Interferon prodrug

Abstract

This disclosure relates to methods and compositions for treating cancer using inducible IFN alpha-prodrugs. When an inducible IFN alpha-prodrug is present at a site of interest (such as the tumor microenvironment), a protease-cleaving linker is cleaved by an active protease at the site of interest, releasing an unattenuated form of IFN alpha. The method generally involves administering an effective amount of the inducible IFN alpha-prodrug to a subject who requires it. The inducible IFN alpha-prodrug may be compound 1, compound 2, compound 3, compound 4, or compound 5.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 490,240 filed on 14 March 2023 and U.S. Provisional Patent Application No. 63 / 493,802 filed on 3 April 2023, the entirety of which disclosures are incorporated herein by reference. [Background technology]

[0002] 1. Background Cancer immunotherapy is rapidly establishing itself as the fourth pillar of cancer treatment, primarily due to the clinical success of checkpoint inhibitors. Despite the achievement of lasting responses in some patients using these novel therapies, the response rate remains relatively low and is limited to only a few cancer types. Tumor mutational burden, the presence or absence of T-cell infiltration into the tumor, and the overall immunosuppressive tumor microenvironment significantly influence the response to immunotherapy. While immune checkpoint blockade can block the physiological arrest signals that arise in response to immune activation, other approaches can be used to actively stimulate the anti-tumor immune response. One approach involves the use of immune-activating cytokines. Numerous preclinical and clinical studies have demonstrated the potential of cytokine therapy to enhance anti-tumor immunity. In fact, these were some of the earliest cancer immunotherapies approved for clinical use. However, systemic toxicity and poor pharmacokinetic properties have limited their clinical application.

[0003] Interferons ("IFNs") are a family of related signaling proteins classified into three main types: alpha, beta, and gamma. When they bind to specific receptors, they activate signaling pathways, thereby activating a wide range of genes. These genes are now known to be involved not only in antiviral activity but also in immunomodulatory and antiproliferative activity.

[0004] IFN is a potent immune antagonist and has been considered a promising therapeutic agent for oncology. However, IFN is very potent and has a short serum half-life, which has been shown to result in a narrow therapeutic range. As a result, therapeutic administration of IFN leads to undesirable systemic effects and toxicity. This is exacerbated by the need to administer large amounts of cytokine (i.e., IFN) to achieve the desired level of cytokine at the intended site of action (e.g., the tumor microenvironment). Unfortunately, due to the inability to effectively target and control the biology of cytokines and their activity, cytokines have not achieved the expected clinical benefits in treating tumors.

[0005] Inducible IFN alpha prodrug constructs are described in International Patent Applications PCT / US2019 / 032320, PCT / US2020 / 060624, and PCT / US2022 / 040564, respectively, to overcome the toxicity and short half-life issues that limit the clinical use of IFN alpha in oncology. The aforementioned inducible IFN alpha prodrug constructs comprise a polypeptide chain comprising IFN and an antigen-binding polypeptide that binds to human serum albumin or human serum albumin, which may also extend the half-life. [Overview of the project]

[0006] 2. Overview This disclosure relates to compositions and methods for treating cancer using inducible IFN alpha-prodrugs. The methods generally involve administering an effective amount of the inducible IFN alpha-prodrug to a subject in need. The inducible IFN alpha-prodrug may be compound 1, compound 2, compound 3, compound 4, or compound 5. The inducible IFN alpha-prodrug comprises an IFN alpha polypeptide, an IFN alpha blocking element (e.g., a steric blocking element), and a protease-cleaving polypeptide linker. The IFN alpha-prodrug may further comprise a half-life extension element if desired.

[0007] Inducible IFN alpha prodrugs are conditionally active. When an inducible IFN alpha prodrug is not present at the site of interest (e.g., the tumor microenvironment), the prodrug typically remains intact. Intact prodrugs exhibit attenuated IFN alpha receptor agonist activity. When an inducible IFN alpha prodrug is present at the site of interest (e.g., the tumor microenvironment), the protease-cleaving linker is cleaved by an active protease at the site of interest, releasing an unattenuated form of IFN alpha. This conditional activity maintains immunostimulatory effects while limiting the systemic toxicity associated with non-inducible IFN alpha therapy. Intact inducible IFN alpha prodrugs may, if desired, contain elements that extend their half-life, but do not contain the unattenuated form of IFN alpha after cleavage. Consequently, the short half-life of IFN alpha effectively limits toxicity outside the site of interest.

[0008] This disclosure relates to methods for selectively activating effector CD8+ T cells in the tumor microenvironment and methods for selectively activating tumor-infiltrating lymphocytes. These methods involve administering an effective amount of an inducible IFN alpha-prodrug to a subject requiring such activation. The inducible IFN alpha-prodrug is typically administered systemically and activated by cleavage by a protease that has higher activity in the tumor microenvironment than at other sites. The methods result in a significantly higher frequency of granzyme B+ and / or IFN-gamma-producing CD8+ T cells within the tumor compared to peripheral tissue. These methods can significantly increase the tumor-reactive CD8+ / Treg ratio in the tumor microenvironment. These methods can decrease the frequency of bone marrow-derived suppressor cells and / or Treg cells. The methods can upmodulate the expression of MHC class I and MHC class II. The methods can prolong the activation of natural killer cells.

[0009] Another general aspect of this application relates to a method for modulating a tumor microenvironment, comprising administering an effective amount of an inducible interferon-alpha (IFN-alpha) prodrug to a subject in need thereof, wherein the inducible IFN-alpha prodrug is systemically administered and activated by cleavage by a protease having higher activity in the tumor microenvironment than at other sites, wherein the method yields at least one effect selected from the group consisting of selective activation of effector CD8+ T cells in the tumor microenvironment, selective activation of tumor-infiltrating lymphocytes, an increase in the tumor-reactive CD8+ / Treg ratio in the tumor microenvironment, a decrease in the frequency of bone marrow-derived suppressor cells and / or Treg cells in the tumor microenvironment, an increase in the expression of immune checkpoint proteins in the tumor microenvironment, an increase in the expression of MHC class I and MHC class II, and / or an extension of natural killer cell activation. In a particular embodiment, the method provides at least one effect over a period of at least about 7 days (such as at least about 7, 8, 9, 10, 11, 12, 13, 14, or 15 days) after the last dose of the inducible IFN alpha-prodrug.

[0010] In embodiments of the methods of this disclosure, the inducible IFN alpha-prodrug may be administered at a frequency of about twice a week or less, once a week or less, or about once every two weeks or less. In certain embodiments, the inducible IFN alpha-prodrug may be administered about once every two weeks.

