IL-12 Prodrugs

Abstract

The present disclosure relates to methods and compositions for treating cancer using inducible IL-12 prodrugs.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 328,708, filed April 7, 2022, and U.S. Application No. 63 / 382,694, filed November 7, 2022, the entire contents of each of which are incorporated by reference herein. [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. Although some patients have achieved durable responses using these new therapies, the percentage of responders remains relatively low and limited to only a few cancer types. The tumor mutation burden, the presence or absence of T cell infiltration in the tumor, and the overall immunosuppressive microenvironment of the tumor greatly influence the response to immunotherapy. Although immune checkpoint inhibition 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 therapy to enhance antitumor immunity. In fact, these were some of the first cancer immunotherapies approved for clinical use. However, poor systemic toxicity and pharmacokinetic profiles have limited their clinical application.

[0003] Interleukin-12 (IL-12) is a heterodimeric 70 kDa cytokine composed of two covalently linked glycosylated subunits (p35 and p40) (Lieschke et al., 1997; Jana et al., 2014). It is a potent immune agonist and is considered a promising therapeutic agent in oncology. However, IL-12 has been shown to have a narrow therapeutic window due to its very strong potency and short serum half-life. As a result, therapeutic administration of IL-12 produces undesirable systemic effects and toxicity. This is exacerbated by the need to administer large amounts of cytokines (e.g., IL-12) to achieve desired cytokine levels at the intended site of cytokine action (e.g., tumor microenvironment). Unfortunately, cytokine biology and the inability to effectively target and control its activity have prevented cytokines from achieving the expected clinical benefits in tumor treatment. Inducible forms of IL-12 that are conditionally activated by protease cleavage in the tumor microenvironment to release fully active native IL-12 cytokine within the tumor to stimulate a potent anti-tumor immune response have been described in International Application Nos. PCT / US2019 / 032320, PCT / US2019 / 032322, and PCT / US2021 / 033014. These IL-12 prodrugs contain a native IL-12 molecule linked via a protease-cleavable linker to a half-life extending domain (e.g., an anti-human serum albumin antibody binding fragment, e.g., a VH domain) and an IL-12 blocking element (e.g., an anti-IL-12 antibody binding fragment, e.g., a Fab or scFv) that blocks IL-12 binding to the IL-12Rβ1 or IL-12Rβ2 receptors on peripheral normal tissues. Upon cleavage of the protease-cleavable linker, fully active native IL-12 is released within the tumor, stimulating a potent anti-tumor immune response. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Application No. PCT / US2019 / 032320 [Patent Document 2] International Application No. PCT / US2019 / 032322 [Patent Document 3] International Application No. PCT / US2021 / 033014 Summary of the Invention

[0005] The present disclosure relates to compositions and methods for treating cancer using inducible IL-12 prodrugs. The inducible IL-12 prodrugs contain attenuated IL-12 and have a longer half-life compared to naturally occurring IL-12. Optionally, the IL-12 can be muteinized. The IL-12 mutein can be non-glycosylated or partially non-glycosylated. The inducible IL-12 prodrugs disclosed herein include two or more polypeptide chains, and the inducible IL-12 prodrugs include IL-12 subunits p35 and p40, a half-life extension element, an IL-12 blocking element, and a protease cleavable linker.

[0006] The inducible IL-12 prodrug may comprise two different polypeptides. The first polypeptide may comprise an IL-12 subunit and, optionally, an IL-12 blocking element. The IL-12 blocking element, if present, is operably linked to the IL-12 subunit via a first protease-cleavable linker. The second polypeptide chain may comprise an IL-12 subunit operably linked to a half-life extension element via a second protease-cleavable linker and, optionally, an IL-12 blocking element. The IL-12 blocking element, if present, may be operably linked to the IL-12 subunit via a protease-cleavable linker or may be operably linked to the half-life extension element via a linker that is optionally protease-cleavable. Only one of the first and second polypeptides comprises an IL-12 blocking element. If the IL-12 subunit in the first polypeptide is p35, then the IL-12 subunit in the second polypeptide is p40, and if the IL-12 subunit in the first polypeptide is p40, then the IL-12 subunit in the second polypeptide is p35. The preferred blocking element of the inducible IL-12 prodrug is a single chain antibody that binds to IL-12 or an antigen-binding fragment thereof. The cleavable linkers in the inducible IL-12 prodrug can be the same or different.

[0007] An inducible IL-12 prodrug may comprise three different polypeptides. Typically, one polypeptide chain comprises either the p35 or p40 IL-12 subunit, but not both, a second polypeptide comprises the other IL-12 subunit, and a third polypeptide comprises at least a portion of a blocking element. The first polypeptide may comprise an IL-12 subunit and, optionally, a half-life extension element. The half-life extension element, if present, is operably linked to the IL-12 subunit via a protease-cleavable linker.

[0008] The second polypeptide may comprise an IL-12 subunit, at least an antigen-binding portion of an antibody light chain or an antigen-binding portion of an antibody heavy chain, and optionally a half-life extension, where the half-life extension, if present, is operably linked to the IL-12 subunit via a protease-cleavable linker, and the antibody heavy or light chain is a) operably linked to the IL-12 subunit via a second protease-cleavable linker, or b) optionally operably linked to the half-life extension, via a cleavable linker.

[0009] The third polypeptide may comprise an antigen-binding portion of an antibody heavy chain complementary to the light chain of the second polypeptide, or an antibody light chain complementary to the heavy chain of the second polypeptide, forming an IL-12 binding site with the light chain. If the IL-12 subunit in the first polypeptide is p35, the IL-12 subunit in the second polypeptide is p40, and if the IL-12 subunit in the first polypeptide is p40, the IL-12 subunit in the second polypeptide is p35. In this inducible IL-12 prodrug, the IL-12 blocking element is preferably an antigen-binding fragment of an antibody. The antigen-binding fragment comprises, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain. The protease-cleavable linkers in this inducible IL-12 prodrug may be the same or different.

[0010] The inducible IL-12 prodrug may comprise two different polypeptides in which p35 and p40 are located on the same polypeptide chain. The first polypeptide chain may comprise p35, p40, a half-life extension and at least an antigen-binding portion of an antibody light chain. p35 and p40 may be operably linked, the half-life extension may be operably linked to p40 via a first protease cleavable linker, and the antigen-binding portion of the antibody light chain may be operably linked to p35 via a protease cleavable linker. Alternatively, the half-life extension may be operably linked to p35 via a protease cleavable linker, and the antigen-binding portion of the antibody light chain may be operably linked to p40 via a protease cleavable linker. The second polypeptide comprises at least an antigen-binding portion of an antibody heavy chain that is complementary to the light chain in the second polypeptide and forms an IL-12 binding site with the light chain. The protease-cleavable linkers in the inducible IL-12 prodrugs can be the same or different.

[0011] In an alternative format, the first polypeptide chain may comprise p35, p40, a half-life extension, and at least an antigen-binding portion of an antibody heavy chain. p35 and p40 may be operably linked, the half-life extension may be operably linked to p40 via a protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain may be operably linked to p35 via a protease-cleavable linker. Alternatively, the half-life extension may be operably linked to p35 via a protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain may be operably linked to p40 via a second protease-cleavable linker. The second polypeptide comprises at least an antigen-binding portion of an antibody light chain that is complementary to the heavy chain of the second polypeptide and forms an IL-12 binding site with the light chain. The protease-cleavable linkers in this inducible IL-12 prodrug may be the same or different.

[0012] In one example, an inducible IL-12 prodrug comprises a first polypeptide that does not contain a blocking element and a second polypeptide having the formula [A]-[L1]-[B]-[L3]-[D] or [D]-[L3]-[B]-[L1]-[A] or [B]-[L1]-[A]-[L2]-[D] or [D]-[L1]-[A]-[L2]-[B], where A is an IL-12 subunit; L1 is a first protease-cleavable linker; L2 is a second protease-cleavable linker; L3 is an optional cleavable linker; B is a half-life extending element; and D is a blocking element.

[0013] In another example, a first polypeptide comprises the formula [A]-[L1]-[D] or [D]-[L1]-[A] and a second polypeptide has the formula [A']-[L2]-[B] or [B]-[L2]-[A'], where A and A' are independently either p35 or p40, and if A is p35, A' is p40, and if A is p40, A' is p35, L1 is a first protease-cleavable linker; L2 is a second protease-cleavable linker; B is a half-life extending element; and D is a blocking element.

[0014] The present disclosure relates to a method of inducing immune memory against a tumor, comprising administering to a subject in need thereof an effective amount of an inducible IL-12 prodrug as described herein, wherein the inducible IL-12 prodrug is administered systemically and activated upon cleavage by a protease that has a higher activity in the tumor microenvironment than elsewhere. The present disclosure relates to a method for selectively activating effector CD8+ T cells in the tumor microenvironment and / or a method for selectively activating tumor infiltrating lymphocytes, comprising administering to a subject in need thereof an effective amount of an inducible IL-12 prodrug as described herein, wherein the inducible IL-12 prodrug is administered systemically and activated upon cleavage by a protease that has a higher activity in the tumor microenvironment than elsewhere, resulting in a significantly higher frequency of CD8+ T cells producing TNF, IFN gamma, and / or granzyme B in the tumor.

[0015] In embodiments of the disclosed methods, the inducible IL-12 prodrug can be administered about twice a week or less frequently, once a week or less frequently, or about once every two weeks or less frequently. In certain embodiments, the inducible IL-12 prodrug can be administered about once every two weeks.

[0016] A preferred inducible IL-12 prodrug for use in the disclosed method is chimeric compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, or an amino acid sequence variant of any of the above, where chimeric compound 1 comprises a first polypeptide chain of SEQ ID NO: 1 and a second polypeptide chain of SEQ ID NO: 7, and the amino acid sequence variant of chimeric compound 1 may comprise a first polypeptide chain having at least about 80% identity to SEQ ID NO: 1 and a second polypeptide chain having at least about 80% identity to SEQ ID NO: 7. Compound 2 comprises a first polypeptide chain of SEQ ID NO: 2 and a second polypeptide chain of SEQ ID NO: 7, and the amino acid sequence variant of compound 2 may 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: 7. Compound 3 comprises a first polypeptide chain of SEQ ID NO:3 and a third polypeptide chain of SEQ ID NO:8, and an amino acid sequence variant of compound 3 may 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:8. Compound 4 comprises a first polypeptide chain of SEQ ID NO:4 and a fourth polypeptide chain of SEQ ID NO:8, and an amino acid sequence variant of compound 4 may 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:8. Compound 5 comprises a first polypeptide chain of SEQ ID NO:5 and a fifth polypeptide chain of SEQ ID NO:8, and an amino acid sequence variant of compound 5 may 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:8. Compound 5 comprises a first polypeptide chain of SEQ ID NO:5 and a fifth polypeptide chain of SEQ ID NO:8, and an amino acid sequence variant of compound 5 may 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:8.Compound 6 comprises a first polypeptide chain of SEQ ID NO:6 and a sixth polypeptide chain of SEQ ID NO:8, and an amino acid sequence variant of compound 6 may 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:8. [Brief description of the drawings]

[0017] The drawings are not necessarily to scale or exhaustive. Instead, emphasis is generally placed on illustrating the principles of the invention described herein. The accompanying drawings, which form a part of this specification, illustrate several embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the disclosure. In the drawings:

[0018] [Figure 1] 1 is a graph showing the in vitro activity of chimeric compound 1 in the IL-12 HEK-Blue reporter assay, comparing intact chimeric compound 1 (squares) and truncated chimeric compound 1 (triangles) with chimeric IL-12 (circles).

[0019] [Diagram 2] AB show that chimeric compound 1 is well tolerated and induces tumor regression in a cleavage-dependent manner. A is a graph showing the antitumor activity of various doses of chimeric compound 1 in a mouse model. Chimeric compound 1 was administered intraperitoneally twice weekly for 2 weeks at 7 μg / dose and 43 μg / dose, and the NC (non-cleavable) version of chimeric compound 1 was administered at 43 μg / dose. B is a graphical depiction of the calculated therapeutic window of chimeric IL-12 and chimeric compound 1 per mole using the same tumor model (MC38) based on identifying effective and toxic dose levels of both treatments.

[0020] [Figure 3A]3A-3E are graphs showing that chimeric compound 1 produces antitumor immunity and protective memory in multiple syngeneic tumor models. The antitumor activity of chimeric compound 1 at various doses in various mouse syngeneic tumor models, CT26 model (FIG. 3A), B16-F10 model (FIG. 3B), EMT-6 model (FIG. 3C), A20 model (FIG. 3D), and EG7.OVA model (FIG. 3E), is shown. Mice were dosed twice weekly for a total of 2 weeks at the doses listed in the figure legends. [Figure 3B] 3A-3E are graphs showing that chimeric compound 1 produces antitumor immunity and protective memory in multiple syngeneic tumor models. The antitumor activity of chimeric compound 1 at various doses in various mouse syngeneic tumor models, CT26 model (FIG. 3A), B16-F10 model (FIG. 3B), EMT-6 model (FIG. 3C), A20 model (FIG. 3D), and EG7.OVA model (FIG. 3E), is shown. Mice were dosed twice weekly for a total of 2 weeks at the doses listed in the figure legends. [Figure 3C] 3A-3E are graphs showing that chimeric compound 1 produces antitumor immunity and protective memory in multiple syngeneic tumor models. The antitumor activity of chimeric compound 1 at various doses in various mouse syngeneic tumor models, CT26 model (FIG. 3A), B16-F10 model (FIG. 3B), EMT-6 model (FIG. 3C), A20 model (FIG. 3D), and EG7.OVA model (FIG. 3E), is shown. Mice were dosed twice weekly for a total of 2 weeks at the doses listed in the figure legends. [Figure 3D] 3A-3E are graphs showing that chimeric compound 1 produces antitumor immunity and protective memory in multiple syngeneic tumor models. The antitumor activity of chimeric compound 1 at various doses in various mouse syngeneic tumor models, CT26 model (FIG. 3A), B16-F10 model (FIG. 3B), EMT-6 model (FIG. 3C), A20 model (FIG. 3D), and EG7.OVA model (FIG. 3E), is shown. Mice were dosed twice weekly for a total of 2 weeks at the doses listed in the figure legends. [Figure 3E]3A-3E are graphs showing that chimeric compound 1 produces antitumor immunity and protective memory in multiple syngeneic tumor models. The antitumor activity of chimeric compound 1 at various doses in various mouse syngeneic tumor models, CT26 model (FIG. 3A), B16-F10 model (FIG. 3B), EMT-6 model (FIG. 3C), A20 model (FIG. 3D), and EG7.OVA model (FIG. 3E), is shown. Mice were dosed twice weekly for a total of 2 weeks at the doses listed in the figure legends. [Figure 3F] 3A-3G are graphs showing that chimeric compound 1 induces anti-tumor immunity and protective memory in multiple syngeneic tumor models. Graphs showing tumor volume in EMT6 (FIG. 3F) and MC38 (FIG. 3G) models rechallenged with the same tumor on the opposite flank over time. This demonstrates that treatment with chimeric compound 1 induces immune memory against the same tumor type. [Figure 3G] 3A-3G are graphs showing that chimeric compound 1 induces anti-tumor immunity and protective memory in multiple syngeneic tumor models. Graphs showing tumor volume in EMT6 (FIG. 3F) and MC38 (FIG. 3G) models rechallenged with the same tumor on the opposite flank over time. This demonstrates that treatment with chimeric compound 1 induces immune memory against the same tumor type.

[0021] [Figure 4A] Figure 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and induces activation of intratumoral effector cells (NK and CD8+ T cells) in the MC38 model. Heatmap of transcripts with statistically significant differences between the two treatments obtained from NanoString analysis of bulk RNA from tumor samples. Transcripts with average normalized counts below 50 were excluded from the heatmap. Each lane represents an individual animal. [Figure 4B] Figure 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and induces activation of intratumoral effector cells (NK and CD8+ T cells) in the MC38 model. Volcano plots of differentially expressed transcripts between chimeric compound 1 and vehicle treated mice are shown. [Figure 4C] FIG. 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and induces activation of intratumoral effector cells (NK and CD8+ T cells) in the MC38 model. FIG. 2 shows the frequency of tumor-infiltrating NK cells producing IFNγ or granzyme B. [Figure 4D] FIG. 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and induces activation of intratumoral effector cells (NK and CD8+ T cells) in the MC38 model. FIG. 2 shows the frequency of tumor-infiltrating NK cells producing IFNγ or granzyme B. [Figure 4E] FIG. 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and induces activation of intratumoral effector cells (NK and CD8+ T cells) in the MC38 model. FIG. 2 shows flow cytometry images showing the frequency of tetramer-positive CD8+ T cells producing IFNγ and / or TNF. [Figure 4F] FIG. 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and induces activation of intratumoral effector cells (NK and CD8+ T cells) in the MC38 model. FIG. 2 shows the frequency of polyfunctional tetramer-positive CD8+ T cells as measured by co-expression of IFNγ, TNF, and granzyme B.

[0022] [Figure 5A] Figure 2 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. Figure 2 shows a heatmap of transcripts with statistically significant differences in expression between the two treatments obtained from NanoString analysis of bulk RNA from tumor samples. Transcripts with an average normalized count below 50 were excluded from the heatmap. Each lane represents an individual animal. [Figure 5B] FIG. 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. FIG. 1 shows a volcano plot of differentially expressed transcripts between chimeric compound 1-treated and vehicle-treated mice. [Figure 5C] FIG. 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. Graphs of pathway scores for vehicle and chimeric compound 1 for antigen processing, interferon, MHC, and NK cell function are shown. [Figure 5D] FIG. 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. FIG. 1 shows normalized counts from individual transcripts for vehicle and chimeric compound 1. [Figure 5E] FIG. 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. FIG. 2 shows flow cytometry plots showing the frequency of tetramer+CD8+ T cells producing IFN-gamma and / or granzyme B. [Figure 5F] 1 shows that treatment with chimeric compound 1 remodels the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. FIG. 2 shows the frequency of polyfunctional tetramer-positive CD8+ T cells as measured by co-expression of IFN-gamma, TNF, and granzyme B by flow cytometry.

