IL-12 prodrug, method of use, and pharmaceutical composition

Abstract

This disclosure relates to methods and compositions for treating cancer, including advanced solid tumors, metastatic solid tumors, or lymphoma, using an inducible IL-12 prodrug. This disclosure relates to compositions and methods for treating cancer using an inducible IL-12 prodrug. This inducible IL-12 prodrug contains attenuated IL-12 and has a longer half-life compared to naturally occurring IL-12. Optionally, IL-12 may be mutein. IL-12 mutein may be aglycosylated or partially aglycosylated.

Description

[Technical Field]

[0001] This application claims the benefits of U.S. Provisional Patent Application No. 63 / 494,517, filed on April 6, 2023, and U.S. Provisional Patent Application No. 63 / 495,899, filed on April 13, 2023, which are incorporated herein by reference as a whole. [Background technology]

[0002] 1. Background Cancer immunotherapy has rapidly established itself as the fourth pillar of cancer treatment, largely due to the clinical success of checkpoint inhibitors. While some patients have achieved a durable response with these new therapies, the responder rate remains relatively low and is limited to only certain cancer types. Tumor mutational load, the presence or absence of T cell infiltration in the tumor, and the overall immunosuppressive microenvironment of the tumor significantly influence the response to immunotherapy. Blocking immune checkpoints can prevent physiological arrest signals that arise in response to immune activation, but other approaches may also be used to favorably stimulate the anti-tumor immune response. One approach involves the use of immune-activating cytokines. Numerous preclinical and clinical trials have demonstrated the potential of cytokine therapies that enhance anti-tumor immunity. In fact, these were some of the first cancer immunotherapies to be approved for clinical use. However, their clinical application has been limited due to systemic toxicity and inadequate pharmacokinetic profiles.

[0003] Interleukin-12 (IL-12) is a 70 kDa heterodimeric 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 range due to its high potency and short serum half-life. As a result, therapeutic administration of IL-12 can lead to undesirable systemic effects and toxicity. This is exacerbated by the need to administer large amounts of cytokine (i.e., IL-12) to achieve desired cytokine levels at the site where cytokine action is intended (e.g., the tumor microenvironment). Unfortunately, due to the biological properties of cytokines and the inability to effectively target and control their activity, cytokines have not achieved the expected clinical benefits in tumor treatment. Inducible IL-12 molecules that are conditionally activated by protease cleavage in the tumor microenvironment to release fully active native IL-12 cytokines within the tumor, thereby stimulating a potent antitumor immune response, are described in international applications PCT / US2019 / 032320, PCT / US2019 / 032322, and PCT / US2021 / 033014. These IL-12 prodrugs contain a native IL-12 molecule conjugated via a protease-cleavable linker to a half-life extension domain (e.g., an anti-human serum albumin antibody-binding fragment such as a VH domain) and an IL-12 blocking element (e.g., an anti-IL-12 antibody-binding fragment such as Fab or scFv) that blocks the binding of IL-12 to IL-12Rβ1 or IL-12Rβ2 receptors in peripheral normal tissues. When the protease-cleavable linker is cleaved, 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 Publication No. 2019 / 222294 [Patent Document 2] International Publication No. 2019 / 222296 [Patent Document 3] International Publication No. 2021 / 236676 [Overview of the project] [Means for solving the problem]

[0005] 2. Overview This disclosure relates to compositions and methods for treating cancer using inducible IL-12 prodrugs. These inducible IL-12 prodrugs contain weakened IL-12 and have a longer half-life compared to naturally occurring IL-12. Optionally, IL-12 may be mutein. IL-12 mutein may be aglycosylated or partially aglycosylated. The inducible IL-12 prodrugs disclosed herein comprise two or more polypeptide chains, and each inducible IL-12 prodrug comprises IL-12 subunits p35 and p40, a half-life extension element, an IL-12 blocking element, and a protease-cleavable linker.

[0006] An inducible IL-12 prodrug may contain two different polypeptides. The first polypeptide may contain an IL-12 subunit and optionally contain an IL-12 blocking element. If present, this IL-12 blocking element is operably linked to the IL-12 subunit via a first protease-cleavable linker. The second polypeptide chain may contain an IL-12 subunit operably linked to a half-life extension element via a second protease-cleavable linker and optionally contain an IL-12 blocking element. If present, this IL-12 blocking element may be operably linked to the IL-12 subunit via a protease-cleavable linker, and optionally operably linked to a half-life extension element via a protease-cleavable linker. Only one of the first and second polypeptides contains the 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 this inducible IL-12 prodrug is a single-chain antibody or its antigen-binding fragment that binds to IL-12. The cleavable linkers in this inducible IL-12 prodrug may be the same or different.

[0007] An inducible IL-12 prodrug may contain three different polypeptides. Typically, 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 (component) of the blocking element. The first polypeptide may contain an IL-12 subunit and optionally a half-life extension element. This half-life extension element, if present, is operably linked to the IL-12 subunit via a protease-cleavable linker.

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

[0009] The third polypeptide may include an antigen-binding moiety of an antibody heavy chain complementary to the light chain in the second polypeptide, or an antibody light chain complementary to the heavy chain in the second polypeptide and forming an IL-12 binding site together 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, as separate components, includes at least an antigen-binding moiety of an antibody light chain and at least an antigen-binding moiety of a complementary antibody heavy chain. The protease-cleavable linkers in this inducible IL-12 prodrug may be the same or different.

[0010] An inducible IL-12 prodrug may contain two different polypeptides, p35 and p40, located on the same polypeptide chain. The first polypeptide chain may include p35, p40, a half-life extension element, and at least an antigen-binding moiety of the antibody light chain. p35 and p40 may be operably linked, the half-life extension element may be operably linked to p40 via a first protease-cleavable linker, and the antigen-binding moiety of the antibody light chain may be operably linked to p35 via a protease-cleavable linker. Alternatively, the half-life extension element may be operably linked to p35 via a protease-cleavable linker, and the antigen-binding moiety of the antibody light chain may be operably linked to p40 via a protease-cleavable linker. The second polypeptide includes at least an antigen-binding moiety of the antibody heavy chain, which is complementary to the light chain in the second polypeptide and forms an IL-12 binding site together with the light chain. The protease-cleavable linkers in this inducible IL-12 prodrug may be the same or different.

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

[0012] In one example, the inducible IL-12 prodrug comprises a first polypeptide and does not comprise a blocking element. The second polypeptide has 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], wherein A is an IL-12 subunit, L1 is a first protease-cleavable linker, L2 is a second protease-cleavable linker, L3 is an optionally cleavable linker, B is a half-life extension element, and D is a blocking element.

[0013] In another example, the first polypeptide comprises the formula: [A]-[L1]-[D] or [D]-[L1]-[A], and the second polypeptide has the formula: [A’]-[L2]-[B] or [B]-[L2]-[A’], wherein A is either p35 or p40. When A is p35, A’ is p40; when A is p40, A’ is p35. A’ is either p35 or p40, L1 is a first protease-cleavable linker, L2 is a second protease-cleavable linker, B is a half-life extension element, and D is a blocking element.

[0014] The present disclosure relates to a method for treating a progressive solid tumor, a metastatic solid tumor, or a lymphoma, the method comprising administering to a subject in need thereof an effective amount of an inducible IL-12 prodrug.

[0015] The inducible IL-12 prodrug to be administered can be compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14, compound 15, compound 16, compound 17, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 24, compound 25, compound 26, compound 27, compound 28, compound 29, compound 30, compound 31, compound 32, compound 33, compound 34, compound 35, compound 36, or an amino acid sequence variant of any of the foregoing.

[0016] The IL-12 prodrug can be administered orally, parenterally, intravenously, intra-articularly, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, intrahepatically, intracranially, by spray / inhalation, by bronchoscopic placement, or intratumorally.

[0017] Typically, the inducible IL-12 prodrug is administered intravenously, at a frequency of about twice a week or lower, for example, once every two weeks. The inducible IL-12 prodrug can be administered at a dose of about 0.016 mg / kg to about 500 mg / kg per administration, for example, about 0.016 mg / kg, about 0.032 mg / kg, about 0.056 mg / kg, about 0.084 mg / kg, about 0.126 mg / kg, about 0.190 mg / kg, about 0.290 mg / kg, or about 0.440 mg / kg (in each case per administration). A dose of about 1 mg to about 500 mg of the IL-12 prodrug can be administered per administration. For example, doses of about 1 mg, about 3 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 100 mg, or about 200 mg can be administered per administration.

[0018] In some embodiments, the treated patients have not achieved a complete response to prior or ongoing treatment (typically treatment with immune checkpoint inhibitors such as anti-PD-1, anti-PD-L1, or anti-CTLA4). In various embodiments, the inducible IL-12 prodrug administered is an amino acid sequence variant of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or any of the aforementioned.

[0019] This method can be used to treat any desired advanced solid tumor, metastatic solid tumor, or lymphoma. For example, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, cancer of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, adenomatous carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer. Cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, gliomas, head and neck cancers, hepatocellular carcinomas, histiocytic hyperplasia, hypopharyngeal cancers, intraocular melanomas, islet cell tumors, Kaposi's sarcoma, kidney cancers, Langerhans cell histiocytosis, laryngeal cancers, lip and oral cancers, liver cancers, lobular carcinoma in situ, lung cancers, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary origin, midline tract carcinoma involving the NUT gene (gene), oral cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal cavity / paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis / ureteral cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratomatous tumor, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0020] Advanced solid tumors or metastatic solid tumors may be colon cancer, lung cancer, melanoma, renal cell carcinoma, or breast cancer.

[0021] Advanced solid tumors, metastatic solid tumors, or lymphomas may include melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, high microsatellite instability or mismatch repair deficiency cancers, high microsatellite instability or mismatch repair deficiency colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial cancer, high tumor mutational load cancers, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), or esophageal cancer.

[0022] The methods disclosed herein are particularly suitable for treating colorectal cancer.

[0023] This disclosure also relates to a pharmaceutical composition comprising an inducible IL-12 prodrug, citric acid and / or citrate, a disaccharide, and a surfactant. In various embodiments, the citrate is sodium citrate, magnesium citrate, or potassium citrate; the disaccharide is sucrose, trehalose, lactose, or maltose; and the surfactant is a nonionic surfactant selected from polysorbate 80, polysorbate 20, Span®-80, castor oil, or poloxamer. The pharmaceutical composition may be a liquid (e.g., an aqueous liquid for injection or infusion) or a solid (e.g., a lyophilized or spray-dried formulation). An exemplary pharmaceutical composition comprises about 1 mg / mL to about 100 mg / mL of an inducible IL-12 prodrug, about 5 mM to about 500 mM of sodium citrate, about 20 mM to about 500 mM of sucrose, and about 0.001% to about 2% of polysorbate 80.

[0024] If the pharmaceutical composition is an aqueous liquid, the pharmaceutical composition has a pH of approximately 5.0 to approximately 8.0.

[0025] 3. Brief explanation of the drawing The drawings are not necessarily to scale or exhaustive. Rather, the focus is generally on illustrating the principles of the inventions described herein. The accompanying drawings constitute part of this specification, illustrate several embodiments consistent with this disclosure, and, together with this specification, help to illustrate the principles of this disclosure. [Brief explanation of the drawing]

[0026] [Figure 1] This graph shows the in vitro activity of chimeric compound 1 in the IL-12 HEK-Blue reporter assay, comparing intact chimeric compound 1 (square) and cleaved chimeric compound 1 (triangle) with chimeric IL-12 (circle).

[0027] [Figure 2] Figures A and B demonstrate that chimeric compound 1 is well-tolerated and induces cleavage-dependent tumor regression. Figure A is a graph showing the antitumor activity of chimeric compound 1 at various doses in a mouse model. Chimeric compound 1 was administered intraperitoneally at 7 μg / dose and 43 μg / dose twice weekly for two weeks, while the NC (non-cleavable) version of chimeric compound 1 was administered at 43 μg / dose. Figure B is a graphical representation of the therapeutic range calculated molarly for chimeric IL-12 and chimeric compound 1 using the same tumor model (MC38), based on the identification of active and toxic dose levels for both treatments.

[0028] [Figure 3A] This graph shows that chimeric compound 1 induces antitumor immunity and protective memory in a syngeneic tumor model. It illustrates the antitumor activity of various doses of chimeric compound 1 in the CT26 mouse syngeneic tumor model. Mice were administered the doses indicated in the legend of the figure twice a week for a total of two weeks. [Figure 3B]This graph shows that chimeric compound 1 induces antitumor immunity and protective memory in a syngeneic tumor model. It illustrates the antitumor activity of various doses of chimeric compound 1 in the B16-F10 mouse syngeneic tumor model. Mice were administered the doses indicated in the legend of the figure twice a week for a total of two weeks. [Figure 3C] This graph shows that chimeric compound 1 induces antitumor immunity and protective memory in a syngeneic tumor model. It illustrates the antitumor activity of various doses of chimeric compound 1 in the EMT-6 mouse syngeneic tumor model. Mice were administered the doses indicated in the legend of the figure twice a week for a total of two weeks. [Figure 3D] This graph shows that chimeric compound 1 induces antitumor immunity and protective memory in a syngeneic tumor model. It illustrates the antitumor activity of various doses of chimeric compound 1 in the A20 model, a mouse syngeneic tumor model. Mice were administered the doses indicated in the legend of the figure twice a week for a total of two weeks. [Figure 3E] This graph shows that chimeric compound 1 induces antitumor immunity and protective memory in a syngeneic tumor model. It illustrates the antitumor activity of various doses of chimeric compound 1 in the EG7.OVA mouse syngeneic tumor model. Mice were administered the doses indicated in the legend of the figure twice a week for a total of two weeks. [Figure 3F] This graph shows that chimeric compound 1 induces antitumor immunity and protective memory in a syngeneic tumor model. The graph also shows tumor volume over time in an EMT6 model where the same tumor was re-challenged on the contralateral flank, demonstrating that treatment with chimeric compound 1 induces immunological memory against the same tumor type. [Figure 3G] This graph shows that chimeric compound 1 induces antitumor immunity and protective memory in a syngeneic tumor model. The graph also shows the tumor volume over time in an MC38 model where the same tumor was re-challenged on the contralateral flank, demonstrating that treatment with chimeric compound 1 induces immunological memory against the same tumor type.

[0029] [Figure 4A]This study demonstrates that treatment with chimeric compound 1 alters the tumor microenvironment and induces activation of intratumor effector cells (NK and CD8+ T cells) in the MC38 model. The heatmap shows transcripts with statistically significant differences between the two treatments, derived from NanoString analysis of bulk RNA from tumor samples. Transcripts with a normalized mean count of less than 50 were excluded from the heatmap. Each lane represents an individual animal. [Figure 4B] This study demonstrates that treatment with chimeric compound 1 alters the tumor microenvironment in the MC38 model and induces activation of tumor effector cells (NK and CD8+ T cells). A volcano plot of differentially expressed transcripts between chimeric compound 1-treated mice and vehicle-treated mice is shown. [Figure 4C] This study demonstrates that treatment with chimeric compound 1 alters the tumor microenvironment in the MC38 model and induces activation of tumor effector cells (NK and CD8+ T cells). It also shows the relative abundance of tumor-infiltrating NK cells producing IFNγ or granzyme B. [Figure 4D] This study demonstrates that treatment with chimeric compound 1 alters the tumor microenvironment in the MC38 model and induces activation of tumor effector cells (NK and CD8+ T cells). It also shows the relative abundance of tumor-infiltrating NK cells producing IFNγ or granzyme B. [Figure 4E] This study demonstrates that treatment with chimeric compound 1 alters the tumor microenvironment in the MC38 model and induces activation of intratumor effector cells (NK and CD8+ T cells). The flow cytometry image shows the relative abundance of tetramer-positive CD8+ T cells producing IFNγ and / or TNF. [Figure 4F] This study demonstrates that treatment with chimeric compound 1 alters the tumor microenvironment in the MC38 model and induces activation of intratumor effector cells (NK and CD8+ T cells). The pie chart shows the relative abundance of multifunctional tetramer-positive CD8+ T cells, measured by examining the co-expression of IFNγ, TNF, and granzyme B.

[0030] [Figure 5A] This study demonstrates that treatment with chimeric compound 1 alters the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. A heatmap of transcripts with statistically significant differences in expression between the two treatments, derived from NanoString analysis of bulk RNA from tumor samples, is shown. Transcripts with a normalized mean count of less than 50 were excluded from the heatmap. Each lane represents an individual animal. [Figure 5B] This study demonstrates that treatment with chimeric compound 1 alters the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. A volcano plot of differentially expressed transcripts between mice treated with chimeric compound 1 and those treated with the vehicle is shown. [Figure 5C] This study demonstrates that treatment with chimeric compound 1 alters the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. Graphs showing pathway scores for the vehicle and chimeric compound 1 regarding antigen processing, interferon, MHC, and NK cell function are presented. [Figure 5D] This shows that treatment with chimeric compound 1 alters the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. The graph shows the normalized counts from individual transcripts for the vehicle and chimeric compound 1. [Figure 5E] This shows that treatment with chimeric compound 1 alters the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. The flow cytometry diagram shows the relative abundance of tetramer+CD8+ T cells producing IFN-gamma and / or granzyme B. [Figure 5F] This shows that treatment with chimeric compound 1 alters the tumor microenvironment and activates B16-F10 tumor-infiltrating NK cells and CD8+ T cells. The pie graph shows the relative abundance of multifunctional tetramer-positive CD8+ T cells, measured by flow cytometry examining the co-expression of IFN-gamma, TNF, and granzyme B.

[0031] [Figure 6]This pie graph demonstrates that treatment with chimeric compound 1 induces a sustained multifunctional CD8+ T cell response. Mice were transplanted with EMT6 cells and randomized to the treatment group. Mice received the treatment twice weekly for two weeks, and tumors were harvested at the time shown in the graph. The proportion of multifunctional 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.

[0032] [Figure 7A] This study demonstrates that systemic administration of chimeric compound 1 leads to CD8+ T cell infiltration and activation in tumors, as evaluated by immunofluorescence staining, and increases IL-12 and IFN-gamma signaling by tumor-infiltrating CD8+ T cells. Mice were transplanted with EMT6 cells and randomized to the treatment group. Mice received the treatment twice a week for two weeks, and tumors were collected on day 11. NanoString GeoMX analysis was performed on FFPE tumor tissue. Immunofluorescence images of tumor-infiltrating CD8+ T cells in the vehicle and in chimeric compound 1 are shown. [Figure 7B] This study demonstrates that systemic administration of chimeric compound 1 leads to CD8+ T cell infiltration and activation in tumors, as evaluated by immunofluorescence staining, and increases IL-12 and IFN-gamma signaling by tumor-infiltrating CD8+ T cells. Mice were transplanted with EMT6 cells and randomized to the treatment group. Mice received the treatment twice a week for two weeks, and tumors were collected on day 11. NanoString GeoMX analysis was performed on FFPE tumor tissue. The graph shows differential gene expression analysis of tumor-infiltrating CD8+ T cells. [Figure 7C] This study demonstrates that systemic administration of chimeric compound 1 leads to CD8+ T cell infiltration and activation in tumors, as assessed by immunofluorescence staining, and increases IL-12 and IFN-gamma signaling by tumor-infiltrating CD8+ T cells. Mice were transplanted with EMT6 cells and randomized to the treatment group. Mice received the compound twice weekly for two weeks, and tumors were collected on day 11. NanoString GeoMX analysis was performed on FFPE tumor tissue. The heatmap shows genes related to IL-12 signaling. [Figure 7D] This study demonstrates that systemic administration of chimeric compound 1 leads to CD8+ T cell infiltration and activation in tumors, as assessed by immunofluorescence staining, and increases IL-12 and IFN-gamma signaling by tumor-infiltrating CD8+ T cells. Mice were transplanted with EMT6 cells and randomized to the treatment group. Mice received the treatment twice weekly for two weeks, and tumors were collected on day 11. NanoString GeoMX analysis was performed on FFPE tumor tissue. The heatmap shows genes associated with IFN-gamma signaling.

[0033] [Figure 8] Graphs A and B show the antitumor activity of chimeric compound 1 in various tests using a syngeneic mouse MC38 tumor model. Graph A shows tumor growth over time in MC38 tumor-bearing mice treated with or without daily FTY720 treatment (+ / -). Graph B shows tumor growth over time in MC38 tumor-bearing mice administered twice weekly with or without chimeric compound 1.

[0034] [Figure 9A] This study demonstrates that chimeric compound 1 is preferentially activated within the tumor mesenter (TME), expanding its therapeutic range compared to chimeric IL-12. The graph shows the presence of total chimeric compound 1 or free chimeric IL-12 over time in the plasma of MC38 tumor-bearing mice treated with chimeric compound 1. The area under the curve was calculated to determine the ratio of total chimeric compound 1 to free chimeric IL-12. [Figure 9B] This study demonstrates that chimeric compound 1 is preferentially activated within the tumor mesenter (TME), expanding its therapeutic range compared to chimeric IL-12. The graph shows the presence of total chimeric compound 1 or free chimeric IL-12 over time in tumors of MC38 tumor-bearing mice treated with chimeric compound 1. The area under the curve was calculated to determine the ratio of total chimeric compound 1 to free chimeric IL-12. [Figure 9C]This shows that chimeric compound 1 is preferentially activated within the TME, expanding its therapeutic range compared to chimeric IL-12. The pie chart graph shows the proportion of pluripotent CD8+ T cells in tumors, peripheral blood, tumor-inflow lymph nodes, or non-tumor-inflow lymph nodes of MC38 tumor-bearing mice administered chimeric compound 1 twice. The proportion of pluripotent CD8+ T cells was measured by examining the co-expression of IFN-gamma, TNF, and granzyme B after PMA / ionomycin restimulation.