[0011] Furthermore, this disclosure relates in general to a method for increasing the expression of immune checkpoint proteins, the method comprising administering an effective amount of an inducible interferon alpha (IFN alpha) prodrug to a subject requiring such increase, wherein the inducible IFN alpha prodrug is systemically administered and activated by cleavage by a protease having higher activity in the tumor microenvironment than at other sites. In certain embodiments, the method increases the expression of at least 1, 2, 3, 4, 5, 6, or more immune checkpoint proteins. In certain embodiments, the immune checkpoint protein is PD-L1. In certain embodiments, the immune checkpoint protein is PD-1. In certain embodiments, the immune checkpoint protein is TIGIT and / or PVR. In certain embodiments, the immune checkpoint protein is CTLA-4. In certain embodiments, the immune checkpoint protein is LAG-3. In certain embodiments, the method increases the expression of immune checkpoint proteins in the tumor microenvironment.

[0012] In a particular embodiment, the inducible interferon alpha (IFN alpha) prodrug comprises a fusion polypeptide having the formula:[D]-[L1]-[A]-[L2']-[H], where, [A] is interferon alpha (IFN alpha) polypeptide, its mutain, or active fragment. [D] is the blocking part, [H] is the half-life extension portion, [L1] is a protease-cleaving polypeptide linker containing the amino acid sequence of SEQ ID NO: 6, 9, or 12. [L2'] is a protease-cleaving polypeptide linker containing the amino acid sequence of SEQ ID NOs: 6, 9, or 12. Here, the blocking portion and the half-life extension portion each independently contain human serum albumin (HSA) or an antibody or antibody fragment that binds to HSA.

[0013] In certain embodiments, the inducible IFN alpha-prodrug for use in the methods of the present disclosure essentially consists of compound 1 (SEQ ID NO: 1), compound 2 (SEQ ID NO: 2), compound 3 (SEQ ID NO: 3), compound 4 (SEQ ID NO: 4), compound 5 (SEQ ID NO: 5), or any amino acid sequence variant of the above. A preferred inducible IFN alpha-prodrug for use in the methods of the present disclosure is compound 1 (SEQ ID NO: 1), compound 2 (SEQ ID NO: 2), compound 3 (SEQ ID NO: 3), compound 4 (SEQ ID NO: 4), compound 5 (SEQ ID NO: 5), or any amino acid sequence variant of the above.

[0014] This disclosure further relates to a method for treating cancer by administering a combination therapy to a subject in need thereof. The combination therapy may include compound 1 (SEQ ID NO: 1), compound 2 (SEQ ID NO: 2), compound 3 (SEQ ID NO: 3), compound 4 (SEQ ID NO: 4), compound 5 (SEQ ID NO: 5), or any amino acid sequence variant of the above, and an anti-PD-1 antibody, any anti-PD-L1 antibody, or an anti-CTL-4 antibody, anti-TIGIT antibody, anti-PVR antibody, anti-LAG3 antibody, or any antigen-binding fragment of the above, or any other checkpoint inhibitor. The method comprises administering an effective dose of the combination therapy to a subject.

[0015] In various embodiments, an anti-PD-1 antibody can be administered. Anti-PD-1 antibodies include AMP-224 (AstraZenica), 609A (3SBio), 704 (3SBio), 705 (3SBio), ABBV-181 (AbbVie), ADU-1503 / bion-004 (Chinook Therapeutics), AGEN2034 / balstilimab (Agenus), AK103 (Akeso), AK104 (Akeso), AK112 (Akeso), AK123 (Akeso), AMG256 (Amgen), AMG404 (Amgen), ANB030 (AnaptysBio), ANKEBIO anti-PD1 preparation (Anhui Anke Biotechnology), anti-PD-1 / anti-CD47 (DiNonA), ASKG915 (Ask Gene Pharmaceuticals), AV-MEL-1 (Aivita Biomedical), BCD-100 (Biocad CJSC), BI754091 (Boehringer Ingelheim), BiCKI-IL-7 (OSE Immunotherapeutics), Boehringer-PD-1-Unknown (Boehringer Ingelheim), BSK-050K01 (Biosion), Camrelizumab (Jiangsu Hengrui Medicine), CB201 (Crescendo Biologics), CB213 (Crescendo Biologics), CC-90006 (AnaptsBio), Setrelimab (J&J), chPD1 (Kiromic Biopharma), CMAB819 (Mabpharm), CS1003 (CStone Pharmaceuticals), CS17938 (Shenzhen Chipscreen Biosciences), CTX-8371 (Compass Therapeutics), CX-072 (CytomX (Therapeutics), CX-188 (CytomX Therapeutics), Ciparizumab (Harbin Gloria Pharmaceuticals), DB004 (DotBio), EMB02 (EpimAb Biotherapeutics), Geptambrimab / Genolimuzumab (Apollomics), GS19 (Suzhou Zelgen)Biopharmaceuticals, HLX10 (Shanghai Henlius Biotech), HX008 (Taizhou HanZhong Pharmaceuticals), HY003 (Juventus Cell Therapy), IBI315 / BH2950 (Innovent Biologics), IBI318 (Innovent Biologics, IBI319 (Innovent Biologics), IMM1802 (ImmuneOnco Biopharma), IMT200 (TrueBinding), Jemperli / AnaptysBio (AnaptysBio), JTX-4014 (Jounce Therapeutics, Keytruda / Basin (Merck), LBL-006 (Nanjing Leads Biolabs), Libtayo / Nanjing-rwlc (Regeneron Pharmaceuticals), LVGN3616 (Lyvgen). Biopharma, LXF821 (Novartis), LY01015 (Luye Pharma Group), LY3462817 (Eli Lilly), MCLA-134 (Merus). NV) MEDI5752 (AstraZenica) NIR178 (Novartis) ONCR-177 (Oncorus) ONO-4685 (Ono Pharmaceutical) Pharmaceutical, MGD019 (MacroGenius), PD1-GDT CAR-T (Kiromic Biopharma). Biotherapeutics) PT-001 (Merck) PT627 (Merck) RB-M1 (Refuge). Biotechnologies) RG6139 (Roche), RG6279 (Roche), RTX-002 (RubrYc). Therapeutics), Schervier (Pfizer), Servier-PD1xLAG3-Download (Servier), SL-279252 / TAK-252 (Shattuck Labs), Sofusa PD1 (Sorrento).Therapeutics may select from the group consisting of spartalizumab (Novartis), SSI-361 (Lyvgen Biopharma), Sym021 (Servier), teboterimab (MacroGenics), tislerizumab (BeiGene), TSR-075 (AnaptsBio), Tuhura-DO / PD-1-unknown (Tuhura Biopharma), tripalimab (Shanghai Junshi Biosciences), cintilimab (Innovent Biologics), Unicar-CAR-T&PD-1-unknown (Shanghai Unicar-Therapy Bio-Medicine Technology), Xdivane (Xbrane Biopharma), XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y-Biologics), and zimbererimab (Arcus Biosciences).