[0023] [Figure 6] 1 is a pie chart showing that treatment with chimeric compound 1 induces sustained polyfunctional CD8+ T cell responses. Mice were implanted with EMT6 cells and randomly assigned to treatment groups. Mice were dosed twice weekly for two weeks and tumors were harvested at the indicated time points. The frequency of polyfunctional tumor-infiltrating CD8+ T cells was measured by examining the co-expression of IFN-gamma, TNF, and granzyme B. All animals in the vehicle group were removed from the study by day 21 due to tumor burden.

[0024] [Figure 7A]Figure 1 shows that systemic administration of chimeric compound 1 results in infiltration and activation of CD8+ T cells in tumors as assessed by immunofluorescence staining, and increased IL-12 and IFN gamma signaling by tumor-infiltrating CD8+ T cells. Mice were implanted with EMT6 cells and randomized into treatment groups. Mice were dosed twice weekly for 2 weeks, and tumors were harvested on day 11. Nanostring GeoMX analysis was performed on FFPE tumor tissue. Immunofluorescence images of tumor-infiltrating CD8+ T cells in vehicle and chimeric compound 1. [Figure 7B] Figure 1 shows that systemic administration of chimeric compound 1 results in infiltration and activation of CD8+ T cells in tumors as assessed by immunofluorescence staining, and increased IL-12 and IFN gamma signaling by tumor-infiltrating CD8+ T cells. Mice were implanted with EMT6 cells and randomized into treatment groups. Mice were dosed twice weekly for 2 weeks, and tumors were harvested on day 11. Nanostring GeoMX analysis was performed on FFPE tumor tissues. Figure 1 shows differential gene expression analysis of tumor-infiltrating CD8+ T cells. [Figure 7C] FIG. 7 shows that systemic administration of chimeric compound 1 results in infiltration and activation of CD8+ T cells in tumors as assessed by immunofluorescence staining, and increased IL-12 and IFN gamma signaling by tumor-infiltrating CD8+ T cells. Mice were implanted with EMT6 cells and randomized into treatment groups. Mice were dosed twice weekly for 2 weeks, and tumors were harvested on day 11. Nanostring GeoMX analysis was performed on FFPE tumor tissue. Heatmap showing genes associated with IL-12 (FIG. 7C) and IFN gamma (FIG. 7D) signaling. [Figure 7D]FIG. 7 shows that systemic administration of chimeric compound 1 results in infiltration and activation of CD8+ T cells in tumors as assessed by immunofluorescence staining, and increased IL-12 and IFN gamma signaling by tumor-infiltrating CD8+ T cells. Mice were implanted with EMT6 cells and randomized into treatment groups. Mice were dosed twice weekly for 2 weeks, and tumors were harvested on day 11. Nanostring GeoMX analysis was performed on FFPE tumor tissue. Heatmap showing genes associated with IL-12 (FIG. 7C) and IFN gamma (FIG. 7D) signaling.

[0025] [Figure 8] 1A-B are graphs showing the antitumor activity of chimeric compound 1 in various studies of a mouse syngeneic MC38 tumor model. A is a graph showing tumor growth over time in MC38 tumor-bearing mice treated with chimeric compound 1 (+ / -) daily FTY720 treatment. B is a graph showing tumor growth over time in MC38 tumor-bearing mice administered CD4, CD8, and NK cell depleting antibodies twice weekly in combination with chimeric compound 1.

[0026] [Figure 9A] 9A-9B show that chimeric compound 1 is preferentially activated in the TME and expands the therapeutic window compared to chimeric IL-12. Graphs showing the presence of total chimeric compound 1 or free chimeric IL-12 over time from the plasma (FIG. 9A) or tumor (FIG. 9B) of MC38 tumor-bearing mice treated with chimeric compound 1. The area under the curve was calculated and the ratio of total chimeric compound 1 to free chimeric IL-12 was calculated. [Figure 9B] 9A-9B show that chimeric compound 1 is preferentially activated in the TME and expands the therapeutic window compared to chimeric IL-12. Graphs showing the presence of total chimeric compound 1 or free chimeric IL-12 over time from the plasma (FIG. 9A) or tumor (FIG. 9B) of MC38 tumor-bearing mice treated with chimeric compound 1. The area under the curve was calculated and the ratio of total chimeric compound 1 to free chimeric IL-12 was calculated. [Figure 9C]Figure 1 shows that chimeric compound 1 is preferentially activated within the TME and expands the therapeutic window compared to chimeric IL-12. Figure 2 shows pie charts depicting the frequency of polyfunctional CD8+ T cells in tumors, peripheral blood, tumor-draining or non-tumor-draining lymph nodes of MC38 tumor-bearing mice treated twice with chimeric compound 1. The frequency of polyfunctional CD8+ T cells was measured by examining the co-expression of IFN-gamma, TNF, and granzyme B after PMA / ionomycin restimulation.

[0027] [Figure 10A] FIG. 1 shows that chimeric compound 1 activates tumor-infiltrating immune cell populations in the MC38 syngeneic tumor model. Representative flow plots of CD11b+ and CD103+ tumor-infiltrating dendritic cells. [Figure 10B] Figure 2 shows that chimeric compound 1 activates tumor-infiltrating immune cell populations in the MC38 syngeneic tumor model. Figure 3 shows the ratio of CD11b+ and CD103+ tumor-infiltrating dendritic cells. [Figure 10C] FIG. 1 shows that chimeric compound 1 activates tumor-infiltrating immune cell populations in the MC38 syngeneic tumor model. FIG. 2 shows the frequency of CD4+ T conventional cells with a TH1 phenotype (Tbet+IFNgamma+TFN+). [Figure 10D] FIG. 1 shows that chimeric compound 1 activates tumor-infiltrating immune cell populations in the MC38 syngeneic tumor model. FIG. 1 is a representative flow plot showing the frequency of tumor-infiltrating FoxP3+ Tregs producing IFN-gamma and TNF. [Figure 10E] Figure 10 shows that chimeric compound 1 activates tumor-infiltrating immune cell populations in MC38 syngeneic tumor models. Graphs showing the frequency of tumor-infiltrating FoxP3+ Tregs producing IFN-gamma and TNF (Figure 10E) and Tbet (Figure 10F). Data are presented as mean ± SD unless otherwise stated, and P values ​​are derived from t-test (**, p<0.01; ***, p<0.001; ****, p<0.00001). [Figure 10F]Figure 10 shows that chimeric compound 1 activates tumor-infiltrating immune cell populations in MC38 syngeneic tumor models. Graphs showing the frequency of tumor-infiltrating FoxP3+ Tregs producing IFN-gamma and TNF (Figure 10E) and Tbet (Figure 10F). Data are presented as mean ± SD unless otherwise stated, and P values ​​are derived from t-test (**, p<0.01; ***, p<0.001; ****, p<0.00001).

[0028] [Figure 11A] FIG. 1 shows that systemic treatment with chimeric compound 1 expands novel TCR clones and increases the overall clonality of the TCR repertoire. FIG. 2 shows heat maps depicting intratumoral CD8+ T cell downstream TCR signaling following treatment with vehicle and chimeric compound 1. [Figure 11B] Figure 1 shows that systemic treatment with chimeric compound 1 expands new TCR clones and increases the overall clonality of the TCR repertoire. Figure 2 shows the clonal frequency of individual VDJ recombinations on the TCR-beta chain in EMT-6 tumor models treated with vehicle and chimeric compound 1. On day 11, live T cells were isolated from EMT-6 tumors and TCR sequencing was performed. [Figure 11C] Figure 1 shows that systemic treatment with chimeric compound 1 expands new TCR clones and increases the overall clonality of the TCR repertoire. Figure 2 shows the clonal frequency of individual VDJ recombinations on the TCR-beta chain in EMT-6 tumor models treated with vehicle and chimeric compound 1. On day 11, live T cells were isolated from EMT-6 tumors and TCR sequencing was performed. [Figure 11D] 1 shows that systemic treatment with chimeric compound 1 expands new TCR clones and increases the overall clonality of the TCR repertoire. [Figure 11E]Figure 1 shows that systemic treatment with chimeric compound 1 expands new TCR clones and increases the overall clonality of the TCR repertoire. Figure 2 shows the frequency of the top 50 TCR clones plotted for each animal.

[0029] [Figure 12A] FIG. 1 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. FIG. 2 is a heat map of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. [Figure 12B] 1 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. FIG. 2 shows the uptake of 2-NDBG in EMT-6 TILs from either vehicle or chimeric compound 1 treated animals. [Figure 12C] 1 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. FIG. 2 shows the uptake of 2-NDBG in EMT-6 TILs from either vehicle or chimeric compound 1 treated animals. [Figure 12D] FIG. 12D shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. FIG. 12F shows heat maps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (FIG. 12D), mitochondrial biogenesis (FIG. 12E), and mitochondrial translation (FIG. 12F). [Figure 12E] FIG. 12D shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. FIG. 12F shows heat maps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (FIG. 12D), mitochondrial biogenesis (FIG. 12E), and mitochondrial translation (FIG. 12F). [Figure 12F] FIG. 12D shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. FIG. 12F shows heat maps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (FIG. 12D), mitochondrial biogenesis (FIG. 12E), and mitochondrial translation (FIG. 12F). [Figure 12G]12A-12C show that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs depicting EMT-6 infiltrating CD8+ T cells derived from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (FIGS. 12G and 12H), TMRM (FIGS. 12K, 12L), and MitoSOX (FIGS. 12O-12P). [Figure 12H] 12A-12C show that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs depicting EMT-6 infiltrating CD8+ T cells derived from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (FIGS. 12G and 12H), TMRM (FIGS. 12K, 12L), and MitoSOX (FIGS. 12O-12P). [Figure 12I] Figure 12 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs showing EMT-6 infiltrating NK cells from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (Figures 12I and 12J), TMRM (Figures 12M-12N), and MitoSOX (Figures 12Q-12R). Data are presented as mean ± SD, and P values ​​are derived from t-test (*, p<0.05, **p<0.01, ***, p<0.001, ****, p<0.0001). [Figure 12J] Figure 12 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs showing EMT-6 infiltrating NK cells from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (Figures 12I and 12J), TMRM (Figures 12M-12N), and MitoSOX (Figures 12Q-12R). Data are presented as mean ± SD, and P values ​​are derived from t-test (*, p<0.05, **p<0.01, ***, p<0.001, ****, p<0.0001). [Figure 12K]12A-12C show that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs depicting EMT-6 infiltrating CD8+ T cells derived from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (FIGS. 12G and 12H), TMRM (FIGS. 12K, 12L), and MitoSOX (FIGS. 12O-12P). [Figure 12L] 12A-12C show that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs depicting EMT-6 infiltrating CD8+ T cells derived from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (FIGS. 12G and 12H), TMRM (FIGS. 12K, 12L), and MitoSOX (FIGS. 12O-12P). [Figure 12M] Figure 12 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs showing EMT-6 infiltrating NK cells from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (Figures 12I and 12J), TMRM (Figures 12M-12N), and MitoSOX (Figures 12Q-12R). Data are presented as mean ± SD, and P values ​​are derived from t-test (*, p<0.05, **p<0.01, ***, p<0.001, ****, p<0.0001). [Figure 12N] Figure 12 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs showing EMT-6 infiltrating NK cells from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (Figures 12I and 12J), TMRM (Figures 12M-12N), and MitoSOX (Figures 12Q-12R). Data are presented as mean ± SD, and P values ​​are derived from t-test (*, p<0.05, **p<0.01, ***, p<0.001, ****, p<0.0001). [Figure 12O]12A-12C show that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs depicting EMT-6 infiltrating CD8+ T cells derived from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (FIGS. 12G and 12H), TMRM (FIGS. 12K, 12L), and MitoSOX (FIGS. 12O-12P). [Figure 12P] 12A-12C show that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs depicting EMT-6 infiltrating CD8+ T cells derived from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (FIGS. 12G and 12H), TMRM (FIGS. 12K, 12L), and MitoSOX (FIGS. 12O-12P). [Figure 12Q] Figure 12 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs showing EMT-6 infiltrating NK cells from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (Figures 12I and 12J), TMRM (Figures 12M-12N), and MitoSOX (Figures 12Q-12R). Data are presented as mean ± SD, and P values ​​are derived from t-test (*, p<0.05, **p<0.01, ***, p<0.001, ****, p<0.0001). [Figure 12R] Figure 12 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Graphs showing EMT-6 infiltrating NK cells from animals treated with vehicle or chimeric compound 1 stained with MitoTracker Red (Figures 12I and 12J), TMRM (Figures 12M-12N), and MitoSOX (Figures 12Q-12R). Data are presented as mean ± SD, and P values ​​are derived from t-test (*, p<0.05, **p<0.01, ***, p<0.001, ****, p<0.0001). [Figure 12S]FIG. 1 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. FIG. 2 shows that chimeric compound 1 preferentially expands new clones over pre-existing clones. FIG. 3 shows that, from left to right, SEQ ID NOs: 450-457 and 458-465 are disclosed, which are TCR-derived sequences and define T cell clones. FIG. 4 shows the percentage of the TCR repertoire for the top 50 shared clones. [Figure 12T] FIG. 1 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. FIG. 2 shows that chimeric compound 1 preferentially expands new clones over pre-existing clones. FIG. 3 shows that, from left to right, SEQ ID NOs: 450-457 and 458-465 are disclosed, which are TCR-derived sequences and define T cell clones. FIG. 4 shows the percentage of the TCR repertoire for the top 50 shared clones. [Figure 12U] Figure 2 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 3 shows that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12V] Figure 2 shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 3 shows that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells.

[0030] [Figure 13A] Compound 36 is inducible, stable in human serum, and selectively processed by dissociated primary human tumor samples. Western blot analysis of compound 36 diluted in healthy human serum from n=6 donors and incubated at 37° C. for 24 or 72 hours before analysis is shown. [Figure 13B]Figure 2 shows that compound 36 is inducible, stable in human serum, and selectively processed by dissociated primary human tumor samples. Figure 2 shows a graph of protein cleavage after incubation with primary human dissociated tumor samples (n=88) or primary human healthy cells (n=13) exposed to compound 36 for 48 hours before protein cleavage was measured by activity in the human Tblast assay. Box plots represent the 25th and 75th percentiles, and lines represent the median for each indication. Whiskers represent the minimum and maximum values ​​within a given display.

[0031] [Figure 14A] 1 is a graph showing IFN-gamma production by intracellular cytokine staining with or without ex vivo restimulation from TILs of mice treated with either vehicle or chimeric compound 1. [Figure 14B] 1 is a graph showing IFN-gamma production by intracellular cytokine staining with or without ex vivo restimulation from TILs of mice treated with either vehicle or chimeric compound 1.

[0032] [Figure 15A] 1 shows selective activation of tumor-infiltrating immune cells by chimeric compound 1. FIG. 2 is a graph showing the frequency of IFNγ- and TNF-producing conventional CD4+ T cells (FoxP3−) in tumor tissues compared to peripheral tissues. [Figure 15B] 1 shows selective activation of tumor-infiltrating immune cells by chimeric compound 1. FIG. 2 is a graph showing the frequency of NK cells producing IFNγ and TNF in tumor tissues compared to peripheral tissues.

[0033] [Figure 16A]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16B] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16C] Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16D] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16E]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16F] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16G] Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16H] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16I]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16J] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16K] Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16L] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16M]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16N] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16O] Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16P] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16Q]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16R] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16S] Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16T] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16U]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16V] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16W] Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Fig. 16X] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16Y]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16Z] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16ZA] Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16ZB] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16ZC]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16ZD] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16ZE] Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16ZF] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16ZG]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16ZH] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16ZI] 1 is a graph showing the activity of IL-12 prodrugs in a HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate the activity of the intact inducible IL-12 prodrug and triangles indicate the activity of the in vitro protease-activated (truncated) inducible IL-12 prodrug. Circles represent the activity of the control chimeric IL-12. The respective EC50 values ​​are shown in the table (ND=not determined). The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results confirmed that the inducible IL-12 prodrugs were active and inducible. 2 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16ZJ] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16ZK]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16ZL] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16ZM] Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16ZN] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase. [Figure 16ZO]Graph showing activity of IL-12 prodrugs in HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares indicate activity of intact inducible IL-12 prodrugs and triangles indicate activity of in vitro protease activated (truncated) inducible IL-12 prodrugs. Circles represent activity of control chimeric IL-12. Respective EC50 values ​​are shown in the table (ND=not determined). Analysis was performed based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirmed that the inducible IL-12 prodrugs were active and inducible. [Figure 16ZP] 1 is an image of an SDS-PAGE gel showing the results of a protein cleavage assay with elastase.