[0035] [Figure 10A] This study demonstrates that chimeric compound 1 activates tumor-infiltrating immune cell populations in the MC38 syngeneic tumor model. Representative flow plots of CD11b+ tumor-infiltrating dendritic cells and CD103+ tumor-infiltrating dendritic cells are shown. Unless otherwise noted, data are expressed as mean ± SD, and p-values ​​are derived from t-tests (** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.00001). [Figure 10B] This study demonstrates that chimeric compound 1 activates tumor-infiltrating immune cell populations in the MC38 syngeneic tumor model. The graph shows the ratio of CD11b+ tumor-infiltrating dendritic cells to CD103+ tumor-infiltrating dendritic cells. Unless otherwise noted, data are expressed as mean ± SD, and p-values ​​are derived from t-tests (** for p<0.01, *** for p<0.001, **** for p<0.00001). [Figure 10C] This shows that chimeric compound 1 activates the tumor-infiltrating immune cell population in the MC38 syngeneic tumor model. The graph shows the relative abundance of conventional CD4+ T cells with the TH1 phenotype (Tbet+IFN-gamma+TFN+). Unless otherwise noted, data are expressed as mean ± SD, and p-values ​​are derived from t-tests (** for p<0.01, *** for p<0.001, **** for p<0.00001). [Figure 10D]This shows that chimeric compound 1 activates tumor-infiltrating immune cell populations in the MC38 syngeneic tumor model. A representative flow plot shows the relative abundance of tumor-infiltrating FoxP3+ Treg cells that produce IFN-gamma and TNF. Unless otherwise noted, data are expressed as mean ± SD, and p-values ​​are derived from t-tests (** for p<0.01, *** for p<0.001, **** for p<0.00001). [Figure 10E] This study demonstrates that chimeric compound 1 activates tumor-infiltrating immune cell populations in the MC38 syngeneic tumor model. The graph shows the relative abundance of tumor-infiltrating FoxP3+ Treg cells that produce IFN-gamma and TNF. Unless otherwise noted, data are expressed as mean ± SD, and p-values ​​are derived from t-tests (** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.00001). [Figure 10F] This shows that chimeric compound 1 activates tumor-infiltrating immune cell populations in the MC38 syngeneic tumor model. The graph shows the proportion of tumor-infiltrating FoxP3+ Treg cells that produce Tbet. Unless otherwise noted, data are expressed as mean ± SD, and p-values ​​are derived from t-tests (** for p<0.01, *** for p<0.001, **** for p<0.00001).

[0036] [Figure 11A] This shows that systemic treatment with chimeric compound 1 increases novel TCR clones and enhances the overall clonality of the TCR repertoire. The heatmap shows downstream TCR signaling in tumor CD8+ T cells after treatment with the vehicle and chimeric compound 1. [Figure 11B] This study demonstrates that systemic treatment with chimeric compound 1 increases the number of novel TCR clones and enhances the overall clonality of the TCR repertoire. The graph shows the clonal frequency of individual VDJ recombinations on the TCR-beta chain in a vehicle-treated EMT-6 tumor model. Live T cells were isolated from the EMT-6 tumor on day 11 and TCR sequencing was performed. [Figure 11C]This study demonstrates that systemic treatment with chimeric compound 1 increases the number of novel TCR clones and enhances the overall clonality of the TCR repertoire. The graph shows the clonal frequency of individual VDJ recombinations on the TCR-beta chain in an EMT-6 tumor model treated with chimeric compound 1. Live T cells were isolated from the EMT-6 tumor on day 11 and TCR sequencing was performed. [Figure 11D] This study demonstrates that systemic treatment with chimeric compound 1 increases the number of novel TCR clones and enhances the overall clonality of the TCR repertoire. The graph shows the clonality index scores for the vehicle and chimeric compound 1. [Figure 11E] This study demonstrates that systemic treatment with chimeric compound 1 increases the number of novel TCR clones and enhances the overall clonality of the TCR repertoire. The graph shows the frequencies of the top 50 TCR clones plotted for each animal.

[0037] [Figure 12A]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12B]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12C]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12D]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12E]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12F]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12G]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12H]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12I]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12J]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12K]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12L]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12M]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12N]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12O]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12P]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12Q]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12R]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12S]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12T]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12U]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells. [Figure 12V]This shows that treatment with chimeric compound 1 promotes increased mitochondrial respiration and fitness. Figure 12A is a heatmap of tumor-infiltrating CD8+ T cells showing genes related to glycolysis. Figures 12B-12C are graphs showing 2-NDBG uptake in EMT-6 TILs in either vehicle-treated or chimeric compound 1-treated animals. Figures 12D-12F are heatmaps of tumor-infiltrating CD8+ T cells showing genes related to the TCA cycle (Figure 12D), mitochondrial biosynthesis (Figure 12E), and mitochondrial translation (Figure 12F). Figures 12G, 12H, 12K, 12L, 12O, and 12P are graphs showing EMT-6-infiltrating CD8+ T cells in either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12G and 12H), TMRM (Figures 12K and 12L), and MitoSOX (Figures 12O-12P). Figures 12I, 12J, 12M, 12N, 12Q, and 12R are graphs showing EMT-6 infiltrating NK cells from either vehicle-treated or chimeric compound 1-treated animals stained with mitotracker red (Figures 12I and 12J), TMRM (Figures 12M-12N), or MitoSOX (Figures 12Q-12R). Data are expressed as mean ± SD, and p-values ​​are derived from t-tests (* for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001). Figures 12S-12T show that chimeric compound 1 preferentially increases new clones over previously existing clones. Figure 12S discloses sequence numbers 450-457 in the order they are listed. Figure 12T discloses sequence numbers 458-465 in the order they are listed. Figures 17A–17B are graphs showing the proportion of the top 50 common clones in the TCR repertoire. Figures 12U–12V show that treatment with chimeric compound 1 increases mitochondrial mass and fitness in tumor-infiltrating immune cells.

[0038] [Figure 13A]This study demonstrates that 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 prior to analysis is shown. [Figure 13B] This shows that compound 36 is inducible, stable in human serum, and selectively processed by dissociated primary human tumor samples. The graph shows the protein cleavage measured by activity in a human Tblast assay after incubation with primary human dissociated tumor samples (n=88) or primary human healthy cells (n=13) exposed to compound 36 for 48 hours prior to protein cleavage. The box plots represent the 25th and 75th percentiles, and the lines represent the median for each indication. The whiskers represent the minimum and maximum values ​​for a given indication.

[0039] [Figure 14] A and B are graphs showing IFN-gamma production in mouse-derived TILs treated with either the vehicle or chimeric compound 1, with and without ex vivo restimulation, as measured by intracellular cytokine staining.

[0040] [Figure 15] A and B show the selective activation of tumor-infiltrating immune cells. A is a graph showing the relative abundance of conventional CD4+ conventional T cells (FoxP3-) that produce IFNγ and TNF in tumor tissue compared to peripheral tissue. B is a graph showing the relative abundance of NK cells that produce IFNγ and TNF in tumor tissue compared to peripheral tissue.

[0041] [Figure 16A]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16B]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16C]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16D]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16E]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16F]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16G]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16H]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16I]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16J]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16K]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16L]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16M]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16N]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16O]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16P]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16Q]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16R]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16S]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16T]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16U]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16V]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16W]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16X]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16Y]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16Z]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZA]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZB]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZC]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZD]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZE]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZF]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZG]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZH]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZI]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZJ]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZK]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZL]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZM]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZN]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZO]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase. [Figure 16ZP]Figures 16A, 16C, 16E, 16G, 16I, 16K, 16M, 16O, 16Q, 16S, 16U, 16W, 16Y, 16ZA, 16ZC, 16ZE, 16ZG, 16ZI, 16ZK, 16ZM, and 16ZO are graphs showing the activity of inducible IL-12 prodrugs in the HEK-Blue IL-12 reporter assay in the presence of human serum albumin (HSA). Squares represent the activity of intact inducible IL-12 prodrugs, triangles represent the activity of in vitro protease-activated (cleaved) inducible IL-12 prodrugs, and circles represent the activity of control chimeric IL-12. The EC50 values ​​for each are shown in the table (ND = not determined). The analysis was performed based on the quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the inducible IL-12 prodrug as being active and inducible. Figures 16B, 16D, 16F, 16H, 16J, 16L, 16N, 16P, 16R, 16T, 16V, 16X, 16Z, 16ZB, 16ZD, 16ZF, 16ZH, 16ZI, 16ZJ, 16ZL, 16ZN, and 16ZP are images of SDS-PAGE gels showing the results of protein cleavage assays using elastase.

[0042] [Figure 17-1] Graphs A, C, E, G, and I show the results of analyzing inducible IL-12 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 5 μg, 50 μg, and 500 μg of each inducible IL-12 prodrug twice weekly. The data indicate that tumor volume was suppressed over time in a dose-dependent manner. Graphs B, D, F, H, and J show the group mean body weight over time. [Figure 17-2]Graphs A, C, E, G, and I show the results of analyzing inducible IL-12 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 5 μg, 50 μg, and 500 μg of each inducible IL-12 prodrug twice weekly. The data indicate that tumor volume was suppressed over time in a dose-dependent manner. Graphs B, D, F, H, and J show the group mean body weight over time. [Figure 17-3] Graphs A, C, E, G, and I show the results of analyzing inducible IL-12 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 5 μg, 50 μg, and 500 μg of each inducible IL-12 prodrug twice weekly. The data indicate that tumor volume was suppressed over time in a dose-dependent manner. Graphs B, D, F, H, and J show the group mean body weight over time. [Figure 17-4] Graphs A, C, E, G, and I show the results of analyzing inducible IL-12 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 5 μg, 50 μg, and 500 μg of each inducible IL-12 prodrug twice weekly. The data indicate that tumor volume was suppressed over time in a dose-dependent manner. Graphs B, D, F, H, and J show the group mean body weight over time. [Figure 17-5] Graphs A, C, E, G, and I show the results of analyzing inducible IL-12 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 5 μg, 50 μg, and 500 μg of each inducible IL-12 prodrug twice weekly. The data indicate that tumor volume was suppressed over time in a dose-dependent manner. Graphs B, D, F, H, and J show the group mean body weight over time.

[0043] [Figure 18A] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18B]This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18C] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18D] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18E] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18F] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18G] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18H] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18I] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18J] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18K] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18L] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18M] This is a schematic diagram showing an inducible IL-12 prodrug. [Figure 18N] This is a schematic diagram showing an inducible IL-12 prodrug. [Modes for carrying out the invention]

[0044] 4. Detailed explanation This disclosure relates to compositions and methods for treating cancer using inducible IL-12 prodrugs.

[0045] A. IL-12 Prodrug This disclosure relates to an inducible IL-12 prodrug containing weakened IL-12 and having a longer half-life compared to naturally occurring IL-12. The inducible IL-12 prodrugs disclosed herein contain at least one polypeptide chain and may optionally contain two or more polypeptides. The two or more polypeptide chains disclosed herein are distinct, 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 extension element, an IL-12 blocking element, and a protease-cleavable linker. The p35 and p40 subunits associate to form an IL-12 heterodimer having endogenous IL-12 receptor agonist activity. In the inducible IL-12 prodrug, the IL-12 receptor agonist activity is weakened and the circulating half-life is extended. IL-12 receptor agonist activity is attenuated via blocking elements. Half-life extension elements can also contribute to attenuation, for example, through steric effects. Blocking elements can block all or part of the IL-12 receptor agonist activity by steric blockage and / or non-covalent binding to IL-12 (e.g., to the p35, p40, or p35p40 complex). When the protease-cleavable linker is cleaved, an active form of IL-12 (e.g., more active than the inducible IL-12 prodrug) is released from the inducible IL-12 prodrug. Typically, the released IL-12 is at least 10 times more active than the inducible IL-12 prodrug. Preferably, the released IL-12 has activity 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 than that of the inducible IL-12 prodrug.

[0046] The form of IL-12 released when an inducible IL-12 prodrug is cleaved typically has a short half-life, often substantially similar to that of naturally occurring IL-12. While the half-life of the inducible IL-12 prodrug is prolonged, toxicity is reduced or eliminated because the inducible IL-12 prodrug in the circulating blood is attenuated and the target of active IL-12 is directed to the desired site (e.g., the tumor microenvironment).

[0047] As will be understood by those skilled in the art, the number of polypeptide chains, as well as the positions of the p35 and p40 subunits, half-life extension elements, protease-cleavable linkers, and blocking elements (and components of such elements, such as VH domains or VL domains) on the polypeptide chains, can vary and are often a matter of design preference. All such variations are encompassed in this disclosure.

[0048] In various embodiments, the inducible IL-12 prodrug comprises two distinct polypeptide chains. Typically, the first polypeptide chain contains p35, and the second polypeptide chain contains p40. The p35 and p40 subunits associate to form a biologically active heterodimer. The p35p40 heterodimer complex may be covalently bonded, for example, via disulfide bonds.

[0049] In various embodiments, either the first or second polypeptide may include an IL-12 blocking element (e.g., 13zac bound to IL-12) operably linked to an IL-12 subunit via a protease-cleavable linker. The other polypeptide chain may further include a half-life extension element operably linked to an IL-12 subunit via a protease-cleavable linker. Preferably, the inducible IL-12 prodrug includes one functional blocking element and one functional half-life extension element. For example, if the first polypeptide chain includes an IL-12 blocking element, the second polypeptide chain does not include 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 an optional protease-cleavable linker. The protease-cleavable linkers in the first and second polypeptide chains may be the same or different. Preferably, the protease-cleavable linkers in the first and second polypeptide chains are the same. The blocking element in this inducible IL-12 prodrug may be a single-chain antibody. Any single-chain antibody that has binding specificity to IL-12 can be used as the blocking element. Preferably, the blocking element is scFv.

[0050] The inducible IL-12 prodrugs disclosed herein preferably contain one half-life extension element and one blocking element, but such elements may contain two or more components present on the same polypeptide chain or on different polypeptide chains. For illustrative purposes, and as disclosed and illustrated 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 scenario, these components can associate in the inducible IL-12 prodrug to form an antigen-binding site such as a Fab that binds to IL-12 and weakens IL-12 activity.

[0051] In various embodiments, the p35 subunit and the p40 subunit may be located on the same polypeptide chain, or they may be linked via an optional protease-cleavable linker. In such embodiments of double-chain or polychain prodrugs, at least one of the half-life extension element, the blocking element, or a component of the half-life extension element or the blocking element is located on a separate polypeptide. For example, the first polypeptide may contain p35 and p40 linked via an optional 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 the antibody light chain. The second polypeptide contains a portion of the antibody heavy chain that is complementary to the antibody light chain. The portion of the antibody light chain, together with the complementary heavy chain, associates in the inducible IL-12 prodrug to form an IL-12 binding site. In another example, the first polypeptide comprises a p35 subunit, a p40 subunit, a half-life extension element, and a portion of the antibody heavy chain. In this example, the second polypeptide contains a portion of the 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 an inducible IL-12 prodrug to form an IL-12 binding site. In these inducible IL-12 prodrugs, the p35 and p40 subunits may be operably linked via an optional protease-cleavable linker. Preferably, the p35 and p40 subunits are operably linked by a non-cleavable linker.

[0052] In the inducible IL-12 prodrugs disclosed herein, the half-life extension element is preferably operably linked to either a p35 subunit or a p40 subunit via a protease-cleavable linker. For example, an 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, an 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 the blocking element) via an optional protease-cleavable linker. In such exemplary embodiments, the inducible IL-12 prodrug comprises at least one additional polypeptide comprising an IL-12 subunit (p40 or p35) that is not present on the first polypeptide. Further arrangements of elements in the inducible IL-12 prodrug are envisioned and are included in this disclosure. For example, a blocking element may be operably coupled to either the p35 subunit or the p40 subunit via a protease-cleavable linker. Either the half-life extension element or the blocking element may be operably coupled to the p35 subunit, and the other half-life extension element or the blocking element may be operably coupled to the p40 subunit. If the half-life extension element is operably coupled to the p35 subunit, the blocking element may be operably coupled to the p40 subunit. If the half-life extension element is operably coupled to the p40 subunit, the blocking element may be operably coupled to the p35 subunit. The blocking element in this inducible IL-12 prodrug is preferably Fab.

[0053] An inducible IL-12 prodrug may contain three polypeptide chains. Typically, 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 (component) of the blocking element. If the IL-12 subunit in the first polypeptide is p35, then the IL-12 subunit in the second polypeptide is p40. If the IL-12 subunit in the first polypeptide is p40, then the IL-12 subunit in the second polypeptide is p35. Upon expression and folding of the polypeptides, the p35 and p40 subunits can associate to form a biologically active heterodimer. The p35p40 heterodimer complex may be covalently bonded, for example, via disulfide bonds.

[0054] In some embodiments, the first polypeptide may further include a half-life extension element, which, if present, is operably linked to an IL-12 subunit via a protease-cleavable linker. The second polypeptide may further include a portion of the blocking element, and the third polypeptide may include the remainder of the blocking element. In such an inducible IL-12 prodrug, the IL-12 blocking element may be an antigen-binding fragment of an antibody formed by the interaction of polypeptide 2 and polypeptide 3, e.g., a Fab fragment. In some embodiments, the second polypeptide may include at least an antigen-binding portion of the antibody light chain. Alternatively, the second polypeptide may include at least an antigen-binding portion of the 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 an 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 further, the half-life extension element may be operably linked to a portion of a blocking element (e.g., the antigen-binding portion of the antibody light chain or the antigen-binding portion of the heavy chain) via an optional protease-cleavable linker. If a half-life extension element is present and operably linked to the IL-12 subunit, the antibody heavy chain or light chain may be operably linked to the IL-12 subunit via a protease-cleavable linker, or alternatively, if a half-life extension element is present and operably linked to the IL-12 subunit, the antibody heavy chain or light chain may be operably linked to the IL-12 subunit via an optional cleavable linker. The protease-cleavable linkers in the first, second, and / or polypeptide chains may be the same or different.

[0055] Compounds 1, 2, 3, 4, 5, and 6 are specific examples of inducible IL-12 prodrugs comprising two polypeptide chains for use as relating to this disclosure. Further details regarding compounds 1, 2, 3, 4, 5, and 6, as well as their activities, are disclosed in International Application PCT / US2021 / 33014.

[0056] Compounds 7, 8, 17, 18, 21-28, 34, and 35 are specific examples of inducible IL-12 prodrugs containing one polypeptide chain for use according to the Disclosure. Compounds 9-13, 15, 19, 20, 29-31, and 36 are specific examples of inducible IL-12 prodrugs containing two polypeptide chains for use according to the Disclosure. Compounds 14, 16, 32, and 33 are specific examples of inducible IL-12 prodrugs containing three polypeptide chains for use according to the Disclosure. [Table 1-1] [Table 1-2]

[0057] As described above, IL-12 may optionally be mutaine. IL-12 mutaine retains IL-12 activity, such as endogenous IL-12 receptor agonist activity. The p35 and / or p40 subunits of IL-12 may be mutaine.

[0058] The present invention also relates to certain single-chain IL-12-inducible polypeptides. The single-chain IL-12 polypeptides disclosed herein comprise IL-12, a blocking element, a half-life extension element, and a protease-cleavable linker. IL-12 has receptor agonist activity against its homologous IL-12 receptors. The IL-12 receptor activating activity is attenuated when the blocking element binds to IL-12. When the protease-cleavable linker is cleaved, the active IL-12 polypeptide is released. Single-chain inducible IL-12 polypeptides are disclosed in International Applications PCT / US2019 / 032320 and PCT / US2019 / 032322.

[0059] B. Half-life extension element This specification details domains that extend the half-life of inducible IL-12 prodrugs. Extending the in vivo half-life of therapeutic molecules with short natural half-lives allows for more tolerable and manageable dosing regimens without sacrificing efficacy.

[0060] Half-life extension elements extend the in vivo half-life, resulting in alterations to the pharmacokinetics and pharmacokinetics of inducible IL-12 prodrugs. While not theoretically bound, half-life extension elements alter the pharmacokinetic properties of inducible IL-12 prodrugs, including alterations to tissue distribution, permeation, and diffusion. In some embodiments, half-life extension elements can improve tissue targeting, tissue permeation, intra-tissue diffusion, and enhanced efficacy compared to proteins without half-life extension elements. While not theoretically bound, exemplary methods for improving the pharmacokinetics of polypeptides involve the expression of elements in polypeptide chains that bind to receptors that are recycled to the cell's plasma membrane rather than being degraded by lysosomes, such as FcRn receptors and transferrin receptors on endothelial cells. Three proteins, e.g., human IgG, HSA (or fragments), and transferrin, persist in human serum much longer than would be predicted from their size alone, and this is due to their binding ability to receptors that are recycled rather than degraded by lysosomes. These proteins or fragments are typically linked to other polypeptides to retain FcRn binding ability and to extend their serum half-lives. HSAs may also be directly bound to pharmaceutical compositions or linked via short linkers. HSA fragments may also be used. HSAs and their fragments can function as both blocking elements and half-life extending elements. Human IgG and Fc fragments can also perform similar functions.

[0061] Serum half-life extension elements may also be antigen-binding polypeptides that bind to long-lived serum proteins such as serum albumin and transferrin. Examples of such polypeptides include antibodies and their fragments, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies (e.g., heavy-chain variable domain (VH), light-chain variable domain (VL), and camel-type nanobody variable domain (VHH)), and dAbs. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as antikalin, affilin, affibody molecules, affimers, afitins, alpha bodies, avimers, DARPin, fynomers, Knitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of antigen-binding polypeptides include ligands for a desired receptor, ligand-binding moieties of receptors, lectins, and peptides that bind to or associate with one or more target antigens.

[0062] The half-life extension elements provided herein are preferably a human serum albumin (HSA) binding domain and an antigen-binding polypeptide or fragment thereof that binds to human serum albumin or immunoglobulin Fc.

[0063] The half-life extension element for the inducible IL-12 prodrug extends the half-life of the inducible IL-12 prodrug to 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 longer. In some embodiments, the half-life extension element extends the half-life of the inducible IL-12 prodrug to at least 2-3 days, 3-4 days, 4-5 days, 5-6 days, 6-7 days, 7-8 days, or longer.

[0064] C. Shielding element The blocking element may be any element that binds to IL-12 and inhibits the ability of an inducible IL-12 prodrug to bind to its receptor and / or activate it. The blocking element can inhibit the receptor binding ability and / or receptor activation ability of IL-12 by, for example, steric blockage and / or non-covalent binding to the IL-12 prodrug. The blocking elements disclosed herein may bind to p19, p35, p40, p35p40 heterodimer complex, or p19p40 heterodimer complex.