[0016] The active ingredients are A167(Sichuan Kelun), ABL501(ABL Bio), and ABL503(ABL). Bio). Biopharma) 、BMS-936559 / MDX-1105(BMS) 、APL-502 / TQB2450(Apollomics) 、Arbutus-PD-L1-release (Arbutus Biopharma) 、ASC22(Ascletis). Pharma)、ATG-101(Antengene)、AVA-004(Avacta Group)、AVA021(Avacta Group)、AVA027(Avacta Group)、AVA-040-100(Avacta Group)、AVA04-Vbp(Avacta Group, Bavencio / Baven (Merck), BCD-135 (Biocad CJSC), BGB-A333 (BeiGene), Bintrafusp alfa / GSK4045154 (Merck), CA-170 / aupm-170 (Dr.Reddy's). Laboratories, CCX559 (ChemoCentryx), CDR101 (CDR-Life), Checkpoint Therapeutics, CTX-8371 (Compass). Therapeutics) 、DiNonA-protein complex-promoter (DiNonA) DR30207(Zhejiang Doer Biologics) 、DuoBody-PD-L1x4-1BB(Ligand Pharmaceuticals) エバフォ(Alphamab Oncology) EPIM-001(Elpis Biopharmaceuticals) ES101(Elpiscience Biopharma, INBRX-105 (Inhibrx), FAZ053 (Novartis), FS118 (F-Star Therapeutics), GB262 (GenorBiopharma, GS-4224 (Gilead), GT900008 (Kintor Pharmaceuticals), GX-P2 (Genexine), Hamni-PS-L1 / CD47-derivative (Hanmi Pharmaceutical), HBM7015 (HBM Holdings), HBM9167 (HBM). Holdings, HLX20 (Shanghai Henlius Biotech), HTI-1088 (Jiangsu Hengrui Medicine), IBI318 (Innovent Biologics), IBI322 (Innovent Biologics), IBI323 (Innovent Biologics), IGM-7354 (IGM). Biosciences) 、IMC-001(Sorrento Therapeutics) 、Imfinzi / デルルル(AstraZenica) 、IMM25(ImmuneOnco Biopharma) 、IMM2502(ImmuneOnco Biopharma) 、IMM2503(ImmuneOnco). Biopharma, IMM2504 (ImmuneOnco Biopharma), INCB86550 (Incyte), IO103 (IO Biotech), JS003 (Shanghai Junshi Biosciences), Jubilant-PD-L1 inhibitor (Jubilant Therapeutics), KD033 (Kadmon). Holdings, KN046 (Alphamab Oncology), KY1003 (Sanofi), KY1043 (Sanofi), LY3300054 (Eli Lilly), LY3415244 (Eli Lilly), MRNA-6981 (Moderna), MSB2311 (Transcenta). Holding). Biotherapeutics)、PDL-GEX(GlycotopeIt may be selected from the group consisting of GmbH), PMC-122 (PharmAbcine), PMI06 (D&D Pharmatech), Protheragen-RV-scFv-PDL1-unknown (Protheragen), PRS-344 (Pieris Pharmaceuticals), Q-1802 (Merck), RC98 (Yantai Rongchang Pharmaceutical), RV-scFv-PDL1 (Protheragen), SenI_TAAx22P (Hebei Senlang Biotechnology), SHC020 (Nanjing Sanhome Pharmaceutical), Sugemalimab (Ligand Pharmaceuticals), Atezolizumab (Roche), TST005 (Transcenta Holding), TT-01 (Topmunnity Therapeutics), TTX-siPDL1 (TransCode Therapeutics), UniCAR-T-PD-L1 (GEMoaB monoclonals), Vaximm (VXM10), and YBL-013 (Y-Biologics).

[0017] Also, the present disclosure relates to a method of treating cancer, which comprises administering to a subject in need thereof an inducible interferon alpha (IFN alpha) prodrug comprising a checkpoint inhibitor and a fusion polypeptide having the formula: [D]-[L1]-[A]-[L2’]-[H], wherein [A] is an interferon alpha (IFNa) polypeptide, a mutein thereof, or an active fragment, [D] is a blocking moiety, [H] is a half-life extension moiety, [L1] is a protease-cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 6, 9, or 12, [L2’] is a protease-cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 6, 9, or 12, Here, each of the blocking portion and the half-life extension portion independently includes human serum albumin (HSA) or an antibody or antibody fragment that binds to HSA.

[0018] In certain embodiments, the checkpoint inhibitor is an anti-PD-1 antibody or a fragment thereof. In certain embodiments, it is an anti-PD-L1 antibody or a fragment thereof. In certain embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody or a fragment thereof. In certain embodiments, the checkpoint inhibitor is an anti-LAG3 antibody or a fragment thereof. In certain embodiments, the checkpoint inhibitor is a TIGIT antibody or a fragment thereof. In certain embodiments, the checkpoint inhibitor is a PVR antibody or a fragment thereof.

[0019] In certain embodiments, the IFN alpha polypeptide includes mouse interferon alpha 1 (mIFNa1), mouse interferon alpha 11 (mIFNa11), human interferon alpha 2b (IFNA2b), mouse interferon alpha 11 (mIFNa11), interferon alpha 8 (IFNA8), interferon alpha 14 (IFNA14), interferon alpha 16 (IFNA16), or a mutein thereof.

[0020] In certain embodiments, the IFN alpha polypeptide includes the amino acid sequences of SEQ ID NOs: 234-237.

[0021] In certain embodiments, each of the blocking portion and the half-life extension portion includes HSA.

[0022] In certain embodiments, each of the blocking portion and the half-life extension portion includes an antibody or antibody fragment that binds to HSA.

[0023] In certain embodiments, one of the blocking portion and the half-life extension portion includes HSA, and the other of the blocking portion and the half-life extension portion includes an antibody or antibody fragment that binds to HSA.

[0024] In a particular embodiment, at least one of the blocking portion and the half-life extension portion comprises an antibody or antibody fragment that binds to HSA, wherein the antibody or antibody fragment has the amino acid sequence of residues 1 to 116 of SEQ ID NO: 5. In a particular embodiment, each of [L1] and [L2'] includes sequence numbers 6, 9, or 12.

[0025] In a particular embodiment, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5 and 238-257. In certain embodiments, the inducible interferon alpha (IFN alpha) prodrug is activated in the tumor microenvironment of bladder cancer, glioblastoma multiforme, head and neck cancer, gastric cancer, colorectal cancer, cervical cancer, endometrial cancer, melanoma, kidney cancer, non-small cell lung cancer-adenocarcinoma (NSCLC-Ad), non-small cell lung cancer-adenocarcinoma (NSCLC-Sq), ovarian cancer, or uterine cancer. 3. Brief explanation of the drawing [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1 is a graph showing the antitumor activity of compound 1 in the mouse MC38 model. Compound 1 at a dose of 100 μg and the vehicle (control) were administered intraperitoneally twice a week for two weeks.

