potency and antitumor activity of high-dose IL-2. It was therefore important to directly compare the activity of compound 1 to native IL-2. MC38 tumor-bearing mice were treated with titrated doses of compound 1 (twice weekly for 2 weeks) as before, or with rhIL-2 administered twice daily for 2 weeks (dosing regimen: 5 days on, 2 days off schedule for 2 weeks). The difference in dosing schedule reflects the poor in vivo pharmacokinetic properties of rhIL-2 in both humans (15) and mice (16) and is analogous to the dosing of high-dose IL-2 patients in the clinic. Because the two treatments are administered at different dosing regimens, the correct way to compare treatment groups is to compare the total amount of IL-2 delivered during the dosing period. Treatment of MC38 tumor-bearing mice with a total of 5.04 μM compound 1 resulted in complete tumor rejection in 8 of 8 mice. In contrast, even when mice were treated with 15.5 μM native IL-2 (three times the total amount of IL-2 administered with compound 1), only five of eight mice completely rejected the tumor (Figure 2D).

[0170] As mentioned above, the pharmacokinetic profile of Proleukin treatment (t 1 / 2 <1 h), resulting in an impractical dosing schedule in which many patients receive high doses up to 15 times every 8 h (17). Similarly, in mice, rhIL-2 is rapidly cleared from the circulation (16). We hypothesized that the increased activity of compound 1 compared to native IL-2 is due to its extended half-life and pharmacokinetic profile. To confirm this, tumor-bearing mice were dosed once on day 0 and once on day 4, and drug exposure was measured in plasma and within tumors at various time points. In contrast to rhIL-2, administration of compound 1 extended exposure in plasma, with a half-life of approximately 20 h and exposure maintained over 4 days (Figure 2E). Furthermore, intraperitoneal administration of compound 1 extended drug exposure within the tumor itself, demonstrating tissue penetration by compound 1 (Figure 2F). Total compound 1 levels were C in plasma at 6 h after dosing. maxand peaked in the tumor at 12 hours post-injection.

[0171] Compound 1 was designed to limit the systemic activity of IL-2 while delivering fully active IL-2 locally to tumors using a cleavable linker. To test whether systemic administration of compound 1 resulted in local delivery of rhIL-2 to tumors, plasma and tumor samples were collected at various time points after administration and analyzed for the presence of free human IL-2 (i.e., not bound to blocking Fab) released by enzymatic processing. To specifically measure human IL-2 released from the IL-2 prodrug by proteolytic processing, we identified an ELISA kit specific for human IL-2 (Figure 13E), where the blocking domain of compound 1 prevented the binding of the ELISA detection reagent (Figure 13F). This allowed us to assess the levels of free human IL-2, which may only be present in the mouse system through in vivo processing of compound 1. Systemic administration of compound 1 resulted in almost no detectable free IL-2 in plasma (Figure 2G). In contrast, systemic administration of compound 1 prolonged the levels of detectable free IL-2 in tumors (Figure 2H), demonstrating that tumor-dependent processing promotes increased exposure of fully active IL-2 in tumors following systemic delivery of compound 1.

[0172] To better quantify the differences between plasma and tumors with respect to compound 1 processing, the area under the exposure curve (Figure 2E-H) was measured and is reported in Table 3. Notably, while the amount of total compound 1 in plasma was approximately 18-fold higher than that detected in tumors, the amount of free IL-2 in tumors was still more than 5-fold higher than that detected in plasma, suggesting an approximately 93-fold increase in compound 1 processing in tumors compared to plasma. [Table 3-2]

[0173] The therapeutic window (TW) of a therapy is defined as the ratio of the maximum tolerated dose to the lowest effective dose, which identifies the difference between activity and serious adverse events. In the clinic, the TW of Proleukin is relatively small. Similarly, the TW of rhIL-2 in MC8 tumor-bearing mice was calculated to be less than one-fourth in our model (Figure 9B). The half-life extension element of WW0177 makes it a more active version of IL-2, so less WW0177 is needed to achieve full efficacy compared to recombinant hIL-2. However, the maximum tolerated dose of WW0177 is also low, resulting in a TW of less than 2. However, compound 1 was significantly more active than equimolar amounts of rhIL-2, and no toxicity was observed in tumor-bearing mice receiving up to 960 μg / dose of compound 1, so the TW of compound 1 is at least greater than 20, which represents at least a 5-fold improvement compared to rhIL-2 (Figure 13G). These data demonstrate that compound 1 is not simply an attenuated IL-2 molecule, but rather a unique inducible IL-2 prodrug that enhances the activity of a payload while limiting its systemic activity.