[0065] Examples of suitable blocking elements include the full-length or IL-12-binding fragment or mutain of the IL-12 congener receptor. Antibodies and their antigen-binding fragments can also be used, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies (e.g., heavy-chain variable domain (VH), light-chain variable domain (VL), and camel-type nanobody variable domain (VHH)), dAbs, etc. Other suitable antigen-binding domains that bind to IL-12 can also be used, including non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as antikalin, affilin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, finomers, Knitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block the binding of IL-12 to its homologous receptor. Advantageously, such moieties can also function as half-life extension elements. For example, peptides modified by binding to a water-soluble polymer such as PEG can sterically inhibit or prevent the binding of cytokines to their receptors. Alternatively, polypeptides or fragments thereof with long serum half-lives, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, and fragments of such polypeptides and mutaine, can also be used.

[0066] Preferred IL-12 blocking elements are single-chain variable fragments (scFv) or Fab fragments. scFv blocking elements contain the amino acid sequences shown in SEQ ID NOs. 144-188. Alternatively, Fab blocking elements contain the amino acid sequences shown in SEQ ID NOs. 189-194. The IL-12 antibody fragments encompassed in SEQ ID NOs. 144-194 are optimized to enhance the potential for development of the inducible IL-12 prodrugs disclosed herein.

[0067] Preferred antibody light chain blocking elements include SEQ ID NOs: 192-193. These preferred components can be located on one polypeptide chain, and the complementary antigen-binding portion of the heavy chain can be located on a second polypeptide chain. Preferred heavy chain blocking elements include SEQ ID NOs: 189-191 and 194. These preferred components can be located on one polypeptide chain, and the complementary light chain can be located on a second polypeptide chain. The antibody light chain and antibody heavy chain together form the IL-12 binding site.

[0068] In some embodiments, the IL-12 blocking element includes an amino acid sequence that is identical to, for example, SEQ ID NOs. 144-194 by 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%, and at least about 99% across the entire length of SEQ ID NOs. Typically, the amino acid sequence of the CDR remains unchanged, and amino acid substitutions reside in the framework region.

[0069] This disclosure also relates to functional variants of IL-12 blocking elements, including SEQ ID NOs. 144-194. These functional variants of IL-12 blocking elements, including SEQ ID NOs. 144-194, generally differ from SEQ ID NOs. 144-194 by one or several amino acids (including substitutions, deletions, insertions, or combinations thereof) and substantially retain the ability to bind to IL-12 polypeptides (e.g., p35 subunit, p40 subunit, or p35p40 complex) and inhibit the binding of IL-12 to its homologous receptors.

[0070] A functional variant may include at least one amino acid substitution, deletion, or insertion compared to the IL-12 blocking elements including SEQ ID NOs. 144-194. A functional variant may include one, two, three, four, five, six, seven, eight, nine, or ten amino acid changes compared to the IL-12 blocking elements including SEQ ID NOs. 144-194. In some preferred embodiments, a functional variant differs from the IL-12 blocking elements including SEQ ID NOs. 144-194 by fewer than ten, eight, five, four, three, two, or one amino acid change, e.g., an amino acid substitution or deletion. In other embodiments, a functional variant may include one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions compared to SEQ ID NOs. 144-194. The amino acid substitutions may be conservative or non-conservative, but are preferably conservative.

[0071] In other embodiments, functional variants 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 containing SEQ ID NOs. 144-194. Non-conservative amino acid substitutions are recognizable to those skilled in the art. Functional variants of the isolated portion preferably contain one or fewer, two or fewer, three or fewer, four or fewer, or five or fewer amino acid deletions.

[0072] This specification also discloses an inducible IL-12 prodrug comprising a blocking element having specificity for IL-12 and a half-life extension element. The blocking element is an antibody or antigen-binding fragment having binding specificity for IL-12, in particular for the IL-12 subunit beta precursor (p40) as defined by SEQ ID NO: 421 disclosed herein. This antibody or antigen-binding fragment comprises an antigen-binding domain that binds to the residues of SEQ ID NO: 421 shown in Table 2. This disclosure relates to an antibody or antigen-binding fragment that binds to an IL-12 epitope defined by the amino acid residues shown in Table 2, an inducible IL-12 prodrug comprising such antibody or antigen-binding fragment, and the use of such antibody or antigen-binding fragment for the preparation of an inducible IL-12 prodrug or a drug comprising such inducible IL-12 prodrug.

[0073] [Table 2]

[0074] D. Protease-cleavable linker As disclosed herein, an inducible IL-12 prodrug comprises one or more linker sequences. The linker sequences help to provide flexibility between polypeptides, for example, allowing a blocking element to inhibit the activity of IL-12. The linker may be located between IL-12 subunits, half-life extension elements, and / or blocking elements. As described herein, an inducible IL-12 prodrug comprises a protease-cleavable linker. The protease-cleavable linker may comprise one or more cleavage sites for one or more desired proteases. Preferably, the desired protease is enriched or selectively expressed at a 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.

[0075] Preferred linkers are typically less than about 100 amino acids. Such linkers can be of various lengths, such as 1 amino acid (e.g., Gly) to 30 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 50 amino acids, 1 amino acid 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 has an amino acid length of 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. Preferred linkers are typically about 5 amino acids to about 30 amino acids.

[0076] Preferably, the linker length is in the range of 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 linked domains. In a preferred embodiment, the linker is cleavable by a cleavage agent, such as an enzyme. Preferably, the separated portion contains a protease cleavage site. Optionally, the separated portion contains one or more cleavage sites. The separated portion may contain a single protease cleavage site. The separated portion may also contain two or more protease cleavage sites, for example, two, three, four, five, or more. If the separated portion contains two or more protease cleavage sites, these cleavage sites may be cleaved by the same protease or different proteases. A separated portion containing two or more cleavage sites is called a "tandem linker". Two or more cleavage sites can be positioned in any desired configuration, including, but not limited to, a configuration where one cleavage site is adjacent to another, a configuration where one cleavage site overlaps another, or a configuration where one cleavage site is followed by another with an amino acid interposed between the two cleavage sites.

[0077] Of particular note in the present invention is the disease-specific protease-cleavable linker. Furthermore, a protease-cleavable linker that is preferentially cleaved at a desired location within the body, such as the tumor microenvironment, compared to peripheral circulation, is also preferred. For example, the rate at which a protease-cleavable linker is cleaved within the tumor microenvironment may be at least about 10 times, at least about 100 times, at least about 1000 times, or at least about 10,000 times faster in a desired location within the body, such as the tumor microenvironment, compared to peripheral circulation (e.g., plasma).

[0078] Proteases known to be associated with abnormal cells or tissues include serine proteases, cysteine ​​proteases, aspartate proteases, threonine proteases, glutamate proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, cathepsins B, C, D, E, G, K, L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidine, bromelain, calpain, caspases, and caspase- 3. This includes, but is not limited to, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, plasmmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAPα), dipeptidyl peptidase, meprin, granzyme, and dipeptidyl peptidase IV (DPPIV / CD26). Proteases capable of cleaving linker amino acid sequences (which may be encoded by chimeric nucleic acid sequences provided herein) may be selected from the group consisting of, for example, prostate-specific antigen (PSA), matrix metalloproteinases (MMPs), ADAM (A Disintigrin and a Metalloproteinase), plasminogen activator, cathepsins, caspases, tumor cell surface proteases, and elastases. MMPs may be, for example, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), or matrix metalloproteinase 14 (MMP14).Furthermore, or alternatively, the linker may be cleaved by cathepsins such as cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, and / or cathepsin L. Preferably, the linker may be cleaved by MMP14 or cathepsin L.

[0079] Table 3 shows proteases useful for linker cleavage and for use in the inducible IL-12 prodrugs disclosed herein, and Table 4 shows exemplary proteases and their cleavage sites. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

[0080] 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. In particular, MMP9-cleavable linkers, ADAM-cleavable linkers, CTSL1-cleavable linkers, FAPα-cleavable linkers, and cathepsin-cleavable linkers are examples. Several preferred protease-cleavable linkers are cleaved by MMPs and / or cathepsins.

[0081] The separation portions disclosed herein are typically less than 100 amino acids. Such separation portions can be of various lengths, such as 1 amino acid (e.g., Gly) to 30 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 50 amino acids, 1 amino acid 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 has an amino acid length of 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. Preferred linkers are typically about 5 amino acids to about 30 amino acids.

[0082] Preferably, the linker length is in the range of 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 domains being linked.

[0083] In some embodiments, the separation portion is the sequence GPAGLYAQ (sequence number 195), GPAGMKGL (sequence number 196), PGGPAGIG (sequence number 197), ALFKSSFP (sequence number 198), ALFFSSPP (sequence number 199), LAQRLRSS (sequence number 200), LAQKLKSS (sequence number 201), GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (sequence number 202), RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (sequence number 203), KG GGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO: 204), RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO: 205), KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO: 206), SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO: 207), RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (SEQ ID NO: 208), GGGALFKSSFPLAQKLKSSPGGPAGIGGGR (SEQ ID NO: 209), RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (SEQ ID NO: 210), RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (SEQ ID NO: 211), RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO: 212), GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO: 213), GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO: 214), RGGALFKSSFPLAQKLKS Includes SGPAGLYAQGGK (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).

[0084] Certain preferred isolates include the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). Isolates disclosed herein may contain one or more identical or different cleavage motifs or functional variants. Isolates may contain one, two, three, four, five, or more cleavage motifs or functional variants. Isolates containing 30 amino acids may contain two cleavage motifs or functional variants, three cleavage motifs or functional variants, or more. The “functional variants” of an isolate retain the ability to be cleaved efficiently at a target site (e.g., a tumor microenvironment expressing high levels of protease) but are not cleaved or are cleaved inefficiently at a peripheral site (e.g., serum). For example, the functional variant retains at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95%, or more of the cleavage efficiency of the separation portion containing any one of sequence numbers 195-220 or 447-448.

[0085] The isolated portion containing multiple cleavage motifs may be selected from SEQ ID NOs: 195-201 or 447-448, and combinations thereof. A preferred isolated portion containing multiple cleavage motifs contains amino acids selected from SEQ ID NOs: 202-220.

[0086] The isolation portion may contain both ALFKSSFP (sequence number 198) and GPAGLYAQ (sequence number 195). The isolation portion may contain two cleavage motifs, each having the sequence GPAGLYAQ (sequence number 195). Alternatively, or further, the isolation portion may contain two cleavage motifs, each having the sequence ALFKSSFP (sequence number 198). The isolation portion may contain the same or a different third cleavage motif.

[0087] 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 about 99% identical to sequence numbers 195–220 or 447–448 over the entire length of sequence numbers 195–220 or 447–448.

[0088] This disclosure also relates to functional variants of the isolates including SEQ ID NOs. 195-220 or 447-448. Functional variants of the isolates including SEQ ID NOs. 195-220 or 447-448 generally differ from SEQ ID NOs. 195-220 or 447-448 by one or several amino acids (including substitutions, deletions, insertions, or combinations thereof) and substantially retain the ability to be cleaved by proteases.

[0089] A functional variant may contain at least one amino acid substitution, deletion, or insertion compared to the isolated portion containing SEQ ID NOs. 195-220 or 447-448. A functional variant may contain one, two, three, four, five, six, seven, eight, nine, or ten amino acid changes compared to the isolated portion containing SEQ ID NOs. 195-220 or 447-448. In some preferred embodiments, a functional variant differs from the isolated portion containing SEQ ID NOs. 195-220 by fewer than ten, eight, five, four, three, two, or one amino acid change, e.g., an amino acid substitution or deletion. In other embodiments, a functional variant may contain one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions compared to SEQ ID NOs. 195-220 or 447-448. The amino acid substitution may be either a conservative or non-conservative substitution, but a conservative substitution is preferred.

[0090] In other embodiments, the functional variant of the isolated portion may contain one, two, three, four, or five or more non-conservative amino acid substitutions compared to the isolated portion containing SEQ ID NOs. 195-220 or 447-448. Non-conservative amino acid substitutions are recognizable to those skilled in the art. The functional variant of the isolated portion preferably contains one or fewer, two or fewer, three or fewer, four or fewer, or five or fewer amino acid deletions.

[0091] The disclosed amino acid sequence in the isolated portion can be described by its relative linear position in the isolated portion with respect to easily cleavable bonds. As will be well understood by those skilled in the art, an isolated portion containing an 8-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', and P4', with the easily cleavable bond located between P1 and P1'. For example, the amino acid positions in the isolated portion containing the sequence GPAGLYAQ (SEQ ID NO: 195) can be described as follows: [Table 7]

[0092] The amino acid positions of the isolated region containing the sequence ALFKSSFP (sequence number 198) can be described as follows: [Table 8]

[0093] Preferably, the amino acids surrounding the cleavage site (for example, the P1 and P1' positions of sequence numbers 195-201 or 447-448) are not substituted.

[0094] In various embodiments, the isolated 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, the functional variants of GPAGLYAQ (SEQ ID NO: 447) or ALFKSSFP (SEQ ID NO: 198) may contain one or more amino acid substitutions and may substantially retain the ability to be cleaved by proteases. In particular, the functional variant of GPAGLYAQ (SEQ ID NO: 195) is cleaved by MMP14, and the functional variant of ALFKSSFP (SEQ ID NO: 198) is cleaved by captepsin L (CTSL1). The functional variants also retain the ability to be cleaved efficiently at target sites (e.g., tumor microenvironments expressing high levels of proteases). For example, functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95%, or more of the cleavage efficiency of the isolation portion containing the amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.

[0095] Preferably, the functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) contains one or fewer, two or fewer, three or fewer, four or fewer, or five or fewer 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 unsubstituted. 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.

[0096] The functional variant of GPAGLYAQ (SEQ ID NO: 195) may preferably include one or more of the following: a) an amino acid substitution of arginine at the P4 position, b) an amino acid substitution of leucine, valine, asparagine, or proline at the P3 position, c) an amino acid substitution of asparagine at the P2 position, d) an amino acid substitution of histidine, asparagine, or glycine at the P1 position, e) an amino acid substitution of asparagine, isoleucine, or leucine at the P1' position, f) an amino acid substitution of tyrosine or arginine at the P2' position, g) an amino acid substitution of glycine, arginine, or alanine at the P3' position, or h) an amino acid substitution of serine, glutamine, or lysine at the P4' position. In functional variants of GPAGLYAQ (SEQ ID NO: 195), amino acid substitutions of a) arginine or isoleucine at the P3 position, b) alanine at the P2 position, c) valine at the P1 position, d) arginine, glycine, asparagine, or threonine at the P1' position, e) aspartic acid or glutamic acid at the P2' position, f) isoleucine at the P3' position, and g) valine at the P4' position are undesirable. In some embodiments, functional variants of GPAGLYAQ (SEQ ID NO: 195) do not contain amino acid substitutions at the P1 and / or P1' positions.

[0097] The amino acid substitutions of the functional variant of GPAGLYAQ (SEQ ID NO: 195) preferably include amino acid substitutions at the P4 and / or P4' positions. For example, the functional variant of GPAGLYAQ (SEQ ID NO: 195) may include leucine at the P4 position, or serine, glutamine, lysine, or phenylalanine at the P4 position. Alternatively, or further, the functional variant of GPAGLYAQ (SEQ ID NO: 195) may include glycine, phenylalanine, or proline at the P4' position.

[0098] In some embodiments, amino acid substitutions at the P2 or P2' position of GPAGLYAQ (SEQ ID NO: 195) are undesirable.

[0099] In some embodiments, functional variants of GPAGLYAQ (SEQ ID NO: 195) include amino acid sequences selected from SEQ ID NOs: 221-295. Specific functional variants of GPAGLYAQ (SEQ ID NO: 195) include GPGLYAQ (SEQ ID NO: 259) and GPAGLKGA (SEQ ID NO: 249).

[0100] Functional variants of LFKSSFP (SEQ ID NO: 448) preferably include hydrophobic amino acid substitutions. Functional variants of LFKSSFP (SEQ ID NO: 448) preferably include (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at the P4 position; (b) lysine, histidine, glycine, proline, asparagine, or phenylalanine at the P3 position; (c) arginine, leucine, alanine, glutamine, or histatine at the P2 position; (d) phenylalanine, histidine, threonine, alanine, or glutamine at the P1 position; and histidine, leucine, or lysine at the P1' position. (f) Phenylalanine, leucine, isoleucine, lysine, alanine, glutamic acid, or proline at the P2' position; (g) Phenylalanine, leucine, glycine, serine, valine, histidine, alanine, or asparagine at the P3' position; and may contain one or more of phenylalanine, histidine, glycine, alanine, serine, valine, glutamine, lysine, or leucine.

[0101] The inclusion of aspartic acid and / or glutamic acid in the functional variant of SEQ ID NO: 448 is generally undesirable and should be avoided. In the functional variant of LFKSSFP (SEQ ID NO: 448), amino acid substitutions of (a) alanine, serine, or glutamic acid at the P3 position, (b) proline, threonine, glycine, or aspartic acid at the P2 position, (c) proline at the P1 position, (d) proline at the P1' position, (e) glycine at the P2' position, (f) lysine or glutamic acid at the P3' position, and (g) aspartic acid at the P4' position are also undesirable.

[0102] The amino acid substitutions of the functional variant of LFKSSFP (SEQ ID NO: 448) preferably include amino acid substitutions at the P4 and / or P1 positions. In some embodiments, amino acid substitutions of the functional variant of LFKSSFP (SEQ ID NO: 448) at the P4' position are undesirable.

[0103] In some embodiments, functional variants of LFKSSFP (SEQ ID NO: 448) include amino acid sequences selected from SEQ ID NOs: 296-374. Specific functional variants of LFKSSFP (SEQ ID NO: 448) include ALFFSSPP (SEQ ID NO: 199), ALFKSFPP (SEQ ID NO: 346), ALFKSLPP (SEQ ID NO: 347), ALFKHSPP (SEQ ID NO: 335), ALFKSIPP (SEQ ID NO: 348), ALFKSSLP (SEQ ID NO: 356), or SPFRSSRQ (SEQ ID NO: 297).

[0104] The isolated portion disclosed herein can form a complex that is stable under physiological conditions with the amino acid sequence (e.g., domain) to which the isolated portion links, and can also be cleaved by a protease. For example, the isolated portion is stable in circulating blood (e.g., not cleaved or cleaved with low efficiency) and cleaved with higher efficiency at a target site (i.e., the tumor microenvironment). Thus, the fusion polypeptide comprising the linker disclosed herein may, if desired, have an extended circulating half-life and / or lower circulating blood bioactivity compared to the components of the fusion polypeptide as separate molecular entities. Furthermore, when at a desired site (e.g., the tumor microenvironment), the linker can be efficiently cleaved to release the components linked together by the linker, restoring or nearly restoring the half-life and bioactivity of the components as separate molecular entities.

[0105] The separated portion preferably remains stable in the circulating blood for at least 2 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.

[0106] In some embodiments, the separated portion is cleaved in the circulating blood by less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 20%, less than 5%, or less than 1% compared to the target site. The separated portion is also stable in the absence of an enzyme that can cleave the linker. However, upon exposure to a suitable enzyme (i.e., a protease), the separated portion is cleaved, and separation of the linked domain occurs.

[0107] E. Pharmaceutical compositions This specification also provides a pharmaceutical composition comprising an inducible IL-12 prodrug described herein, a vector containing a polynucleotide encoding the inducible IL-12 prodrug or a host cell transformed by such vector, and at least one pharmaceutically acceptable carrier.

[0108] This specification provides pharmaceutical formulations or compositions comprising the inducible IL-12 prodrug described herein and a pharmaceutically acceptable carrier. Compositions comprising the inducible IL-12 prodrug described herein are suitable for in vitro or in vivo administration. The term “pharmaceutically acceptable carrier” includes, but is not limited to, carriers that do not impair the efficacy of the biological activity of the component and are not toxic to the subject to which it is administered. Examples of suitable pharmaceutically acceptable carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, and sterile solutions. Such carriers can be formulated by conventional methods and administered to the subject in a suitable dose. Preferably, the compositions are sterile. These compositions may contain adjuvants such as preservatives, emulsifiers, and dispersants. By including various antibacterial and antifungal agents, prevention of microbial action can be ensured.

[0109] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21. st This is described in Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make it isotonic, but the formulation may be hypertonic or hypotonic as desired. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffers such as Ringer's solution, and dextrose solutions. The pH of the solutions is generally about 5 to about 8 or about 7 to 7.5. Other carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing immunogenic polypeptides. The matrix may be in the form of molded articles such as films, liposomes, or microparticles. Certain carriers may be more preferred depending, for example, on the route of administration and the concentration of the composition to be administered. Carriers are suitable for administering nucleic acid sequences encoding IL-12 or inducible IL-12 prodrugs to humans or other subjects.

[0110] This disclosure also relates to pharmaceutical formulations containing the IL-12 prodrug described herein. The formulations are preferably aqueous liquids, more preferably aqueous liquids suitable for injection or infusion. The formulations may also preferably be dry solid formulations, such as lyophilized or spray-dried formulations. In various embodiments, the formulation is a lyophilized (lyophilized cake). Preferred formulations include the IL-12 prodrug described herein, citric acid and / or citrate, disaccharides, and surfactants.

[0111] In such liquid formulations, the IL-12 prodrug is present in concentrations of approximately 1 mg / mL to 100 mg / mL, for example, approximately 50 mg / mL to 100 mg / mL, approximately 50 mg / mL to 75 mg / mL, approximately 75 mg / mL to 100 mg / mL, approximately 25 mg / mL to 50 mg / mL, approximately 1 mg / mL to 50 mg / mL, approximately 1 mg / mL to 25 mg / mL, approximately 1 mg / mL to 20 mg / mL, approximately 1 mg / mL to 15 mg / mL, approximately 1 mg / mL to 10 mg / mL, approximately 5 mg / mL to 25 mg / mL, approximately 5 mg / mL to 20 mg / mL, and approximately 5 mg / mL to 10 mg / mL. The levels may be 15 mg / mL, approximately 5 mg / mL to approximately 10 mg / mL, approximately 1 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, approximately 11 mg / mL, approximately 12 mg / mL, approximately 13 mg / mL, approximately 14 mg / mL, approximately 15 mg / mL, approximately 16 mg / mL, approximately 17 mg / mL, approximately 18 mg / mL, approximately 19 mg / mL, approximately 20 mg / mL, approximately 21 mg / mL, approximately 22 mg / mL, approximately 23 mg / mL, approximately 24 mg / mL, or approximately 25 mg / mL. Aqueous liquids may have a pH of approximately 5.0 to approximately 8.0, for example, approximately 5.0 to approximately 7.0, approximately 5.5 to approximately 7.0, approximately 5.0, approximately 5.5, approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, or approximately 8.0.