[0027] [Figure 2] Figure 2 is a graph showing the presence of compound 1 or vehicle in the periphery of MC38 tumor-bearing mice treated with compound 1 or vehicle over time.

[0028] [Figure 3-1] Figures 3A-3B are graphs showing that CD8+ T cells (CD8+ T cells (Figure 3A) and tetrameric CD8+ T cells (Figure 3B)) constitute approximately 50% of the infiltrating immune cells. Administration of compound 1 results in long-term infiltration of CD8+ T cells. [Figure 3-2]Figures 3C-3E are graphs showing that treatment with compound 1 leads to a significant increase in the frequency of CD8+ T cells, coupled with a decrease in the frequency of immunosuppressive cell populations (PMN-MDSCs (Figure 3C), G-MDSCs (Figure 3D)) and Tregs (Figure 3E) within the TIL. [Figure 3-3] Figures 3F-3G show that compound 1 increases the CD8+ ratio to Treg (Figure 3F) and the tetramer CD8+ ratio to Treg over time in the MC38 syngeneic tumor model. Unless otherwise indicated, data are expressed as mean ± SD, and p-values ​​are derived from t-tests (**, p<0.01; ***, p<0.001; ****, p<0.00001).

[0029] [Figure 4] Figures 4A and 4B are graphs showing that treatment with compound 1 reduces the frequency of Tregs among all CD4+ T cells in the tumor. Figure 4A is a graph showing the percentage of conventional CD4+ T cells over time in an MC38 syngeneic tumor model treated with compound 1 and vehicle. Figure 4B is a graph showing the percentage of FoxP3+ T cells in the CD4+ T cell population. Unless otherwise indicated, data are expressed as mean ± SD, and p-values ​​are derived from t-tests (**, p<0.01; ***, p<0.001; ****, p<0.00001).

[0030] [Figure 5] Figures 5A–5D are graphs showing that treatment with compound 1 activates tumor-infiltrating tetramer-CD8+ T cells. Figures 5A–5D show the frequencies of IFN-gamma and / or granzymes in tetramer-CD8+ T cells and whole-CD8+ T cells.

[0031] [Figure 6] Figure 6 is a pie chart showing the frequency of intratumoral polyfunctional tetramer-positive CD8+ T cells in MC38 tumor-bearing mice treated with compound 1 or the vehicle.

[0032] [Figure 7]Figures 7A-7C are graphs showing that PD-1 is expressed by tumor-infiltrating T cells (Treg (%)PD-1 (Figure 7A), total CD8+%PD-1+ (Figure 7B), CD4+T conventional %PD-1+ (Figure 7C)) when treated with compound 1 in the MC38 syngeneic tumor model.

[0033] [Figure 8-1] Figures 8A-8E are graphs showing the increased expression of PD-L1 on various intratumor immune cell populations, including B cells (Figure 8A), CD11b+ dendritic cells (Figure 8B), CD103+ dendritic cells (Figure 8C), M1 macrophages (Figure 8D), and M2 macrophages (Figure 8E), when treated with compound 1 in an MC38 syngeneic tumor model. [Figure 8-2] Same as above.

[0034] [Figure 9-1] Figures 9A–9F are graphs showing that treatment with compound 1 in the MC38 syngeneic tumor model increases the upregulation of MHC class I and MHC class II cells in the intratumor antigen-presenting cell population, specifically B cells MHC class I (Figure 9A), CD11b+ dendritic cells MHC class I (Figure 9B), CD103+ dendritic cells MHC class I (Figure 9C), B cells MHC class II (Figure 9D), CD11b+ DC: MHC class II (Figure 9E), and CD103+ DC MHC class II (Figure 9F). [Figure 9-2] Same as above.

[0035] [Figure 10] Figures 10A and 10B are graphs showing that treatment with compound 1 increases and prolongs intratumoral NK cell activation in an MC38 syngeneic tumor model. Figure 10A shows the percentage of tumor-infiltrating NK cells producing IFNγ, and Figure 10B shows the percentage of tumor-infiltrating NK cells producing granzyme B+.

[0036] [Figure 11-1]Figures 11A–11D are heatmaps of transcripts over time in mice treated with the vehicle. Pathway analysis shows that the transcriptional profile of vehicle-treated animals progresses over time from immune activation to cancer progression in the MC38 model. [Figure 11-2] Same as above.

[0037] [Figure 12] Figure 12 is a graph showing that treatment with compound 1 results in the accumulation of a sustained transcriptional difference in the tumor microenvironment for approximately one week after the final dose (e.g., day 7), and then plateaus over the remainder of the treatment period.

[0038] [Figure 13] Figure 13 is a heatmap showing that enriched transcripts at various time points largely overlap and increase in intensity, rather than being distinct signatures at different time points.

[0039] [Figure 14] Figures 14A-14B are heatmaps and pathway analyses showing that treatment with compound 1 enriches transcripts related to IFN alpha signaling at an early stage (day 5 after treatment) and at subsequent time points.

[0040] [Figure 15-1] Figures 15A–15F are a series of graphs showing that treatment with compound 1 increases immune activation via the interferon pathway, driving cytotoxic cell infiltration and activation. Figure 15A shows that treatment with compound 1 provides robust and sustained IFN-alpha signaling in the tumor microenvironment. Figures 15B–15F are graphs showing pathway analysis scores for increased infiltration by activated cytotoxic cells (Figure 15A), adoptive immunity (Figure 15B), innate immunity (Figure 15C), apoptosis (Figure 15D), NK cell function (Figure 15E), and T cell function (Figure 15F).

[0041] [Figure 15-2] Same as above.

[0042] [Figure 16] Figures 16A–16C are graphs showing transcriptional analysis of PD-1 expression (Figure 16A), PD-L1 (Figure 16B), and PD-L2 (Figure 16C) in tumors of mice treated with compound 1 or the vehicle in the MC38 syngeneic mouse model. The graphs show that treatment with compound 1 increases the expression of PD-1 and PD-L1 in the tumor microenvironment.

[0043] [Figure 17] Figures 17A–17C are graphs showing the transcriptional analysis expression of TIGIT (Figure 17A), PVR (Figure 17B), and PVRL2 (Figure 17C) in mice treated with compound 1 or the vehicle in the MC38 syngeneic mouse model. The graphs show that treatment with compound 1 increases the expression of TIGIT and PVR in the tumor microenvironment.