[0174] Compound 1 treatment of MC38 tumor-bearing mice induces immunological memory One of the hallmarks of immunological rejection of tumors is the development of protective memory against subsequent tumor rechallenge. To test whether compound 1 treatment resulted in tumor-specific memory following tumor rejection, mice were implanted with MC38 tumor cells, randomly assigned to vehicle or compound 1 treatment, and tumor growth was measured. As in previous studies, compound 1 treatment resulted in tumor rejection, whereas control tumors continued to grow.

[0175] To examine whether tumor rejection in mice treated with compound 1 resulted in immunological memory, spleens from mice were examined for the presence of tumor-specific memory CD8+ T cells 6 months after the initial MC38 implantation (MC38 CR mice) (Figure 3A). Previous studies have confirmed that a peptide derived from the murine leukemia virus protein gp70 (KSPWFTTL) (SEQ ID NO: 7) is an antigen presented by MC38 tumors, and 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-naive mice (Figure 3A-B). Furthermore, tetramer-positive cells from tumor-naive mice maintained a predominantly naive phenotype, whereas cells from MC38 CR mice maintained a predominantly effector memory phenotype (CD44 hi CD62L low ) (Figure 3C-D). Furthermore, upon restimulation, tetramer-positive cells from MC38 CR mice secreted more effector cytokines TNF and IFNγ (Figure 3E-F). Coexpression of two or more effector cytokines is known as polyfunctionality and is associated with greater cytolytic activity in T cells (19). WTX-124 treatment also significantly increased the frequency of polyfunctional T cells after restimulation (Figure 3G). These data are consistent with the idea that compound 1 treatment results in immune-mediated tumor rejection, which then translates into immunological memory.

[0176] Although the phenotype of these splenocytes suggests the generation of tumor-specific memory, the ultimate test of a memory response is protection against rechallenge. Therefore, compound 1-induced MC38 CR mice were rechallenged with MC38 tumor cells 60 days after the initial implantation (Figure 3H). Importantly, no treatment was administered during the rechallenge. Unlike tumor-naive mice, none of the MC38 CR mice developed tumors (Figure 3I), demonstrating that tumor rejection induced by inducible IL-2 protein treatment results in immunological memory and protection against subsequent tumor rechallenge.

[0177] Compound 1 treatment enhanced MC38 tumor infiltration and induced immune cell activation To better understand the mechanism by which Compound 1 treatment induces antitumor immunity, MC38 tumor-bearing mice were randomly assigned to treatment groups on day 0 and treated with either vehicle or Compound 1 on days 1 and 4. Tumors were harvested 24 hours after the second dose. Total RNA was extracted from single cell suspensions and analyzed using the NanoString nCounter® PanCancer Mouse Immune Profiling Panel. Compound 1 treatment led to clear changes in the transcriptional profile, with 437 of 770 genes in the panel showing statistically significant differences in expression compared to the control group (Figure 4A-B). NanoString nSolver™ pathway analysis of this dataset revealed a set 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 (Figure 4C). Interestingly, the expression of several transcripts associated with immune checkpoint proteins, such as PD-1, TIGIT, and CTLA-4, was also increased after compound 1 treatment (Figure 4D), which may reflect an overall increase in immune cell activation among tumor-infiltrating lymphocytes (TILs), as many checkpoint proteins are upregulated during a typical immune response.

[0178] In addition to NanoString analysis, we also performed immune cell profiling by flow cytometry. Just 5 days after the first dose, compound 1 treatment significantly increased the density of infiltrating immune cells, including tumor-specific tetramer-positive CD8+ T cells (increased by about 19.8-fold) and, to a lesser extent, Tregs (increased by about 2.5-fold) (Figure 4E). The effect of IL-2-based treatment on Tregs has been a topic of major debate in the scientific community, partly due to concerns that IL-2 treatment may result in counterproductive Treg proliferation, hindering immunotherapy in the clinic. This has led to the development of several IL-2 mutant molecules designed to avoid Treg engagement. In contrast, we assume that the activity of fully active IL-2 on cytolytic cells overcomes any possible Treg activation associated with the treatment, and therefore compound 1 is not specifically designed to avoid Tregs. In support of this hypothesis, the increase in tetramer-positive CD8+ T cells after compound 1 treatment far exceeded the increase in Tregs, resulting in a significant increase in the tetramer-positive CD8+ / Treg ratio after compound 1 treatment (Figure 4F). This finding, coupled with the highly potent antitumor activity produced by Compound 1, suggests that Treg activation does not significantly impair the efficacy of Compound 1.