[0034] [Figure 17A] 1 is a graph showing the results of analyzing inducible IL-12 prodrugs in a syngeneic MC38 mouse tumor model. They show the average tumor volume over time for mice treated with each inducible IL-12 prodrug (5 μg, 50 μg, and 500 μg) administered every two weeks. The data show that tumor volume was inhibited over time in a dose-dependent manner. [Figure 17B] Graph showing group mean body weight over time. [Figure 17C] 1 is a graph showing the results of analyzing inducible IL-12 prodrugs in a syngeneic MC38 mouse tumor model. They show the average tumor volume over time for mice treated with each inducible IL-12 prodrug (5 μg, 50 μg, and 500 μg) administered every two weeks. The data show that tumor volume was inhibited over time in a dose-dependent manner. [Figure 17D] Graph showing group mean body weight over time. [Figure 17E]1 is a graph showing the results of analyzing inducible IL-12 prodrugs in a syngeneic MC38 mouse tumor model. They show the average tumor volume over time for mice treated with each inducible IL-12 prodrug (5 μg, 50 μg, and 500 μg) administered every two weeks. The data show that tumor volume was inhibited over time in a dose-dependent manner. [Figure 17F] Graph showing group mean body weight over time. [Figure 17G] 1 is a graph showing the results of analyzing inducible IL-12 prodrugs in a syngeneic MC38 mouse tumor model. They show the average tumor volume over time for mice treated with each inducible IL-12 prodrug (5 μg, 50 μg, and 500 μg) administered every two weeks. The data show that tumor volume was inhibited over time in a dose-dependent manner. [Figure 17H] Graph showing group mean body weight over time. [Figure 17I] 1 is a graph showing the results of analyzing inducible IL-12 prodrugs in a syngeneic MC38 mouse tumor model. They show the average tumor volume over time for mice treated with each inducible IL-12 prodrug (5 μg, 50 μg, and 500 μg) administered every two weeks. The data show that tumor volume was inhibited over time in a dose-dependent manner. [Figure 17J] Graph showing group mean body weight over time.

[0035] [Figure 18A] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18B] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18C] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18D] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18E] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18F]FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18G] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18H] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18I] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18J] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18K] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18L] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18M] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. [Figure 18N] FIG. 1 is a schematic diagram showing various inducible IL-12 prodrugs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] A. IL-12 Prodrugs The present disclosure relates to an inducible IL-12 polypeptide prodrug that contains attenuated IL-12 and has a longer half-life compared to naturally occurring IL-12. The IL-12 polypeptide prodrug disclosed herein comprises at least one polypeptide chain, and may optionally comprise two or more polypeptides. The two or more polypeptide chains disclosed herein are different, i.e., the complex may be a heterodimer, a heterotrimer, etc. The inducible IL-12 prodrug comprises a p35 IL-12 subunit, a p40 IL-12 subunit, a half-life extending element, an IL-12 blocking element, and a protease cleavable linker. The p35 subunit and the p40 subunit associate to form an IL-12 heterodimer that has intrinsic IL-12 receptor agonist activity. In the context of the inducible IL-12 prodrug, the activity of the IL-12 receptor agonist is attenuated and the circulating half-life is extended. The activity of the IL-12 receptor agonist is attenuated by the blocking element. The half-life extending element may also contribute to the attenuation, for example, through steric effects. The blocking element may block all or some of the receptor agonist activity of IL-12 by sterically blocking and / or non-covalently binding to IL-12 (e.g., p35, p40, or p35p40 complex). Upon cleavage of the protease-cleavable linker, a form of IL-12 is released from the inducible IL-12 prodrug that is active (e.g., more active than the inducible IL-12 prodrug). Typically, the released IL-12 is at least 10-fold more active than the inducible IL-12 prodrug. Preferably, the released IL-12 is at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 500 times, at least 1000 times, at least about 10,000 times, or more, more active than the inducible IL-12 prodrug.

[0037] The form of IL-12 released upon cleavage of the inducible IL-12 prodrug typically has a short half-life, often substantially similar to that of naturally occurring IL-12. Although the half-life of the inducible IL-12 prodrug is extended, toxicity is reduced or eliminated because circulating inducible IL-12 prodrug is attenuated and active IL-12 is targeted to the desired site (e.g., the tumor microenvironment).

[0038] It will be understood by those of skill in the art that the number of polypeptide chains and the location on the polypeptide chains of the p35 and p40 subunits, half-life extension elements, protease cleavable linker(s), and blocking elements (and components of such elements, e.g., VH or VL domains) can vary and are often a matter of design preference. All such variations are encompassed by the present disclosure.

[0039] In embodiments, the inducible IL-12 prodrug comprises two different polypeptide chains. Typically, the first polypeptide chain comprises p35 and the second polypeptide chain comprises p40. The p35 and p40 subunits associate to form a biologically active heterodimer. The p35p40 heterodimer complex can be covalently linked, for example, via a disulfide bond.

[0040] In embodiments, either the first or second polypeptide may comprise an IL-12 blocking element (e.g., an scFV that binds IL-12) operably linked to the IL-12 subunit via a protease-cleavable linker. The other polypeptide chain may further comprise a half-life extension element operably linked to the IL-12 subunit via a protease-cleavable linker. Preferably, the inducible IL-12 prodrug comprises one functional blocking element and one functional half-life extension element. For example, if the first polypeptide chain comprises an IL-12 blocking element, the second polypeptide chain does not comprise an IL-12 blocking element. In other embodiments, one polypeptide chain comprises either p35 or p40, and further comprises a half-life extension element and a blocking element, each of which is operably linked to p35 or p40 via a protease cleavable linker (e.g., one or more protease cleavable linkers), and the other polypeptide comprises a complementary IL-12 subunit (e.g., either p40 or p35). The IL-12 blocking element on the second polypeptide may be operably linked to the IL-12 subunit via a protease cleavable linker. Alternatively, the IL-12 blocking element may be operably linked to the half-life extension element via any protease cleavable linker. The protease cleavable linkers on the first and second polypeptide chains may be the same or different. Preferably, the protease cleavable linkers on the first and second polypeptide chains are the same. The blocking element of this inducible IL-12 prodrug may be a single chain antibody. Any single chain antibody that has binding specificity for IL-12 can be the blocking element. Preferably, the blocking element is an scFv.

[0041] The inducible IL-12 prodrugs disclosed herein preferably contain one half-life extending element and one blocking element, although such elements may contain two or more components present on the same or different polypeptide chains. To illustrate this, as disclosed and exemplified herein, the components of the blocking element may be present on separate polypeptide chains. For example, the first polypeptide chain may contain an antibody light chain (VL+CL) or a light chain variable domain (VL), and the second polypeptide may contain an antibody heavy chain Fab fragment (VH+CH1) or a heavy chain variable domain (VH) complementary to the VL+CL or VL on the first polypeptide. In such a situation, these components may associate within the inducible IL-12 prodrug to form an antigen binding site (e.g., Fab) that binds to IL-12 and attenuates the activity of IL-12.

[0042] In embodiments, the p35 subunit and the p40 subunit are located on the same polypeptide chain, and may be optionally linked via a protease-cleavable linker. In such embodiments of a two-chain or multi-chain prodrug, at least one of the half-life extension element, the blocking element, or the components of the half-life extension element or the blocking element is present on another polypeptide. For example, a first polypeptide includes p35 and p40 linked via an optionally cleavable polypeptide chain, and the other elements of the inducible IL-12 prodrug are located on a second polypeptide chain. In another example, the first polypeptide chain includes the p35 subunit, the p40 subunit, the half-life extension element, and a portion of an antibody light chain. The second polypeptide contains a portion of an antibody heavy chain that is complementary to the light chain of the antibody. The portion of the antibody light chain together with the complementary heavy chain associates in the inducible IL-12 prodrug to form a binding site for IL-12. In another example, the first polypeptide comprises a p35 subunit, a p40 subunit, a half-life extension element, and a portion of an antibody heavy chain. In this example, the second polypeptide contains a portion of an antibody light chain that is complementary to the antibody heavy chain. The portion of the antibody heavy chain together with the complementary light chain associates in the inducible IL-12 prodrug to form a binding site for IL-12. In these inducible IL-12 prodrugs, the p35 subunit and the p40 subunit can be operably linked via any protease cleavable linker. Preferably, the p35 subunit and the p40 subunit are operably linked with a non-cleavable linker.

[0043] In the inducible IL-12 prodrugs disclosed herein, the half-life extension element is preferably operably linked to either the p35 subunit or the p40 subunit via a protease cleavable linker. For example, the inducible IL-12 prodrug may comprise a first polypeptide in which p35 or p40 is operably linked to the half-life extension element via a protease cleavable linker. In another example, the inducible IL-12 prodrug may comprise a first polypeptide in which p35 or p40 is operably linked to the half-life extension element via a protease cleavable linker, and the half-life extension element is further operably linked to a blocking element (or a component of a blocking element) optionally via a protease cleavable linker. In such an exemplary embodiment, the inducible IL-12 prodrug comprises at least one additional polypeptide comprising an IL-12 subunit (p40 or p35) that is not present in the first polypeptide. Additional arrangements of the elements of the inducible IL-12 prodrug are envisioned and are encompassed by the present disclosure. For example, the blocking element can be operably linked to either the p35 subunit or the p40 subunit via a protease cleavable linker. One of the half-life extension element or the blocking element can be operably linked to the p35 subunit, and the other of the half-life or extension element or the blocking element can be operably linked to the p40 subunit. When the half-life extension element is operably linked to the p35 subunit, the blocking element can be operably linked to the p40 subunit. When the half-life extension element is operably linked to the p40 subunit, the blocking element can be operably linked to the p35 subunit. The blocking element of the inducible IL-12 prodrug is preferably a Fab.

[0044] The inducible IL-12 prodrug may comprise three polypeptide chains. Usually, one polypeptide chain contains either the p35 or p40 IL-12 subunit, but not both, a second polypeptide contains the other IL-12 subunit, and a third polypeptide contains at least a portion of the blocking element. If the IL-12 subunit on the first polypeptide is p35, the IL-12 subunit on the second polypeptide is p40. If the IL-12 subunit on the first polypeptide is p40, the IL-12 subunit on the second polypeptide is p35. When the polypeptide is expressed and folded, the p35 and p40 subunits can associate to form a biologically active heterodimer. The p35p40 heterodimer complex can be covalently linked, for example, via a disulfide bond.

[0045] In some embodiments, the first polypeptide, if present, may further comprise a half-life extension element operably linked to the IL-12 subunit via a protease cleavable linker. The second polypeptide may further comprise a portion of the blocking element, and the third polypeptide may comprise the remainder of the blocking element. In such an inducible IL-12 prodrug, the IL-12 blocking element may be an antigen-binding fragment (e.g., a Fab fragment) of an antibody formed upon interaction of polypeptide 2 and polypeptide 3. In embodiments, the second polypeptide may comprise at least an antigen-binding portion of an antibody light chain. Alternatively, the second polypeptide may comprise at least an antigen-binding portion of an antibody heavy chain. The antigen-binding portion of the antibody light chain or the antigen-binding portion of the heavy chain may be operably linked to the IL-12 subunit via a protease cleavable linker. In some embodiments, the second polypeptide may contain a half-life extension element. If the second polypeptide contains a half-life extension element, the first polypeptide does not contain a half-life extension element. The half-life extension element may be operably linked to the IL-12 subunit via a protease cleavable linker. Alternatively or additionally, the half-life extension element may be operably linked to a portion of the blocking element (e.g., the antigen-binding portion of the antibody light chain or the antigen-binding portion of the heavy chain) via any protease cleavable linker. When the half-life extension element is present and operably linked to the IL-12 subunit, the heavy or light chain of the antibody may be operably linked to the IL-12 subunit via a protease cleavable linker. Alternatively, when the half-life extension element is present and operably linked to the IL-12 subunit, the heavy or light chain of the antibody may be operably linked to the IL-12 subunit, optionally via a cleavable linker. The protease cleavable linkers on the first chain, the second chain, and / or the polypeptide chain may be the same or different.

[0046] Chimeric Compound 1 and Compounds 2, 3, 4, 5, and 6 are illustrative examples of inducible IL-12 prodrugs comprising two polypeptide chains for use in accordance with the present disclosure. Additional details regarding Chimeric Compound 1 and Compounds 2, 3, 4, 5, and 6 and their activities are disclosed in International Application No. PCT / US2021 / 33014.

[0047] Compounds 7, 8, 17, 18, 21-28, 34, and 35 are specific examples of inducible IL-12 prodrugs comprising one polypeptide chain for use according to the present disclosure. Compounds 9-13, 15, 19, 20, 29-31, and 36 are specific examples of inducible IL-12 prodrugs comprising two polypeptide chains for use according to the present disclosure. Compounds 14, 16, 32, and 33 are specific examples of inducible IL-12 prodrugs comprising three polypeptide chains for use according to the present disclosure. [Table 1-1] [Table 1-2]

[0048] As described above, IL-12 can be optionally a mutein. An IL-12 mutein retains the activity of IL-12, e.g., intrinsic IL-12 receptor agonist activity. The IL-12 subunits, p35 and / or p40, can be muteins.

[0049] The present invention also relates to certain single-chain IL-12 inducing polypeptides. The single-chain IL-12 polypeptides disclosed herein comprise IL-12, a blocking element, a half-life extension element, and a protease-cleavable linker. IL-12 has receptor agonist activity for the cognate IL-12 receptor. When the blocking element binds to IL-12, IL-12 receptor activating activity is attenuated. Upon cleavage of the protease-cleavable linker, active IL-12 polypeptide is released. Single-chain inducing IL-12 polypeptides are disclosed in International Application No. PCT / US2019 / 032320 and International Application No. PCT / US2019 / 032322.

[0050] B. Half-life extension elements Domains that increase the half-life of inducible IL-12 prodrugs are contemplated herein. Increasing the in vivo half-life of therapeutic molecules that have naturally short half-lives allows for more acceptable and manageable dosing regimens without sacrificing efficacy.

[0051] Half-life extending elements increase the in vivo half-life and alter the pharmacodynamics and pharmacokinetics of inducible IL-12 prodrugs. Without being bound by theory, half-life extending elements change the pharmacodynamic properties of inducible IL-12 prodrugs, including changes in tissue distribution, penetration, and diffusion. In some embodiments, half-life extending elements may improve tissue targeting, tissue penetration, diffusion in tissues, and improved efficacy compared to proteins without half-life extending elements. Without being bound by theory, an exemplary method of improving the pharmacokinetics of a polypeptide is by expressing elements within the polypeptide chain that bind to receptors that are recycled to the plasma membrane of cells rather than being degraded in lysosomes (e.g., FcRn receptors and transferrin receptors on endothelial cells). Three types of proteins, e.g., human IgG, HSA (or fragments), and transferrin, persist in human serum much longer than would be predicted by their size, which is a function of their ability to bind to receptors that are recycled rather than degraded in lysosomes. These proteins or fragments are usually linked to other polypeptides that retain FcRn binding and extend serum half-life. HSA can be directly linked to pharmaceutical compositions or linked via short linkers. Fragments of HSA can also be used. HSA and its fragments can function as both blocking and half-life extension elements. Human IgG and Fc fragments can also perform similar functions.

[0052] The serum half-life extending element can also be an antigen-binding polypeptide that binds to a protein with a long serum half-life, such as serum albumin, transferrin, etc. Examples of such polypeptides include antibodies and fragments thereof (e.g., polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single chain variable fragments (scFv), single domain antibodies (e.g., heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid nanobodies (VHH)), dAbs, and the like. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies (e.g., binding domains based on anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, kunitz domain peptides, monobodies, and other engineered scaffolds (e.g., SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds)). Further examples of antigen-binding polypeptides include ligands for a desired receptor, ligand-binding portions of receptors, lectins, and peptides that bind to or associate with one or more target antigens.

[0053] The half-life extending elements provided herein are preferably human serum albumin (HSA) binding domains and antigen-binding polypeptides that bind to human serum albumin or immunoglobulin Fc or fragments thereof.

[0054] The half-life extending element of an inducible IL-12 prodrug extends the half-life of the inducible IL-12 prodrug by at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days or more. In some embodiments, the half-life extending element extends the half-life of the inducible IL-12 prodrug by at least 2-3 days, 3-4 days, 4-5 days, 5-6 days, 6-7 days, 7-8 days or more.

[0055] C. Blocking Elements A blocking element can be any element that binds IL-12 and inhibits the ability of an inducible IL-12 prodrug to bind and activate its receptor. A blocking element can inhibit the ability of IL-12 to bind and / or activate its receptor, for example, by sterically blocking and / or by non-covalently binding to the IL-12 prodrug. The blocking elements disclosed herein can bind to p19, p35, p40, the p35p40 heterodimer complex, or the p19p40 heterodimer complex.

[0056] Examples of suitable blocking elements include full length or IL-12 binding fragments or muteins of the cognate receptor for IL-12. Antibodies and antigen-binding fragments thereof that bind IL-12 can also be used, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single chain variable fragments (scFv), single domain antibodies (e.g., heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid nanobodies (VHH), dAbs, etc.). Other suitable antigen-binding domains that bind IL-12 can also be used, including binding domains based on non-immunoglobulin proteins that mimic the binding and / or structure of antibodies (e.g., anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, kunitz domain peptides, monobodies, and other engineered scaffolds (e.g., SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds). Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block the binding of IL-12 to its cognate receptor. Advantageously, such moieties can also function as half-life extension elements. For example, peptides modified by conjugation to water-soluble polymers such as PEG can sterically inhibit or prevent the binding of cytokine to its receptor. Polypeptides or fragments thereof with long serum half-lives can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, etc., and fragments and muteins of such polypeptides.

[0057] A preferred IL-12 blocking element is a single chain variable fragment (scFv) or a Fab fragment. The scFv blocking element comprises the amino acid sequence set forth in SEQ ID NOs: 144-188. Alternatively, the Fab blocking element comprises the amino acid sequence set forth in SEQ ID NOs: 189-194. The IL-12 antibody fragments encompassed by SEQ ID NOs: 144-194 have been optimized to enhance the developability of inducible IL-12 prodrugs as disclosed herein.