[0044] [Figure 18] Figures 18A-18C are graphs showing the transcriptional expression of CTLA4 (Figure 18A), CD80 (Figure 18B), and CD86 (Figure 18C) in mice treated with compound 1 or the vehicle in the MC38 syngeneic mouse model. The graphs show that treatment with compound 1 has a limited effect on CTLA-4 expression in the tumor microenvironment.

[0045] [Figure 19] Figures 19A-19C show graphs illustrating the transcriptional expression of LAG-3 (Figure 18A), TIM-3 (Figure 18B), and NRP-1 (Figure 18C) in mice treated with compound 1 or the vehicle in the MC38 syngeneic mouse model. The graphs show that treatment with compound 1 increased the expression of LAG-3 in the tumor microenvironment.

[0046] [Figure 20-1]Figures 20A-20D are graphs showing the combined activity of compound 1 with several CPIs in a CT26 syngeneic tumor model. The combinations are compound 1 with an anti-PD1 inhibitor (Figure 20A), an anti-PD-L1 antibody, an anti-CTLA-4 antibody (Figure 20C), or an anti-LAG3 antibody (Figure 20D). Figure 20A shows the tumor volume over time in mice treated with 50 μg / animal compound 1, 200 μg / animal compound 1, 50 μg / animal compound 1 with an anti-PD1 inhibitor, 200 μg / animal compound 1 with an anti-PD1 inhibitor, 200 μg / animal anti-PD1 inhibitor, and the vehicle. Figure 20B shows the tumor volume over time in mice treated with 50 μg / animal compound 1, 200 μg / animal compound 1, a combination of 50 μg / animal compound 1 and an anti-PD-L1 inhibitor, a combination of 200 μg / animal compound 1 and an anti-PD-L1 inhibitor, a 200 μg / animal anti-PD-L1 inhibitor, and a vehicle. Figure 20C shows the tumor volume over time in mice treated with 50 μg / animal compound 1, 200 μg / animal compound 1, a combination of 50 μg / animal compound 1 and an anti-CTLA-4 antibody, a combination of 200 μg / animal compound 1 and an anti-CTLA-4 antibody, a 200 μg / animal anti-PD1 inhibitor, and a vehicle in the CT26 mouse model. Figure 20D shows tumor volume over time in mice treated with 50 μg / animal compound 1, 200 μg / animal compound 1, 50 μg / animal compound 1 in combination with anti-LAG3 antibody, 200 μg / animal compound 1 in combination with anti-LAG3 antibody, 200 μg / animal anti-PD1 inhibitor, and vehicle in the CT26 mouse model. Figure 20E shows data from individual mice. Combination therapy with compound 1 and anti-PD-1 antibody and / or anti-CTLA-4 antibody showed improved tumor control compared to compound 1 or anti-PD-1 antibody and / or anti-CTLA-4 antibody monotherapy. [Figure 20-2] Same as above. [Figure 20-3] Same as above.

[0047] [Figure 21-1]Figures 21A-21D are graphs showing the combined activity of compound 1 with several CPIs in an MC38 syngeneic tumor model. The combinations are compound 1 with an anti-PD1 inhibitor (Figure 21A), an anti-PD-L1 inhibitor (Figure 21B), an anti-CTLA-4 antibody (Figure 21C), or an anti-LAG3 antibody (Figure 21D). Figure 21A shows the tumor volume over time in mice treated with 50 μg / animal compound 1, 200 μg / animal compound 1, 50 μg / animal compound 1 with an anti-PD1 inhibitor, 200 μg / animal compound 1 with an anti-PD1 inhibitor, 200 μg / animal anti-PD1 inhibitor, and the vehicle. Figure 21B shows the tumor volume over time in mice treated with 50 μg / animal compound 1, 200 μg / animal compound 1, a combination of 50 μg / animal compound 1 and an anti-PD-L1 inhibitor, a combination of 200 μg / animal compound 1 and an anti-PD-L1 inhibitor, a 200 μg / animal anti-PD-L1 inhibitor, and a vehicle. Figure 21C shows the tumor volume over time in mice treated with 50 μg / animal compound 1, 200 μg / animal compound 1, a combination of 50 μg / animal compound 1 and an anti-CTLA-4 antibody, a combination of 200 μg / animal compound 1 and an anti-CTLA-4 antibody, a 200 μg / animal anti-PD1 inhibitor, and a vehicle in the CT26 mouse model. Figure 21D shows the tumor volume over time in mice treated with 50 μg / animal compound 1, 200 μg / animal compound 1, 50 μg / animal compound 1 in combination with anti-LAG3 antibody, 200 μg / animal compound 1 in combination with anti-LAG3 antibody, 200 μg / animal anti-PD1 inhibitor, and vehicle in the CT26 mouse model. Figure 21E shows data from individual mice. Combination therapy with compound 1 and anti-PD-1 antibody and / or anti-CTLA-4 antibody showed improved tumor control compared to either compound 1 or anti-PD-1 antibody and / or anti-CTLA-4 antibody monotherapy. Combination therapy with 50 μg of compound 1 and anti-PD-1 antibody showed a significant improvement compared to compound 1 treatment with 50 μg alone. Combination therapy with 200 μg of compound 1 and anti-PD-1 antibody, and combination therapy with 200 μg of compound 1 and anti-CTLA4 antibody both showed a significant improvement compared to compound 1 treatment alone.

[0048] [Figure 21-2] Same as above. [Figure 21-3] Same as above.

[0049] [Figure 22-1] Figures 22A-E are graphs showing the time course of tumor volume in mice treated with compound 1 at doses of 10 μg / dose, 50 μg / dose, and 200 μg / dose, or with a vehicle, in various tumor cell models. Figure 22A shows the time course of tumor growth in the MC38 mouse model. Figure 22B shows the time course of tumor growth in the EMT6 mouse model. Figure 22C shows the time course of tumor growth in the B16-F10 mouse model. Figure 22D shows the time course of tumor growth in the A20 mouse model. Figure 22E shows the time course of tumor growth in the EG7 mouse model. [Figure 22-2] Same as above. [Figure 22-3] Same as above.

[0050] [Figure 23] Figure 23 shows the activation of compound 5 by human tumor samples compared to human healthy cells.

[0051] [Figure 24] Figure 24 is a graph showing the tumor volume over time in syngeneic mouse tumor models (mEER models) of HPV-induced oral squamous cell carcinoma (OSCC) in mice treated with compound 1 (WW0610) at doses of 100 μg or 400 μg / dose, along with vehicle (PBS), twice a week for two weeks, or in mice treated with 35 μg of free IFNα (WW0126) administered intraperitoneally twice daily for five days, followed by a two-day rest period for two weeks.