[0179] To assess the activation state of tumor-infiltrating T cells, samples from TILS were restimulated and assessed for production of IFNγ, TNF, and granzyme B. Compound 1 treatment significantly increased the frequency of tetramer-positive CD8 T cells producing IFNγ (Figure 4G-H), TNF (Figure 10A), and granzyme B (Figure 10B). Compound 1 treatment also significantly increased the polyfunctionality of tetramer-positive CD8+ T cells, with a higher frequency of tetramer-positive CD8+ T cells producing any two or all three of these effector cytokines compared to the control group (Figure 4I).

[0180] Recent data have demonstrated that under certain circumstances, Tregs can also produce effector cytokines such as TNF and IFNγ, a phenomenon known as “Treg fragility” (20). Importantly, production of effector cytokines by Tregs is associated with a loss of their suppressive activity. Interestingly, while Tregs from control tumors produced little IFNγ or TNF, a subpopulation of Tregs from tumors treated with compound 1 produced both of these effector cytokines (Figures 4J-M). Taken together, these data demonstrate that compound 1 treatment increases tumor infiltration, activates tumor-specific CD8+ T cells, and causes phenotypic instability of Tregs.

[0181] Tumor-specific activation of immune cells by compound 1 leads to tumor rejection To confirm that the effects of compound 1 systemic treatment are selective to the tumor microenvironment, we compared effector cytokine production by T cells derived from tumors, spleens, peripheral blood, and draining lymph nodes following compound 1 treatment using the same treatment schedules described above. Because tetramer+ populations are selectively enriched among CD8+ T cells within tumors, including these cells in the analysis could bias comparisons between different sites. Therefore, tetramer-negative CD8+ T cells were specifically examined across various tissues. Similar to previous data, compound 1 induced significantly higher frequencies of IFNγ-producing CD8+ T cells and CD4+ non-Tregs within tumors compared to the relatively low levels of activity seen in the peripheral tissues examined (Figures 5A-B). Taken together with previous toxicity data, these data indicate that compound 1 treatment does not result in widespread peripheral T cell activation.

[0182] Although the peripheral CD8+ T cell activation seen with compound 1 treatment was limited, it remained possible that this low level of peripheral activity still played a role in generating antitumor immunity in this model. To test whether tumor-specific activation was sufficient to generate antitumor immunity, mice were implanted with MC38 tumors that had grown to approximately 100–150 mm3, after which some mice were treated with fingolimod or FTY720. FTY720 is a small molecule that blocks sphingosine-1-phosphate receptors, thereby preventing lymphocyte egress from the thymus and secondary lymphoid tissues (21). Thus, any antitumor activity seen in mice treated with FTY720 would derive from immune cells that were already infiltrating the tumor at the start of treatment, and not from activation and subsequent trafficking of additional lymphocytes from secondary immune tissues. Daily FTY720 treatment did not affect the antitumor activity of compound 1 (Figure 5C), indicating that activation of TILs alone was sufficient to reject MC38 tumors. Taken together, these data demonstrate that systemic administration of compound 1 to tumor-bearing animals results in tumor processing of inducible IL-2 molecules and preferential activation of tumor-infiltrating immune cells sufficient to generate potent antitumor immunity.

[0183] In cancer patients, the presence of a pre-existing TIL population (referred to as "hot" tumors) correlates with response to immunotherapy, whereas the absence of a pre-existing TIL population (known as "cold" tumors) shows an inverse correlation. Mouse syngeneic tumor models differ in baseline immune infiltration and response to immunotherapy. For example, in MC38 tumors, approximately 20% of TILs are CD8+ T cells, compared with only 2.5% in B16-F10 tumors (Figures 12A-B).

[0184] To test the activity of compound 1 in a poorly immunogenic tumor model, mice were injected subcutaneously with B16-F10 melanoma cells. Tumors were grown to an average volume of 100 mm 3After allowing the cells to grow to 100 μg / ml, mice were randomized to receive either PBS or various doses of WTX-124, using the same dosing schedule as before. Compound 1 infiltrated tumors in a tetramer-positive manner (Figure 6A), whereas anti-PD-1 treatment alone was ineffective in this model (Figure 11). Interestingly, combining low doses of Compound 1 with PD-1 blockade showed combinatorial activity. However, at high doses of Compound 1, no additional benefit of PD-1 blockade was observed.