[0112] In such liquid formulations, citrate (e.g., monosodium citrate, disodium citrate, and trisodium citrate) is present in concentrations of approximately 5 mM to approximately 500 mM, for example, 5 mM to approximately 300 mM, 5 mM to approximately 250 mM, 5 mM to approximately 200 mM, 5 mM to approximately 150 mM, 5 mM to approximately 100 mM, 10 mM to approximately 100 mM, 2 It exists at concentrations of 0 mM to approximately 100 mM, 20 mM to approximately 90 mM, 20 mM to approximately 80 mM, 30 mM to approximately 80 mM, 30 mM to approximately 70 mM, 40 mM to approximately 70 mM, 40 mM to approximately 60 mM, approximately 20 mM, approximately 30 mM, approximately 40 mM, approximately 50 mM, approximately 60 mM, approximately 70 mM, approximately 80 mM, approximately 90 mM, or approximately 100 mM.

[0113] In such liquid formulations, disaccharides (e.g., sucrose, trehalose, lactose, maltose) are present in concentrations of approximately 20 mM to 500 mM, for example, approximately 20 mM to 300 mM, 20 mM to 250 mM, 100 mM to 300 mM, 100 mM to 250 mM, approximately 100 mM, approximately 120 mM, approximately 140 mM, approximately 150 mM, approximately 160 mM, approximately 170 mM, and approximately It is present at concentrations of approximately 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 370 mM, 390 mM, or 400 mM.

[0114] In such liquid formulations, surfactants (e.g., polysorbate 80, polysorbate 20, span®-80, poloxamer) are present in amounts of approximately 0.001% to approximately 2%, for example, approximately 0.001% to approximately 1%, approximately 0.001% to approximately 0.1%, approximately 0.01% to approximately 1%, approximately 0.01% to approximately 0.1%, approximately 0.002% to approximately 0.2%, approximately 0.005%, approximately 0.006%, approximately 0.007%, approximately 0.008%, approximately 0.009%, approximately 0.01%, approximately 0.02%, approximately 0.03%, approximately 0.04%, approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, approximately 0.1%, or approximately 0.2%.

[0115] Suitable citrates are known in the art and include sodium citrate (monosodium citrate, disodium citrate, and trisodium citrate, etc.), magnesium citrate, potassium citrate, etc. Suitable disaccharides are known in the art and include sucrose, trehalose, lactose, maltose, etc. Suitable surfactants are known in the art and include ionic surfactants, such as fatty acids and fatty acid salts (e.g., sodium stearate, magnesium stearate), alkyl sulfates and their salts (e.g., sodium dodecyl sulfate), and certain water-soluble quaternary ammonium salts. Suitable nonionic surfactants include polysorbate 20, polysorbate 80, Span®-80, castor oil, poloxamer, etc.

[0116] The formulation may contain the IL-12 prodrug described herein, citric acid and / or sodium citrate (e.g., monosodium citrate, disodium citrate, and trisodium citrate), disaccharides (e.g., sucrose, trehalose, lactose, maltose), and nonionic surfactants (polysorbate 20, polysorbate 80, Span®-80, castor oil, poloxamer). The formulation may contain the IL-12 prodrug described herein, citric acid and / or sodium citrate (e.g., monosodium citrate, disodium citrate, and trisodium citrate), sucrose, and polysorbate 80.

[0117] In certain embodiments, the formulation is an aqueous liquid for injection and comprises the IL-12 prodrug described herein at a concentration of about 1 mg / mL to about 100 mg / mL, sodium citrate (e.g., monosodium citrate, disodium citrate, and trisodium citrate) at a concentration of about 5 mM to about 500 mM, sucrose at a concentration of about 20 mM to about 500 mM, and polysorbate 80 at a concentration of about 0.001% to about 2%, with a pH of about 5.0 and about 8.0. Such formulation also comprises water, for example, water for injection (USP).

[0118] Preferably, the concentration of the IL-12 prodrug is approximately 50 mg / mL to 75 mg / mL, 75 mg / mL to 100 mg / mL, 25 mg / mL to 50 mg / mL, 1 mg / mL to 50 mg / mL, 1 mg / mL to 25 mg / mL, 1 mg / mL to 20 mg / mL, 1 mg / mL to 15 mg / mL, 1 mg / mL to 10 mg / mL, 5 mg / mL to 25 mg / mL, 5 mg / mL to 20 mg / mL, 5 mg / mL to 15 mg / mL, 5 mg / mL to 10 mg / mL, 1 mg / mL, 2 mg / mL, and 3 mg / mL. g / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, approximately 11 mg / mL, approximately 12 mg / mL, approximately 13 mg / mL, approximately 14 mg / mL, approximately 15 mg / mL, approximately 16 mg / mL, approximately 17 mg / mL, approximately 18 mg / mL, approximately 19 mg / mL, approximately 20 mg / mL, approximately 21 mg / mL, approximately 22 mg / mL, approximately 23 mg / mL, approximately 24 mg / mL, or approximately 25 mg / mL, and sodium citrate (e.g., monosodium citrate, disodium citrate, and trisodium citrate) The concentrations of (m) are approximately 5mM to 300mM, 5mM to 250mM, 5mM to 200mM, 5mM to 150mM, 5mM to 100mM, 10mM to 100mM, 20mM to 100mM, 20mM to 90mM, 20mM to 80mM, 30mM to 80mM, 30mM to 70mM, 40mM to 70mM, 40mM to 60mM, 20mM to 30mM, 40mM to 40mM, 40mM to 60mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, or 100mM, and the concentrations of sucrose are approximately 20mM to 300mM, 20mM to 250mM, The concentrations range from 100mM to approximately 300mM, 100mM to approximately 250mM, approximately 100mM, approximately 120mM, approximately 140mM, approximately 150mM, approximately 160mM, approximately 170mM, approximately 180mM, approximately 190mM, approximately 200mM, approximately 210mM, approximately 220mM, approximately 230mM, approximately 240mM, approximately 250mM, approximately 260mM, approximately 270mM, approximately 280mM, approximately 290mM, approximately 300mM, approximately 310mM, approximately 320mM, approximately 330mM, approximately 340mM, approximately 350mM, approximately 370mM, approximately 390mM, or approximately 400mM, and the concentration of polysorbate 80 is approximately 0.001% to approximately 1%, approximately 0.The concentrations are approximately 0.01% to 0.1%, 0.01% to 1%, 0.01% to 0.1%, 0.002% to 0.2%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or 0.2%, and the pH is approximately 5.0 to 7.0, 5.5 to 7.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.

[0119] The formulation may also be a dry solid formulation, such as a lyophilized (lyophilized cake) or spray-dried powder, of any of the liquid formulations described herein. Such a dry solid formulation can be reconstituted, for example, with water for injection (USP) to make a liquid formulation for injection or infusion. The dry solid formulations of this disclosure are not necessarily anhydrous and may optionally contain some water.

[0120] In some embodiments of the pharmaceutical composition, the inductive IL-12 prodrug described herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposomes, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical composition, the inductive IL-12 prodrug is bound to liposomes. In some cases, the inductive IL-12 prodrug is bound to the surface of liposomes. In some cases, the inductive IL-12 prodrug is encapsulated within the shell of liposomes. In some cases, the liposomes are cationic liposomes.

[0121] The inducible IL-12 prodrugs described herein are intended for use as pharmaceuticals. Administration can be carried out by various methods, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The administration regimen is determined by the attending physician and other clinical factors. The dose for one patient depends on many factors, including the patient's size, body surface area, age, sex, the specific compound administered, the time and route of administration, the type of therapy, overall health status, and other drugs administered concurrently. "Effective dose" refers to the amount of active ingredient that is sufficient to affect the course and severity of the disease and to reduce or eliminate such condition, and can be determined using known methods.

[0122] Optionally, an inducible IL-12 prodrug or a nucleic acid sequence encoding an inducible IL-12 prodrug is administered by a vector. Numerous compositions and methods exist 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 broadly classified into two types: viral and nonviral delivery systems. Such methods are well known in the art and are readily adaptable for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express and preferably secrete an encoded chimeric polypeptide, or to deliver nucleic acids therapeutically. The components of the IL-12 polypeptides disclosed herein are typically functionally linked in frame to encode a fusion protein.

[0123] As used herein, a plasmid or viral vector is an active factor containing a promoter that transfers the nucleic acid of this disclosure into a cell without degradation and brings about the expression of nucleic acid molecules and / or polypeptides in the target cell. Viral vectors include, for example, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, polioviruses, Sindbis, and other RNA viruses, including those with an HIV backbone. Virus families that share common characteristics and are therefore suitable for use as vectors are also preferred. Retroviral vectors in general and methods for their preparation are described in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). The construction of replication-deficient adenoviruses has been reported previously (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 advantage and use of these viruses as vectors is that while they can replicate within the cells they initially infect, they cannot form new infectious viral particles, thus limiting their spread to other cell types. Recombinant adenoviruses have been shown to achieve high efficiency after direct in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and several other tissue sites. Other useful systems include, for example, replicated vaccinia virus vectors and host-limited non-replicated vaccinia virus vectors.

[0124] The inducible IL-12 prodrug and / or nucleic acid molecule provided can be delivered via virus-like particles. Virus-like particles (VLPs) consist of viral proteins (or more) derived from the structural proteins of a virus. Methods for constructing and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0125] The inducible IL-12 prodrugs disclosed herein may be delivered by subviral densities (DBs). DBs transfer proteins into target cells by membrane fusion. Methods for constructing and using DBs are described, for example, in Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003). The polypeptides provided may be delivered by envelope aggregates. Methods for constructing and using envelope aggregates are described in International Publication WO2006 / 110728.

[0126] Nonviral delivery methods include expression vectors, which contain nucleic acid molecules and nucleic acid sequences encoding polypeptides, with the nucleic acids operably linked to expression regulatory sequences. Suitable vector backbones include plasmids, artificial chromosomes, BACs, YACs, or PACs, which are typically used in the art. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions. Regulatory regions include, 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 start sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to produce inducible IL-12 prodrugs by expression in suitable host cells, such as CHO cells.

[0127] Preferred promoters for controlling transcription from vectors in mammalian host cells can be obtained from various sources, such as the genomes of viruses including polyomaviruses, Simian virus 40 (SV40), adenoviruses, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, such as the β-actin promoter or EF1α promoter, or from hybrid or chimeric promoters (e.g., a CMV promoter fused to the β-actin promoter). Naturally, promoters derived from host cells or related species are also useful herein.

[0128] An enhancer generally refers to a DNA sequence that functions at an indeterminate distance from the transcription start site and can be either 5' or 3' relative to the transcription unit. Furthermore, enhancers can be located within introns or within the coding sequence itself. They are typically 10–300 base pairs (bp) in length and function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response sequences that mediate transcriptional regulation. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), but typically, enhancers from eukaryotic viruses are used for general expression. Preferred examples include the SV40 enhancer on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancer.

[0129] Promoter and / or enhancer regions may be inducible (e.g., chemically or physically modulated). Chemically modulated promoters and / or enhancers may be modulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically modulated promoters and / or enhancers may be modulated, for example, by environmental factors such as temperature and light. Optionally, promoter and / or enhancer regions may act as constitutive promoters and / or enhancers to maximize the expression of the region of the transcription unit being transcribed. In certain vectors, promoter and / or enhancer regions may be active in a cell-type-specific manner. Optionally, in certain vectors, promoter and / or enhancer regions may be active in all eukaryotic cells, regardless of cell type. Preferred promoters of this type include the CMV promoter, SV40 promoter, β-actin promoter, EF1α promoter, and retroviral long terminal repeats (LTRs).

[0130] Vectors may include, for example, origins of replication and / or markers. Marker genes can confer a selectable phenotype to cells, such as antibiotic resistance. Marker products are used to determine whether the vector has been delivered to cells and whether it is expressed after delivery. Examples of selective markers in mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blastosidine. If such selective markers are successfully transferred to mammalian host cells, the transformed mammalian host cells can survive under selective pressure. Other examples of markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. Furthermore, expression vectors may include tag sequences designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or the FLAG® tag (Kodak; New Haven, Conn.), are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including either a carboxyl terminus or an amino terminus.

[0131] F. Application to treatment This specification also provides methods and uses for the treatment of a disease, disorder, or condition related to 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 diseases, immunological disorders, autoimmune diseases, and infectious diseases (i.e., bacterial, viral, or parasitic diseases). Preferably, the disease, disorder, or condition is cancer.

[0132] Any suitable cancer can be treated with the inducible IL-12 prodrugs provided herein. Suitable cancers for illustrative purposes include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, bronchial tumor, cancer of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, adenoid carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, and 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, histiocytic hyperplasia, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, Melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary origin, midline tract cancer involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal cavity / paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytosis Examples include tumors, pituitary tumors, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureteral cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratomatoid tumor, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor. In some embodiments, the cancer is melanoma or breast cancer.

[0133] In some embodiments, the methods provided herein involve administering an effective amount of the inducible IL-12 prodrug provided herein to subjects requiring an enhancement of the immune response. The enhanced immune response may prevent, delay, or treat the development of cancer, tumors, or viral diseases. While not limited to theory, inducible IL-12 prodrugs enhance 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 prodrugs provided herein may induce IFNγ release from natural killer cells as well as CD4+ T cells and CD8+ T cells.

[0134] IL-12 prodrugs can be administered to targets requiring them in combination with immune checkpoint inhibitors. Examples of immune checkpoint proteins include PD-1 (binding to ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273)), CTLA-4 (CD152) (binding to B7-1 (CD80) and B7-2 (CD86)), LAG3 (CD223) (binding to Galectin3, LSECtin, and FGL1); and TIM3 (HAVCR2) (binding to ligands Ceacam1 and Galectin3). Examples include CD134 (TNRFSR4, OX40) which binds to n9; TIGIT (VSTM3, WUCAM) (which binds to CD112 and CD155); BTLA (CD272), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4) which bind to HVEM; and CD160 (BY55) which binds to HVEM; and CD134 (TNRFSR4, OX40) which binds to CD252 (OX-40L). Therapeutic agents such as antibodies that bind to immune checkpoint proteins and inhibit their immunosuppressive activity include the anti-PD1 antibodies pembrolizumab (KEYTRUDA), dostallimab (JEMPERLI), semiprimab-rwlc (LIBATYO), nivolumab (OPDIVO), camrelizumab, tislerizumab, tripalimab, and cintilimab (TYVYT), the anti-PD-L1 antibodies avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ), and the anti-CTLA-4 antibody ipilimumab (YERVOY).

[0135] This method may further include the administration of one or more additional agents for treating cancer, such as chemotherapeutic agents (e.g., Adriamycin, Seruvidine, Bleomycin, Alkeran, Verban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisanthren, Noantrone, Thiguanine, Cytaribine, Procarabizine), tumor immunotherapy agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), cell therapies (e.g., CAR-T, T-cell therapy), and oncolytic viruses. Non-limited examples of anticancer drugs that can be used include: Asibicin; Akurarubicin; Acodazole hydrochloride; Acronin; Adzeresin; Aldesleukin; Altretamine; Ambomycin; Amethantrone acetate; Aminoglutethimide; Amsacrin; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batymastam; Benzodepa; Bicalutamide; Bisanthren hydrochloride; Bisnafide dimesylate; Bizeresin; Bleomycin sulfate; Brequinal sodium; Bropyrimin; Busulfan; Kakutinomycin; Carsterone; Calasemide; Carvethymer; Carboplatin; Carmustine; Carbicin hydrochloride; Carzeresin; Sedefingol; Chlorambucil; Ciloremycin; Cisplatin; Cladribine; Crisnator mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine mesylate; Diadicon; Docetaxel; Doxorubicin; Doxorubicin hydrochloride; Doroxifene; Doroxifene citrate; Dromostanolone propionate; Duazomycin; Edatrexate; Eflornithine hydrochloride; Elsamitrusine; Enloplatin; Empromart; Epipropidine; Epirubicin hydrochloride; Elbrozol; Esolubicin hydrochloride; Estramustine; Estramustine sodium phosphate; Etanidazole; Etoposide; Etoposide phosphate; Etoprine; Fadrozol hydrochloride; Fazarabine; Fenretinide; Furoxuridine; Fludarabine phosphate; Fluorouracil; Fluocitabine; Fosquidone;Fostriesin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; irmofosin; interleukin II (including recombinant interleukin II, i.e., rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-nl; interferon alpha-n3; interferon beta-Ia; interferon gamma-Ib; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; rialozol hydrochloride Lometrexol sodium; Lomustine; Rosoxantrone hydrochloride; Masoprocol; Mytansine; Mechloretamine hydrochloride; Megestrol acetate; Melengestrol acetate; Melphalan; Menogalil; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprin; Metsuredepa; Mitindomide; Mitocalcin; Mitochromin; Mitogiline; Mitomarcin; Mitomycin; Mitospel; Mitotan; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole; Nogaramycin; Olmaplatin; Oxythran; Paclitaxel Lu; Peguaspargase; Periomycin; Pentamustine; Peplomycin sulfate; Perphosphamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; Plicamycin; Promestan; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofulin; Ribopurine; Logretimide; Safingol; Safingol hydrochloride; Semustine; Simtrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine ;Spiroplatin;Streptonigrin;Streptozocin;Surofenull;Talisomycin;Tecogalan sodium;Tegafur;Teloxantrone hydrochloride;Temoporfin;Teniposide;Teloxylone;Testolactone;Thiamipurine;Thioguanine;Thiotepa;Thiazofulin;Tirapazamine;Tremifene citrate;Trestron acetate;Triciribin phosphate;Trimethrexate;Trimethrexate glucuronide;Triptorelin;Tubrozol hydrochloride;Uracil mustard;Uredepah;Bupreotide;Verteporfin;Vinblastine sulfate;Examples include vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; binricinate sulfate; vinoleulosin sulfate; vinorelbine tartrate; vinozolidine sulfate; vinozolidine sulfate; borozol; zeniplatin; dinostatin; and zolubicin hydrochloride.

[0136] In some embodiments of the methods described herein, the inducible IL-12 prodrug is administered in combination with an action factor for the treatment of a specific disease, disorder, or condition. The action factor includes, but is not limited to, therapies including antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiotherapy (gamma rays, C rays, and / or radioisotopes, microwaves, UV radiation, etc., with directional delivery), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapies. In some embodiments, the inducible IL-12 prodrug is administered in combination with an antidiarrheal, antiemetic, analgesic, and / or nonsteroidal anti-inflammatory drug.

[0137] This disclosure relates to a method for treating cancer using the inducible IL-12 prodrug described herein. The method disclosed herein comprises administering a therapeutically effective amount of the inducible IL-12 prodrug described herein to a target.

[0138] Inducible IL-12 prodrugs can be administered in doses ranging from approximately 0.016 mg / kg to approximately 500 mg / kg per administration. For example, approximately 0.016 mg / kg, approximately 0.017 mg / kg, approximately 0.018 mg / kg, approximately 0.019 mg / kg, approximately 0.020 mg / kg, approximately 0.021 mg / kg, approximately 0.022 mg / kg, approximately 0.023 mg / kg, approximately 0.024 mg / kg, approximately 0.025 mg / kg, approximately 0.026 mg / kg, approximately 0.027 mg / kg, approximately 0.028 mg / kg, approximately 0.029 mg / kg, approximately 0.030 mg / kg, approximately 0.031 mg / kg, approximately 0.032 mg / kg, approximately 0.033 mg / kg, approximately 0.034 mg / kg, approximately 0.0 35 mg / kg, approximately 0.036 mg / kg, approximately 0.037 mg / kg, approximately 0.038 mg / kg, approximately 0.039 mg / kg, approximately 0.040 mg / kg, approximately 0.041 mg / kg, approximately 0.042 mg / kg, approximately 0.043 mg / kg, approximately 0.044 mg / kg, approximately 0.045 mg / kg, approximately 0.046 mg / kg, approximately 0.047 mg / kg, approximately 0.048 mg / kg, approximately 0.049 mg / kg, approximately 0.050 mg / kg, approximately 0.051 mg / kg, approximately 0.052 mg / kg, approximately 0.053 mg / kg, approximately 0.054 mg / kg, Approximately 0.055 mg / kg, approximately 0.056 mg / kg, approximately 0.057 mg / kg, approximately 0.058 mg / kg, approximately 0.059 mg / kg, approximately 0.060 mg / kg, approximately 0.061 mg / kg, approximately 0.062 mg / kg, approximately 0.063 mg / kg, approximately 0.064 mg / kg, approximately 0.065 mg / kg, approximately 0.066 mg / kg, approximately 0.067 mg / kg, approximately 0.068 mg / kg, approximately 0.069 mg / kg, approximately 0.070 mg / kg, approximately 0.071 mg / kg, approximately 0.072 mg / kg, approximately 0.073 mg / kg, approximately 0.074 mg g / kg, about 0.075mg / kg, about 0.076mg / kg, about 0.077mg / kg, about 0.078mg / kg, about 0.079mg / kg, about 0.080mg / kg, about 0.081mg / kg, about 0.082mg / kg, about 0.083mg / kg, about 0.08 4mg / kg, approximately 0.085mg / kg, approximately 0.086mg / kg, approximately 0.087mg / kg, approximately 0.088mg / kg, approximately 0.089mg / kg, approximately 0.090mg / kg, approximately 0.091mg / kg, approximately 0.092mg / kg, approximately 0.093mg / kg, approximately 0.0.94 mg / kg, approximately 0.095 mg / kg, approximately 0.096 mg / kg, approximately 0.097 mg / kg, approximately 0.098 mg / kg, approximately 0.099 mg / kg, or approximately 0.100 mg / kg (in all cases, per administration).