[0052] [Figure 25-1]Figures 25A-E are graphs showing that treatment with compound 1 activates tumor-infiltrating CD8+ T cells in a syngeneic mEER tumor model. Figure 25A shows the amount of CD25-positive (IL-2R) cells in the total CD8+ T cell population. Figure 25B shows the quantification of granzyme B (GRZB)-positive cells in the total CD8+ T cell population. Figure 25C shows the quantification of interferon-gamma (IFNg)-positive cells in the total CD8+ T cell population. Figure 25D shows the quantification of tumor necrosis factor (TNF)-positive cells in the total CD8+ T cell population. Figure 25E shows the quantification of T-box transcription factor (Tbet)-positive cells in the total CD8+ T cell population. [Figure 25-2] Same as above.

[0053] [Figure 26] Figures 26A and 26B are graphs showing that treatment with compound 1 (WW0610) activates tumor-infiltrating NK cells in a syngeneic mEER tumor model. Figure 26A shows the quantification of the percentage of granzyme B (GRZB)-positive NK cells among the total NK cells collected. Figure 26A also shows the quantification of the percentage of interferon-gamma (IFNg)-positive NK cells among the total NK cells collected.

[0054] [Figure 27] Figures 27A and 27B are graphs showing that treatment with compound 1 (WW0610) upregulates MHC class I on macrophages in a syngeneic mEER tumor model. Figure 27A shows the quantification of mean fluorescence intensity (MFI) of MHC class I on macrophages. Figure 27B shows the quantification of MFI of MHC class I on CD19-positive B cells.

[0055] [Figure 28-1]Figures 28A-F are graphs showing that treatment with compound 1 (WW0610) dose-dependently upregulates cytokine expression in a syngeneic mEER tumor model. Figure 28A shows the quantification of plasma interferon-gamma (IFNγ). Figure 28B shows the quantification of plasma tumor necrosis factor alpha (TNFα). Figure 28C shows the quantification of plasma CXCL10. Figure 28D shows the quantification of plasma interleukin-10 (IL-10). Figure 28E shows the quantification of serum interleukin-6 (IL-6). Figure 28F shows the quantification of serum interleukin-5 (IL-5). [Figure 28-2] Same as above. [Modes for carrying out the invention]

[0056] 4. Detailed explanation A. Inducible interferon prodrugs This disclosure relates to an inducible IFN-alpha prodrug containing attenuated IFN-alpha and having a longer half-life compared to naturally occurring IFN-alpha.

[0057] This disclosure relates to an inducible IFN alpha prodrug comprising at least one polypeptide chain, and preferably containing two or more polypeptide chains.

[0058] Inducible IFN-alpha prodrugs include IFN-alpha, an IFN-alpha blocking element, a protease-cleaving linker, and, if necessary, a half-life-extending element. IFN-alpha may include human IFN-alpha 1, human IFN-alpha 2, human IFN-alpha 4, human IFN-alpha 5, human IFN-alpha 6, human IFN-alpha 7, human IFN-alpha 8, human IFN-alpha 10, human IFN-alpha 13, human IFN-alpha 14, human IFN-alpha 16, human IFN-alpha 17, and human IFN-alpha 2.

[0059] The inducible IFN alpha prodrugs of this disclosure have attenuated IFN alpha receptor agonist activity and an extended circulating half-life. The IFN alpha receptor agonist activity is attenuated via a blocking element. The half-life extending element may also contribute to attenuation, for example, due to steric effects. Furthermore, the half-life extending element can act as a blocking element capable of blocking all or part of the receptor agonist activity of IFN alpha. For example, the half-life extending element can contribute to blockage when it is adjacent to the IFN alpha polypeptide.

[0060] The blocking element can block all or part of the receptor agonist activity of IFN alpha by sterically blocking non-covalent binding and / or receptor binding to IFN alpha. When the protease-cleaving linker is cleaved, an active form of IFN alpha is released (e.g., more active than the inducible IFN alpha prodrug). Typically, the released IFN alpha is at least 10 times more active than the inducible IFN alpha prodrug. Preferably, the released IFN alpha is at least 20 times, at least 30 times, at least 50 times, at least 1000 times, at least 200 times, at least 300 times, at least 500 times, at least 1000 times, at least about 10,000 times, or more more active than the inducible IFN alpha prodrug.

[0061] The form of IFN alpha released upon cleavage of an inducible IFN alpha prodrug typically has a short half-life, which is often substantially similar to that of naturally occurring IFN alpha. Even if the half-life of the inducible IFN alpha prodrug is prolonged, the agonist activity of the circulating inducible IFN alpha prodrug is weakened, and the active IFN alpha is targeted to the desired site of activity (e.g., the tumor microenvironment), thus reducing or eliminating toxicity.

[0062] The number of polypeptide chains, as well as the positions of elements on the polypeptide chains, half-life extension elements, protease-cleaving linkers (or more), and blocking elements (and components of such elements, such as VH or VL domains), can vary and are often design priorities, as will be understood by those skilled in the art. All such variations are incorporated herein by reference.

[0063] Typically, when the inducible IFN alpha prodrug is a single polypeptide chain, the single polypeptide chain comprises at least one IFN alpha polypeptide [A], a blocking element [D], a protease-cleaving linker [L], and an optional half-life-extending element [H]. In embodiments where an optional half-life-extending element is absent, the blocking element may preferably also function as a half-life-extending element as described herein (e.g., an antigen-binding fragment of an antibody that binds to human serum albumin ("HSA") and sterically inhibits the binding of IFN alpha in the prodrug to the IFN alpha receptor). The IFN alpha polypeptide [A] may be operably linked by the protease-cleaving linker to the blocking element, the half-life-extending element (if present), or both the blocking element and the half-life-extending element (if present). Typically, a single polypeptide chain comprises one IFN alpha polypeptide or two IFN alpha polypeptides. The IFN alpha polypeptides may be positioned at any desired position within the single polypeptide chain.

[0064] A single polypeptide may contain two or more blocking elements that also function as half-life extension elements (e.g., antibody fragments that bind to HSA). When two or more such blocking elements are present in an inducible IFN alpha prodrug, they can block all or part of the receptor agonist activity of IFN alpha and also extend its serum half-life. When two or more such blocking elements are present in an IFN alpha prodrug, separate half-life extension elements or separate blocking elements are optional and typically absent.

[0065] For example, an inducible IFN alpha-prodrug may have formula XII:[D]-[L1]-[A]-[L1']-[D']. In formula XII, [A] is an IFN alpha polypeptide, [L1] is a protease-cleaving polypeptide linker, [L1'] is a protease-cleaving polypeptide linker, and [D] and [D'] are IFN alpha-blocking elements (such as HSA or an anti-HSA antibody or a fragment thereof). Preferably, the blocking elements function as half-life extension elements. [D] and [D'] may have the same or different amino acid sequences. [L1] and [L1'] may preferably have the same or different amino acid sequences and / or protease-cleaving sites (if L2 is protease-cleaving). The protease-cleaving linker may include, for example, the sequence GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9). Examples of preferred inducible IFN alpha-prodrugs are compounds 1-5. Further information regarding these activities is disclosed in International Application No. PCT / US2020 / 060624.