[0058] Preferred antibody light chain blocking elements include SEQ ID NOs: 192-193. These preferred elements may be located on one polypeptide chain and the complementary antigen-binding portion of the heavy chain may be located on a second polypeptide chain. Preferred heavy chain blocking elements include SEQ ID NOs: 189-191 and 194. These preferred elements may be located on one polypeptide chain and the complementary light chain may be located on a second polypeptide chain. The light and heavy chains of the antibody together form the binding site for IL-12.

[0059] In some embodiments, an IL-12 blocking element comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to SEQ ID NOs: 144-194, e.g., over the entire length of SEQ ID NOs: 144-194. Typically, the amino acid sequences of the CDRs are not altered and amino acid substitutions are present in the framework regions.

[0060] The present disclosure also relates to functional variants of IL-12 blocking elements comprising SEQ ID NOs: 144-194. Functional variants of IL-12 blocking elements comprising SEQ ID NOs: 144-194 generally differ from SEQ ID NOs: 144-194 by one or a few amino acids (including substitutions, deletions, insertions, or any combination thereof) and substantially retain the ability to bind to an IL-12 polypeptide (e.g., the p35 subunit, the p40 subunit, or the p35p40 complex) and inhibit binding of IL-12 to its cognate receptor.

[0061] A functional variant may contain at least one or more amino acid substitutions, deletions, or insertions relative to an IL-12 blocking element comprising SEQ ID NO: 144-194. A functional variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to an IL-12 blocking element comprising SEQ ID NO: 144-194. In some preferred embodiments, a functional variant differs from an IL-12 blocking element comprising SEQ ID NO: 144-194 by less than 10, less than 8, less than 5, less than 4, less than 3, less than 2, or by 1 amino acid modification (e.g., amino acid substitution or deletion). In other embodiments, a functional variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NO: 144-194. The amino acid substitutions may be conservative or non-conservative, but are preferably conservative.

[0062] In other embodiments, the functional variant of the IL-12 blocking element may contain one, two, three, four, or five or more non-conservative amino acid substitutions compared to the IL-12 blocking element comprising SEQ ID NOs: 144-194. Non-conservative amino acid substitutions will be recognized by those skilled in the art. The functional variant of the separation portion preferably contains no more than one, two, three, four, or five amino acid deletions.

[0063] Also disclosed herein are inducible IL-12 prodrugs that contain a blocking element with specificity for IL-12 and that contain a half-life extending element. The blocking element is an antibody or antigen-binding fragment that has binding specificity for IL-12, specifically, the IL-12 subunit beta precursor (p40) as defined in SEQ ID NO: 421 as disclosed herein. The antibody or antigen-binding fragment comprises an antigen-binding domain that binds to the residues set forth in Table 2 of SEQ ID NO: 421. The present disclosure relates to antibodies or antigen-binding fragments that bind to an IL-12 epitope as defined by the amino acid residues set forth in Table 2, and inducible IL-12 prodrugs containing such antibodies or antigen-binding fragments, as well as the use of such antibodies or antigen-binding fragments for the preparation of inducible IL-12 prodrugs, or medicaments that contain such inducible IL-12 prodrugs.

[0064] [Table 2]

[0065] D. Protease-Cleavable Linkers As disclosed herein, the inducible IL-12 prodrug comprises one or more linker sequences. The linker sequence serves to provide flexibility between the polypeptides, for example, a blocking element can inhibit the activity of IL-12. The linker can be located between the IL-12 subunit, the half-life extension element, and / or the blocking element. As described herein, the inducible IL-12 prodrug comprises a protease-cleavable linker. The protease-cleavable linker can include one or more cleavage sites for one or more desired proteases. Preferably, the desired proteases are concentrated or selectively expressed in the desired target site of IL-12 (e.g., the tumor microenvironment). Thus, the inducible IL-12 prodrug is preferentially or selectively cleaved at the target site of the desired IL-12 activity.

[0066] Suitable linkers are typically less than about 100 amino acids. Such linkers can be of different lengths, such as 1 amino acid (e.g., Gly) to 30 amino acids, 1 to 40 amino acids, 1 to 50 amino acids, 1 to 60 amino acids, 1 to 70 amino acids, 1 to 80 amino acids, 1 to 90 amino acids, and 1 to 100 amino acids. In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acids in length. Preferred linkers are typically about 5 amino acids to about 30 amino acids.

[0067] Preferably, the length of the linker varies from 2 to 30 amino acids and is optimized for each condition so that the linker does not impose constraints on the conformation or interaction of the linking domains. In a preferred embodiment, the linker is cleavable by a cleavage agent (e.g., an enzyme). Preferably, the separation portion comprises a protease cleavage site. In some cases, the separation portion comprises one or more cleavage sites. The separation portion may comprise a single protease cleavage site. The separation portion may also comprise two or more protease cleavage sites. For example, two cleavage sites, three cleavage sites, four cleavage sites, five cleavage sites, or more. When the separation portion comprises two or more protease cleavage sites, the cleavage sites can be cleaved with the same protease or different proteases. A separation portion comprising two or more cleavage sites is called a "tandem linker". The two or more cleavage sites can be positioned in any desired orientation (including but not limited to one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, or one cleavage site followed by another cleavage site with intervening amino acids between the two cleavage sites).

[0068] Of particular interest in the present invention is disease-specific protease-cleavable linker.Protease-cleavable linker that is preferentially cleaved at desired location in the body, such as tumor microenvironment, relative to peripheral circulation is also preferred.For example, the rate at which protease-cleavable linker is cleaved in tumor microenvironment can be at least about 10 times, at least about 100 times, at least about 1000 times, or at least about 10,000 times faster at desired location in the body (e.g., in tumor microenvironment) compared with peripheral circulation (e.g., in plasma).

[0069] Proteases known to be associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine ​​proteases, aspartic acid proteases, threonine proteases, glutamic acid proteases, metalloproteases, aspartic peptide lyases, serum proteases, cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, cathepsin L, kallikrein, hKl, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidain, bromelain, cal Pain, caspase, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloprotease (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAPa), dipeptidyl peptidase, meprin, granzyme, and dipeptidyl peptidase IV (DPPIV / CD26). The protease capable of cleaving the linker amino acid sequence (which may be encoded by the chimeric nucleic acid sequences provided herein) may be selected from the group consisting of, for example, prostate specific antigen (PSA), matrix metalloproteinase (MMP), A discintigrin and metalloproteinase (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase. The MMP may be, for example, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 14 (MMP14).Additionally or alternatively, the linker can be cleaved by a cathepsin, e.g., cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, and / or cathepsin L. Preferably, the linker is cleavable by MMP14 or cathepsin L.

[0070] Proteases useful for cleaving the linker and for use in the inducible IL-12 prodrugs disclosed herein are shown in Table 3, and exemplary proteases and their cleavage sites are shown in Table 4. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

[0071] Exemplary protease-cleavable linkers include, but are not limited to, kallikrein-cleavable linkers, thrombin-cleavable linkers, chymase-cleavable linkers, carboxypeptidase A-cleavable linkers, cathepsin-cleavable linkers, elastase-cleavable linkers, FAP-cleavable linkers, ADAM-cleavable linkers, PR-3-cleavable linkers, granzyme M-cleavable linkers, calpain-cleavable linkers, matrix metalloproteinase (MMP)-cleavable linkers, plasminogen activator-cleavable linkers, caspase-cleavable linkers, tryptase-cleavable linkers, or tumor cell surface proteases. Specifically, MMP9-cleavable linkers, ADAM-cleavable linkers, CTSL1-cleavable linkers, FAPα-cleavable linkers, and cathepsin-cleavable linkers. Some preferred protease-cleavable linkers are cleaved by MMPs and / or cathepsins.

[0072] The separating moieties disclosed herein are typically less than 100 amino acids. Such separating moieties can be of different lengths, such as 1 amino acid (e.g., Gly) to 30 amino acids, 1 to 40 amino acids, 1 to 50 amino acids, 1 to 60 amino acids, 1 to 70 amino acids, 1 to 80 amino acids, 1 to 90 amino acids, and 1 to 100 amino acids. In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acids in length. Preferred linkers are typically about 5 amino acids to about 30 amino acids.

[0073] Preferably, the length of the linker varies from 2 to 30 amino acids and is optimized for each condition so that the linker does not impose constraints on the conformation or interactions of the linking domains.

[0074] In some embodiments, the separation portion comprises the sequence: GPAGLYAQ (SEQ ID NO: 195); GPAGMKGL (SEQ ID NO: 196); PGGPAGIG (SEQ ID NO: 197); ALFKSSFP (SEQ ID NO: 198); ALFFSSPP (SEQ ID NO: 199); LAQRLRSS (SEQ ID NO: 200); LAQKLKSS (SEQ ID NO: 201); GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (SEQ ID NO: 202); RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (SEQ ID NO: 203); SEQ ID NO:203);KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO:204);RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO:205);KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO:206);SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO:207);RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (SEQ ID NO:208);GGGALFKSSFPLAQKLKSSPGGP AGIGGGR (SEQ ID NO:209); RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (SEQ ID NO:210); RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (SEQ ID NO:211); RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO:212); GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO:213); GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO:214); RGGALFKSSFPLA QKLKSSGPAGLYAQGGK (SEQ ID NO: 215); RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (SEQ ID NO: 216); SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (SEQ ID NO: 217); KGGPGGPAGIGPLAQRLRSSALFKSSFPGR (SEQ ID NO: 218); KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (SEQ ID NO: 219); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (SEQ ID NO: 220).

[0075] Certain preferred separation portions include the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). The separation portions disclosed herein may include one or more cleavage motifs or functional variants, which may be the same or different. The separation portion may include one, two, three, four, five, or more cleavage motifs or functional variants. A separation portion that includes 30 amino acids may contain two cleavage motifs or functional variants, three or more cleavage motifs or functional variants, or more. A "functional variant" of a separation portion retains the ability to be cleaved with high efficiency at the target site (e.g., a tumor microenvironment that expresses high levels of proteases) and is not cleaved or is cleaved with low efficiency in the periphery (e.g., serum). For example, functional variants retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation portion comprising any one of SEQ ID NOs: 195-220 or 447-448.

[0076] The separation portion comprising multiple cleavage motifs may be selected from SEQ ID NOs: 195-201 or 447-448 and combinations thereof. Preferred separation portions comprising multiple cleavage motifs comprise amino acids selected from SEQ ID NOs: 202-220.

[0077] The separation portion may contain both ALFKSSFP (SEQ ID NO: 198) and GPAGLYAQ (SEQ ID NO: 195). The separation portion may contain two cleavage motifs, each having the sequence GPAGLYAQ (SEQ ID NO: 195). Alternatively or additionally, the separation portion may contain two cleavage motifs, each having the sequence ALFKSSFP (SEQ ID NO: 198). The separation portion may contain a third cleavage motif, which may be the same or different.

[0078] In some embodiments, the separated portion comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NO:195-220 or SEQ ID NO:447-448 over the entire length of SEQ ID NO:195-220 or SEQ ID NO:447-448.

[0079] The present disclosure also relates to functional variants of isolated portions comprising SEQ ID NOs: 195-220 or 447-448. Functional variants of isolated portions comprising SEQ ID NOs: 195-220 or 447-448 generally differ from SEQ ID NOs: 195-220 or 447-448 by one or a few amino acids (including substitutions, deletions, insertions, or any combination thereof) and substantially retain their ability to be cleaved by a protease.

[0080] A functional variant may include at least one or more amino acid substitutions, deletions, or insertions relative to the isolated portion including SEQ ID NO: 195-220 or 447-448. A functional variant may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the isolated portion including SEQ ID NO: 195-220 or 447-448. In some preferred embodiments, a functional variant differs from the isolated portion including SEQ ID NO: 195-220 by less than 10, less than 8, less than 5, less than 4, less than 3, less than 2, or one amino acid modification (e.g., amino acid substitution or deletion). In other embodiments, a functional variant may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NO: 195-220 or 447-448. The amino acid substitutions may be conservative or non-conservative, but are preferably conservative.

[0081] In other embodiments, the functional variant of the separated portion may contain one, two, three, four, or five or more non-conservative amino acid substitutions compared to the separated portion containing SEQ ID NO: 195-220 or 447-448. Non-conservative amino acid substitutions can be recognized by those skilled in the art. The functional variant of the separated portion preferably contains no more than one, two, three, four, or five amino acid deletions.

[0082] The amino acid sequences disclosed in the separation portion can be described by their relative linear positions in the separation portion with respect to the scissile bond. As will be appreciated by those skilled in the art, a separation portion that includes an eight amino acid protease substrate (e.g., SEQ ID NOs: 195-201 or 447-448) contains amino acids at positions P4, P3, P2, P1, P1', P2', P3', P4', with the scissile bond being between P1 and P1'. For example, the amino acid positions of a separation portion that includes the sequence GPAGLYAQ (SEQ ID NO: 195) can be described as follows: [Table 6]

[0083] The amino acid positions of the split portion comprising the sequence ALFKSSFP (SEQ ID NO: 198) can be written as follows: [Table 7]

[0084] Preferably, the amino acids surrounding the cleavage site (eg, positions P1 and P1' of SEQ ID NOs: 195-201 or 447-448) are not substituted.

[0085] In an embodiment, the separated portion comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) or a functional variant of SEQ ID NO: 195 or a functional variant of SEQ ID NO: 198. As described herein, a functional variant of PAGLYAQ (SEQ ID NO: 447) or ALFKSSFP (SEQ ID NO: 198) may include one or more amino acid substitutions and substantially retain the ability to be cleaved by a protease. Specifically, a functional variant of GPAGLYAQ (SEQ ID NO: 195) is cleaved by MMP14, and a functional variant of ALFKSSFP (SEQ ID NO: 198) is cleaved by capthepsin L (CTSL1). The functional variant also retains the ability to be cleaved with high efficiency at the target site (e.g., a tumor microenvironment expressing high levels of proteases). For example, a functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retains at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95%, or more of the cleavage efficiency of a separation portion comprising the amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.

[0086] Preferably, a functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) comprises no more than 1, 2, 3, 4, or 5 conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). Preferably, the amino acids at positions P1 and P1' are not substituted. The amino acids at positions P1 and P1' of SEQ ID NO: 195 are G and L, and the amino acids at positions P1 and P1' of SEQ ID NO: 198 are K and S.

[0087] Functional variants of GPAGLYAQ (SEQ ID NO: 195) may preferably include one or more of the following: a) an arginine amino acid substitution at position P4; b) a leucine, valine, asparagine, or proline amino acid substitution at position P3; c) an asparagine amino acid substitution at position P2; d) a histidine, asparagine, or glycine amino acid substitution at position P1; e) an asparagine, isoleucine, or leucine amino acid substitution at position P1'; f) a tyrosine or arginine amino acid substitution at position P2'; g) a glycine, arginine, or alanine amino acid substitution at position P3'; h) a serine, glutamine, or lysine amino acid substitution at position P4'. In functional variants of GPAGLYAQ (SEQ ID NO: 195), the following amino acid substitutions are disfavored: a) arginine or isoleucine at position P3; b) alanine at position P2; c) valine at position P1; d) arginine, glycine, asparagine, or threonine at position P1'; e) aspartic acid or glutamic acid at position P2'; f) isoleucine at position P3'; g) valine at position P4'. In some embodiments, functional variants of GPAGLYAQ (SEQ ID NO: 195) do not include amino acid substitutions at positions P1 and / or P1'.

[0088] The amino acid substitution of a functional variant of GPAGLYAQ (SEQ ID NO: 195) preferably comprises an amino acid substitution at position P4 and / or P4'. For example, a functional variant of GPAGLYAQ (SEQ ID NO: 195) may comprise a leucine at position P4, or a serine, glutamine, lysine, or phenylalanine at position P4. Alternatively, or in addition, a functional variant of GPAGLYAQ (SEQ ID NO: 195) may comprise a glycine, phenylalanine, or proline at position P4'.

[0089] In some embodiments, amino acid substitutions at positions P2 or P2' of GPAGLYAQ (SEQ ID NO: 195) are not preferred.

[0090] In some embodiments, a functional variant of GPAGLYAQ (SEQ ID NO: 195) comprises an amino acid sequence selected from SEQ ID NOs: 221-295. Particular functional variants of GPAGLYAQ (SEQ ID NO: 195) include GPLGLYAQ (SEQ ID NO: 259), and GPAGLKGA (SEQ ID NO: 249).

[0091] The functional variant of LFKSSFP (SEQ ID NO: 448) preferably contains a hydrophobic amino acid substitution. The functional variant of LFKSSFP (SEQ ID NO: 448) may preferably contain one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, phenylalanine at position P3; (c) arginine, leucine, alanine, glutamine, or histatine at position P2; (d) phenylalanine, histidine, threonine, alanine, or glutamine at position P1; (e) P1' (f) phenylalanine, leucine, isoleucine, lysine, alanine, glutamine, or proline at position P2'; (g) phenylalanine, leucine, glycine, serine, valine, histidine, alanine, or asparagine at position P3'; and phenylalanine, histidine, glycine, alanine, serine, valine, glutamine, lysine, or leucine.

[0092] Inclusion of aspartic acid and / or glutamic acid in functional variants of SEQ ID NO: 448 is generally not preferred and is avoided. The following amino acid substitutions are also not preferred in functional variants of LFKSSFP (SEQ ID NO: 448): (a) alanine, serine, or glutamic acid at position P3; (b) proline, threonine, glycine, or aspartic acid at position P2; (c) proline at position P1; (d) proline at position P1'; (e) glycine at position P2'; (f) lysine or glutamic acid at position P3'; (g) aspartic acid at position P4'.

[0093] The amino acid substitution of a functional variant of LFKSSFP (SEQ ID NO: 448) preferably comprises an amino acid substitution at position P4 and / or P1. In some embodiments, an amino acid substitution of a functional variant of LFKSSFP (SEQ ID NO: 448) at position P4' is not preferred.