[0139] For example, approximately 0.100 mg / kg, approximately 0.105 mg / kg, approximately 0.110 mg / kg, approximately 0.115 mg / kg, approximately 0.120 mg / kg, approximately 0.125 mg / kg, approximately 0.130 mg / kg, approximately 0.135 mg / kg, approximately 0.140 mg / kg, approximately 0.145 mg / kg, approximately 0.150 mg / kg, approximately 0.155 mg / kg, approximately 0.160 mg / kg, approximately 0.165 mg / kg, approximately 0.170 mg / kg, approximately 0.175 mg / kg, approximately 0.180 mg / kg, approximately 0.185 mg / kg, approximately 0.190 mg / kg, approximately 0.195 mg / kg kg, about 0.200mg / kg, about 0.205mg / kg, about 0.210mg / kg, about 0.215mg / kg, about 0.220mg / kg, about 0.230mg / kg, about 0.235mg / kg, about 0.240mg / kg, about 0.245mg / kg, about 0.250mg / k g, about 0.255mg / kg, about 0.260mg / kg, about 0.265mg / kg, about 0.270mg / kg, about 0.275mg / kg, about 0.280mg / kg, about 0.285mg / kg, about 0.290mg / kg, about 0.295mg / kg, about 0.300mg / kg , about 0.305mg / kg, about 0.310mg / kg, about 0.315mg / kg, about 0.320mg / kg, about 0.325mg / kg, about 0.330mg / kg, about 0.340mg / kg, about 0.345mg / kg, about 0.350mg / kg, about 0.355mg / kg, About 0.360mg / kg, about 0.365mg / kg, about 0.370mg / kg, about 0.375mg / kg, about 0.380mg / kg, about 0.385mg / kg, about 0.390mg / kg, about 0.395mg / kg, about 0.400mg / kg, about 0.405mg / kg, about 0.410 mg / kg, approximately 0.415 mg / kg, approximately 0.420 mg / kg, approximately 0.425 mg / kg, approximately 0.430 mg / kg, approximately 0.435 mg / kg, approximately 0.440 mg / kg, approximately 0.445 mg / kg, approximately 0.450 mg / kg, approximately 0.455 mg / kg, approximately 0.460 mg / kg, approximately 0.465 mg / kg, approximately 0.470 mg / kg, approximately 0.475 mg / kg, approximately 0.480 mg / kg, approximately 0.485 mg / kg, approximately 0.490 mg / kg, approximately 0.495 mg / kg, or approximately 0.500 mg / kg (in all cases, per administration).

[0140] Typically, IL-12 prodrugs are administered in doses of approximately 0.032 mg / kg, 0.056 mg / kg, 0.084 mg / kg, 0.126 mg / kg, 0.190 mg / kg, 0.290 mg / kg, or 0.440 mg / kg (in all cases, per administration).

[0141] In such methods, IL-12 prodrugs may be administered orally, parenterally, intravenously, intra-articular, intraperitoneal, intramuscular, subcutaneous, intracavitary, percutaneous, intrahepatic, intracranial, by spray / inhalation, by bronchoscopic placement, or within a tumor. Typically, IL-12 prodrugs are administered intravenously.

[0142] Inducible IL-12 prodrugs may be administered approximately twice a week or less frequently, for example, once every two weeks.

[0143] G. Definition All published documents and patents cited in this disclosure are incorporated herein by reference as a whole. In the event of any conflict or inconsistency between any incorporated document and this disclosure, this disclosure shall prevail. No reference to any document herein constitutes prior art of this disclosure. Where a range of values ​​is expressed, embodiments using any specific value within that range are included. Furthermore, references to values ​​described in a range include all values ​​within that range. All ranges include their endpoints and are combinable. Where a value is expressed as an approximation by the preceding use of “about,” it should be understood that a specific value forms another embodiment. References to a specific numerical value include at least that specific value unless otherwise clearly indicated by the context. The use of “or” means “and / or” unless otherwise specified by the specific context of its use.

[0144] Various terms relating to aspects of this specification are used throughout this specification and the claims. Unless otherwise stated, such terms are given their common meanings in the art. Other terms specifically defined shall be construed in accordance with the definitions provided herein. The techniques and procedures described or referenced herein are generally well understood by those skilled in the art, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 4. th Conventional methods, such as the widely used molecular cloning techniques described in (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, are commonly used. Procedures involving the use of commercially available kits and reagents, where necessary, are generally carried out according to the manufacturer's defined protocols and conditions, unless otherwise specified.

[0145] As used herein, the singular forms "a," "an," and "the" include the plural form unless otherwise clearly indicated by the context. Terms such as "includes" and "etc." are intended to indicate unrestricted inclusion unless otherwise specified.

[0146] Unless otherwise stated, the terms “at least,” “less than,” and “about,” or similar terms preceding a series or range of elements should be understood to refer to all elements within that series or range. Those skilled in the art will recognize many equivalents to specific embodiments of the inventions described herein, or can verify them simply by using standard experiments. Such equivalents are intended to be covered within the following claims.

[0147] As used herein, the terms “activatable,” “activate,” “inducible,” and “inducible” refer to polypeptide complexes having attenuated forms of activity (e.g., attenuated receptor binding and / or agonist activity) and active forms. Polypeptide complexes are activated by protease cleavage of the linker, which dissociates the blocking element and half-life extension element from the polypeptide complex. Induced / activated polypeptide complexes can bind to the IL-12 receptor with increased affinity / avidity.

[0148] The terms “antibody” and “immunoglobulin” are used synonymously herein. As used herein, antibody or immunoglobulin is intended to refer to an immunoglobulin molecule containing two heavy (H) chains. Typically, antibodies in mammals (e.g., humans, rodents, and monkeys) contain four polypeptide chains interconnected by disulfide bonds, namely two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (hereinafter abbreviated 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 highly variable regions called complementarity-determining regions (CDRs), and between the CDRs are highly conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. Antibodies may include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, polyspecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, or tetrameric antibodies containing two heavy chain molecules and two light chain molecules. Those skilled in the art will recognize the existence of other forms of antibodies (e.g., camelid antibodies and shark antibodies).

[0149] As used herein, the term “attenuated” refers to an IL-12 receptor agonist having reduced receptor agonist activity compared to the naturally occurring agonist of the IL-12 receptor. Attenuated IL-12 agonists may have agonist activity at least about 10 times, at least about 50 times, at least about 100 times, at least about 250 times, at least about 500 times, at least about 1000 times, or lower compared to the naturally occurring agonist of their receptor. When an IL-12 polypeptide complex containing IL-12 described herein is described as “attenuated” or having “attenuated activity”, it means that the IL-12 polypeptide complex is an attenuated IL-12 receptor agonist.

[0150] The term "cancer" refers to a physiological condition in mammals characterized by uncontrolled proliferation, immortality, metastatic ability, rapid growth and proliferation rates, and / or certain morphological features. Often, cancer can take the form of a tumor or mass, but it may also exist alone in a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. The term cancer encompasses all types of cancer and metastases, 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 detailed 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 cancer (e.g., serous) or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, and various types of head and neck cancers. Triple-negative breast cancer refers to breast cancer in which the expression of the estrogen receptor (ER), progesterone receptor (PR), and Her2 / neu genes is negative.

[0151] As used herein, a “conservative” amino acid substitution generally refers to a substitution in which one amino acid residue is replaced by another amino acid residue belonging to a recognized group, thereby altering the structure of the peptide but substantially preserving its biological activity. Conservative amino acid substitutions are known to those skilled in the art. Conservative amino acid substitutions may include, but are not limited to, substitutions made between amino acids in the 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, it is reasonably expected by those skilled in the art that a single substitution of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of structurally related amino acids will not have a significant effect on the biological activity of the resulting molecule.

[0152] As used herein, the term “half-life extension element” in the context of polypeptide complexes disclosed herein means a chemical element, preferably a polypeptide, that extends the serum half-life and improves pK by, for example, modifying its size (e.g., to exceed the renal filtration cutoff value), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and excretion.

[0153] As used herein, the term “operably linked” in the context of polypeptide complexes refers to the positioning of the components of the polypeptide complex that enables the components to function as intended. For example, a polypeptide containing an IL-12 subunit and an IL-12 blocking element are operably linked by a protease-cleavable linker in the polypeptide complex if the IL-12 blocking element is capable of inhibiting the IL-12 receptor activating activity of the IL-12 polypeptide; however, if the protease-cleavable linker is cleaved, the IL-12 blocking element may, for example, become detached from IL-12, thus reducing or eliminating the inhibition of the IL-12 receptor activating activity of the IL-12 polypeptide by the IL-12 blocking element.

[0154] 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 synonymously herein to refer to an amino acid sequence. It should be noted that the term polypeptide is not used herein to suggest a specific size or number of amino acids contained in a molecule, and that the peptides of the present invention may contain up to several or more amino acid residues.

[0155] In this specification, the term "subject" refers to any animal, including but not limited to any mammal, including humans, non-human primates, rodents, etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0156] As used herein, the term “therapeutic dose” refers to the amount of the compound described herein (i.e., the IL-12 polypeptide complex) that is sufficient to achieve the desired pharmacological or physiological effect under the conditions of administration. For example, a “therapeutic dose” 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 does not need to be complete or curative, as long as some benefit is provided to the subject. The therapeutic dose of a pharmaceutical composition may vary depending on factors such as the individual’s medical condition, age, sex, and weight, as well as the ability of the pharmaceutical composition to induce the desired response in the individual. A clinician of ordinary skill can determine, based on these and other considerations, the appropriate dose to be administered to achieve the desired therapeutic benefit.

[0157] 5. Equal parts Those skilled in the art will readily see that other suitable modifications and applications of the methods of the present invention described herein are obvious and can be carried out using suitable equivalents without departing from the scope of this disclosure or embodiments. Having described specific compounds and methods in detail so far, the same will be more clearly understood by referring to the following examples. The examples are provided for illustrative purposes only and are not intended to limit the scope. [Examples]

[0158] 6. Examples The following are examples of the methods and compositions of the present invention. Considering the overview provided herein, it will be understood that various other embodiments may be implemented.

[0159] Example 1. 1.1 Materials and Methods 1. HEK-Blue IL-12 Reporter Assay HEK-Blue IL-12 cells (InvivoGen) were suspended and seeded at a density of 50,000 cells / well in medium containing or without 15 mg / ml or 40 mg / ml of human serum albumin (HSA), and stimulated at 37°C and 5% CO2 for 20–24 hours with recombinant hIL-12, chimeric IL-12 (mouse p35 / human p40), activatable chimeric IL-12, or a dilution series of activatable hIL-12. The activity of uncleaved and cleaved activatable hIL-12 was tested. Cleaved inducible hIL-12 was generated by incubation with active MMP9 or CTSL-1. IL-12 activity was evaluated by quantitative determination of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric assay. The results support the activity and inducibility of the IL-12 fusion protein. The results are shown in Figure 1.

[0160] 2. Cell lines All cell lines were grown and maintained at Charles River Laboratories (Morrisville, NC and Worcester, MA) according to ATCC guidelines and kept in culture for less than two weeks. Frozen cells were thawed and maintained for 1-3 passages before transplantation. Cell lines B16-F10, CT26, and EMT-6 were cultured in RPMI-1640 containing L-glutamine (Gibco, 11875-085) and 10% heat-inactivated fetal bovine serum (Gibco, 35-015-CV), while MC38 was cultured in Dulbecco's modified Eagle medium (Gibco, 1966-025) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, 16000-044). Cells were washed twice with PBS and counted before tumor transplantation.

[0161] 3. MC38 model tumor transplantation All in vivo mouse work was performed at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of the Animal Care Committee (IACUC) and in accordance with current regulations and standards as well as NIH. The flanks of 6-8 week old female C57B1 / 6 mice from Charles River Laboratories were shaved one day before tumor cell transplantation. A total of 5 × 10⁶ mice were examined. 5 Individual MC38 cells were subcutaneously injected, and tumor growth was monitored. These cells were transplanted into reserve mice to obtain tumors of sufficient size for randomization. The group average tumor size was 100–150 mm. 3Tumor volume was monitored until [specific time], and mice were randomized to the treatment group on day 0. Mice receiving chimeric compound 1 were administered twice weekly for 2 weeks (days 1, 4, 8, and 11), unless otherwise noted. The inducible IL-12 prodrugs used in these studies included chimeric compound 1. Mice receiving recombinant chimeric IL-12 (chimeric IL-12 or WW0295) were administered twice daily for 5 days, followed by a 2-day rest period (5 / 2 regimen), and this cycle was repeated for a total of 2 weeks. All treatments were administered by intraperitoneal injection. Both body weight and tumor volume were measured twice weekly during the study period. Two dimensions of the tumor were measured using calipers, and the formula was: tumor volume (mm²). 3 The volume was calculated using the formula ) = [(w² × l) / 2], where w = tumor width and l = tumor length (in mm). The mouse experiment continued until the tumor reached 1500 mm³ or until the experiment was completed on day 45. See Figures 2A-28, 3G, and 4A-4F. 4. B16-F10 model All in vivo mouse work was performed at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of the Animal Care Committee (IACUC) and in accordance with current regulations and standards as well as NIH. The flanks of 6-8 week old female C57B1 / 6 mice from Charles River Laboratories were shaved one day before tumor cell transplantation. Total 1 × 10⁶ 5 Individual B16-F10 cells were subcutaneously injected, and tumor growth was monitored. These cells were transplanted into reserve mice to obtain tumors of sufficient size for randomization. The group average tumor size was 50–100 mm. 3 Tumor volume was monitored until [specific condition], and mice were randomized to the treatment group on day 0. Mice receiving chimeric compound 1 were administered on days 1 and 4, and tumors were collected 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 Figures 3B and 5A-5F.

[0162] 5. EMT-6 Model All in vivo work with mice was performed at Charles River Laboratories (Morrisville, NC and Worcester, MA) with approval from the Institutional Animal Care and Use Committee (IACUC) and in accordance with current regulations and guidelines and the NIH. The flanks of 6 - 8 week-old female C57B1 / 6 mice from Charles River Laboratories were shaved one day prior to tumor cell transplantation. A total of 1×10 5 EMT6 cells were injected subcutaneously, and tumor growth was monitored. Transplantation was performed on pre - mice to obtain tumors of sufficient size for randomization. Tumor volume was monitored until the group average reached 50 - 100 mm 3 . Mice were randomized into treatment groups on day 0. Mice receiving chimeric compound 1 were administered twice weekly for two weeks (days 1, 4, 8, and 11) unless otherwise noted. The inducible IL - 12 prodrugs used in these tests included chimeric compound 1. All treatments were administered by intraperitoneal injection. In some experiments, mice that had previously rejected their tumors were rechallenged with 1×10 5 EMT6 cells in the contralateral flank four months after the initial rejection reaction. In these experiments, age - matched tumor - naive animals were used as controls. In some experiments, tumor samples were collected, incubated in 5 - 10 mL of 10% neutral buffered formalin for at least 72 hours, then embedded in paraffin and mounted on slides. Unstained slides were sent to NanoString, and immunofluorescent staining and geospatial transcriptomic analysis using the NanoString GeoMX DSP system were performed. See Figures 3C, 3F, 6, and 7A - 7D.

[0163] 6. CT26 Model All in vivo mouse work was performed at Charles River Laboratories (Morrisville, NC and Worcester, MA) with the approval of the Animal Care Committee (IACUC) and in accordance with current regulations and standards as well as NIH. The flanks of 6-8 week old female Balb / C mice from Charles River Laboratories were shaved one day before tumor cell transplantation. A total of 3 × 10⁶ mice were transplanted. 5 Individual CT26 cells were subcutaneously injected, and tumor growth was monitored. These cells were transplanted into reserve mice to obtain tumors of sufficient size for randomization. The group average tumor size was 30–60 mm. 3 Tumor volume was monitored until [specific time], and mice were randomized to the treatment group on day 0. Mice receiving chimeric compound 1 were administered twice weekly for 2 weeks (days 1, 4, 8, and 11), unless otherwise noted. The inducible IL-12 prodrugs 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 during the study period. Two dimensions of the tumor were measured using calipers, and the formula was: Tumor volume (mm²). 3 )=[(w 2 The volume was calculated using [w = width of tumor, l = length of tumor (in mm)]. In the formula, w = width of tumor, and l = length of tumor. The tumor was 1500 mm. 3 The mouse trials continued until the target was reached, or until the trial reached its endpoint on day 45. See Figure 3A.

[0164] 7. EG7.OVA Model All in vivo mouse work was performed in Covance (Ann Arbor, MI) with the approval of the Animal Care Committee (IACUC) and in accordance with current regulations and standards as well as the NIH. The flanks of 6-8 week old female C57B1 / 6 mice from Charles River Laboratories were shaved one day before tumor cell transplantation. Total 1 × 10 6 Individual EG7.OVA cells were subcutaneously injected, and tumor growth was monitored. These were transplanted into reserve mice to obtain tumors of sufficient size for randomization. The group average was approximately 93 mm. 3Tumor volume was monitored until [specific time], and mice were randomized to the treatment group on day 0. Mice receiving chimeric compound 1 were administered twice weekly for 2 weeks (days 1, 4, 8, and 11), unless otherwise noted. The inducible IL-12 prodrugs 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 during the study period. Two dimensions of the tumor were measured using calipers, and the formula was: Tumor volume (mm²). 3 )=[(w 2 The volume was calculated using [w = width of tumor, l = length of tumor (in mm)]. In the formula, w = width of tumor, and l = length of tumor. The tumor was 1500 mm. 3 The mouse trials continued until the target was reached, or until the trial reached its endpoint on day 45. See Figure 3E.

[0165] 8. A20 Model All in vivo mouse work was performed at Covance (Ann Arbor, MI) with the approval of the Animal Care Committee (IACUC) and in accordance with current regulations and standards as well as the NIH. The flanks of 6-8 week old female Balb / C mice from Charles River Laboratories were shaved one day before tumor cell transplantation. A total of 5 × 10⁶ mice were transplanted. 5 Individual A20 cells were subcutaneously injected, and tumor growth was monitored. These cells were transplanted into reserve mice to obtain tumors of sufficient size for randomization. The group average tumor size was approximately 90–130 mm. 3 Tumor volume was monitored until [specific time], and mice were randomized to the treatment group on day 0. Mice receiving chimeric compound 1 were administered twice weekly for 2 weeks (days 1, 4, 8, and 11), unless otherwise noted. The inducible IL-12 prodrugs 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 during the study period. Two dimensions of the tumor were measured using calipers, and the formula was: Tumor volume (mm²). 3 )=[(w 2 The volume was calculated using [w = width of tumor, l = length of tumor (in mm)]. In the formula, w = width of tumor, and l = length of tumor. The tumor was 1500 mm. 3The mouse trials continued until the target was reached, or until the trial reached its endpoint on day 45. See Figure 3D.

[0166] 9. Tumor digestion and NanoString analysis MC38 tumors and B16-F10 tumors were found in phenol-free RPMI-1640 (ThermoFisher) in small fragments (<5 mm). 3 After being chopped into pieces, the tumor was enzymatically digested with collagenase IV (3 mg / mL, Gibco, 17104019) with shaking at 37°C for 35 minutes. After digestion, the tumor samples were mechanically dissociated using a 70 μM cell strainer. EMT6 tumors were processed using Miltenyi Biotech's gentleMACS™ C tubes (130-093-237). Briefly, the tumors were chopped into pieces (<5 mm) in HBSS containing 1.25 mg / mL collagenase IV (Gibco, 17104019), 0.0025 mg / mL hyaluronidase (Sigma-Aldrich, H3506), and 0.01 mg / mL DNASE I (Worthington, LS002004). 3 The sample was cut into ) sections. The sample was placed in a gentleMACS® Octo Dissociator and processed using program 37C_m_TDK_1, after which the sample was passed through a 70 μM cell strainer to remove undigested tumor fragments. The single-cell suspension was 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 immunoprofiling code set panel with the nCounter FLEX analysis system. NanoString analysis was performed using nSolver® software with the Advanced Analysis module installed.

[0167] 10. Flow cytometry All cells were stained in a 96-well round-bottom plate using FAC buffer (PBS + 0.5% BSA) or 1× permeabilization buffer (eBioscience, 00-5223-56) (where appropriate). Cells were first treated with FC blocks (BioLegend, 101320) at room temperature, followed by tetramer staining for 20 minutes. After tetramer staining, cells were stained with an extracellular antibody master mix at 4°C for 20 minutes, and then fixed / permeabilized overnight using the eBioscience® Foxp3 transcription factor staining buffer set according to the manufacturer's protocol. The following day, samples were washed with Perm buffer and stained with intracellular markers at 4°C for 20 minutes. Cells were analyzed using a Cytek Aurora system. Fluorescence minus one (FMO) and single-stain controls (cells or OneComp ebeads® (Thermofisher, 01-1111-42)) were stained in parallel with the cells. Unless otherwise noted, when effector cytokine production was evaluated using flow cytometry, cells were re-stimulated in complete medium at 37°C for 4 hours 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) and then stained. Cells used in the 2-NDBG assay were starved in glucose-free RPMI-1640 medium (Gibco, 11879-020) for 1 hour, then incubated with 2-NDBG (Cayman Chemical, #186689-07-6) at 37°C / 5% CO2 for 1 hour, and subsequently stained for extracellular markers.Cells were stained with MitoTracker Deep Red FM (ThermoFisher, #M46753), MitoTracker Green FM (ThermoFisher, M46750), MitoSOX Red (ThermoFisher, M36008), and TMRM (ThermoFisher, T668) in RPMI1640 medium (Gibco, A10491-01) containing 10% heat-inactivated FBS (Gibco, 10082-147) and penicillin / streptomycin (Gibco, 15140-122) at 37°C / 5% CO2 for 1 hour. Cells were then washed with FAC buffer and stained for extracellular markers. Specific antibody clones are described below. I purchased the following protein-specific fluorescent dye-conjugated antibodies from BioLegend: CD8α APC, clones 53-67; CD4 BV650, clones RM4-5; CD3 AF700, clone 17A2; CD45 BV605, clones 30-F11; CD49b APC / Cy7, clone DX5; CD25 BV421, clone PC61; CD25 APC / Fire 750, clone PC61; Ki67 PeCy7, clone 16A8; Ki67 AF700, clone 16A8; Granzyme B FITC, clone GB11; IFNγ PE, clone XMG1.2; F4 / 80 Pe / Dazzle 594, clone BM8; CD3 complex PeCy7, clone 17A2; FC block, clone 93. We purchased the following protein-specific fluorescent dye-conjugated antibodies from eBioscience: CD45 BUV395, clon30-F11; CD4 BUV496, clon GK1.5; CD8 BUV563, 53.6-7; TNF BV750, clon MP6-XT22; CD49B Pe-Cy5, clon DX5; FoxP3 AF488, clon FJK-16s; FoxP3 eFlour450, clon FJK-16s. The fluorescent dye-conjugated tetramer for MulV p15E peptide KSPWFTTL (SEQ ID NO: 449) was purchased from ThermoFisher Scientific (50-168-9385). The Live / Dead blue dye was also purchased from ThermoFisher Scientific (L23105).