[0066] In certain embodiments, each of the blocking portion and the half-life extension portion contains HSA. In certain embodiments, each of the blocking portion and the half-life extension portion contains an antibody or antibody fragment that binds to HSA. In certain embodiments, one of the blocking portion and the half-life extension portion contains HSA, and the other of the blocking portion and the half-life extension portion contains an antibody or antibody fragment that binds to HSA. In certain embodiments, at least one of the blocking portion and the half-life extension portion contains an antibody or antibody fragment that binds to HSA, and the antibody or antibody fragment has the amino acid sequence of residues 1 to 116 of SEQ ID NO: 5.

[0067] In certain embodiments, the IFN alpha polypeptide comprises mouse interferon alpha 1 (mIFNa1), mouse interferon alpha 11 (mIFNa11), human interferon alpha 2b (IFNA2b), mouse interferon alpha 11 (mIFNa11), interferon alpha 8 (IFNA8), interferon alpha 14 (IFNA14), interferon alpha 16 (IFNA16), or their mutaines. In certain embodiments, the IFN alpha polypeptide comprises the amino acid sequences of SEQ ID NOs. 234-237. Table 1. Polypeptide sequences of interferon alpha [Table 1-1] [Table 1-2] Table 2. Inducible IFN alpha-prodrugs [Table 2]

[0068] The IFN alpha polypeptide and the blocking element and / or half-life extending element (if present) can be operably linked by a protease-cleaving polypeptide. For example, an inducible IFN alpha prodrug may be any of formulas (I) to (IX): [A]-[L1]-[H]-[L2]-[D](I); [D]-[L2]-[H]-[L1]-[A](II); [A]-[L1]-[D]-[L2]-[H](III); [H]-[L2]-[D]-[L1]-[A](IV); [H]-[L1]-[A]-[L2']-[D](V); [D]-[L1]-[A]-[L2']-[H](VI); [H]-[L]-[D]-[L2]-[A]-[L3]-[D'](VII); [D]-[L]-[A]-[L2]-[D']-[L3]-[H](VIII); [D]-[L]-[H]-[L2]-[D']-[L3]-[A](IX).

[0069] In formulas (I) to (IX), [A] is an IFN alpha polypeptide, [D] is an IFN alpha blocking element (e.g., the extracellular portion of INF alpha receptor 1 (IFNAR1) or IFN alpha receptor 2 (IFNAR2), or an antibody or antigen-binding fragment), [D'] is either INF alpha receptor 1 (IFNAR1) or IFN alpha receptor 2 (IFNAR2) that is not present in [D], [H] is a half-life extension element, [L1] is a protease-cleaving polypeptide linker, [L2] is a polypeptide linker that is optionally protease-cleaving, and [L2'] is a protease-cleaving polypeptide linker. [L1] and [L2] or [L1] and [L2'] may, if desired, have the same or different amino acid sequences and / or protease-cleaving sites (if L2 is protease-cleaving). [H] may also optionally provide blocking. The protease-cleaving linker may include, for example, the sequence GPAGLYAQ (sequence number 6) or ALFKSSFP (sequence number 9). In certain embodiments, each of [L1] and [L2'] includes sequence numbers 6, 9, or 12.

[0070] The present invention also relates to a particular inducible IFN alpha prodrug comprising two or more polypeptide chains. Such an inducible IFN alpha prodrug comprises at least one IFN alpha polypeptide [A], a blocking element [D], a protease-cleaving linker [L], and optionally a half-life extension element [H], which may be located on the same polypeptide chain or different polypeptide chains. The blocking element and the half-life extension element (if present) may include two or more components located on the same polypeptide chain or different polypeptide chains. As an example, and as disclosed and illustrated herein, the components of the blocking element may be located on separate polypeptide chains. For example, the first polypeptide chain may include the light chain (VL+CL) or light chain variable domain (VL) of an antibody, and the second polypeptide may include the VL+CL or a heavy chain Fab fragment (VH+CH1) or heavy chain variable domain (VH) of an antibody complementary to VL on the first polypeptide. In such circumstances, these components can associate within the peptide complex to form antigen-binding sites, such as Fab, which bind to IFN-alpha and attenuate IFN-alpha activity.

[0071] For example, an inducible IFN alpha prodrug may have a first polypeptide of formula (X-XI). Formula X:[D]-[L]-[A]-[L2]-[H] or Formula XI:[H]-[L]-[A]-[L2]-[D]. In formulas (X)-(XI), [A] is the IFN alpha polypeptide, [D] is the heavy chain Fab fragment (VH+CH1) or heavy chain variable domain (VH) of the IFN alpha antibody, [H] is the half-life extension element, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that is optionally protease-cleavable, and [L2'] is a protease-cleavable polypeptide linker. [L1] and [L2] or [L1] and [L2'] may, if desired, have the same or different amino acid sequences and / or protease-cleavage sites (if L2 is protease-cleavable). The inducible IFN alpha-prodrug may have a light chain (VL+CL) or light chain variable domain (VL) of a second polypeptide antibody complementary to VH+CH1 or VH. The protease-cleaving linker may contain the sequence GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9).

[0072] In various embodiments, the inducible IFN alpha prodrug may comprise a first polypeptide chain containing an IFN alpha polypeptide and an antibody heavy chain Fab fragment (VH+CH1) or heavy chain variable domain (VH), and the second polypeptide may comprise a half-life extension element and an antibody light chain (VL+CL) or light chain variable domain (VL) complementary to VH+CH1 or VH on the first polypeptide.

[0073] An inducible IFN alpha-prodrug containing two or more polypeptide chains is described in International Application No. PCT / US2022 / 040564.

[0074] B. Half-life extension element Domains that extend the half-life of inducible IFN alpha-prodrugs are intended herein. By increasing the in vivo half-life of therapeutic molecules that have a short half-life in nature, more tolerable and manageable dosing regimens become possible without sacrificing efficacy.