[0185] To further explore the mechanism of tumor growth inhibition, total tumor RNA was extracted from mice treated with compound 1 24 hours after the second dose and analyzed using the NanoString nCounter® PanCancer Mouse Immune Profiling Panel. Compound 1 treatment resulted in a large transcriptional shift, with 184 transcripts out of 770 examined being statistically different after compound 1 treatment (Figure 6B). Interestingly, flow cytometric immune profiling of B16-F10 TILs did not show the significant increase in immune cells observed in MC38 tumors after compound 1 treatment, which may explain the difference in antitumor efficacy between these two models at this dose (data not shown). However, compound 1 treatment did induce proliferation and granzyme B production by tumor-infiltrating tetramer-positive CD8+ T cells (Figures 6C-D) and NK cells (Figures 6E-F). Furthermore, compound 1 was more effective than equimolar amounts of recombinant hIL-2 in inducing CD25 expression and proliferation by tumor-infiltrating NK cells, CD4+ non-Tregs, total CD8+ T cells, and tetramer+ CD8+ T cells. This may be due to the enhanced PK profile of the IL-2 prodrug compared to the free cytokine. Finally, similar to the MC38 model, compound 1 treatment of B16-F10 tumor-bearing mice caused a small subset of tumor-infiltrating FoxP3+ Tregs to produce proinflammatory cytokines such as TNF and IFNγ (Figures 6G-H), suggesting that compound 1 treatment also induces Treg vulnerability in this model. Taken together, these data demonstrate that compound 1 treatment can induce TIL activation and inhibition of tumor growth in a low-inflammation, low-temperature tumor model.

[0186] Compound 1 is stable in human serum and processed by human tumors Human tumor samples are heterogeneous in nature and display varying degrees of protease dysregulation and expression (15). Therefore, a screen was performed to identify potential INDUKINE™ molecular linkers based on their stability in systemic circulation and processing by most tumor types. The basis of this screen was a protease-independent approach, and rather than using linkers based on specific target proteases, linkers that are specifically cleaved by primary human tumor samples were selected. This resulted in the selection of linker sequences that separate the different domains of compound 1.

[0187] As a test of the peripheral stability of compound 1, the protein was incubated with human serum from healthy donors (n=3) for up to 72 hours prior to processing and measured by Western blot. Consistent with the mouse plasma experiments, compound 1 was not processed by human serum in any of the donors tested (Figure 7A). To test whether compound 1 was processed by tumor samples, an ex vivo processing assay was developed. Briefly, dissociated human tumor samples were incubated with compound 1 or a control protein for 48 hours, after which the resulting IL-2 activity was measured. Activity induced by incubation of dissociated human tumor samples with compound 1 was normalized to a range of 0-100% activation, using compound 1-NC as a surrogate for the baseline activity of the intact compound 1 prodrug and precut compound 1 as representative of the fully activated molecule. In some cases, primary human tumor samples also contained viable TILs that had the capacity to consume a portion of the free IL-2 in the precut compound 1 positive control group. This may result in artificial suppression of the activity of the positive control in certain samples, while some samples may record greater than 100% activity compared to the positive control. Despite this issue, the assay is sufficient to be used as a binary analysis of whether compound 1 is processed by primary human tumor samples.

[0188] To investigate how well compound 1 was processed by various tissue samples, we examined healthy primary human cells (n=13) and primary human tumor samples (n=97) for their ability to cleave compound 1. The healthy primary cells were derived from a variety of tissues, and the tumor samples covered a wide range of tumor types and stages. Importantly, exposure of compound 1 to healthy primary cells did not produce any evidence of cleavage, again suggesting that the protein is stable in the patient periphery (Figure 7B). In contrast, the majority of tumor samples tested were able to process compound 1. These data suggest that compound 1 is stable when exposed to human serum or healthy primary cells, but is efficiently activated by most human tumor samples, supporting its further development as a novel immunotherapy for cancer patients.