[0168] 11. Pharmacokinetic Analysis Plasma and tumor samples were collected at the time indicated by Charles River Laboratories and stored at -80°C. MC38 tumor lysates were produced by homogenizing each tumor using a Qiagen TissueRuptor (Qiagen) with disposable probes in ice-cold lysis buffer (diH2O containing 1× Tris buffered saline (Sigma-Aldrich, T5912-1L), 1 mM EDTA (Sigma-Aldrich, 3690-100mL), 1% Triton® X-100 (Sigma-Aldrich, X100-1000mL), and a protease inhibitor (Sigma-Aldrich, P8340-1L)). Plasma and tumor samples were analyzed using sandwich ELISA on an MSD platform that 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 the MSD MESO® QuickPlex SQ 120 system. Data acquisition and analysis were performed using MSD Workbench 4.0.12, and pharmacokinetic parameters were calculated using Phoenix WinNonlin version 8.1.

[0169] 12. Stability of Compound 36 in Human Serum Compound 36 was incubated in double-decker sets of human serum (BioIVT) from a healthy donor at each time point. The sample at 0 hours (T0) was 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. The stability of compound 36 was evaluated by Western blot analysis using a 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 (primary (R&D Systems, AB-219-NA) and secondary (Jackson Labs, 805-035-180) of anti-IL-12 were loaded into a 12-230 kDA Jess separation module, and the analysis was performed using a Jess system set to standard settings for chemiluminescence, with a modified standard protocol for non-reducing conditions. The resulting Western blots were analyzed using Compass for Simple Western software (v4.1.0).

[0170] 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 subsequent 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, seeded in 96-well round-bottom plates, and incubated with titrated amounts of intact or protease-activated (cleaved) INDUKINE® protein or chimeric IL-12. After 72 hours, IFNγ production was measured using the human IFNγ-specific AlphaLisa kit (Perkin Elmer, AL217C) according to the manufacturer's protocol, and Enspire Manager software (V4.13.3005.1482) was run using the Perkin Elmer Enspire Alpha Reader.

[0171] 14. Ex vivo inducible IL-12 prodrug protein processing assay Primary human healthy cells were purchased from either ATCC, Lonza, or Zen-Bio and cultured according to the manufacturer's protocol. Dissociated human tumor samples were purchased from Discovery Life Sciences. To investigate the processing of the inducible IL-12 prodrug, the samples were thawed, washed, and counted. The cells were then resuspended for 48 hours in a medium containing either intact compound 36, an uncleavable variant of compound 36, or pre-cleaved compound 36, after which the cell culture supernatant was collected and frozen for subsequent analysis. The cell culture supernatant was then used to stimulate previously activated human Tblasts using the assays detailed above. To assess the level of processing, IFNγ production was normalized to a control using the following formula. Percent to full activity = (1 - ((sample - non - cleavable control)) / ((cleaved control - non - cleavable control)))×100

[0172] 15. Data representation, bioinformatics analysis, and statistics Flow cytometry plots were generated using FlowJo software (v10.5.30). These are representative samples. All quantitative plots were generated using GraphPad Prism 8 software (64 - bit) for Windows® (San Diego, CA). In in vitro activity assays, data were analyzed using a non - linear sigmoidal 4 - parameter logistic curve fitting model without constraints. Statistical analyses were also performed using GraphPad Prism software (San Diego, CA). Student's t - test was used for comparison of two samples, and analysis of variance (ANOVA) test and multiple comparisons were used for comparison of three or more groups. A mixed - effects model was used to analyze the anti - tumor effect over time. For NanoString datasets, statistical analyses were performed using nSolver™ software with the Advanced Analysis module installed. Pathway analysis was performed using Partek software (v10.0.22.0428) based on transcripts that were significantly different after mWTX - 330 with a FDR step - up of 0.05.

[0173] 1.2 Results 1. Chimeric compound 1 is an inducibly activated IL - 12 prodrug that generates a robust cleavage - dependent anti - tumor immune response 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 (cleaved) chimeric compound 1, HEK-Blue IL-12 reporter cells were incubated with either intact or protease-activated chimeric compound 1, and IL-12 signaling was evaluated. In this assay, the activity of intact chimeric compound 1 was 1 / 175th that of either cleaved 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 treated with titrations of chimeric compound 1, and tumor growth was monitored over time. This study also included a variant of chimeric compound 1 with an uncleavable linker (Figure 1) at the highest dose as a control. In this model, even the lowest test dose of chimeric compound 1 (7 μg / dose) produced statistically significant inhibition of tumor growth, and 43 μg / dose was sufficient to induce complete tumor rejection (Figure 2A). In contrast, the non-cleavable (NC) variant of chimeric compound 1 showed lower activity even compared to the lowest dose of chimeric compound 1, indicating that the full potency of chimeric compound 1 depends on the in vivo processing of the molecule. Furthermore, treatment with chimeric compound 1 generated robust antitumor immunity in syngeneic tumor models with less immune cell infiltration ("colder"), including CT26 (Figure 3A), B16-F10 (Figure 3B), and EMT-6 (Figure 3C), demonstrating the broad activity of this molecule in vivo. While the use of the non-cleavable control indicated that processing is required for full activity, these data did not directly demonstrate that processing occurs in the tumor mesenteric necrosis (TME). FTY720 is a small molecule inhibitor of sphingosine-1-phosphate receptor-1 that prevents lymphocyte escape from secondary lymphoid tissue and effectively isolates TILs from a normal immune cell population recirculating in vivo. Animals co-treated with chimeric compound 1 / FTY720 retained potent initial antitumor activity associated with chimeric compound 1 treatment, but the completeness of tumor control decreased after discontinuation of INDUKINE™ molecule administration (Figure 8A).

[0174] These data indicate that the systemically administered chimeric compound 1 is processed in the TME and that the local release of this IL-12 is sufficient to cause initial tumor growth inhibition.

[0175] To identify the effector cell populations involved in the anti-tumor immunity induced by chimeric compound 1, individual effector cell populations were depleted with antibodies in conjunction with treating MC38 tumor-bearing mice with chimeric compound 1.

[0176] Interestingly, depletion of the CD8+ T cell population did not inhibit initial tumor control, but chimeric compound-treated mice without CD8+ T cells ultimately could not control tumor growth (Figure 8B). In contrast, depletion of either NK cells or total CD4+ T cells alone did not inhibit the anti-tumor activity induced by chimeric compound 1 (Figure 8B). However, in mice depleted of all three populations, there was little anti-tumor activity with chimeric compound 1 treatment, suggesting that these cell types act together to reject the MC38 tumor in response to treatment. Furthermore, rechallenging chimeric compound 1-treated mice that had previously rejected either the MC38 tumor (Figure 3F) or the EMT-6 tumor (Figure 3G) protected 100% of the animals from tumor growth. Taken together, these data suggest that treatment with chimeric compound 1 generates a robust and durable anti-tumor immune response that depends on in vivo cleavage / activation of the molecule.

[0177] 2. The inducible IL-12 prodrug design of chimeric compound 1 improves its pharmacokinetic profile and expands the therapeutic window of chimeric IL-12 In clinical settings, the pharmacokinetic profiles of free cytokines are insufficient, and their short half-lives lead to rapid clearance and insufficient exposure in patients, resulting in irrational administration schedules. To investigate whether the design of chimeric compound 1 improves the molecular half-life and exposure, 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 separately measure the amount of total IL-12 (blocked + unblocked) 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 nearly 16 hours (Figure 9A). Furthermore, only about 2% of the chimeric compound 1 found in plasma was in the form of a cleaved molecule. In contrast, when the same analysis was performed on tumor samples (Figure 9B), nearly 45% of the molecules were exposed IL-12, and intertumor exposure was maintained far beyond the exposure achieved by treatment with chimeric IL-12. To further confirm the selective processing of chimeric compound 1 in tumors, the activation status of tumor-infiltrating CD8+ T cells, CD4+ conventional T cells, and NK cells was compared with the same populations in tumor-inflowing and non-inflowing lymph nodes and peripheral blood after treatment with chimeric compound 1. As a result of treatment with chimeric compound 1, the proportion of multifunctional CD8+ T cells in MC38 tumors was significantly increased, but no such increase was observed in lymph nodes or peripheral blood (Figure 9C). Similarly, in CD4+ conventional T cells (FoxP3-) (Figure 15A) and NK cells (Figure 15B), chimeric compound 1 preferentially increased the proportion of effector cytokine-producing cells in tumors compared to peripheral tissues.

[0178] 3. Treatment with chimeric compound 1 activates various TIL populations in the MC38 model. To better understand the mechanism by which chimeric compound 1 treatment generates antitumor immunity, MC38 tumor-bearing mice were randomized to a treatment group on day 0 and treated with either the vehicle or chimeric compound 1 on days 1 and 4. Tumors were harvested 24 hours after the second administration and analyzed by flow cytometry or NanoString analysis using the PanCancer mouse immunoprofiling panel. Systemic treatment with chimeric compound 1 significantly affected the transcriptional profile of TMEs, with 364 of the 770 transcripts examined showing 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, chimeric compound 1 treatment resulted in a significant increase in the proportion of tumor-infiltrating NK cells producing IFNγ, TNF, and granzyme B (Figure 4D). Interestingly, treatment with chimeric compound 1 resulted in NK cells, NKT cells, CD4+ conventional T cells, and CD8+ T cells producing high levels of IFNγ, 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 the antigen presentation of exogenous peptides and / or antigens in either MHC class I or MHC class II proteins. Presentation of exogenously obtained antigens in MHC class I proteins is a phenomenon known as cross-presentation, which is mediated exclusively by a unique population of dendritic cells identified by CD103 expression (25). Consistent with bioinformatics analysis, flow cytometry analysis showed that treatment with chimeric compound 1 significantly increased the proportion of the cross-presenting CD103+ DC population among all DCs (Figures 10A-10B).

[0179] Considering the role of CD8+ T cells in tumor rejection mediated by chimeric compound 1 (Figure 8A) and the finding that chimeric compound 1 increases tumor invasion by cross-presenting DCs, it was considered highly likely that treatment with chimeric compound 1 also enhanced CD8+ T cell activation. Indeed, differential expression analysis of total RNA showed that treatment with chimeric compound 1 significantly increased the expression of many transcripts associated with the activation of cytotoxic CD8+ T cells, including Ifnγ, granzyme B, perforin, and Tnf, in addition to several chemotactic molecules (Figure 4B). Although chimeric compound 1 did not increase the proportion of tumor-specific CD8+ T cells at this initial stage, robust activation of the tumor-specific CD8+ T cell population occurred as a result of the treatment, as indicated by the increased proportion of tetramer-positive + multifunctional CD8+ T cells (Figure 4F), with nearly 100% of tumor-specific T cells producing IFNγ. Similar results were observed when considering not only tetramer-positive cells but also the entire tumor-invading CD8+ T cell population. Furthermore, in CD4+ T cells, treatment with chimeric compound 1 significantly increased the proportion of conventional CD4+ T cells with the TH1 phenotype (Tbet+IFNγ+TNF+) (Figure 10C). Finally, recent published literature has highlighted the role of IFNγ in the phenomenon known as Treg vulnerability, in which Tregs lose their regulatory activity and are converted to the effector phenotype. As a result of treatment with chimeric compound 1, a significant portion of the FoxP3+ Treg population co-produced the effector cytokines TNF and IFNγ (Figures 10D-10F) and expressed Tbet (Figure 10F). This indicates that systemic treatment with chimeric compound 1 can induce Treg instability in TMEs. In summary, these data indicate that systemic administration of chimeric compound 1 results in transcriptional reprogramming of TMEs and subsequent activation of various tumor-infiltrating effector cell populations.

[0180] 4. Treatment with chimeric compound 1 increases the number of unique TCR clones in TME and enhances TCR clonality. In the MC38 tumor model, treatment with chimeric compound 1 induces rapid tumor rejection, making it technically difficult to fully investigate the dynamics of immune activation. In contrast, treatment of the EMT-6 tumor model with chimeric compound 1 induces complete rejection over a longer period, which is preferable for a more detailed analysis of the ongoing CD8+ T cell response (Figure 3C). Therefore, mice carrying established EMT-6 tumors were randomized to treatment groups and administered either the vehicle or chimeric compound 1 twice a week for two weeks. Tumors and plasma were then collected at various time points. Interestingly, in the control animals, the proportion of pluripotent CD8+ T cells increased during the experiment but ultimately decreased along with final tumor growth. In contrast, treatment with chimeric compound 1 increased the proportion of pluripotent CD8+ T cells compared to the control animals as early as 5 days after the start of treatment (Figure 6), and this proportion continued to increase even after exposure to chimeric compound 1 became undetectable.

[0181] To better understand the transcriptional effects of treatment with chimeric compound 1, particularly on tumor-infiltrating CD8+ T cell populations, geospatial NanoString analysis was performed on tumor samples at day 11. This technique allows for transcriptional analysis of specific cell populations while preserving spatial information that would otherwise be lost during tissue dissociation, by integrating immunofluorescence with whole-transcriptome analysis of specific cells. In the control group, the majority of CD8+ T cells were confined to the tumor periphery (Figure 7A). In contrast, treatment with chimeric compound 1 induced significant invasion of EMT-6 tumors, with CD8+ T cells penetrating deeply into the tumor tissue (Figure 7A). Whole-transcriptome analysis of tumor-infiltrating CD8+ T cells showed that substantial transcriptional reprogramming of these cells occurred as a result of treatment with chimeric compound 1, including upregulation of many genes associated with T cell activation, such as Tbet, IFNγ, Cd25, and chemoattractants known to be involved in the recruitment of additional immune cells (Figure 7B).

[0182] In tumor-infiltrating CD8+ T cells, both IL-12 (Figure 7C) and IFNγ signaling (Figure 7D) were significantly upregulated by treatment with chimeric compound 1, supporting local release of exposed IL-12 and subsequent IFNγ production in the tumor mesenter. Treatment with chimeric compound 1 also increased the expression of transcripts downstream of TCR signaling (Figure 11A). Based on the effects on cross-presenting DCs, we hypothesized that treatment with chimeric compound 1 could activate new T cell clones and generate anti-tumor immunity. To test this, T cells were isolated from EMT-6 tumors after treatment with chimeric compound 1 or a vehicle and subjected to TCR sequencing. While many low-frequency clones dominated the tumor-infiltrating TCR repertoire in control animals, treatment with chimeric compound 1 promoted robust growth of several TCR clones (Figure 11B), resulting in a significant increase in the overall clonality of the tumor-infiltrating TCR repertoire (Figure 11C). In fact, when examining the overall frequency of the top 50 clones in each group, treatment with chimeric compound 1 resulted in a significant increase in the number of clones constituting more than 1% of the total repertoire in several animals (Figure 11D). Further analysis of this subset revealed that only one of the eight clones common to both treatment groups increased at least tenfold with treatment with chimeric compound 1 (Figure 12SA). In contrast, all of the clones unique to the chimeric compound 1 treatment group increased more than tenfold compared to the control group (Figure 12T). This suggests that treatment with chimeric compound 1 enhances the clonality of the T cell population not by increasing the frequency of already dominant clones, but primarily by increasing clones that were previously under-presented.

[0183] 5. Chimeric compound 1 significantly increases mitochondrial activity in tumor-infiltrating CD8+ T cells and NK cells. Newly activated CD8+ T cells have high energy demands and rely heavily on glucose uptake and glycolysis to rapidly generate the energy needed to perform effector functions, subsequently converting to mitochondrial-dependent oxidative phosphorylation as they develop into long-lived memory cells. However, recent published literature has shown that tumor-infiltrating CD8+ T cells often fail to induce significant mitochondrial respiration compared to those activated in the spleen or lymph nodes, suggesting that TME (tumor-mediated respiration) negatively impacts the metabolic health of effector cells. In tumor-infiltrating CD8+ T cells, treatment with chimeric compound 1 resulted in significant enrichment of glycolysis-related transcripts (Figure 12A). Therefore, we hypothesized that treatment with chimeric compound 1 may lead to increased glycolysis by increasing glucose uptake in tumor-infiltrating CD8+ T cells. 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-derived animals (Figures 12B-12C). Therefore, treatment with chimeric compound 1 did not simply increase glucose uptake by tumor-infiltrating CD8+ T cells, but rather reprogrammed these cells to utilize glucose more efficiently than those from vehicle-treated animals.

[0184] Treatment with chimeric compound 1 not only promoted increased glycolysis but also enriched transcripts related to the TCA cycle, mitochondrial biosynthesis, and mitochondrial translation. This suggests that treatment with chimeric compound 1 may improve mitochondrial activity and the health of tumor-infiltrating effector cells (Figures 12D-12F). To test this, TILs were isolated from animals treated with vehicle or chimeric compound 1 and mitochondrial phenotyping was performed by flow cytometry. Mitotracker red is a pH-sensitive dye that specifically stains actively respirating mitochondria. While there was limited evidence of ongoing active mitochondrial respiration in tumor-infiltrating CD8+ T cells from vehicle-treated animals, levels of active respiration were significantly elevated in animals treated with chimeric compound 1 (Figures 12G-12H). Interestingly, this finding also applied to NK cells (Figures 12I-12J) and all CD4+ T cells (Figure 12U). This increase was not simply due to an increase in total mitochondrial mass, but primarily to an increase in mitochondrial activity. This is because treatment with chimeric compound 1 only slightly increased the total mitochondrial mass of tumor-infiltrating NK cells, CD8+ T cells, and all CD4+ T cells (Figure 12V). Furthermore, TMRM staining also revealed that treatment with chimeric compound 1 significantly increased 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 shown to be involved in both NFAT signaling and subsequent IL-2 production (31), as well as IFNγ production by CD4+ memory 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).

[0185] Oxidative phosphorylation is a major energy source for memory T cells, and increased reliance on this pathway has been associated with superior antitumor immunity and a “stem cell-like” phenotype. In tumor-infiltrating CD8+ T cells from mice treated with chimeric compound 1, the expression of genes associated with T cell stem cell characteristics, including Tcf7, Cxcr3, and Il2rγ, was significantly upregulated, while the expression of several genes associated with CD8+ T cell exhaustion, including Pdcd1, Havcr2, and Lag3, was significantly downregulated. In summary, these data indicate that systemic administration of chimeric compound 1 is sufficient to restore mitochondrial respiration in tumor-infiltrating CD8+ T cells and NK cells, leading to a more “stem cell-like” phenotype in the CD8+ T cell population, which may result in superior antitumor immunity.

[0186] 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, for clinical development, a fully human IL-12 payload is used. Compound 36 is identical to chimeric compound 1 except that it contains fully human IL-12 as its payload. Like the mouse surrogate molecule, intact compound 36 showed significantly lower activity than either cleaved 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, the activity of intact compound 36 was, on average, 1 / 61st compared to the cleaved molecule. In both of these in vitro assays, cleaved compound 36 exhibited 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 detected after 72 hours at 37°C (Figure 13A), confirming the molecular stability. To investigate whether compound 36 is selectively processed by primary human tumor samples, an in vitro cleavage assay was developed. Briefly, primary human dissociative tumor samples from various indications, or primary human cells derived from healthy tissue, were incubated for 48 hours with either compound 36, pre-cleaved compound 36, or an uncleavable variant of compound 36. The cell culture supernatant containing the processed inducible IL-12 prodrug was then collected. Since human T-blasts can distinguish between intact compound 36 and the cleaved compound 36, primary human T-blasts were exposed to the cell culture supernatant, and IFNγ production was used as a surrogate marker for compound 36 processing. The results of this assay were then normalized against an uncleavable negative control (0% processing rate) and a cleaved positive control (100% processing rate). Of the n=88 primary human tumor samples evaluated, compound 36 was efficiently processed in all indications tested (Figure 13B). In contrast, when compound 36 was incubated with primary human cells (n=13) derived from various healthy tissues, no evidence of processing was obtained.These data suggest that compound 36 is efficiently and selectively processed by primary human tumor samples, supporting the continued clinical development of this molecule.

[0187] 1.3 Discussion IL-12 is a cytokine that has long attracted considerable interest 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). However, despite this interest, its use in clinical settings has been hindered by its inadequate pharmacokinetic properties and the unacceptable levels of toxicity associated with its systemic administration (9, 10, 24, 36). To address these concerns, we developed compound 36, an inducible IL-12 prodrug. Compound 36 is a prodrug molecule designed to provide a low-frequency systemic delivery therapy through targeted intratumor activation that releases native IL-12 into the tumor microenvironment. Our data for chimeric compound 1 demonstrated antitumor activity in the MC38 tumor model that was dependent on in vivo cleavage of the inducible IL-12 prodrug by the tumor. Furthermore, chimeric compound 1 was a highly potent monotherapy in several mouse tumor models with varying baseline invasion levels, including a complete response in a model refractory to anti-PD-1 treatment (EMT-6). These complete responses led to robust immunological memory for subsequent rechallenges with the same tumor cell line, highlighting the role of the immune system in tumor rejection. In the MC38 model, long-term efficacy depended on the presence of CD8+ T cells, but overall tumor growth inhibition was driven by contributions from three main effector cell types: CD8+ T cells, CD4+ conventional T cells, and NK cells. The design of the inducible IL-12 prodrug also increased exposure in tumor tissue compared to plasma, favoring a ratio of active IL-12 to blocked prodrug molecules, which correlated with selectively localized pharmacodynamic changes (effector cell pluripotency) observed in tumors compared to peripheral tissues. Importantly, chimeric compound 1 was demonstrated to be well-tolerated in mice compared to recombinant chimeric IL-12 treatment, while maintaining its potential to induce complete tumor regression, resulting in a nearly 10-fold improvement in the therapeutic range compared to unblocked cytokines.Improvement in therapeutic range is a key characteristic of inducible IL-12 prodrugs and is necessary to facilitate the clinical development of potent cytokines for oncological therapy.