[0094] In some embodiments, a functional variant of LFKSSFP (SEQ ID NO: 448) comprises an amino acid sequence selected from SEQ ID NOs: 296 to 374. Particular functional variants of LFKSSFP (SEQ ID NO: 448) include ALFFSSPP (SEQ ID NO: 199), ALFKSFPP (SEQ ID NO: 346), ALFKSLPP (SEQ ID NO: 347), ALFKHSPP (SEQ ID NO: 335), ALFKSIPP (SEQ ID NO: 348), ALFKSSLP (SEQ ID NO: 356), or SPFRSSRQ (SEQ ID NO: 297).

[0095] The detached moieties disclosed herein may form stable complexes with the amino acid sequences (e.g., domains) to which they are attached under physiological conditions, while being cleaved by proteases. For example, the detached moieties are stable (e.g., not cleaved or cleaved with low efficiency) in circulation, but are cleaved with higher efficiency at the target site (i.e., tumor microenvironment). Thus, the fusion polypeptides comprising the linkers disclosed herein may, if desired, have an extended circulating half-life and / or a lower biological activity in circulation, compared to the components of the fusion polypeptide as separate molecular entities. However, when the linker can be efficiently cleaved to release the components attached by the linker at the desired location (e.g., tumor microenvironment), the half-life and biological activity of the components as molecular entities are restored or nearly restored.

[0096] The separated portion desirably remains stable in circulation for at least 2 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or more.

[0097] In some embodiments, the separation portion is cleaved in circulation by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% compared to the target position.The separation portion is also stable in the absence of enzymes capable of cleaving the linker.However, when exposed to a suitable enzyme (i.e., a protease), the separation portion is cleaved, resulting in the separation of the linked domains.

[0098] E. Pharmaceutical Compositions Also provided herein is a pharmaceutical composition comprising an inducible IL-12 prodrug described herein, a vector comprising a polynucleotide encoding an inducible IL-12 prodrug, or a host cell transformed with this vector, and at least one pharma- ceutically acceptable carrier.

[0099] Provided herein is a pharmaceutical formulation or composition containing an inducible IL-12 prodrug as described herein and a pharma- ceutically acceptable carrier. The compositions containing the inducible IL-12 prodrug as described herein are suitable for administration in vitro or in vivo. The term "pharma- ceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the components and is not toxic to the subject to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, emulsions (e.g., water, oil / water emulsions), various types of wetting agents, and sterile solutions. Such carriers can be formulated in conventional manner and administered to the subject in an appropriate dosage. Preferably, the compositions are sterile. These compositions may also contain auxiliary agents, such as preservatives, emulsifying agents, and dispersing agents. Various antibacterial and antifungal agents can be included to ensure that the action of microorganisms is prevented.

[0100] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21 st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to make the formulation isotonic, but the formulation can be made hypertonic or hypotonic, if necessary. Examples of pharma- cetically acceptable carriers include, but are not limited to, sterile water, saline, buffers (e.g., Ringer's solution), and dextrose solution. The pH of the solution is generally about 5 to about 8, or about 7 to 7.5. Other carriers include sustained release formulations, such as semipermeable matrices of solid hydrophobic polymers containing an immunogenic polypeptide. The matrices are in the form of shaped articles (e.g., 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 preferable. The carrier is one suitable for administering a nucleic acid sequence encoding IL-12 or an inducible IL-12 prodrug to a human or other subject.

[0101] In some embodiments of the pharmaceutical composition, the inducible IL-12 prodrug described herein is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a fullerene, a liquid crystal, a liposome, a quantum dot, a superparamagnetic nanoparticle, a dendrimer, or a nanorod. In other embodiments of the pharmaceutical composition, the inducible IL-12 prodrug is bound to a liposome. In some cases, the inducible IL-12 prodrug is conjugated to the surface of the liposome. In some cases, the inducible IL-12 prodrug is encapsulated within the shell of the liposome. In some cases, the liposome is a cationic liposome.

[0102] The inducible IL-12 prodrugs described herein are intended for use as medicines. Administration can be performed in a variety of ways, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the route of administration will depend on the type of treatment and the type of compound contained in the pharmaceutical composition. The dosing schedule will be determined by the attending physician and other clinical factors. The dosage per patient will vary depending on many factors, such as the patient's size, body surface area, age, sex, the specific compound to be administered, the time and route of administration, the type of treatment, general health, and other drugs administered concomitantly. An "effective dose" refers to the amount of active ingredient that is sufficient to affect the course and severity of the disease, leading to the reduction or remission of such pathology, and can be determined using known methods.

[0103] Optionally, the inducible IL-12 prodrug or the nucleic acid sequence encoding the inducible IL-12 prodrug is administered by a vector. There are numerous compositions and methods that can be used to deliver nucleic acid molecules and / or polypeptides to cells, either in vitro or in vivo, for example, via expression vectors. These methods and compositions can be primarily categorized into two classes: viral-based delivery systems and non-viral-based delivery systems. Such methods are well known in the art and can be readily adapted for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo (e.g., to generate cell lines that express and preferably secrete the encoded chimeric polypeptide, or to deliver the nucleic acid therapeutically to a subject). The components of the IL-12 polypeptides disclosed herein are typically operably linked in frame to encode a fusion protein.

[0104] As used herein, a plasmid or viral vector is an agent that transports the disclosed nucleic acid into a cell without degradation and includes a promoter that causes expression of the nucleic acid molecule and / or polypeptide in the cell to which it is delivered. Viral vectors are, for example, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus, Sindbis, and other RNA viruses, including those viruses with an HIV backbone. Also preferred are any virus families that share the properties of these viruses that make them suitable for use as vectors. In general, retroviral vectors and methods for their production are described in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). The construction of replication-deficient adenoviruses has been described in the following: (Berkner et al., J. Virol. 61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virol. 57:267-74 (1986); Davidson et al., J. Virol. 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)). The use and advantage of these viruses as vectors is that they can replicate within the initially infected cell, but are unable to form new initially infectious viral particles, thus limiting the extent to which they can spread to other cell types. Recombinant adenoviruses have been shown to achieve high efficiency following direct in vivo delivery to airway epithelia, hepatocytes, vascular endothelium, CNS parenchyma, and many other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.

[0105] The provided inducible IL-12 prodrugs and / or nucleic acid molecules can be delivered via virus-like particles. Virus-like particles (VLPs) consist of viral protein(s) derived from viral structural proteins. Methods for making and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0106] The inducible IL-12 prodrugs disclosed herein can be delivered in subviral dense particles (DBs). DBs transport proteins to target cells by membrane fusion. Methods for making and using DBs are described, for example, in Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003). The provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO2006 / 110728.

[0107] Non-viral based delivery methods may include expression vectors that contain nucleic acid sequences encoding nucleic acid molecules and polypeptides, the nucleic acid being operably linked to an expression control sequence. Suitable vector backbones include those routinely used in the art (e.g., plasmids, artificial chromosomes, BACs, YACs, or PACs). Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clonetech (Pallo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen / Life Technologies (Carlsbad, CA). Vectors usually contain one or more regulatory regions. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to produce inducible IL-12 prodrugs by expressing them in suitable host cells (e.g., CHO cells).

[0108] Preferred promoters controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, such as the genomes of viruses (e.g., polyoma, Simian Virus 40 (SV40), adenovirus, retrovirus, Hepatitis B virus, and most preferably, cytomegalovirus (CMV)), or heterologous mammalian promoters (e.g., the β-actin promoter or the EF1α promoter), or hybrid or chimeric promoters (e.g., the CMV promoter fused to the β-actin promoter). Of course, promoters from the host cell or related species are also useful herein.

[0109] Enhancers generally refer to DNA sequences that function at any distance from the transcription start site and can be located either 5' or 3' of a transcription unit. Additionally, enhancers can be located within introns and within the coding sequence itself. They are usually 10-300 base pairs (bp) in length and function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers may also contain response elements that mediate regulation of transcription. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), and eukaryotic viral enhancers will usually be used for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0110] The promoter and / or enhancer may be inducible (e.g., chemically or physically regulated). Chemically regulated promoters and / or enhancers may be regulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically regulated promoters and / or enhancers may be controlled, for example, by environmental factors (e.g., temperature and light). Optionally, the promoter and / or enhancer region may function as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region may be cell type-specifically active. Optionally, in certain vectors, the promoter and / or enhancer region may be active in all eukaryotic cells, regardless of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the β-actin promoter, the EF1α promoter, and retroviral long terminal repeats (LTRs).

[0111] A vector may also include, for example, an origin of replication and / or a marker. A marker gene may confer a selectable phenotype (e.g., antibiotic resistance) to a cell. A marker product is used to determine whether a vector has been delivered to a cell and, once delivered, whether it is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully introduced into a mammalian host cell, the transformed mammalian host cell can survive if placed under selection pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector may include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences (e.g., GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak, New Haven, Conn.) sequences) are usually expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at the carboxyl or amino terminus.

[0112] F. Therapeutic Applications Also provided herein are methods and uses for treating a disease, disorder, or condition associated with a target antigen, comprising administering an inducible IL-12 prodrug described herein to a subject in need thereof. The disease, disorder, or condition includes, but is not limited to, cancer, inflammatory disease, immune disease, autoimmune disease, infectious disease (i.e., bacterial, viral, or parasitic disease). Preferably, the disease, disorder, or condition is cancer.

[0113] Any suitable cancer can be treated with the inducible IL-12 prodrugs provided herein. Examples of suitable cancers include, for example, 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, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, neuroepithelioma. , fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular 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 cancer, liver cancer, lobular carcinoma, lung cancer, macroglobulinemia, malignant Fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary site, midline carcinoma with NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid carcinoma, 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 carcinoma, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor. In an embodiment, the cancer is melanoma or breast cancer.

[0114] In some embodiments, provided herein are methods of enhancing an immune response in a subject in need thereof by administering to the subject an effective amount of an inducible IL-12 prodrug provided herein. Enhanced immune response may prevent, delay, or treat the onset of cancer, tumor, or viral disease. Without being bound by theory, the inducible IL-12 prodrug enhances the immune response by activating innate and adaptive immunity. In some embodiments, the methods described herein enhance the activity of natural killer cells and T lymphocytes. In some embodiments, the inducible IL-12 prodrug provided herein may induce IFNγ release from natural killer cells and CD4+ and CD8+ T cells.

[0115] The present disclosure relates to a method of selectively activating tumor infiltrating lymphocytes comprising administering to a subject in need thereof an effective amount of an inducible interleukin-12 (IL-12) prodrug as described herein. Typically, the inducible IL-12 prodrug is administered systemically and is activated upon cleavage by proteases that have higher activity in the tumor microenvironment than elsewhere, resulting in a significantly higher frequency of IFN-gamma and / or granzyme-producing CD8+ T cells.

[0116] The present disclosure relates to a method of inducing immune memory against a tumor comprising administering to a subject in need thereof an effective amount of an inducible interleukin-12 (IL-12) prodrug as described herein. Typically, the inducible IL-12 prodrug is administered systemically and is activated upon cleavage by a protease that has higher activity in the tumor microenvironment than in other regions.

[0117] The present disclosure relates to a method of selectively activating effector CD8+ T cells in a tumor microenvironment comprising administering to a subject in need thereof an effective amount of an inducible interleukin-12 (IL-12) prodrug as described herein. Typically, the inducible IL-12 prodrug is administered systemically and activated upon cleavage by a protease that has higher activity in the tumor microenvironment than elsewhere, resulting in a significantly higher frequency of TNF- and / or IFN-gamma-producing CD8+ T cells in the tumor compared to peripheral tissues.

[0118] The method may further include administration of one or more additional agents to treat the cancer (e.g., chemotherapeutic agents (e.g., adriamycin, cervidin, bleomycin, alkeran, velban, oncovin, fluorouracil, thiotepa, methotrexate, bisantrene, noanthrone, tiguanine, citalibine, procarabidine), immuno-oncology agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), cell therapy (e.g., CAR-T, T cell therapy), oncolytic viruses, etc.). Non-limiting examples of anti-cancer drugs that can be used include: acivicin; aclarubicin; acodazole hydrochloride; acronine; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; visnafide dimesylate; bizeresin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calsterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; ciloremycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide. ;Cytarabine;Dacarbazine;Dactinomycin;Daunorubicin hydrochloride;Decitabine;Dexorumaplatin;Desaguanine;Desaguanine mesylate;Diazicon;Docetaxel;Doxorubicin;Doxorubicin hydrochloride;Droloxifene;Droloxifene citrate;Dromostanolone propionate;Duazomycin;Edatrexate;Eflornithine hydrochloride;Elsamitrucin;Enloplatin;Empromer t;epipropizine;epirubicin hydrochloride;elbrozole;esorubicin hydrochloride;estramustine;estramustine sodium phosphate;etanidazole;etoposide;etoposide phosphate;etoprine;fadrozole hydrochloride;fazarabine;fenretinide;floxuridine;fludarabine phosphate;fluorouracil;flurocitabine;foskidone;fostriecin sodium;gemcitabine;gemcitabine hydrochloride;Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Irmofosine; Interleukin II (including recombinant interleukin II; or rIL2); Interferon alfa-2a; Interferon alfa-2b; Interferon alfa-nl; Interferon alfa-n3; Interferon beta-Ia; Interferon gamma-Ib; Iproplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprolide acetate acid salt;liarozole hydrochloride;lometrexol sodium;lomustine;losoxantrone hydrochloride;masoprocol;maytansine;mechlorethamine hydrochloride;megestrol acetate;melengestrol acetate;melphalan;menogaril;mercaptopurine;methotrexate;methotrexate sodium;metoprine;meturedepa;mitindomide;mitocalcin;mitochromine;mitogillin;mitomarcine;mitomycin;mitospel;mitotane;mitoxantane Santron hydrochloride;Mycophenolic acid;Nocodazole;Nogalamycin;Ormaplatin;Oxisuran;Paclitaxel;Pegaspargase;Periomycin;Pentamustine;Peplomycin sulfate;Perfosfamide;Pipobroman;Piposulfan;Piroxantrone hydrochloride;Plicamycin;Promestane;Porfimer sodium;Porfiromycin;Prednimustine;Procarbazine hydrochloride;Puromycin;Puromycin hydrochloride;Pi Lazofurin;Riboprin;Rogletimide;Safingol;Safingol hydrochloride;Semustine;Simtrazene;Sparfosate sodium;Sparsomycin;Spirogermanium hydrochloride;Spiromustine;Spiroplatin;Streptonigrin;Streptozocin;Sulofenur;Tallysomycin;Tecogalan sodium;Tegafur;Teroxantrone hydrochloride;Temoporfin;Teniposide;Teroxylon;Testolactone;Thiamiprine;Thioguanine;Thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestron acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tuburozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglicinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinzolidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride;

[0119] An IL-12 prodrug can be administered to a subject in need thereof in combination with an immune checkpoint inhibitor. Immune checkpoint proteins include, for example, PD-1 (binds to ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273)), CTLA-4 (CD152) (binds to B7-1 (CD80) and B7-2 (CD86)), LAG3 (CD223) (binds to galectin 3, LSECtin, and FGL1); TIM3 (HAVCR2) (binds to ligands Ceacam1 and Galectin 2); binds to cutin 9); TIGIT (VSTM3, WUCAM) (binds to CD112 and CD155); BTLA (CD272) (binds to HVEM (TNFRSF14)); B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD160 (BY55) (binds to HVEM); CD134 (TNRFSR4, OX40) (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 (LIBATYO), nivolumab (OPDIVO), camrelizumab, tislelizumab, toripalimab, and sintilimab (TYVYT), the anti-PD-L1 antibodies avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ), and the anti-CTLA-4 antibody ipilimumab (YERVOY).

[0120] In some embodiments of the methods described herein, the inducible IL-12 prodrug is administered in combination with an agent for the treatment of a particular disease, disorder, or condition. The agent includes, but is not limited to, an antibody, a small molecule (e.g., a chemotherapeutic agent), a hormone (steroid, peptide, etc.), radiation therapy (direct delivery of gamma rays, C-rays, and / or radioisotopes, microwave, UV radiation, etc.), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapy-related therapies. In some embodiments, the inducible IL-12 prodrug is administered in combination with an antidiarrheal, antiemetic, analgesic, and / or nonsteroidal anti-inflammatory agent.

[0121] G. Definition All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent that material incorporated by reference contradicts or is inconsistent with this specification, the specification will take precedence over any such material. Citation of any reference herein is not an admission that such reference is prior art to this disclosure. Ranges of values, when expressed, include embodiments using any specific value within the range. Furthermore, reference to values ​​specified within a range includes any and all values ​​within that range. All ranges are inclusive of endpoints and are combinable. When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. The use of "or" will mean "and / or" unless the particular context of its use dictates otherwise.

[0122] Throughout the specification and claims, various terms are used in connection with aspects of the present specification. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein. The techniques and procedures described or referenced herein are generally well understood and commonly used by those of skill in the art using conventional methodology, such as the widely used molecular cloning methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4 th 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-specified protocols and conditions unless otherwise noted.

[0123] As used in this document, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. Words such as "including," "for example," and the like are intended to mean including but not limited to, unless the context specifically indicates otherwise.

[0124] Unless otherwise indicated, the terms "at least," "less than," and "about," or similar terms, preceding a series or range of elements, should be understood to refer to every element in the series or range. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0125] As used herein, the terms "activatable," "activate," "induce," and "inducible" refer to a polypeptide complex having an attenuated active form (e.g., attenuated receptor binding and / or agonist activity) as well as an activated form. The polypeptide complex is activated upon protease cleavage of the linker, which releases the blocking element and the half-life extension element from the polypeptide complex. The induced / activated polypeptide complex may bind with high affinity / avidity to the IL-12 receptor.