[0189] 1.3 Discussion High-dose IL-2 therapy was first approved in 1992 for patients with metastatic renal cell carcinoma and in 1998 for patients with advanced melanoma (4). Prior to the advent of the modern field of immuno-oncology, high-dose IL-2 stood out as a treatment with complete responses, albeit in a minority of patients. However, the antitumor potential of inflammatory cytokines such as IL-2 has been hampered by the severe toxicities associated with their systemic delivery and engagement of target cells outside the tumor microenvironment (4). In the case of IL-2 specifically, several pharmaceutical and biotechnology companies have attempted to minimize this problem by creating less active forms of IL-2 (known as non-α molecules) that avoid activation of the IL-2 high affinity receptor (7-10). Unfortunately, these molecular variants still systemically activate cells carrying the intermediate affinity receptor (CD122 / CD132 subunits) involved in cytokine signaling and face similar toxicity issues as fully active IL-2 treatment at doses required to confirm efficacy in preclinical models. Indeed, non-α approaches to IL-2 therapy may merely shift the therapeutic window rather than improving it. Furthermore, newly activated CD8+ T cells upregulate CD25 to form high-affinity receptors, which are required for sustained proliferation in the presence of antigen. For example, a paper using a viral infection model showed that CD8+ T cells lacking CD25 were unable to proliferate in infected tissues, despite expression of intermediate-affinity receptors (22).

[0190] The design of inducible IL-2 addresses the challenges associated with rhIL-2 therapy. Inducible IL-2 contains native IL-2, which maximizes the pharmacological potential of this cytokine in promoting antitumor immunity. The molecule is designed as a prodrug to minimize systemic toxicity and is conditionally activated to release IL-2 selectively in the tumor microenvironment. Compound 1 activity was highly inducible in vitro in human reporter cell assay systems, as well as in human and mouse primary cells. Similarly, compound 1 was effective in mouse syngeneic models, and this efficacy was dependent on tumor-specific processing. The half-life extension domain provides the opportunity for better drug exposure at lower dosing frequencies compared to the traditional dosing schedule of high-dose IL-2 therapy (Proleukin). For example, while complete responses could be reliably obtained in the MC38 mouse model by dosing twice weekly, complete responses could also be achieved in 100% of mice with slightly higher prodrug doses and less frequent dosing once every 2 weeks. Furthermore, the peripheral inactivation provided by the IL-2 inactivation domain allowed this IL-2 prodrug to be safely administered to mice without apparent toxicity at doses more than 20-fold higher than those required for potent efficacy. Between increased efficacy and reduced toxicity, compound 1 has a significantly broader therapeutic window than previously described for high-dose IL-2.

[0191] The data reported in this paper show that the efficacy of compound 1 is driven by the proliferation and activation of effector cells (both T cells and NK cells) within the tumor that are capable of producing effector cytokines such as TNF, granzyme B, and IFN-γ. Indeed, activation of tumor-infiltrating immune cells was sufficient to generate a strong antitumor response. Furthermore, compound 1 treatment increased the frequency of tumor-infiltrating polyfunctional CD8+ T cells, which is associated with greater cytolytic activity in viral models (18, 19). One concern expressed by proponents of non-α IL-2 therapy is that because CD25 is highly expressed on Tregs, Treg proliferation will inhibit the antitumor immunity generated in response to wild-type IL-2. Although compound 1 treatment did indeed result in a small expansion of Tregs, the proliferation of CD8+ T cells far exceeded that of Tregs, resulting in a favorable CD8 / Treg ratio after treatment. Furthermore, WTX-124 treatment led to a tumor-specific increase in the expression of IFN-γ by effector cells. IFN-γ is a fundamental effector cytokine that promotes antitumor effects by amplifying the cellular immune component of the response and biasing CD4+ T cells toward a TH1 phenotype. Also, more recently, IFN-γ has been shown to control Treg mechanistic vulnerability (20). This phenomenon was observed upon treatment with Compound 1, as intratumoral Tregs began to produce cytokines traditionally associated with effector T cells, which may contribute to the overall efficacy of Compound 1.

[0192] A key feature of compound 1 is the selective processing of the prodrug within the tumor, which allows 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 in the circulation and when WTX-124 was exposed to healthy primary human cells or plasma, as shown in mice and non-human primates (data not shown). In contrast, compound 1 was robustly processed by primary human dissociated tumor samples from various types of cancer, demonstrating the potential of systemically administered compound 1 to selectively deliver IL-2 to disease sites and actively contribute to the development of an effective immune response. The clinical benefit and safety of compound 1 treatment will be explored in upcoming Phase I trials testing compound 1 alone or in combination with the anti-PD-1 therapy pembrolizumab, subject to FDA approval.