[0188] Treatment with chimeric compound 1 robustly activated various tumor-infiltrating innate and adaptive effector cell populations. This supports a mechanism in which the infiltration and activation of multiple effector cells play an essential role in early tumor control. Tumor-specific delivery of activated IL-12 and subsequent induction of intratumoral IFNγ also induced Treg vulnerability, which likely contributes to the potent efficacy induced by chimeric compound 1 treatment. Equally important, however, are the effects of chimeric compound 1 treatment on antigen processing and presentation, as well as the observation of increased tumor infiltration by cross-presenting dendritic cells. These cells are responsible for the de novo generation of novel T cell responses to novel tumor antigens, and their importance in generating preclinical antitumor immunity has been identified in several published works (37, 38). Because IL-12 plays two roles—as a direct activator of effector cell populations and as a driver of cross-presentation dendritic cell activation—cytokine-based therapies are likely to have an advantage over other treatments in terms of efficacy in “cold” tumors. Indeed, we observed this effect using a low-invasive EMT-6 model, a “cold” tumor model. NanoString digital spatial profiling showed that systemic treatment with chimeric compound 1 enhanced deep invasion of EMT-6 tumors by CD8+ T cells, supported by increased intratumoral 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 in tumor-infiltrating T cells, driving the increase of several novel clones. This suggests that systemic treatment with chimeric compound 1 resulted in the activation of a de novo T cell response to a unique tumor antigen, which may be key to the previously observed CD8+ T cell-dependent tumor rejection.

[0189] Finally, systemic treatment with chimeric compound 1 significantly affected the metabolism of tumor-infiltrating effector cells, altering not only the metabolic status of activated tumor-infiltrating CD8+ T cells but also the metabolic status of intratumor NK cells. TMEs are known to have several distinct characteristics compared to typical cellular environments, including low pH, hypoxic conditions, and intense competition for extracellular glucose, all of which can impair effector cell activity. Despite evidence of increased glycolysis, we observed a slight decrease in extracellular glucose uptake after chimeric compound 1 treatment. This suggests that chimeric compound 1 treatment does not simply promote increased glucose uptake by tumor-infiltrating cells, but rather facilitates increased metabolic efficiency and enhances oxidative phosphorylation. Recent studies have shown that tumor-infiltrating T cells are often less robust in activating mitochondrial respiration compared to those activated in the spleen or lymph nodes, suggesting that TMEs impair 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 showed little evidence of ongoing mitochondrial respiration, despite efficiently taking up 2-NDBG in vitro. In contrast, effector cells from animals treated with compound 36 robustly upregulated active mitochondrial respiration, mitochondrial membrane potential, and mitochondrial reactive oxygen species. In the highly dysregulated metabolic environment within tumors, where glucose competition is fierce and all molecules must be used to the maximum to support effector cell activation, it may be crucial for these cells to strongly convert to oxidative phosphorylation.

[0190] In addition to the metabolic effects of TME, effector cells also need to cope with exhaustion, another immunomodulatory mechanism. This state is characterized by high co-expression of checkpoint proteins (such as PD-1, LAG-3, TIGIT, and TIM-3) (39), loss of effector cytokine production, and inability to proliferate after restorative stimulation. However, recent publications have shown that some tumor-specific T cells maintain a more stem cell-like phenotype, known as increased "stem cell-likeness," and the transcription factor TCF1 has been identified as playing a crucial role in maintaining this phenotype (40). Furthermore, this increased "stem cell-likeness" has been associated with improved anti-tumor immunity. In addition to metabolic reactivation of effector cells, treatment with chimeric compound 1 likely increased the stem cell-likeness of tumor-infiltrating CD8+ T cells, upregulated TCF7 (an mRNA transcript associated with the protein TCF1), and reduced PD-1 and Tim-3 expression in these cells. In summary, these data suggest that treatment with chimeric compound 1 results in robust and comprehensive reprogramming of the tumor-infiltrating CD8+ T cell response.

[0191] The mechanistic studies described herein not only identified the cell types involved in the antitumor efficacy of chimeric compound 1 treatment but also provide insights into how these effector cells can overcome the inhibitory microenvironment within tumors. Furthermore, these data demonstrate that the inducible IL-12 prodrug design of chimeric compound 1 significantly extended the molecule's half-life compared to recombinant IL-12, enabling selective activation in the tumor mesenteric enzymatic area (TME) after systemic administration, resulting in a significantly expanded therapeutic range. Additional studies using fully human compound 36 showed that the inducible IL-12 prodrug was highly inducible in vitro and cleaved by the majority of human tumor samples, but exhibited stability when incubated with normal primary cells and serum. In summary, these data provide clear evidence for continued preclinical development of this therapeutic molecule and support advancing compound 36 to human clinical trials.

[0192] Example 2. MC38 Experiment (Test MC38-e52) The MC38 cell line, a rapidly growing colon adenocarcinoma cell line, was used. Using this tumor model, the ability of an IL-12 prodrug to affect tumor growth and body weight was investigated.

Table 5-1

[0193] To reduce ulceration, mice were anesthetized with isoflurane when cells were transplanted. 5×10 5 MC38 tumor cells were implanted subcutaneously in the flanks of female C57BL / 6 mice with 0% Matrigel. The cell injection volume was 0.1 mL / mouse. The mice were 8 - 12 weeks old on the start date. When the tumors reached an average size of 100 - 150 mm 3 , pair matching was performed and treatment was initiated. This was designated as day 1 of the test. Body weight was measured at the start and then twice a week until the end. Measurements with calipers were performed twice a week until the end. Any adverse reactions were recorded immediately. Individual animals in which more than 25% weight loss was observed once or more than 20% weight loss was measured three times consecutively were euthanized. Groups in which the average weight loss exceeded 20% or the mortality rate exceeded 10% had dosing discontinued, and the group was not euthanized but allowed to recover. In groups with more than 20% weight loss, individuals that reached the individual weight loss endpoint were euthanized. When weight loss related to group treatment recovered to within 10% of the original weight, dosing was resumed at a lower dose or lower frequency dosing schedule. Exceptions to non-treatment weight % recovery were permitted on a case-by-case basis. The endpoint was tumor growth delay (TGD). The animals were monitored individually. The endpoint of the experiment was either a tumor volume of 1500 mm 3 or day 40, whichever came first. When the endpoint was reached, the animals were euthanized. The results are shown in Figures 17A - 17J. References for Examples 1 and 2 1. Propper DJ,Balkwill FR. Harnessing cytokines and chemokines for cancer therapy. Nat.Rev.Clin.Oncol.2022.page 237-53. 2. Berraondo P,Sanmamed MF,Ochoa MC,Etxeberria I,Aznar MA,Perez-Gracia JL,et al.Cytokines in clinical cancer immunotherapy. Br. J.Cancer. Nature Publishing Group;2019.page 6-15. 3. Conlon KC,Miljkovic MD,Waldmann TA. Cytokines in the Treatment of Cancer. J Interf Cytokine Res.Mary Ann Liebert,Inc.;2019;39:6-21. 4. Nirschl CJ,Brodkin HR,Hicklin DJ,Ismail N,Morris K,Seidel-Dugan C,et al.Discovery of a Conditionally Activated IL-2 that Promotes Antitumor Immunity and Induces Tumor Regression. Cancer Immunol Res.American Association for Cancer Research(AACR);2022;10:581-96. 5. Charych DH,Hoch U,Langowski JL,Lee SR,Addepalli MK,Kirk PB,et al.NKTR-214,an Engineered Cytokine with Biased IL2 Receptor Binding,Increased Tumor Exposure,and Marked Efficacy in Mouse Tumor Models. Clin Cancer Res.American Association for Cancer Research Inc.;2016;22:680-90. 6. Klein C,Waldhauer I,Nicolini VG,Freimoser-Grundschober A,Nayak T,Vugts DJ,et al.Cergutuzumab amunaleukin(CEA-IL2v),a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy:Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines. Oncoimmunology. Taylor and Francis Inc.;2017;6. 7. Lopes JE,Fisher JL,Flick HL,Wang C,Sun L,Ernstoff MS,et al.ALKS 4230:A novel engineered IL-2 fusion protein with an improved cellular selectivity profile for cancer immunotherapy. J Immunother Cancer.BMJ Publishing Group;2020;8:673. 8. Sharma M,Khong H,Fa’ak F,Bentebibel SE,Janssen LME,Chesson BC,et al.Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy. Nat Commun.Nature Research;2020;11. 9. Waldmann TA.Cytokines in cancer immunotherapy. Cold Spring Harb Perspect Biol.Cold Spring Harbor Laboratory Press;2018;10. 10. Nguyen KG,Vrabel MR,Mantooth SM,Hopkins JJ,Wagner ES,Gabaldon TA,et al.Localized Interleukin-12 for Cancer Immunotherapy. Front.Immunol.2020. 11. Del Vecchio M,Bajetta E,Canova S,Lotze MT,Wesa A,Parmiani G,et al.Interleukin-12:biological properties and clinical application. Clin Cancer Res.2007;13:4677-85. 12. Lasek W,Zagozdzon R,Jakobisiak M.Interleukin 12:Still a promising candidate for tumor immunotherapy? Cancer Immunol. Immunother.2014. 13. Tait Wojno ED,Hunter CA,Stumhofer JS. The Immunobiology of the Interleukin-12 Family:Room for Discovery. Immunity.2019. 14. Glassman CR,Mathiharan YK,Jude KM,Su L,Panova O,Lupardus PJ,et al.Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells. Cell.2021; 15. Wang P,Li X,Wang J,Gao D,Li Y,Li H,et al.Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent. Nat Commun.Nature Publishing Group;2017;8:1395. 16. Wittrup KD,Kaufman HL,Schmidt MM,Irvine DJ.Intratumorally anchored cytokine therapy.https: / / doi.org / 101080 / 1742524720222084070. Taylor&Francis;2022;19:1-8. 17. Mirlekar B,Pylayeva-Gupta Y.IL-12 family cytokines in cancer and immunotherapy. Cancers(Basel).2021. 18. 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Matloubian M,Lo CG,Cinamon G,Lesneski MJ,Xu Y,Brinkmann V,et al.Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1.Nature. Nature;2004;427:355-60. 24. Cohen J.IL-12 Deaths:Explanation and a Puzzle. Science(80-).1995; 25. Bedoui S,Whitney PG,Waithman J,Eidsmo L,Wakim L,Caminschi I,et al.Crosspresentation of viral and self antigens by skin-derived CD103+ dendritic cells. Nat Immunol.2009;10:488-95. 26. Overacre-Delgoffe AE,Chikina M,Dadey RE,Yano H,Brunazzi EA,Shayan G,et al.Interferon-γ Drives T reg Fragility to Promote Anti-tumor Immunity. Cell.2017;169:1130-1141.e11. 27. Pearce EL,Poffenberger MC,Chang CH,Jones RG.Fueling immunity:Insights into metabolism and lymphocyte function.Science(80-.).2013. 28. Scharping NE,Menk A V,Moreci RS,Whetstone RD,Dadey RE,Watkins SC,et al.The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction. Immunity. Elsevier;2016;45:374-88. 29. McLane LM,Abdel-Hakeem MS,Wherry EJ.CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu.Rev.Immunol.2019. 30. Siska PJ,Beckermann KE,Mason FM,Andrejeva G,Greenplate AR,Sendor AB,et al.Mitochondrial dysregulation and glycolytic insufficiency functionally impair CD8 T cells infiltrating human renal cell carcinoma. JCI Insight.2017; 31. Sena LA,Li S,Jairaman A,Prakriya M,Ezponda T,Hildeman DA,et al.Mitochondria Are Required for Antigen-Specific T Cell Activation through Reactive Oxygen Species Signaling. Immunity.2013; 32. Rackov G,Tavakoli Zaniani P,Colomo del Pino S,Shokri R,Monserrat J,Alvarez-Mon M,et al.Mitochondrial reactive oxygen is critical for IL-12 / IL-18-induced IFN-γ production by CD4+ T cells and is regulated by Fas / FasL signaling. Cell Death Dis 2022 136. Nature Publishing Group;2022;13:1-14. 33. Mo F,Yu Z,Li P,Oh J,Spolski R,Zhao L,et al.An engineered IL-2 partial agonist promotes CD8+ T cell stemness. Nature.2021; 34. Hong Y,Robbins Y,Yang X,Mydlarz WK,Sowers A,Mitchell JB,et al.Cure of syngeneic carcinomas with targeted IL-12 through obligate reprogramming of lymphoid and myeloid immunity. JCI Insight.2022; 35. Xue D,Moon B,Liao J,Guo J,Zou Z,Han Y,et al.A tumor-specific pro-IL-12 activates preexisting cytotoxic T cells to control established tumors. Sci Immunol.American Association for the Advancement of Science;2022;7. 36. Leonard JP,Sherman ML,Fisher GL,Buchanan LJ,Larsen G,Atkins MB,et al.Effects of single-dose interleukin-12 exposure on interleukin-12 associated toxicity and interferon-γ production. Blood.1997; 37. Hildner K,Edelson BT,Purtha WE,Diamond M,Matsushita H,Kohyama M,et al.Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science.2008;322:1097-100. 38. Broz ML, Binnewies M, Boldajipour B, Nelson AE, Pollack JL, Erle DJ, et al. 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[0194] Example 3. Phase I, first-in-human, multicenter, dose-escalation and expansion study of an inducible IL-12 prodrug. 3.1 Introduction and Background Interleukin-12 Cytokines are small secreted proteins that modulate host immunity through both autocrine and paracrine mechanisms (Waldmann et al., 2018). Interleukin-12 (IL-12) has long been studied for its ability to induce antitumor immune responses (Waldmann et al., 2018, Del Vecchio et al., 2007). The active IL-12 (p70) molecule is a heterodimer composed of a 35kD subunit (p35; encoded by IL12A) covalently bonded to a 40kD subunit (p40; encoded by IL12B), and is structurally similar to IL23 and IL27 (Tait Wojno et al., 2019). Antigen-presenting cells such as dendritic cells, macrophages, and monocytes produce IL-12 upon activation by pathogen-associated molecular patterns, injury-associated molecular patterns, cytokines, and / or intercellular interactions (Del Vecchio et al., 2007). When IL-12 binds to high-affinity heterodimer receptors (IL-12Rβ1 / IL-12Rβ2) on activated T cells, natural killer (NK) cells, and natural killer T (NKT) cells, an intracellular signaling cascade is initiated via tyrosine kinase 2 (TYK2), JAK2, and STAT proteins (especially STAT4), inducing changes in gene expression (Tait Wojno et al., 2019, Bacon et al., 1995).

[0195] IL-12 signaling is pro-inflammatory and can drive productive immune responses against malignant cells through multiple mechanisms. IL-12 was first purified from the supernatant of EBV-transformed B cells and was called a "natural killer cell stimulator" due to its ability to enhance NK cell-mediated cytotoxicity (Kobayashi et al., 1989). This activity of IL-12 is partially mediated by increased transcription of genes encoding granzyme B and perforin (Aste-Amezaga et al., 1994). IL-12 also enhances T cell cytotoxicity and stimulates the proliferation of activated NK cells and T cells, even in the presence of other cytokines and mitogens (Stern et al., 1990, Gately et al., 1992, Perussia et al., 1992, Trinchieri et al., 1994). Interferon-gamma (IFNγ) release from IL-12-stimulated NK cells, CD4+ T cells, CD8+ T cells, and plasmacytoid dendritic cells utilizes several additional antitumor mechanisms (Trinchieri et al., 1994; Berraondo et al., 2018). Among its many beneficial activities, IFNγ directly inhibits cell proliferation / cytotoxicity in tumor cells, enhances MHC I / II expression in both immune and tumor cells, induces the expression of immunotransport chemokines (CXCL9, 10, 11), promotes tumor-killing M1 macrophage polarization, and inhibits angiogenesis (Castro et al., 2018; Berraondo et al., 2018). IL-12 can also direct T cell fate by promoting the differentiation of naive CD4+ T cells into type 1 helper T (Th1) cells (Hsieh et al., 1993). Furthermore, IL-12 provides a crucial third signal to naive CD8+ T cells to promote effector and memory differentiation and clonal growth in the presence of an antigen (signal 1) and a co-stimulatory signal (signal 2) (Curtsinger et al., 1999; Curtsinger et al., 2003; Mescher et al., 2006; Chowdhury et al., 2011).IL-12 also possesses unique signaling activity that can stimulate further IL-12 release in dendritic cells, thereby enhancing antigen presentation to T cells (Grohmann et al., 1998; Bianchi et al., 1999).

[0196] In an attempt to leverage these favorable immune activities for patient benefit, recombinant IL-12 was developed and investigated as an anticancer agent. In multiple mouse models, recombinant mouse IL-12 administered systemically as monotherapy induced regression of subcutaneous and metastatic tumors and extended survival at tolerable doses (Curtsinger et al., 1993, Curtsinger et al., 1996, Nastala et al., 1994, Zou et al., 1995). Mice cured by IL-12 treatment were protected from re-challenge by the same tumor cells, providing evidence of anti-tumor immunological memory (Zou et al., 1995, Brunda et al., 1996). Mechanistically, T cells and IFNγ were required for the antitumor activity of IL-12 in mice, but NK cells were not (Brunda et al., 1993, 1996; Nastala et al., 1994; Zou et al., 1995). Taken together, nonclinical experience has shown that recombinant IL-12 can be delivered via intravenous (IV), intraperitoneal, and intratumoral routes and improves the survival of transplantable mouse tumor models, carcinogen-induced mouse tumor models, and genetically engineered mouse tumor models at tolerable doses (Tugues et al., 2014).

[0197] Despite promising findings in mice, testing recombinant human IL-12 (rhIL-12) in patients has proven far more challenging. In a Phase I dose-escalation study (N=40), rhIL-12 was administered via IV bolus injection to patients with advanced renal cell carcinoma (RCC), melanoma, and colon cancer. This first-in-human (FIH) study identified tolerable doses and schedules, detected activity consistent with the biological properties of IL-12 (e.g., increased circulating IFNγ, NK cell lytic activity, and T cell proliferation), and observed preliminary evidence of antitumor activity (Atkins et al., 1997; Robertson et al., 1999). However, in a subsequent Phase II study (N=17), the same dose unexpectedly caused severe toxicity in patients with advanced RCC, resulting in 12 hospitalizations and 2 deaths (Leonard et al., 1997). Significant differences in toxicity were attributed to the omission of a single "test dose" before initiating continuous daily doses in the Phase 2 trial (Leonard et al., 1997). Exploration of a modified schedule of twice-weekly IV administration in patients with metastatic melanoma and RCC (N=28) showed improvements in safety and tolerability, but only slight antitumor activity was observed, with a partial response (PR) in one RCC patient (Gollob et al., 2000). Subcutaneous (SC) administration of rhIL-12 was also explored in melanoma and RCC, but did not significantly improve the treatment index (TI) in these indications (Bajetta et al., 1998, Motzer et al., 1998, Motzer et al., 2001). In contrast, SC rhIL-12 demonstrated both tolerability and activity in lymphoma, resulting in a clinical response in over 50% of patients with cutaneous T-cell lymphoma (CTCL; N=9) as monotherapy, and in nearly 70% of patients with B-cell non-Hodgkin lymphoma (NHL; N=43) when used in combination with rituximab (Rook et al., 1999, Ansell et al., 2002).rhIL-12 showed activity as a monotherapy in patients with relapsed / refractory NHL (N=32), but the response rate was significantly higher with IV administration than with SC administration (Younes et al., 2004). Ultimately, development of systemically administered rhIL-12 (IV or SC) was discontinued, and this therapy is currently not approved for any indication.

[0198] Interleukin-12 Prodrug Recent developments in immuno-oncological drugs that enhance anti-tumor immunity are rapidly transforming cancer treatment. However, these drugs are not effective for all tumor types, nor are they effective for all patients with a particular tumor type. This gap represents an unmet medical need for novel immunotherapy approaches.

[0199] The IL-12 prodrug in this study is a conditionally activated IL-12 prodrug designed to address several shortcomings of rhIL-12. This IL-12 prodrug is engineered by attaching a protease-cleavable linker to an inactivation domain to inhibit IL-12 binding to its receptor peripherally, and a half-life extension domain to enhance tumor exposure. This prodrug is activated in the tumor mesenteric area (TME) via proteolytic cleavage of the linker, thereby releasing fully active IL-12 cytokines to stimulate a potent antitumor immune response. The preferential activation of the IL-12 prodrug in tumors is designed to both reduce systemic toxicity and enhance antitumor efficacy, thereby maximizing the potential clinical benefit to the patient. The preferred IL-12 prodrug for evaluation is compound 36.

[0200] 3.2 Rationale for the Examination This Phase 1 first-in-human dose-escalation and dose-expansion study will investigate IL-12 prodrugs as monotherapy for patients with relapsed / refractory (r / r) advanced or metastatic solid tumors and lymphomas. Patients enrolled in this study will include those with primary or secondary resistance to immune checkpoint inhibitor (CPI) therapy, as well as those with tumor types for which CPIs are not approved. This first-in-human study will characterize the clinical safety, tolerability, pharmacokinetics (PK), pharmacokinetics (PD), and preliminary antitumor efficacy of IL-12 prodrugs.

[0201] The patients enrolled in this study are those with metastatic malignant diseases for which treatment options are limited.

[0202] The initial clinical dose of 0.016 mg / kg administered intravenously every two weeks (Q2W) was selected based on a comprehensive analysis of available nonclinical safety and pharmacological data regarding IL-12 prodrugs, as well as preliminary knowledge from clinical trials using rhIL-12.