[0126] The terms "antibody" and "immunoglobulin" are used interchangeably herein. Antibody or immunoglobulin as used herein refers to an immunoglobulin molecule consisting of two heavy (H) chains. Typically, mammalian (e.g., human, rodent, and monkey) antibodies comprise four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CHI, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity determining regions (CDRs)) flanked by more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antibody may include, for example, a monoclonal antibody, a recombinantly produced antibody, a single specific antibody, a multispecific antibody (including a bispecific antibody), a human antibody, a humanized antibody, a chimeric antibody, an immunoglobulin, a synthetic antibody, or a tetrameric antibody comprising two heavy chain molecules and two light chain molecules. Those skilled in the art will recognize that other forms of antibodies exist, such as camelid and shark antibodies.

[0127] The term "attenuated" as used herein refers to an IL-12 receptor agonist that has reduced receptor agonist activity compared to the naturally occurring agonist of the IL-12 receptor. An attenuated IL-12 agonist may have 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 naturally occurring agonist of the receptor. When an IL-12 polypeptide complex containing IL-12 described herein is described as having "attenuated" or "attenuated activity", it means that the IL-12 polypeptide complex is an attenuated IL-12 receptor agonist.

[0128] The term "cancer" refers to a physiological condition in mammals in which a population of cells is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or specific morphological features. In many cases, cancer may take the form of a tumor or mass, but may exist alone in a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. The term cancer includes any type of cancer and metastasis, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer (e.g., triple-negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. Triple-negative breast cancer refers to breast cancer in which the gene expression of estrogen receptor (ER), progesterone receptor (PR), and Her2 / neu is negative.

[0129] "Conservative" amino acid substitutions as used herein generally refer to the substitution of one amino acid residue for another within a recognized group, which may change the structure of the peptide, but substantially retain the biological activity of the peptide. Conservative substitutions of amino acids are known to those skilled in the art. Conservative substitutions of amino acids may include, but are not limited to, substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. For example, those skilled in the art can reasonably expect that a single substitution of leucine with isoleucine or valine, a single substitution of aspartate with glutamate, a single substitution of threonine with serine, or a similar substitution of an amino acid with a structurally related amino acid will not significantly affect the biological activity of the resulting molecule.

[0130] As used herein, the term "half-life extending element" in the context of the polypeptide complexes disclosed herein refers to a chemical element, preferably a polypeptide, that extends serum half-life and improves pK, for example, by modifying its size (e.g., above the renal filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and excretion.

[0131] As used herein, the term "operably linked" in the context of a polypeptide complex refers to an orientation of the components of the polypeptide complex that allows the components to function as intended. For example, a polypeptide comprising an IL-12 subunit and an IL-12 blocking element is operably linked in a polypeptide complex with a protease-cleavable linker if the IL-12 blocking element is capable of inhibiting the IL-12 receptor activating activity of the IL-12 polypeptide, but upon cleavage of the protease-cleavable linker, e.g., the IL-12 blocking element diffuses away from the IL-12, thereby reducing or eliminating inhibition of the IL-12 receptor activating activity of the IL-12 polypeptide by the IL-12 blocking element.

[0132] As used herein, the terms "peptide," "polypeptide," or "protein" are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used interchangeably herein to refer to an amino acid sequence. It should be understood that the term polypeptide is not used herein to imply a particular size or number of amino acids comprising the molecule, and that the peptides of the present invention may contain several amino acid residues or more.

[0133] The term "subject" herein refers to any animal, e.g., any mammal, including but not limited to, humans, non-human primates, rodents, etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0134] As used herein, the term "therapeutically effective amount" refers to an amount of a compound (i.e., an IL-12 polypeptide complex) described herein that is sufficient to achieve a desired pharmacological or physiological effect under the conditions of administration. For example, a "therapeutically effective amount" may be an amount sufficient to reduce the signs or symptoms of a disease or condition (e.g., a tumor). Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject. The therapeutically effective amount of a pharmaceutical composition may vary depending on factors (e.g., the individual's condition, age, sex, and weight) and the ability of the pharmaceutical composition to elicit a desired response in an individual. Based on these and other considerations, an ordinary skilled clinician can determine the appropriate amount to administer to achieve the desired therapeutic effect.

[0135] 5. Equivalent It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein will be apparent and may be made using suitable equivalents without departing from the scope of the disclosure or embodiments. While certain compounds and methods have been described in detail, these 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. EXAMPLES

[0136] The following are examples of methods and compositions of the present invention. Given the summary provided herein, it will be understood that other various embodiments may be practiced.

[0137] In the mouse studies described herein, chimeric compound 1, an IL-12 prodrug that contains mouse IL-12 p35 and human IL-12 p40 and is therefore active in mouse models, was used as a surrogate for a human IL-12 prodrug that contains human p35 and p40. Chimeric IL-12, which contained mouse p35 and human p40, was used as a control. Chimeric compound 1 contains mouse IL-12p 35 and is a surrogate for compound 36, which contains human IL-12p 35 and is otherwise identical to chimeric compound 1.

[0138] Example 1. 1.1 Materials and Methods 1. HEK-Blue IL-12 Reporter Assay HEK-Blue IL-12 cells (InvivoGen) in suspension were plated at a density of 50,000 cells / well in medium with or without 15 or 40 mg / ml human serum albumin (HSA) and stimulated with serial dilutions of recombinant hIL-12, chimeric IL-12 (mouse p35 / human p40), activated chimeric IL-12, or activated hIL-12 for 20–24 h at 37°C and 5% CO2. The activity of uncleaved and cleaved activatable hIL-12 was tested. Cleaved inducible hIL-12 was generated upon incubation with active MMP9 or CTSL-1. IL-12 activity was assessed by quantifying secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric-based assay. Results confirmed that the IL-12 fusion proteins were active and inducible. The results are shown in FIG.

[0139] 2. Cell lines All cell lines were cultured and maintained by Charles River Laboratories (Morrisville, NC and Worcester, MA) according to ATCC guidelines, and the culture period was ≤2 weeks. Frozen cells were thawed and passaged 1–3 times before implantation. B16-F10, CT26, and EMT-6 cell lines were cultured in RPMI-1640 containing L-glutamine (Gibco, 11875-085) with 10% heat-inactivated fetal bovine serum (Gibco, 35-015-CV), and MC38 was cultured in Dulbecco's modified Eagle's medium (Gibco, 1966-025) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, 16000-044). Before tumor implantation, cells were washed twice with PBS and counted.

[0140] 3. MC38 Model Tumor Implantation All mouse in vivo experiments were performed at Charles River Laboratories (Morrisville, NC and Worcester, MA) with Institutional Animal Care and Use Committee (IACUC) approval and in accordance with current regulations and standards, as well as the NIH. One day prior to tumor cell implantation, the flanks of 6-8 week-old female C57Bl / 6 mice at Charles River Laboratories were shaved. A total of 5 × 10 5 MC38 cells were injected subcutaneously and tumor growth was monitored. Additional mice were implanted to have tumors large enough for randomization. Group averages ranged from 100 to 150 mm. 3 Tumor volumes were monitored until tumor volume reached 0.001 and mice were randomly assigned to treatment groups on day 0. Unless otherwise noted, mice administered chimeric compound 1 were dosed twice weekly (days 1, 4, 8, and 11) for 2 weeks. The inducible IL-12 prodrug used in these studies included chimeric compound 1. Mice administered recombinant chimeric IL-12 (chimeric IL-12 or WW0295) were dosed twice daily for 5 days, followed by a 2-day rest period (5 / 2 regimen), with the cycle repeated for a total of 2 weeks. All treatments were administered by intraperitoneal injection. Both body weight and tumor volume were measured twice weekly throughout the study. Tumors were measured in two dimensions using calipers and volumes were calculated using the following formula: tumor volume (mm 3 )=[(w2×l) / 2], where w=tumor width (mm), l=tumor length (mm). Mice were continued on study until tumors reached 1500 mm3 or the study reached its endpoint of day 45. See Figures 2A-28, 3G, and 4A-4F.

[0141] 4. B16-F10 model All mouse in vivo experiments were performed at Charles River Laboratories (Morrisville, NC and Worcester, MA) with Institutional Animal Care and Use Committee (IACUC) approval and in accordance with current regulations and standards, as well as the NIH. One day prior to tumor cell implantation, the flanks of 6-8 week-old female C57Bl / 6 mice at Charles River Laboratories were shaved. A total of 1 × 10 5B16-F10 cells were injected subcutaneously and tumor growth was monitored. Additional mice were implanted to have tumors large enough for randomization. Group averages ranged from 50 to 100 mm. 3 Tumor volumes were monitored until tumor volume reached 100%, and on day 0, mice were randomized into treatment groups. Mice receiving chimeric compound 1 were dosed on days 1 and 4, and tumors were harvested 24 hours after the second dose (day 5). The inducible IL-12 prodrug used in these studies included chimeric compound 1. All treatments were administered by intraperitoneal injection. See Figure 3B and Figures 5A-5F.

[0142] 5. EMT-6 model All mouse in vivo experiments were performed with Institutional Animal Care and Use Committee (IACUC) approval at Charles River Laboratories (Morrisville, NC and Worcester, MA) in accordance with current regulations and standards and with the NIH. One day prior to tumor cell implantation, the flanks of 6-8 week-old female C57Bl / 6 mice at Charles River Laboratories were shaved. A total of 1 × 10 5 EMT6 cells were injected subcutaneously and tumor growth was monitored. Additional mice were implanted to have tumors large enough for randomization. Group averages ranged from 50 to 100 mm. 3 Tumor volumes were monitored until tumor volume reached 100% and mice were randomly assigned to treatment groups on day 0. Unless otherwise noted, mice receiving chimeric compound 1 were dosed twice weekly (days 1, 4, 8, and 11) for 2 weeks. The inducible IL-12 prodrug used in these studies included chimeric compound 1. All treatments were administered by intraperitoneal injection. In some experiments, mice that had previously rejected tumors were then injected with 1×10 5The mice were rechallenged with EMT6 cells. In those experiments, age-matched tumor-naive animals were used as controls. In some experiments, tumor samples were harvested and incubated in 10% neutral buffered formalin (5-10 mL) for at least 72 hours before being embedded in paraffin and mounted on slides. Unstained slides were submitted to Nanostring for immunofluorescence staining and geospatial transcription analysis using the Nanostring GeoMX DSP system. See Figures 3C, 3F, 6, and 7A-7D.

[0143] 6.CT26 model All mouse in vivo experiments were performed at Charles River Laboratories (Morrisville, NC and Worcester, MA) with Institutional Animal Care and Use Committee (IACUC) approval and in accordance with current regulations and standards, as well as the NIH. One day prior to tumor cell implantation, the flanks of 6-8 week-old female Balb / C mice at Charles River Laboratories were shaved. A total of 3 × 10 5 CT26 cells were injected subcutaneously and tumor growth was monitored. Additional mice were implanted to have tumors large enough for randomization. Group averages ranged from 30 to 60 mm. 3 Tumor volumes were monitored until tumor volume reached 0.001 and mice were randomized into treatment groups on day 0. Unless otherwise noted, mice receiving Chimeric Compound 1 were dosed twice weekly (days 1, 4, 8, and 11) for two weeks. The inducible IL-12 prodrug used in these studies included Chimeric Compound 1. All treatments were administered by intraperitoneal injection. Both body weight and tumor volume were measured twice weekly throughout the study period. Tumors were measured in two dimensions using calipers and volumes were calculated using the following formula: Tumor Volume (mm 3 )=[(w 2 × l) / 2] (where w = tumor width (mm), l = tumor length (mm)). 3 Mice were continued on study until the IL-16 expression level was reached or until the study reached its endpoint of day 45. See Figure 3A.

[0144] 7.EG7.OVA model All mouse in vivo experiments were performed with Institutional Animal Care and Use Committee (IACUC) approval at Covance (Ann Arbor, MI) in accordance with current regulations and standards, as well as the NIH. One day prior to tumor cell implantation, the flanks of 6-8 week-old female C57Bl / 6 mice from Charles River Laboratories were shaved. A total of 1 × 10 6 EG7. OVA cells were injected subcutaneously and tumor growth was monitored. Additional mice were implanted to have tumors large enough for randomization. Group average was approximately 93 mm 3 Tumor volumes were monitored until tumor volume reached 0.001 and mice were randomized into treatment groups on day 0. Unless otherwise noted, mice receiving Chimeric Compound 1 were dosed twice weekly (days 1, 4, 8, and 11) for two weeks. The inducible IL-12 prodrug used in these studies included Chimeric Compound 1. All treatments were administered by intraperitoneal injection. Both body weight and tumor volume were measured twice weekly throughout the study period. Tumors were measured in two dimensions using calipers and volumes were calculated using the following formula: Tumor Volume (mm 3 )=[(w 2 × l) / 2] (where w = tumor width (mm), l = tumor length (mm)). 3 Mice were continued on study until the IL-16 expression level was reached or the study reached its endpoint of day 45. See Figure 3E.

[0145] 8. A20 model All mouse in vivo experiments were performed with Institutional Animal Care and Use Committee (IACUC) approval at Covance (Ann Arbor, MI) in accordance with current regulations and standards, as well as the NIH. One day prior to tumor cell implantation, the flanks of 6-8 week-old female Balb / C mice at Charles River Laboratories were shaved. A total of 5 × 10 5 A20 cells were injected subcutaneously and tumor growth was monitored. Additional mice were implanted to have tumors large enough for randomization. Group averages ranged from approximately 90 to 130 mm. 3Tumor volumes were monitored until tumor volume reached 0.001 and mice were randomized into treatment groups on day 0. Unless otherwise noted, mice receiving Chimeric Compound 1 were dosed twice weekly (days 1, 4, 8, and 11) for two weeks. The inducible IL-12 prodrug used in these studies included Chimeric Compound 1. All treatments were administered by intraperitoneal injection. Both body weight and tumor volume were measured twice weekly throughout the study period. Tumors were measured in two dimensions using calipers and volumes were calculated using the following formula: Tumor Volume (mm 3 )=[(w 2 × l) / 2] (where w = tumor width (mm), l = tumor length (mm)). 3 Mice were continued on study until the IL-16 expression level was reached or the study reached its endpoint of day 45. See Figure 3D.

[0146] 9. Tumor Digestion and NanoString Analysis MC38 and B16-F10 tumors were cut into small pieces (5 mm 3 After mincing into small pieces (<5 mm), tumors were enzymatically digested with collagenase IV (3 mg / mL, Gibco, 17104019) for 35 min at 37° C. with shaking. After digestion, tumor samples were mechanically dissociated through a 70 μM cell strainer. EMT6 tumors were processed using gentleMACS™ C-Tubes (130-093-237) from Miltenyi Biotech. Briefly, tumors were cut into small pieces (<5 mm) in HBSS containing 1.25 mg / mL collagenase type IV (Gibco, 17104019), 0.0025 mg / mL hyaluronidase (Sigma-Aldrich, H3506), and 0.01 mg / mL DNASE I (Worthington, LS002004). 3The samples were then cut into small pieces (less than 5 × 10). The samples were placed on a gentleMACS™ Octo Dissociator and processed using program 37C_m_TDK_1, after which the samples were passed through a 70 μM cell strainer to remove undigested tumor debris. Single cell suspensions were then counted and analyzed by flow cytometry. For NanoString analysis, 5 × 10 5 Cells were frozen in 100 μL of RLT lysis buffer (Qiagen, 1053393). RNA samples were sent to LakePharma and analyzed using the nCounter Mouse PanCancer Immune Profiling Codeset Panel with the nCounterFLEX analysis system. NanoString analysis was performed using nSolver™ software with the Advanced Analysis module installed.

[0147] 10. Flow Cytometry All cell staining was performed in 96-well round bottom plates using FACs buffer (PBS + 0.5% BSA) or 1x permeabilization buffer (eBioscience, 00-5223-56) as appropriate. First, cells were treated with FC block (BioLegend, 101320) at room temperature, followed by tetramer staining for 20 minutes. Next, after tetramer staining, cells were stained with a master mix of extracellular antibodies for 20 minutes at 4°C, followed by fixation / permeabilization 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 for intracellular markers for 20 minutes at 4°C. Cells were analyzed on a Cytek Aurora system. Fluorescence minus one (FMO) and single stain controls (Cell or OneComp ebeads™ (Thermofisher, 01-1111-42)) were stained together with the cells. Unless otherwise stated, when flow cytometry was used to assess production of effector cytokines, cells were restimulated with PMA (50 ng / mL, Sigma-Aldrich, P1585) and ionomycin (1 μg / mL, Sigma-Aldrich, IO634-1MG) in the presence of 1× Brefeldin A (Thermofisher Scientific, 00-4506-51) in complete medium for 4 h at 37° C. before staining. Cells used for the 2-NDBG assay were starved for 1 h in glucose-free RPMI-1640 medium (Gibco, 11879-020) and then incubated with 2-NDBG (Cayman Chemical, #186689-07-6) for 1 h at 37° C. / 5% CO2 before staining for extracellular markers.MitoTracker Deep Red FM (ThermoFisher, #M46753), MitoTracker Green FM (ThermoFisher, M46750), MitoSOX Red (ThermoFisher, M36008), and TMRM (ThermoFisher, T668) staining was performed for 1 hour at 37°C / 5% CO2 in RPMI1640 medium (Gibco, A10491-01) containing 10% heat-inactivated FBS (Gibco, 10082-147) and penicillin / streptomycin (Gibco, 15140-122). Cells were then washed with FACs buffer and stained for extracellular markers. Specific antibody clones are detailed below. Fluorochrome-conjugated antibodies specific for the following proteins were purchased from BioLegend: CD8α APC, clone 53-67; CD 4BV650, clone RM4-5; CD3 AF700, clone 17A2; CD45 BV605, clone 30-F11; CD49b APC / Cy7, clone DX5; CD25 BV421, clone PC61; CD25 APC / Fire750, clone PC61; Ki67 PeCy7, clone 16A8; Ki67 AF700, clone 16A8; Granzyme B FITC, clone GB11; IFNγ PE, clone XMG1.2; F4 / 80 Pe / Dazzle594, clone BM8; CD3 complex PeCy7, clone 17A2; FC Block, clone 93. Fluorochrome-conjugated antibodies specific for 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: 449) was purchased from ThermoFisher Scientific (50-168-9385).Live / Dead Blue Dye was also purchased from ThermoFisher Scientific (L23105).