[0193] In summary, this study presents the design and mechanistic features of compound 1, a novel conditionally activated IL-2 prodrug that delivers full potency IL-2 in a tumor-selective manner and activates tumor-specific immune cell populations. 1.4 References 1. Wei SC, Duffy CR, Allison JP. Fundamental mechanisms of immune checkpoint blockade therapy. Cancer Discov 2018;8:1069-86. 2.Ribas A, Wolchok JD.Cancer immunotherapy using checkpoint blockade.Science 2018;359:1350-5. 3.Pardoll DM.The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer 2012;12:252-64. 4.Waldmann TA.Cytokines in cancer immunotherapy.Cold Spring Harb Perspect Biol 2018;10. 5.Mitra S,Leonard WJ.Biology of IL-2 and its therapeutic modulation:Mechanisms and strategies.J Leukoc Biol 2018;103:643-55. 6.Malek TR.The biology of interleukin-2.Annu Rev Immunol 2008;26:453-79. 7.Sharma M,Khong H,Fa’ak F,Bentebibel SE,Janssen LME,Chesson BC,et al.Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy.Nat Commun 2020;11:661. 8.Charych DH,Hoch U,Langowski JL,Lee SR,Addepalli MK,Kirk PB,et al.NKTR-214,an engineered cytokine with biased IL2 receptor binding,increased tumor exposure,and marked efficacy in mouse tumor models.Clin Cancer Res 2016;22:680-90. 9.Klein C,Waldhauer I,Nicolini VG,Freimoser-Grundschober A,Nayak T,Vugts DJ,et al.Cergutuzumab amunaleukin(CEA-IL2v),a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy:Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines.Oncoimmunology 2017;6:e1277306. 10.Lopes JE,Fisher JL,Flick HL,Wang C,Sun L,Ernstoff MS,et al.ALKS 4230:a novel engineered IL-2 fusion protein with an improved cellular selectivity profile for cancer immunotherapy.J Immunother Cancer 2020;8:e000673. 11.Mitschke J,Burk UC,Reinheckel T.The role of proteases in epithelial-to-mesenchymal cell transitions in cancer.Cancer Metastasis Rev 2019;38:431-44. 12.Dudani JS,Warren AD,Bhatia SN.Harnessing protease activity to improve cancer care.Annu Rev Cancer Biol 2018;2:353-76. 13.Yost KE,Satpathy AT,Wells DK,Qi Y,Wang C,Kageyama R,et al.Clonal replacement of tumor-specific T cells following PD-1 blockade.Nat Med 2019;25:1251-9. 14.Konrad MW,Hemstreet G,Hersh EM,Mansell PW,Mertelsmann R,Kolitz JE,et al.Pharmacokinetics of recombinant interleukin 2 in humans.Cancer Res 1990;50:2009-17. 15.Sands H,Loveless SE.Biodistribution and pharmacokinetics of recombinant,human 125I-interleukin-2 in mice.Int J Immunopharmacol 1989;11:411-6. 16.Atkins MB,Lotze MT,Dutcher JP,Fisher RI,Weiss G,Margolin K,et al.High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma:analysis of 270 patients treated between 1985 and 1993.J Clin Oncol 1999;17:2105. 17.Ye X,Waite JC,Dhanik A,Gupta N,Zhong M,Adler C,et al.Endogenous retroviral proteins provide an immunodominant but not requisite antigen in a murine immunotherapy tumor model.Oncoimmunology 2020;9:1758602. 18.Wherry EJ,Blattman JN,Murali-Krishna K,Van Der Most R,Ahmed R.Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment.J Virol 2003;77:4911-27. 19.Imai N,Tawara I,Yamane M,Muraoka D,Shiku H,Ikeda H.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-γ 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,Lefrancois L.IL-2 is not required for the initiation of CD8 T cell cycling but sustains expansion.J Immunol 2003;171:5727-35.

Claims

1. 1. A composition for treating cancer, comprising an inducible interleukin-2 (IL-2) prodrug, wherein an effective amount of the inducible IL-2 prodrug is systemically administered to a subject in need of treatment and is activated by cleavage by a protease that has greater activity in a tumor microenvironment than at other sites, such that cleavage of the inducible IL-2 prodrug is at least about 40 times greater in the tumor microenvironment compared to the circulation.