[0203] The HNSTD dose in the GLP toxicity study for IND application in NHP is 0.3 mg / kg. Considering the human equivalent dose (HED) of 0.3 / 3.1 = 0.0968 mg / kg and incorporating a safety factor of 6, the proposed starting dose of the IL-12 prodrug in patients is 0.0968 / 6 = 0.016 mg / kg. The expected PK for the IL-12 prodrug in humans was predicted using allometric scaling of NHP PK with typical scaling factors for clearance and volume. The predicted exposure margins for Cmax and AUC for the 0.3 mg / kg HNSTD dose in NHP are currently estimated to be 20-fold and 19-fold, respectively, compared to the FIH starting dose.

[0204] The expected mean concentration in patients with a starting dose of 0.016 mg / kg IV is approximately 1 / 2.7 of the mean concentration (i.e., the pharmacologically estimated minimum therapeutic dose [MABEL]) that caused approximately 50% tumor regression compared to vehicle controls in the MC38 mouse model. The exposure associated with complete regression in the MC38 model is expected to be over 0.29 mg / kg Q2W in patients, which is approximately 18 times the starting dose.

[0205] Nonclinical data demonstrate low systemic exposure to free IL-12 after administration of IL-12 prodrugs. In NHP GLP toxicity studies, free IL-12 (i.e., IL-12 and / or anti-HSA-IL-12) was detected immediately after IV bolus administration of 0.3 mg / kg of IL-12 prodrug, but the peak concentration was approximately 1 / 580th of the Cmax of the IL-12 prodrug. At a starting dose of 0.016 mg / kg, the expected maximum exposure of free IL-12 in patients is approximately 7 pM, which is about 1 / 20th of the maximum exposure of free IL-12 in HNSTD in NHP (approximately 0.13 nM) and 1 / 27th of the level tolerable after IV bolus administration of 500 ng / kg of rhIL-12 (Cmax 0.19 nM, Atkins et al., 1997).

[0206] In summary, the IL-12 prodrug is designed to minimize exposure to free IL-12 in the systemic circulation. At the starting dose, free IL-12 is predicted to be significantly lower than levels previously associated with systemic toxicity after repeated administration of rhIL-12 in cancer patients. In this Phase 1 First-In, First-Hear trial, initial dose and dose escalation strategies were designed using a Bayesian logistic regression model (BLRM) and dose escalation with overdose control (EWOC) to safely reach the expected therapeutic range and minimize exposure to potentially ineffective dosing regimens. During this Phase 1, First-In, First-Hear trial, newly obtained safety and biomarker data will be used to further guide the dosing strategy for the IL-12 prodrug.

[0207] 3.5 Test Objectives Main purpose The primary purpose of dose escalation. To evaluate the safety and tolerability of IL-12 prodrugs, and

[0208] To determine the maximum tolerated dose (MTD) and / or recommended dose (RDE) in the dose expansion part of the inducible IL-12 prodrug, and

[0209] Evaluate the antitumor activity.

[0210] The main objective of the dose expansion portion of this study. To further characterize the safety and tolerability of IL-12 prodrugs, and

[0211] Evaluate the antitumor activity of IL-12 prodrugs as measured by the overall response rate (ORR; complete response [CR] + partial response [PR]) using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, immuno-ORR (immune-ORR [iORR]; immune CR [iCR] + immune PR [iPR]) using immuno-RECIST (iRECIST), or the Lugano classification of lymphoma (Cheson et al., 2014).

[0212] Primary endpoint. The frequency, severity, and association of therapeutic adverse events (TEAEs) and serious adverse events (SAEs), changes in safety laboratory parameters, and DLTs (if observed) that occurred during treatment.

[0213] Overall response rate (ORR) (complete response [CR] + partial response [PR]), duration of response (DOR) according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, and immunological overall response rate (iORR) according to immuno-RECIST (iRECIST) (complete immunological response [iCR] + partial immunological response [iPR]), or response according to the Lugano classification (lymphoma only, Cheson et al., 2014).

[0214] Secondary purpose The secondary objectives of this examination are as follows: To characterize the PK profile of IL-12 prodrugs (i.e., both the parent compound and free IL-12),

[0215] To evaluate changes in major immunological biomarkers in the blood and in treated tumor biopsies at baseline and after administration of IL-12 prodrugs,

[0216] To evaluate the antitumor activity of IL-12 prodrugs as measured by duration of response (DOR) and progression-free survival (PFS) according to RECIST 1.1, iRECIST, or the Lugano classification of lymphoma (Cheson et al., 2014).

[0217] To evaluate the immunogenicity of IL-12 prodrugs (i.e., their potential to induce an anti-drug antibody [ADA] response), and

[0218] To determine the effect of IL-12 prodrugs on overall survival (OS).

[0219] The secondary endpoints are as follows: Plasma concentration-time profiles and calculated PK parameters of IL-12 prodrugs and free IL-12

[0220] Characterization of baseline-to-baseline changes in peripheral immune cells, including T cell subsets, in response to IL-12 prodrugs.

[0221] Changes in immunological biomarkers in baseline and treated tumor biopsies in response to IL-12 prodrugs, determined by immunohistochemistry (IHC), lymphocyte density, and / or activation status in tumor biopsies.

[0222] Incidence and titer of ADA against IL-12 prodrugs

[0223] ORR (CR+PR) based on Best Overall Response Rate (BOR)

[0224] DOR

[0225] Disease control rates at 3 months, 6 months, and 9 months

[0226] Progression-free survival (PFS) as assessed by overall survival (OS) as measured by the principal investigator, according to RECIST v1.1 (solid tumors) and iRECIST or Lugano classification (lymphoma, Cheson et al., 2014).

[0227] exploratory purpose The exploratory objectives are as follows: To evaluate pharmacokinetics,

[0228] To investigate immunological biomarkers (blood, tumor) that may correlate with treatment outcomes, and

[0229] Evaluate tumor biopsies for potential biomarkers of target binding and immune activation.

[0230] The exploratory endpoints are as follows: Regulation of cytokines including, but not limited to, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-15, IFNγ, IFNγ-induced protein 10 (IP-10), transforming growth factor β (TGFβ), tumor necrosis factor α (TNFα), and C-reactive protein (CRP).

[0231] Changes in the levels of lymphocytes (including lymphocyte subsets) in peripheral blood

[0232] Characterization of intratumor immune cells, including the proportion of T cell subsets and immune cells.

[0233] Changes in gene expression profiles of immune responses in baseline and post-treatment tumor biopsies in response to IL-12 prodrugs.

[0234] Evaluation of free IL-12 in ex-vivo cleavage assay using tumor biopsy.

[0235] 3.6 Exam Content This is a Phase 1 first-in-human, multicenter trial involving dose escalation of IL-12 prodrug (Part 1) followed by dose expansion of IL-12 prodrug (Part 2) in two arms (A and B).

[0236] Dose escalation phase (Part 1) The dose-escalation portion of this study will be conducted in patients with relapsed / refractory (r / r) advanced solid tumors and / or metastatic solid tumors. Patients with primary CNS malignancies are ineligible. Patients with castration-resistant prostate cancer (CRPC) and non-Hodgkin lymphoma (NHL) are eligible for dose expansion (see Arm B below) but ineligible for dose escalation. During dose escalation, the IL-12 prodrug will be administered as monotherapy on days 1 and 15 of a 28-day treatment cycle (i.e., every two weeks, Q2W).

[0237] The starting dose of the IL-12 prodrug is 0.016 mg / kg. The selection of the starting dose in this FIH clinical trial was based on safety and PK data obtained from previous clinical experience with rhIL-12, including non-GLP and GLP-compliant toxicity studies in cynomolgus monkeys, pharmacological studies in tumor-bearing mice, and other data. A total of eight provisional dose levels are established for dose escalation. If the initial dose level is not tolerated, a dose level 50% lower than the starting dose is also included.

[0238] To minimize the number of patients treated at dose levels that may be below therapeutic levels, a minimum of 3 and a maximum of 6 patients will be enrolled in each cohort. In each dose cohort, at least 3 patients must have completed the dose-limiting toxicity (DLT) observation period before enrollment in subsequent cohorts is initiated.

[0239] To ensure patient safety, the first two patients at each dose level should be administered at least 7 days apart, and all subsequent patients should be administered at least 2 days apart. Before recruiting patients to the next dose cohort, the dose cohort's DLT evaluation period must be completed and the data reviewed by the Dose Elevation Committee (DEC).

[0240] Patients may receive IL-12 prodrugs as long as they continue to demonstrate clinical benefit as assessed by the principal investigator, or until disease progression or other treatment discontinuation criteria are met. Intra-patient dose escalation is not permitted.

[0241] A Bayesian logistic regression model (BLRM) employing dose escalation with overdose control (EWOC) is used to guide dose escalation. Data from patients who meet the requirements for inclusion in the dose determination set (DDS) are included in the model. After the completion of the dose cohort or whenever the BLRM is updated, the DEC reviews all available clinical, PK, and laboratory data, and determines the decision to escalate and the actual dose and schedule to be selected, based on the BLRM's recommendations regarding the highest tolerable dose in accordance with EWOC principles.

[0242] Dose escalation will continue until the MTD and / or RDE are determined. Once the MTD / RDE is identified, the dose expansion part (Part 2) of the study will begin. Determining the RDE and selecting the optimal dose will draw on non-clinical pharmacological and toxicological data as well as clinical PK, PD, antitumor activity, and safety data.

[0243] Dose escalation and determination of the MTD and / or recommended dose will be guided by BLRM with overdose control (EWOC). The dose escalation meeting will be held after all patients in the cohort have completed one cycle of the study treatment. Safety assessments, including adverse events and laboratory values, will be closely monitored for all enrolled patients to identify DLTs. Before determining the MTD and / or recommended dose for each group, at least six patients must have been treated with the MTD and / or recommended dose of the IL-12 prodrug.

[0244] Table 5-2 lists some of the starting doses and provisional dose levels that may be evaluated during this study. The doses investigated are not limited to the provisional dose levels listed in the table. The proposed dose escalation scheme for IL-12 prodrugs includes dose level-1, which is 50% lower than the first dose level, in case the first dose level is not tolerated. The dose will be selected based on patient safety data and subject to meeting the EWOC criteria under BLRM. [Table 5-2]

[0245] Dose escalation and determination of maximum tolerated dose (MTD) / recommended dose The MTD (Mean Time Tolerance) is defined as the highest dose of the study drug at which the posterior probability of the true DLT rate in the target interval (0.16–0.33) exceeds 0.50, and at which at least six patients in the confirmatory cohort were treated during the DLT observation period (i.e., the safety review period) of the study drug. AEs and abnormal laboratory values ​​were considered DLTs.

[0246] In dose escalation (Part 1), an adaptive two-parameter BLRM with EWOC is used to select dose levels and estimate the MTD. Each cohort consists of newly enrolled patients who receive escalating doses of IL-12 prodrugs until the MTD is reached.

[0247] The determination of the MTD during dose escalation is based on the estimated probability of DLT in cycle 1 in the dose determination set (DDS). If the MTD is not reached during dose escalation, the RDE is determined as the optimal dose for the therapeutic range of the IL-12 prodrug, based on a review of DLT, AE and SAE, laboratory values, PK and PD data by DEC.

[0248] At all decision points, adaptive BLRMs allow for changes in the rate of dose escalation based on observed toxicity. Therefore, it is possible to skip some dose levels during the study or to add additional intermediate dose levels or schedules. BLRMs recommend doses that should not be exceeded at any decision point during escalation, and the maximum dose increase permitted by the protocol. Dose escalations should not exceed a 100% increase from the current dose being administered. The cohort may be expanded to any dose level below the dose deemed unacceptable for further characterization of safety, tolerability, PK, or PD. Dose escalations may be terminated at any time based on newly arising safety concerns (i.e., without establishing an MTD).

[0249] Dose escalation phase (Part 2) Patients eligible for dose escalation are those with a confirmed diagnosis of locally advanced or metastatic solid tumor or lymphoma (r / r) and who have progressed on standard therapy, are unable to tolerate standard therapy, or for whom no standard therapy has demonstrated benefit. Dose escalation (Part 2) will be conducted in two arms enrolling the following patient populations.

[0250] Arm A: Patients with indications for which CPI is indicated / approved (e.g., cutaneous melanoma, RCC, non-small cell lung cancer [NSCLC], head and neck squamous cell carcinoma [HNSCC], urothelial carcinoma, high microsatellite instability-H tumors, etc.), who have been treated with a CPI regimen, and who exhibit primary or secondary resistance to CPI therapy. Primary resistance is defined as the best response after at least 6 weeks of exposure to a PD-(L)1 inhibitor being disease progression or disease stability (SD) of less than 6 months. Secondary resistance is defined as disease progression more than 6 months after initiation of a PD-(L)1 inhibitor in patients who have achieved clinical benefit (i.e., complete response (CR) or partial response (PR) or SD longer than 6 months). Patients who discontinue CPI therapy (e.g., anti-PD-(L)1) for toxicity or other reasons and do not exhibit primary or secondary resistance to CPI as defined herein are ineligible. Patients with Hodgkin lymphoma are also ineligible.

[0251] Arm B: Patients with tumor types for which CPI therapy is not indicated / approved (e.g., pancreatic cancer, microsatellite-stable [MSS] colorectal cancer, castration-resistant prostate cancer [CRPC], NHL) and who are CPI-naive. NHL patients must have either follicular lymphoma or diffuse large B-cell lymphoma (DLBCL), but other subtypes of NHL, including T-cell lymphoma, may also be considered. All NHL patients must have received at least two prior systemic therapies. Patients with primary CNS malignancies or those who have received anti-PD-(L)1 in clinical trials or off-label are ineligible.

[0252] Additional arms may be added to the dose expansion for specific indications of interest.

[0253] Patients will continue the study treatment until unacceptable toxicity, disease progression of solid tumors according to RECIST (Appendix D, Eisenhauer et al., 2009) or iRECIST (Appendix E), or disease progression of NHL according to the Lugano classification (Cheson et al., 2014) is confirmed (dose expansion part), and / or discontinuation of the treatment at the discretion of the principal investigator, or withdrawal of consent by the patient.

[0254] This study consists of a screening period, a treatment period involving either dose escalation (Part 1) or dose expansion (Part 2), an end-of-treatment (EOT) visit, and a safety follow-up period. The safety follow-up visit will take place 30 days after the last dose of the study drug or before the initiation of a new cancer regimen, whichever comes first. For patients undergoing dose expansion (Part 2), overall survival status will be assessed every 12 weeks (±2 weeks). To assess overall survival, patients will be contacted by telephone until the initiation of a new therapy or death, whichever comes first.

[0255] The end of the trial is defined as when 80% of patients discontinue the trial or complete follow...

Claims

1. A method for treating an advanced solid tumor, a metastatic solid tumor, or a lymphoma, comprising administering to a subject in need of such treatment an inducible IL-12 prodrug comprising compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the foregoing.

2. The method according to claim 1, wherein the administration is oral, parenteral, intravenous, intra-articular, intraperitoneal, intramuscular, subcutaneous, intracavitary, percutaneous, intrahepatic, intracranial, spray / inhalation, bronchoscopy, or intratumor.

3. The method according to claim 2, wherein the administration is performed intravenously.

4. The method according to any one of claims 1 to 3, wherein the inducible IL-12 prodrug is administered about twice a week or less frequently.

5. The method according to any one of claims 1 to 3, wherein the inducible IL-12 prodrug is administered about once a week or less frequently.

6. The method according to any one of claims 1 to 3, wherein the inducible IL-12 prodrug is administered once every two weeks.

7. An embodiment according to any one of the prior claims, wherein approximately 0.016 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered every two weeks.

8. An embodiment according to any one of the prior claims, wherein approximately 0.032 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered every two weeks.

7. An embodiment according to any one of the prior claims, wherein approximately 0.056 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered every two weeks.

8. The present invention relates to any one of the prior claims, wherein approximately 0.084 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the aforementioned, is administered every two weeks.

9. An embodiment according to any one of the prior claims, wherein approximately 0.126 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered every two weeks.

10. An embodiment according to any one of the prior claims, wherein approximately 0.190 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered every two weeks.

11. An embodiment according to any one of the prior claims, wherein approximately 0.290 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered every two weeks.

12. An embodiment according to any one of the prior claims, wherein approximately 0.440 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered every two weeks.

13. An embodiment according to any one of the prior claims, wherein approximately 1 mg to approximately 500 mg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered every two weeks.

14. The method according to any one of the prior claims, wherein approximately 1 mg, approximately 3 mg, approximately 10 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 100 mg, or approximately 200 mg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the aforementioned, is administered every two weeks.

15. According to any one of the prior claims, compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered as a primary therapy.

16. The method according to any one of claims 1 to 14, wherein the subject was unable to achieve a complete response to prior treatment or continued treatment.

17. The method according to claim 16, wherein the prior treatment or the continuing treatment includes treatment with a checkpoint inhibitor.

18. The method according to claim 17, wherein the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

19. The method according to claim 18, wherein the checkpoint inhibitor is an anti-CTL4-A antibody.

20. The aforementioned advanced solid tumor, the aforementioned metastatic solid tumor, or the aforementioned lymphoma is adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, cancer of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, adenoid carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, ocular cancer, Germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytic hyperplasia, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, Skin tumors, metastatic cervical squamous cell carcinoma of unknown primary origin, midline tract cancer involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal cavity / paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumors, pleuropneumonia The method according to any one of the prior claims, wherein the patient is a blastoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureteral cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratomatoid tumor, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

21. The method according to any one of the prior claims, wherein the advanced solid tumor or the metastatic solid tumor is colon cancer, lung cancer, melanoma, renal cell carcinoma, or breast cancer.

22. The method according to any one of the prior claims, wherein the advanced solid tumor, the metastatic solid tumor, or the lymphoma is melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), squamous cell carcinoma of the head and neck (HNSCC), classical Hodgkin lymphoma (cHL), mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, high microsatellite instability or mismatch repair deficiency cancer, high microsatellite instability or mismatch repair deficiency colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial cancer, high tumor mutational load cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), or esophageal cancer.

23. According to any one of the prior claims, compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered in parallel with the anti-PD-1 antibody or its antigen-binding fragment.

24. A pharmaceutical composition comprising an inducible IL-12 prodrug, citric acid and / or citrate, a disaccharide, and a surfactant.

25. The pharmaceutical composition according to claim 24, wherein the citrate is sodium citrate, magnesium citrate, or potassium citrate, the disaccharide is sucrose, trehalose, lactose, or maltose, and the surfactant is a nonionic surfactant selected from polysorbate 80, polysorbate 20, Span®-80, castor oil, or poloxamer.

26. The pharmaceutical composition according to claim 24 or 25, wherein the IL-12 prodrug comprises compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or any amino acid sequence variant of the above.

27. The pharmaceutical composition according to claim 24 or 26, wherein the composition is a liquid or a freeze-dried product.

28. The pharmaceutical composition according to any one of claims 24 to 27, wherein the composition is an aqueous liquid for injection or infusion.

29. The pharmaceutical composition according to claim 28, wherein the composition comprises about 1 mg / mL to about 100 mg / mL of IL-12 prodrug, about 5 mM to about 500 mM of sodium citrate, about 20 mM to about 500 mM of sucrose, and about 0.001% to about 2% of polysorbate 80.

30. The pharmaceutical composition according to claim 29, wherein the composition comprises about 5 mg / mL of IL-12 prodrug, about 50 mM of sodium citrate, about 240 mM of sucrose, and about 0.02% of polysorbate 80.

31. The pharmaceutical composition according to claim 28 or 29, having a pH of approximately 5.0 to approximately 7.

5.

32. The pharmaceutical composition according to claim 30, wherein the pH is approximately 5.

5.

33. A pharmaceutical composition which is a freeze-dried product of the composition described in any one of claims 28 to 31.

34. A method for treating colorectal cancer, comprising administering to a subject in need of such treatment an inducible IL-12 prodrug containing compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the aforementioned.

35. The method according to claim 34, wherein the administration is oral, parenteral, intravenous, intra-articular, intraperitoneal, intramuscular, subcutaneous, intracavitary, percutaneous, intrahepatic, intracranial, spray / inhalation, bronchoscopy, or intratumor.

36. The method according to claim 35, wherein the administration is performed intravenously.

37. The method according to any one of claims 34 to 36, wherein the inducible IL-12 prodrug is administered about twice a week or less.

38. The method according to any one of claims 34 to 36, wherein the inducible IL-12 prodrug is administered about once a week or less frequently.

39. The method according to any one of claims 34 to 36, wherein the inducible IL-12 prodrug is administered once every two weeks.

40. The method according to any one of claims 34 to 39, wherein approximately 0.016 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the foregoing, is administered every two weeks.

41. The present invention according to any one of claims 34 to 39, wherein approximately 0.032 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered every two weeks.

42. The method according to any one of claims 34 to 39, wherein approximately 0.056 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the foregoing, is administered every two weeks.

43. The method according to any one of claims 34 to 39, wherein approximately 0.084 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the aforementioned, is administered every two weeks.

44. An embodiment according to any one of claims 34 to 39, wherein approximately 0.126 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the foregoing, is administered every two weeks.

45. The present invention according to any one of claims 34 to 39, wherein approximately 0.190 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the foregoing, is administered every two weeks.

46. The present invention according to any one of claims 34 to 39, wherein approximately 0.290 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the foregoing, is administered every two weeks.

47. The present invention according to any one of claims 34 to 39, wherein approximately 0.440 mg / kg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the foregoing, is administered every two weeks.

48. An embodiment according to any one of claims 34 to 39, wherein approximately 1 mg to approximately 500 mg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered every two weeks.

49. The method according to any one of claims 34 to 39, wherein approximately 1 mg, approximately 3 mg, approximately 10 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 100 mg, or approximately 200 mg of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the aforementioned, is administered every two weeks.

50. According to any one of claims 34 to 39, compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, 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, or compound 36, or an amino acid sequence variant of the above, is administered as a primary therapy.

51. The method according to any one of claims 34 to 50, wherein the subject failed to achieve a complete response to prior treatment or continued treatment.

52. The method according to claim 51, wherein the prior treatment or the continuing treatment includes treatment with a checkpoint inhibitor.

53. The method according to claim 52, wherein the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

54. The method according to claim 53, wherein the checkpoint inhibitor is an anti-CTL4-A antibody.

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