[0148] 11. Pharmacokinetic Analysis Plasma and tumor samples were collected by Charles River Laboratories at the indicated time points and stored at -80°C. MC38 tumor lysates were obtained by homogenizing each tumor with a Qiagen TissueRuptor using a disposable probe (Qiagen) in ice-cold lysis buffer (1X Tris-buffered saline (Sigma-Aldrich, T5912-1L), 1 mM EDTA (Sigma-Aldrich, 3690-100mL), 1% Triton® X-100 (Sigma-Aldrich, X100-1000mL) with protease inhibitors (Sigma-Aldrich, P8340-1L) in diH2O. Plasma and tumor samples were analyzed using a sandwich ELISA on the MSD platform, which detects both intact chimeric compound 1 and free / released IL-12. Free IL-12 levels were quantified using an in-house developed ECLIA assay on an MSD MESO™ QuickPlex SQ120 system. Data acquisition and analysis were performed using MSD Workbench 4.0.12, and pharmacokinetic parameters were calculated using Phoenix WinNonlin version 8.1.

[0149] 12. Stability of Compound 36 in Human Serum Compound 36 was incubated in human serum from healthy donors (BioIVT) in duplicate at each time point. Time zero (T0) samples were immediately frozen at -80°C. The remaining samples were incubated at 37°C for 24 hours (T24) or 72 hours (T72) and then stored at 80°C. Compound 36 stability was assessed by Western blot analysis using the JESS system (Protein Simple, SM-W004) according to the manufacturer's general protocol. Input controls (intact and protease cleaved) were also analyzed. Samples and antibodies (anti-IL-12 primary antibody (R&D Systems, AB-219-NA)) and secondary antibody (Jackson Labs, 805-035-180) were loaded into a 12-230 kDA Jess separation module and run using the Jess system set to standard settings for chemiluminescence with a modification of the standard protocol for non-reducing conditions. Analysis of the resulting Western blots was performed using Compass for Simple Western Software (v4.1.0).

[0150] 13. Human Primary Cell Assay Human PBMCs were isolated from whole blood (BioIVT) using Ficoll-Paque Plus (GE Healthcare, GE17-1440-03) according to the manufacturer's protocol and frozen in Recovery Cell Culture Freezing Media (Gibco, 12648010) for later use. To generate activated T cells (Tblasts), PBMCs were thawed, counted, stimulated with 5 μg / mL PHA (Sigma-Aldrich, L1668-5MG) for 72 hours, and then frozen. Tblasts were thawed, counted, plated in 96-well round-bottom plates, and incubated with titrated amounts of complete or protease-activated (truncated) INDUKINE™ protein or chimeric IL-12. After 72 hours, IFNγ production was measured using a human IFNγ-specific AlphaLisa kit (Perkin Elmer, AL217C) according to the manufacturer's protocol using a Perkin Elmer Enspire Alpha Reader running Enspire Manager software (V4.13.3005.1482).

[0151] 14. Ex Vivo Inducible IL-12 Prodrug Protein Treatment Assay Primary human healthy cells were purchased from ATCC, Lonza, or Zen-Bio and cultured according to the manufacturer's protocol. Dissociated human tumor samples were purchased from Discovery Life Sciences. To investigate inducible IL-12 prodrug treatment, samples were thawed, washed, and counted. Cells were then resuspended in medium containing either intact compound 36, a non-cleavable variant of compound 36, or pre-cleaved compound 36 for 48 hours, after which cell culture supernatants were collected, frozen, and used for later analysis. Cell culture supernatants were then used to stimulate previously activated human Tblasts using the assay detailed above. To assess treatment levels, IFNγ production was normalized to control using the following formula: Percent full activity=(1-((sample-uncleavable Ctrl)) / ((cleaved Ctrl-uncleavable Ctrl)))*100

[0152] 15. Data Representation, Bioinformatics Analysis, and Statistics Flow cytometry plots were generated using FlowJo software (v10.5.30). This is a representative sample. 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). Comparisons of two samples used Student's t-test, whereas comparisons of more than two groups used an analysis of variance with multiple comparisons (ANOVA). Antitumor effects over time were analyzed using a mixed-effects model. For the NanoString dataset, statistical analysis was performed using nSolverTM software with the Advanced analysis module installed. Pathway analysis was performed using Partek software (v10.0.22.0428) based on transcripts significantly different after mWTX-330 at an FDR step-up of 0.05.

[0153] 1.2 Results 1. Chimeric compound 1 is a selectively activated, inducible IL-12 prodrug that generates potent, cleavage-dependent anti-tumor immune responses in multiple models. To address the clinical shortcomings of free cytokine therapy, we developed a selectively inducible IL-12 prodrug. To measure the difference in activity between intact and protease-activated (truncated) chimeric compound 1, HEK-Blue IL-12 reporter cells were incubated with intact chimeric compound 1 or protease-activated chimeric compound 1 and IL-12 signaling was assessed. In this assay, intact chimeric compound 1 was 175-fold less active than either truncated chimeric compound 1 or chimeric IL-12 (Figure 1). To test whether chimeric compound 1 could generate antitumor immunity in vivo, MC38 tumor-bearing animals were administered titrated doses of chimeric compound 1 and tumor growth was monitored over time. In this study, a variant of chimeric compound 1 containing a non-cleavable linker (Figure 1) is included at the highest dose as a control. In this model, even the lowest tested dose of chimeric compound 1 (7 μg / dose) produced statistically significant tumor growth inhibition, and 43 μg / dose was sufficient to produce complete tumor rejection (Figure 2A). In contrast, the non-cleavable (NC) variant of chimeric compound 1 was less active than the lowest dose of chimeric compound 1. This demonstrates that the full efficacy of chimeric compound 1 is dependent on in vivo processing of the molecule. Treatment with chimeric compound 1 produced potent antitumor immunity even in syngeneic tumor models with low immune cell infiltration ("cold"), including CT26 (Figure 3A), B16-F10 (Figure 3B), and EMT-6 (Figure 3C). This demonstrates the broad activity of the molecule in vivo. Although the use of a non-cleavable control demonstrated the necessity of processing for full activity, these data did not directly demonstrate that processing was occurring within the TME. FTY720 is a small molecule inhibitor of sphingosine 1 phosphate receptor 1 that prevents lymphocyte egress from secondary lymphoid tissues and effectively isolates TILs from normal circulating immune cell populations in vivo. Animals co-treated with chimeric compound 1 / FTY720 retained the potent early antitumor activity associated with treatment with chimeric compound 1, but tumor control was not complete after administration of the INDUKINE™ molecule was discontinued (Figure 8A).

[0154] These data demonstrate that systemically administered chimeric compound 1 is processed within the TME and that this local release of IL-12 is sufficient to produce early tumor growth inhibition.

[0155] To identify effector cell populations involved in chimeric compound 1-induced antitumor immunity, MC38 tumor-bearing mice were treated with chimeric compound 1 in combination with antibody-based depletion of individual effector cell populations.

[0156] Interestingly, depletion of CD8+ T cell populations did not inhibit initial tumor control, but mice treated with chimeric compound without CD8+ T cells ultimately failed to control tumor growth (Figure 8B). In contrast, depletion of either NK cells or total CD4+ T cells alone did not inhibit chimeric compound 1-induced antitumor activity (Figure 8B). However, treatment with chimeric compound 1 in mice depleted of all three populations showed little antitumor activity, suggesting that these cell types rejected MC38 tumors in response to treatment. Furthermore, rechallenge of chimeric compound 1-treated mice, which had previously rejected either MC38 tumors (Figure 3F) or EMT-6 tumors (Figure 3G), protected 100% of the animals from tumor growth. Overall, these data suggest that treatment with chimeric compound 1 generates a strong and durable antitumor immune response that is dependent on in vivo cleavage / activation of the molecule.

[0157] 2. Inducible IL-12 prodrug design of chimeric compound 1 enhances its pharmacokinetic profile and expands the therapeutic window of chimeric IL-12 In the clinic, free cytokines have a short half-life and poor pharmacokinetic profile, leading to fast clearance and insufficient exposure in patients, leading to irrational dosing schedules. To investigate whether the design of chimeric compound 1 enhances the half-life and exposure of the molecule, MC38 tumor-bearing mice were administered a single dose of either chimeric IL-12 or chimeric compound 1, and peripheral blood and tumor samples were collected over time. Using a unique set of detection reagents, it was possible to measure the amount of total IL-12 (blocked + unblocked) separately or selectively measure the amount of unblocked IL-12 present. In the plasma of tumor-bearing mice, the half-life of chimeric IL-12 was only 4 hours, while the half-life of chimeric compound 1 was almost 16 hours (Figure 9A). Moreover, only about 2% of chimeric compound 1 detected in plasma was in the form of the cleaved molecule. In contrast, when the same analysis was performed on tumor samples (Figure 9B), nearly 45% of the molecules were unmasked IL-12, and the exposure between tumors was maintained far beyond that achieved by treatment with chimeric IL-12. To further confirm the selective treatment of chimeric compound 1 in tumors, the activation status of tumor-infiltrating CD8+ T cells, CD4+ T conventional cells, and NK cells was compared with the same populations in tumor-draining and non-draining lymph nodes and peripheral blood after treatment with chimeric compound 1. Treatment with chimeric compound 1 significantly increased the frequency of polyfunctional CD8+ T cells in MC38 tumors, but such an increase was not observed in lymph nodes or peripheral blood (Figure 9C). Similarly, among CD4+ T conventional cells (FoxP3-) (Figure 15A) and NK cells (Figure 15B), chimeric compound 1 preferentially increased the frequency of effector cytokine-producing cells in tumors compared to peripheral tissues.

[0158] 3. Treatment with chimeric compound 1 activates various TIL populations in the MC38 model To better understand the mechanism by which treatment with chimeric compound 1 produces antitumor immunity, MC38 tumor-bearing mice were randomized into treatment groups on day 0 and treated with either vehicle or chimeric compound 1 on days 1 and 4. 24 hours after the second dose, tumors were harvested and analyzed by flow cytometry or NanoString analysis using the PanCancer mouse immune profiling panel. Systemic treatment with chimeric compound 1 had a profound effect on the transcriptional profile of the TME, with 364 of the 770 transcripts surveyed having statistically significant differences in expression after treatment (Figure 4A). Treatment with chimeric compound 1 resulted in significant enrichment of several immune-related signaling pathways, including "PD-L1 expression and PD-1 checkpoint in cancer," "NK cell cytotoxicity," and "TH1 and TH2 differentiation." Consistent with this analysis, treatment with chimeric compound 1 significantly increased the frequency of tumor-infiltrating NK cells producing IFNγ, TNF, and granzyme B (Figures 4C-4D). Interestingly, treatment with chimeric compound 1 resulted in NK, NKT, CD4+ T conventional, and CD8+ T cells producing elevated levels of IFNγ, as measurable by intracellular cytokine staining, without ex vivo restimulation (Figures 14A-14B). However, the signaling pathway with the highest enrichment score after treatment with chimeric compound 1 was “antigen processing and presentation.” Gene set enrichment analysis revealed that treatment with chimeric compound 1 significantly enriched several gene sets related to antigen presentation of exogenous peptides and / or antigens on either MHC class I or MHC class II proteins. Presentation of exogenous antigens on MHC class I proteins is a phenomenon known as cross-presentation, and is mediated exclusively by a unique population of dendritic cells identified by their expression of CD103 (25). Consistent with the bioinformatics analysis, flow cytometry analysis demonstrated that treatment with chimeric compound 1 significantly increased the frequency of the cross-presenting CD103+ DC population among all DCs (FIGS. 10A-10B).

[0159] Given the role of CD8+ T cells in chimeric compound 1-mediated tumor rejection (Figure 8A) and the finding that chimeric compound 1 increases tumor infiltration by cross-presenting DCs, it seems likely that treatment with chimeric compound 1 may also promote CD8+ T cell activation. Indeed, differential expression analysis of total RNA demonstrated that treatment with chimeric compound 1 significantly increased the expression of many transcripts associated with cytotoxic CD8+ T cell activation, including IFNγ, granzyme B, perforin, and TNF, in addition to several chemoattractant molecules (Figure 4B). Although chimeric compound 1 did not increase the frequency of tumor-specific CD8+ T cells at this early time point, treatment led to robust activation of the tumor-specific CD8+ T cell population, as demonstrated by the increased frequency of tetramer+ polyfunctional CD8+ T cells (Figure 4F), with nearly 100% of tumor-specific T cells producing IFNγ. Similar results were seen when considering the entire tumor-infiltrating CD8+ T cell population, not just tetramer-positive cells. Furthermore, among CD4+ T cells, treatment with chimeric compound 1 significantly increased the frequency of CD4+ T conventional cells with a TH1 phenotype (Tbet+IFNγ+TNF+) (Figure 10C). Finally, recent publications have highlighted the role of IFNγ in driving Tregs away from regulatory activity and toward an effector phenotype in a phenomenon known as Treg fragility. Treatment with chimeric compound 1 resulted in a significant subset of the FoxP3+ Treg population that co-produced the effector cytokines TNF and IFNγ (Figures 10D-10F) and expressed Tbet (Figure 10F). This demonstrates that systemic treatment with chimeric compound 1 can induce Treg instability within the TME. Overall, these data demonstrate that systemic administration of chimeric compound 1 results in transcriptional reprogramming of the TME, followed by activation of various tumor-infiltrating effector cell populations.

[0160] 4. Treatment with chimeric compound 1 expands unique TCR clones and increases TCR clonality within the TME. In the MC38 tumor model, treatment with chimeric compound 1 resulted in rapid tumor rejection, making a full investigation of the dynamics of immune activation technically difficult. In contrast, treatment with chimeric compound 1 on the EMT-6 tumor model resulted in complete rejection over a longer period of time, which favors a more thorough analysis of ongoing CD8+ T cell responses (Figure 3C). Thus, mice bearing established EMT-6 tumors were randomly assigned to treatment groups and administered either vehicle or chimeric compound 1 twice a week for two weeks. Tumors and plasma were then harvested at various time points. Interestingly, in control animals, the frequency of polyfunctional CD8+ T cells expanded over the course of the experiment, but eventually regressed in line with tumor growth. In contrast, treatment with chimeric compound 1 increased the frequency of polyfunctional CD8+ T cells over control animals 5 days after the start of treatment (Figure 6), and this frequency continued to expand even after exposure to chimeric compound 1 was no longer detectable.

[0161] To better understand the transcriptional effects of treatment with chimeric compound 1 specifically on tumor-infiltrating CD8+ T cell populations, geospatial NanoString analysis was performed on day 11 tumor samples. This technique merges immunofluorescence with whole-transcriptome analysis of specific cells, allowing transcriptional analysis of specific cell populations while preserving spatial information that is otherwise lost during tissue dissociation. In the control group, CD8+ T cells were primarily restricted to the outer edge of the tumor (Figure 7A). In contrast, treatment with chimeric compound 1 induced significant infiltration of EMT-6 tumors, with CD8+ T cells penetrating deep into the tumor tissue (Figure 7A). Whole-transcriptome analysis of tumor-infiltrating CD8+ T cells demonstrated that treatment with chimeric compound 1 resulted in significant transcriptional reprogramming of these cells, including upregulation of many genes associated with T cell activation (e.g., Tbet, IFNγ, Cd25, and chemoattractants known to be involved in the recruitment of additional immune cells) (Figure 7B).

[0162] Among the pathways activated in tumor-infiltrating CD8+ T cells by treatment with chimeric compound 1, both IL-12 (Figure 7C) and IFNγ signaling (Figure 7D) were significantly upregulated, confirming the local release of unmasked IL-12 and subsequent production of IFNγ within the TME. Treatment with chimeric compound 1 also increased the expression of transcripts downstream of TCR signaling (Figure 11A). Given its effect on cross-presenting DCs, we hypothesized that treatment with chimeric compound 1 may result in the activation of new T cell clones that generate anti-tumor immunity. To test this, T cells were isolated from EMT-6 tumors after treatment with chimeric compound 1 or vehicle and sent for TCR sequencing. The tumor-infiltrating TCR repertoire of control animals was dominated by many low-frequency clones, and treatment with chimeric compound 1 promoted a strong expansion of several TCR clones (Figure 11B) and significantly increased the overall clonality of the tumor-infiltrating TCR repertoire (Figure 11C). Indeed, when examining the overall frequency of the top 50 clones in each group, treatment with chimeric compound 1 significantly increased the number of clones that represented >1% of the total repertoire across animals (Figure 11D). Further analysis of this subset revealed that only one of eight clones common to both treatment groups was expanded by at least 10-fold by treatment with chimeric compound 1 (Figure 12S). In contrast, of the clones unique to the chimeric compound 1 treatment group, all were expanded by more than 10-fold compared to the control group (Figure 12T), suggesting that treatment with chimeric compound 1 increases the clonality of the T cell population primarily by expanding previously underrepresented clones, rather than increasing the frequency of already dominant clones.