2. 2. The composition of claim 1, wherein cleavage of the inducible IL-2 prodrug is at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 93-fold, at least about 95-fold, or at least about 100-fold greater in the tumor microenvironment compared to the circulation.

3. 10. The composition of claim 1, wherein said administration results in an amount of inducible IL-2 prodrug in plasma that is at least about 5 times greater than the amount of inducible IL-2 prodrug in the tumor.

4. 4. The composition of claim 3, wherein the amount of the inducible IL-2 prodrug in the plasma is at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 18-fold, at least about 20-fold, or at least about 25-fold greater than the amount of the inducible IL-2 prodrug in the tumor.

5. The composition of claim 1, wherein said administration of said composition significantly increases the tumor-reactive CD8+ / Treg ratio.

6. A composition for inducing immunological memory against a tumor, comprising an inducible interleukin-2 (IL-2) prodrug, wherein an effective amount of the inducible IL-2 prodrug is systemically administered to a subject in need of treatment and is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other sites.

7. 7. The composition of claim 6, wherein cleavage of the inducible IL-2 prodrug is at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 93-fold, at least about 95-fold, or at least about 100-fold greater in the tumor microenvironment compared to circulation.

8. 7. The composition of claim 6, wherein said administration results in an amount of inducible IL-2 prodrug in plasma that is at least about 5 times greater than the amount of inducible IL-2 prodrug in said tumor.

9. 9. The composition of claim 8, wherein the amount of the inducible IL-2 prodrug in the plasma is at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 18-fold, at least about 20-fold, or at least about 25-fold greater than the amount of the inducible IL-2 prodrug in the tumor.

10. The immunological memory is characterized by a memory phenotype (e.g., CD8+CD44 ｈｉ CD62 ｌｏｗ 7. The composition of claim 6, characterized by tumor-reactive CD8+ cells having:

11. The composition of claim 6, wherein the immunological memory is characterized by tumor-reactive CD8+ cells producing TNF and / or IFNγ upon restimulation.

12. 7. The composition of claim 6, wherein the immunological memory is characterized by polyfunctional tumor-reactive CD8+ cells that produce TNF and IFNγ upon restimulation.

13. The composition of claim 10, wherein the tumor-reactive CD8+ cells further produce granzyme B upon restimulation.

14. 1. A composition for selectively activating effector CD8+ T cells in a tumor microenvironment, comprising an inducible interleukin-2 (IL-2) prodrug, wherein the composition is systemically administered in an effective amount to a subject in need of treatment and is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other sites, resulting in a significantly higher frequency of TNF- and / or IFNγ-producing CD8+ T cells in the tumor compared to peripheral tissues.

15. 1. A composition for selectively activating tumor-infiltrating lymphocytes, comprising an inducible interleukin-2 (IL-2) prodrug, wherein the composition is systemically administered in an effective amount to a subject in need of treatment and is activated by cleavage by a protease having higher activity in the tumor microenvironment than in other sites, resulting in a significantly higher frequency of TNF- and / or IFNγ-producing CD8+ T cells in the tumor compared to peripheral tissues.

16. 15. The composition of claim 14, wherein said administration results in at least about 40-fold greater cleavage of said inducible IL-2 prodrug in the tumor microenvironment compared to the circulation.

17. 17. The composition of claim 16, wherein cleavage of the inducible IL-2 prodrug is at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 93-fold, at least about 95-fold, or at least about 100-fold greater in the tumor microenvironment compared to the circulation.

18. 15. The composition of claim 14, wherein said administration results in an amount of inducible IL-2 prodrug in plasma that is at least about 5 times greater than the amount of inducible IL-2 prodrug in said tumor.

19. 19. The composition of claim 18, wherein the amount of the inducible IL-2 prodrug in the plasma is at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 18-fold, at least about 20-fold, or at least about 25-fold greater than the amount of the inducible IL-2 prodrug in the tumor.

20. 15. The composition of claim 14, wherein said administration of said composition significantly increases the tumor-reactive CD8+ / Treg ratio.

21. The composition of any one of claims 1 to 20, wherein the inducible IL-2 prodrug is Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing.

22. The composition of any one of claims 1 to 20, wherein the inducible IL-2 prodrug is administered no more frequently than about twice a week.

23. The composition of any one of claims 1 to 20, wherein the inducible IL-2 prodrug is administered no more frequently than about once a week.

24. The composition according to any one of claims 1 to 20, wherein the inducible IL-2 prodrug is administered about once every two weeks.

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