[0163] 5. Chimeric compound 1 significantly increases mitochondrial activity in tumor-infiltrating CD8+ T cells and NK cells In recent years, activated CD8+ T cells have been shown to have considerable energy requirements and, as they develop into long-lived memory cells, rely heavily on glucose uptake and glycolysis to rapidly generate the energy required to perform effector functions before transitioning to mitochondrial-dependent oxidative phosphorylation. However, recent publications have demonstrated that tumor-infiltrating CD8+ T cells are often poorly able to induce mitochondrial respiration compared to cells activated in the spleen or lymph nodes, suggesting that the TME negatively impacts the metabolic health of effector cells. In tumor-infiltrating CD8+ T cells, treatment with chimeric compound 1 resulted in a significant enrichment of transcripts related to glycolysis (Figure 12A). Thus, we hypothesized that treatment with chimeric compound 1 could increase glucose uptake by tumor-infiltrating CD8+ T cells, thereby leading to increased glycolysis. However, when these cells were incubated with a non-metabolizable fluorescent glucose analog (2-NDBG), tumor-infiltrating CD8+ T cells from animals treated with chimeric compound 1 actually took up slightly less glucose than those from vehicle animals (Figures 12B-12C). Thus, rather than simply increasing glucose uptake by tumor-infiltrating CD8+ T cells, treatment with chimeric compound 1 instead reprogrammed those cells to utilize glucose more efficiently than those from vehicle-treated animals.

[0164] In addition to promoting increased glycolysis, treatment with chimeric compound 1 also enriched transcripts related to the TCA cycle, mitochondrial biogenesis, and mitochondrial translation, suggesting that treatment with chimeric compound 1 may promote mitochondrial activity and health in tumor-infiltrating effector cells (Figures 12D-12F). To test this, TILs were isolated from vehicle- or chimeric compound 1-treated animals, and mitochondrial phenotyping was performed by flow cytometry. Mitotracker Red is a dye that specifically stains actively respiring mitochondria with pH sensitivity. While tumor-infiltrating CD8+ T cells from vehicle-treated animals showed limited evidence of ongoing active mitochondrial respiration, those from chimeric compound 1-treated animals showed significantly increased levels of active respiration (Figures 12G-12H). Interestingly, this finding also extended to NK cells (Figures 12I-12J) and total CD4+ T cells (Figure 12U). This increase was primarily due to increased mitochondrial activity rather than simply increased mitochondrial mass, as treatment with chimeric compound 1 only slightly increased the total mitochondrial mass in tumor-infiltrating NK cells, CD8+ T cells, and total CD4+ T cells (Figure 12V). Furthermore, TMRM staining also revealed that treatment with chimeric compound 1 significantly increased the mitochondrial membrane potential in both CD8+ T cells (Figures 12K-12L) and NK cells (Figures 12M-12N). Mitochondrial reactive oxygen species (ROS) have been previously linked to both NFAT signaling and the subsequent production of IL-2 (31) and IFNγ production by memory CD4+ T cells and can be detected using the dye MitoSOX Red. Treatment with chimeric compound 1 increased the production of mitochondrial ROS species in both CD8+ T cells (Figures 12O-12P) and NK cells (Figures 12Q-12R).

[0165] Oxidative phosphorylation is the primary energy source for memory T cells, and increased reliance on this pathway is associated with superior antitumor immunity and a "stem cell-like" phenotype. Tumor-infiltrating CD8+ T cells from chimeric compound 1-treated mice also significantly upregulate the expression of genes associated with T cell stemness (e.g., Tcf7, Cxcr3, and Il2rγ), but significantly downregulate the expression of several genes associated with CD8+ T cell exhaustion (e.g., Pdcd1, Havcr2, and Lag3). Overall, these data demonstrate that systemic administration of chimeric compound 1 is sufficient to restore mitochondrial respiration in tumor-infiltrating CD8+ T cells and NK cells, leading to the introduction of a more "stem cell-like" phenotype in the CD8+ T cell population, which can translate into superior antitumor immunity.

[0166] 6. Compound 36, a fully human inducible IL-12 prodrug, is stable in human serum and is preferentially activated by primary human tumor samples. In preclinical mouse studies, it was important to use a surrogate molecule that was active in mice. However, in clinical development, a fully human IL-12 payload will be used. Compound 36 is identical to chimeric compound 1, except that it contains fully human IL-12 as the payload. Similar to the mouse surrogate molecule, intact compound 36 exhibited significantly less activity than truncated compound 36 or recombinant human IL-12 in the HEK-Blue IL-12 reporter assay. Similarly, when exposed to stimulated primary human Tblasts from multiple donors, intact compound 36 was, on average, 61-fold less active than the truncated molecule. In both of these in vitro assays, truncated compound 36 had similar activity to recombinant human IL-12. Furthermore, when compound 36 was incubated in serum from healthy human donors (n=6), no free IL-12 was detectable after 72 hours at 37°C (Figure 13A), confirming the stability of the molecule. To investigate whether compound 36 is selectively processed by primary human tumor samples, an in vitro cleavage assay was developed. Briefly, primary human isolated tumor samples from various indications, or primary human cells from healthy tissues, were incubated with either compound 36, pre-cleaved compound 36, or a non-cleavable variant of compound 36 for 48 hours, after which cell culture supernatants containing the processed inducible IL-12 prodrug were collected. Since human Tblasts can distinguish between intact and cleaved compound 36, primary human Tblasts were exposed to cell culture supernatants and the production of IFNγ was used as a surrogate marker of compound 36 processing. The results of this assay were then normalized to a non-cleavable negative control (0% treatment) and a pre-cleavage positive control (100% treatment). Of n=88 primary human tumor samples evaluated, compound 36 was efficiently processed across all tested indications (Figure 13B). In contrast, incubation of compound 36 with primary human cells derived from a variety of healthy tissues (n=13) gave no evidence of processing. These data suggest that compound 36 is efficiently and selectively processed by primary human tumor samples and support the continued clinical development of this molecule.

[0167] 1.3 Investigation IL-12 is a cytokine that has long been important in oncology due to its potential to induce innate and adaptive immune responses (9, 11) and promising antitumor preclinical data (12, 18, 20, 34, 35). Nevertheless, despite this importance, the use of this cytokine in clinical settings has been hindered by poor pharmacokinetic properties and unacceptable levels of toxicity associated with systemic administration (9, 10, 24, 36). To address these concerns, we developed an inducible IL-12 prodrug, compound 36. The prodrug molecule, compound 36, is designed to be an infrequently administered, systemically administered therapy with targeted intratumoral activation that releases native IL-12 into the tumor microenvironment. Our data on chimeric compound 1 demonstrated antitumor activity in the MC38 tumor model that was dependent on in ivo cleavage of the inducible IL-12 prodrug by the tumor. Furthermore, chimeric compound 1 was a highly potent monotherapy in several mouse tumor models with different baseline infiltration levels, including a complete response in a model (EMT-6) that was unresponsive to anti-PD-1 treatment. These complete responses led to strong immune memory against subsequent rechallenge with the same tumor cell line, highlighting the role of the immune system in tumor rejection. In the MC38 model, long-term efficacy was dependent on the presence of CD8+ T cells, whereas overall tumor growth suppression was driven by the contribution of three major effector cell types (CD8+ T cells, CD4+ T conventional cells, and NK cells). The inducible IL-12 prodrug design also resulted in increased exposure and a favorable ratio of active IL-12 to blocked prodrug molecules in tumor tissues compared to plasma. This correlated with the selectively localized pharmacodynamic changes (effector cell multifunctionality) observed in tumors versus peripheral tissues. Importantly, chimeric compound 1 proved to be well tolerated in mice compared with treatment with recombinant chimeric IL-12, while maintaining the potential to induce complete tumor regression, resulting in an almost 10-fold improvement in the therapeutic window compared with the unblocked cytokine.An improved therapeutic window is a key feature of inducible IL-12 prodrugs and is necessary to facilitate the clinical development of this potent cytokine for tumor treatment.

[0168] Treatment with chimeric compound 1 potently activated various tumor-infiltrating innate and adaptive effector cell populations, supporting a mechanism of action in which infiltration and activation of multiple effector cells play a fundamental role in early tumor control. Tumor-specific delivery of active IL-12 and subsequent induction of intratumoral IFNγ also induced Treg vulnerability, which may contribute to the strong efficacy conferred by treatment with chimeric compound 1. However, equally important was the effect of treatment with chimeric compound 1 on antigen processing and presentation, and the observed increase in tumor infiltration by cross-presenting dendritic cells. These cells are involved in the de novo generation of new T cell responses against novel tumor antigens, and several publications have recognized their importance in generating preclinical antitumor immunity (37, 38). When it comes to inducing efficacy in "cold" tumors, the dual role of IL-12 (as a direct activator of effector cell populations and as a promoter of cross-presenting dendritic cell activation) may give this cytokine-based therapeutic an edge over other treatments. Indeed, we observed this effect using a model of "cold" tumors (the poorly infiltrated EMT-6 model). Systemic treatment with chimeric compound 1 promoted deep infiltration of EMT-6 tumors by CD8+ T cells, and NanoString digital spatial profiling demonstrated intratumoral increases in IL-12 and IFNγ signaling, as well as significant upregulation of transcripts associated with robust CD8+ T cell activation. Treatment also significantly increased the clonality of the TCR repertoire among tumor-infiltrating T cells, promoting the expansion of several novel clones, suggesting that systemic treatment with chimeric compound 1 activated novel T cell responses against unique tumor antigens, which may be key to the previously observed CD8+ T cell-dependent tumor rejection.

[0169] Finally, systemic treatment with chimeric compound 1 had substantial effects on the metabolism of tumor-infiltrating effector cells, altering the metabolic state of not only activated tumor-infiltrating CD8+ T cells but also intratumoral NK cells. The TME is known to have several distinct characteristics compared to representative cellular environments (e.g., lower pH, hypoxia, and significant competition for extracellular glucose), all of which can impair effector cell activity. Despite evidence of increased glycolysis, we observed a slight reduction in extracellular glucose uptake following treatment with chimeric compound 1. This suggests that treatment with chimeric compound 1 not only increases glucose uptake by tumor-infiltrating cells, but instead promotes increased metabolic efficiency and increases oxidative phosphorylation. Recent studies have demonstrated that tumor-infiltrating T cells often fail to robustly activate mitochondrial respiration compared to T cells activated in the spleen or lymph nodes, suggesting that the TME impairs the metabolic health of effector cells (27, 28, 30). Consistent with these studies, EMT-6 tumor-infiltrating CD8+ T cells and NK cells from vehicle-treated animals had little evidence of ongoing mitochondrial respiration, despite efficient uptake of 2-NDBG in vitro. In contrast, effector cells from animals treated with compound 36 strongly upregulated active mitochondrial respiration, mitochondrial membrane potential, and mitochondrial reactive oxygen species. The strong shift of these cells toward oxidative phosphorylation may be important in light of the highly dysregulated metabolic environment within tumors, where there is intense competition for glucose and every molecule must be used to its full potential to support effector cell activation.

[0170] In conjunction with the metabolic effects of the TME, effector cells must also compete with another mechanism of immune regulation: exhaustion. This state is characterized by high co-expression of checkpoint proteins (e.g., PD-1, LAG-3, TIGIT, and TIM-3) (39), loss of production of effector cytokines, and failure to proliferate after restimulation. However, recent publications have demonstrated that some tumor-specific T cells maintain an additional stem cell-like phenotype (referred to as increased "stemness") and recognize a key role for the transcription factor TCF1 in that phenotype (40). Moreover, increased "stemness" is associated with enhanced antitumor immunity. In addition to metabolic activation of effector cells, treatment with chimeric compound 1 may have increased stemness in tumor-infiltrating CD8+ T cells, upregulated TCF7 (an mRNA transcript associated with the protein TCF1), and decreased expression of PD-1 and Tim-3 by these cells. Overall, these data suggest that treatment with chimeric compound 1 results in potent and comprehensive reprogramming of tumor-infiltrating CD8+ T cell responses.

[0171] The mechanistic studies described herein not only identified the cell types involved in the antitumor effects of treatment with chimeric compound 1, but also provide insight into how these effector cells can overcome the suppressive microenvironment within tumors. Furthermore, these data demonstrate that the design of an inducible IL-12 prodrug of chimeric compound 1 significantly increased the half-life of the molecule compared to recombinant IL-12, allowing selective activation within the TME after systemic administration, resulting in a significantly expanded therapeutic window. Additional studies with the fully human compound 36 demonstrated that the inducible IL-12 prodrug was highly inducible and cleaved in the majority of human tumor samples in vitro, yet showed stability when incubated with normal primary cells and serum. Altogether, these data provide clear evidence for the continued preclinical development of this therapeutic molecule and support the progression of compound 36 toward human clinical trials. Example 2. MC38 Experiment (Study MC38-e52)

[0172] The MC38 cell line, a rapidly growing colon adenocarcinoma cell line, was used. This tumor model was used to investigate the ability of IL-12 prodrugs to affect tumor growth and body weight. [Table 5]

[0173] For cell transplantation to reduce ulcers, mice were anesthetized with isoflurane. Female C57BL / 6 mice were anesthetized with 5×10 6 cells in 0% Matrigel in the flank. 5 MC38 tumor cells were injected subcutaneously. The cell injection volume was 0.1 mL / mouse. The age of the mice on the initiation day was 8-12 weeks. The tumors were an average of 100-150 mm 3 Pair-matching was done when the animals reached a size of 0.5 mm and treatment was initiated. This was day 1 of the study. Body weights were measured at the start and then every 2 weeks until termination. Caliper measurements were taken every 2 weeks until completion. Any adverse events were reported immediately. Any individual animal with a single observed weight loss of more than 25% or three consecutive measurements of weight loss of more than 20% was euthanized. Any group with a mean weight loss of more than 20% or mortality of more than 10% was discontinued and the group was allowed to recover rather than euthanized. Within groups with weight loss of more than 20%, individuals who reached individual weight loss endpoints were euthanized. If group treatment related weight loss recovered to within 10% of original weight, dosing was resumed at a lower dose or less frequent dosing schedule. Exceptions to recovery of untreated weight % were allowed on a case-by-case basis. The endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the study was a tumor volume of 1500 mm 3 or 40 days, whichever occurred first. When the endpoint was reached, the animals were euthanized. The results are shown in Figures 17A-17J. 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Table 8-1

Table 8-2

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Claims

1. A composition for selectively activating target tumor-infiltrating lymphocytes, comprising an inducible interleukin-12 (IL-12) prodrug, characterized in that the composition is administered systemically, and the inducible IL-12 prodrug is activated by cleavage by a protease that is more active in the tumor microenvironment than elsewhere, resulting in a significantly increased frequency of CD8+ T cells that produce IFN gamma and / or granzymes.

2. A composition for inducing immunological memory against a tumor in a subject, comprising an inducible interleukin-12 (IL-12) prodrug, characterized in that the composition is administered systemically, and the inducible IL-12 prodrug is activated by cleavage by a protease having higher activity in the tumor microenvironment than other regions.

3. A composition for selectively activating effector CD8+ T cells in a target tumor microenvironment, comprising an inducible interleukin-12 (IL-12) prodrug, characterized in that the composition is administered systemically, the inducible IL-12 prodrug is activated by cleavage by a protease that is more active in the tumor microenvironment than elsewhere, and the frequency of CD8+ T cells producing TNF and / or IFN gamma is significantly increased in the tumor compared to peripheral tissue.

4. The composition according to any one of claims 1 to 3, wherein the inducible IL-12 prodrug yields tumor-responsive CD8+ T cells that produce IL-12 and IFN gamma.

5. A composition for treating cancer, comprising an inducible interleukin-12 (IL-12) prodrug, characterized in that the composition is administered systemically, wherein the inducible IL-12 prodrug is activated in the tumor microenvironment by cleavage by a protease having higher activity in the tumor microenvironment than elsewhere, resulting in cleavage of the inducible IL-12 prodrug in the tumor microenvironment at least about 40 times more than in circulation.

6. The composition according to claim 5, wherein the cleavage of the inducible IL-12 prodrug in the tumor microenvironment is 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 more than the circulation.

7. The composition according to claim 5 or 6, wherein the administration results in an amount of inducible IL-12 prodrug in the plasma that is at least about five times greater than the amount of inducible IL-12 prodrug in the tumor.

8. The composition according to claim 7, wherein the amount of the inducible IL-12 prodrug in the plasma is 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 the inducible IL-12 prodrug in the tumor.

9. The composition according to any one of claims 1 to 3 and 5, wherein the inducible IL-12 prodrug is compound 2, compound 3, compound 4, compound 5, compound 6, or any of the aforementioned amino acid sequence variants.

10. The composition according to any one of claims 1 to 3 and 5, wherein the inducible IL-12 prodrug is compound 7, compound 8, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14, compound 15, compound 16, compound 17, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 24, compound 25, compound 26, compound 27, compound 28, compound 29, compound 30, compound 31, compound 32, compound 33, compound 34, compound 35, compound 36, or any of the aforementioned amino acid sequence variants.

11. The composition according to any one of claims 1 to 3 and 5, characterized in that the composition is administered at a frequency of about twice a week or less.

12. The composition according to any one of claims 1 to 3 and 5, characterized in that the composition is administered at a frequency of about once a week or less.

13. The composition according to any one of claims 1 to 3 and 5, characterized in that the composition is administered approximately once every two weeks.

14. The composition according to any one of claims 1 to 3 and 5, characterized in that the composition is administered in combination with one or more additional agents.

15. The composition according to claim 14, wherein the one or more additional agents are adoptive cell therapy.

16. The composition according to claim 15, wherein the adoptive cell therapy is CAR-T cell therapy or T cell therapy.

17. The composition according to claim 15, wherein the one or more additional agents are checkpoint inhibitors.

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