[0075] Half-life extension elements increase the in vivo half-life and alter the pharmacodynamics and pharmacokinetics of inducible IFN alpha-prodrugs. While not constrained by theory, half-life extension elements alter the pharmacodynamic properties of inducible IFN alpha-prodrugs, including changes in tissue distribution, penetration, and diffusion. In some embodiments, half-life extension elements can improve tissue targeting, tissue penetration, intra-tissue diffusion, and enhanced efficacy compared to proteins without half-life extension elements. While not constrained by theory, an exemplary method for improving the pharmacokinetics of polypeptides is by expressing elements in polypeptide chains that bind to receptors that are recycled to the cell's plasma membrane rather than being degraded in lysosomes, such as FcRn receptors and transferrin receptors on endothelial cells. Three types of proteins, e.g., human IgG, HSA (or fragments), and transferrin, persist in human serum much longer than would be predicted by their size alone, and this is a function of their ability to bind to receptors that are recycled rather than degraded in lysosomes. These proteins or fragments retain FcRn binding and are routinely ligated to other polypeptides to extend their serum half-lives. HSAs may also be directly bound to pharmaceutical compositions or via short linkers. Fragments of HSAs may also be used. HSAs and their fragments can function as both blocking and half-life extending elements. Human IgG and Fc fragments can also perform similar functions.

[0076] Furthermore, serum half-life extension elements may be antigen-binding polypeptides that bind to proteins with long serum half-lives, such as serum albumin and transferrin. Examples of such polypeptides include antibodies and their fragments, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibody single-chain variable fragments (scFv), antigen-binding fragments (Fab), and single-domain antibodies (such as heavy-chain variable domains (VH), light-chain variable domains (VL), and camel-type nanobody variable domains (VHH), and dAb). Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies (such as anticarin, affin, affibody molecules, affimers, afitins, alpha bodies, avimers, DARPin, finomers, Knitz domain peptides, and monobodies), as well as binding domains based on other manipulated scaffolds (such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds). Further examples of antigen-binding polypeptides include ligands for desired receptors, ligand-binding moieties of receptors, lectins, and peptides that bind to or associate with one or more target antigens. Antibodies and fragments thereof can function as both blocking elements and half-life-extending elements.

[0077] Furthermore, half-life extension elements can function as both blocking elements and half-life extension elements. For example, a half-life extension element (e.g., anti-HSA) can function as a blocking element when adjacent to an IFN alpha polypeptide.

[0078] The half-life extension elements provided herein are preferably human serum albumin (HSA) binding domains, antigen-binding polypeptides that bind to human serum albumin, or immunoglobulin Fc or fragments thereof.

[0079] The half-life extension element for inducible IFN alpha-prodrugs extends the half-life of the inducible IFN alpha-prodrug by at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more.

[0080] C. Shielding element The blocking element may be any element that binds to IFN-alpha and / or inhibits the ability of an IFN-alpha polypeptide to bind to and activate its receptor. The blocking element can inhibit the ability of IFN-alpha to bind to and / or activate its receptor, for example, by sterically blocking an inducible IFN-alpha prodrug and / or by non-covalently binding to an inducible IFN-alpha prodrug. Some of the blocking elements disclosed herein can bind to IFN-alpha (e.g., human IFN-alpha 1, human IFN-alpha 2, human IFN-alpha 4, human IFN-alpha 5, human IFN-alpha 6, human IFN-alpha 7, human IFN-alpha 8, human IFN-alpha 10, human IFN-alpha 13, human IFN-alpha 14, human IFN-alpha 16, human IFN-alpha 17, human IFN-alpha 2).

[0081] Examples of suitable blocking elements include the full-length or IFN-alpha binding fragment or mutain of the IFN-alpha homologous receptor. For example, if the IFN-alpha polypeptide is INF-alpha 2a, the blocking element may be the extracellular portion of INF-alpha receptor 1 (IFNAR1) or its interferon-binding portion or mutain, or the extracellular portion of IFN-alpha receptor 2 (IFNAR2) or its interferon-binding portion or mutain.

[0082] Antibodies that bind to IFN alpha and their antigen-binding fragments (including antigen-binding fragments (Fab), polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibody single-chain variable fragments (scFv), single-domain antibodies (heavy-chain variable domain (VH), light-chain variable domain (VL), and camel-type nanobody variable domain (VHH), and dAb, etc.)) may also be used. Other suitable antigen-binding domains that bind to IFN alpha may also be used, including non-immunoglobulin proteins that mimic the binding and / or structure of antibodies (anticarin, affin, affibody molecules, affimers, afitins, alpha bodies, avimers, DARPin, finomers, Knitz domain peptides, monobodies, etc.), as well as binding domains based on other manipulated scaffolds (SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds, etc.).

[0083] Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block the binding of IFN alpha to its homologous receptor. Advantageously, such moieties can also function as half-life extending elements. For example, peptides modified by conjugation to water-soluble polymers such as PEG can sterically inhibit or block the binding of cytokines to their receptors. Polypeptides or fragments thereof with long serum half-lives (e.g., serum albumin (human serum albumin), immunoglobulin Fc, and transferrin) as well as fragments of such polypeptides and mutaines can also be used. For example, antibody and antigen-binding domains that bind to proteins with long serum half-lives (e.g., HSA, immunoglobulin, or transferrin) or receptors recycled to the plasma membrane (e.g., FcRn or transferrin receptors) can also inhibit cytokines, especially when bound to their antigens.

[0084] Suitable IFN alpha blocking elements are single-chain variable fragments (scFv) or Fab fragments.

[0085] Inducible IFN alpha prodrugs comprising an IFN alpha-specific blocking element and further comprising a half-life-extending element are also disclosed herein.

[0086] The blocking element may include two or more components present on the same polypeptide chain or on separate polypeptide chains. The first polypeptide chain may include the antibody light chain (VL+CL) or light chain variable domain (VL), and the second polypeptide may include the antibody heavy chain Fab fragment (VH+CH1) or heavy chain variable domain (VH) complementary to VL+CL or VL on the first polypeptide. In such a scenario, these components may associate in the peptide complex to form antigen-binding sites such as Fab that bind to IFN alpha and attenuate IFN alpha activity.

[0087] D. Protease-cleaving linker As disclosed herein, an inducible IFN alpha prodrug comprises one or more linker sequences. The linker sequences function to provide flexibility between polypeptides, for example, so that a blocking element can inhibit the activity of IFN alpha. The linker may be positioned between IFN alpha subunits, half-life extension elements, and / or blocking elements. As described herein, an inducible IFN alpha prodrug comprises a protease-cleaving linker. The protease-cleaving linker may contain 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 for IFN alpha activity (e.g., the tumor microenvironment). Thus, the inducible IFN alpha prodrug is preferentially or selectively cleaved at the target site for desired IFN alpha activity.

[0088] Preferred linkers are typically less than about 100 amino acids. Such linkers may be of different lengths (from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 to 40 amino acids, from 1 to 50 amino acids, from 1 to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 1 to 100 amino acids, etc.). In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acid lengths. Preferred linkers are typically from about 5 amino acids to about 30 amino acids.

[0089] Preferably, the linker length varies from 2 to 30 amino acids and is optimized for each condition so as not to impose any constraints on the conformation or interaction of the linked domains. In preferred embodiments, the linker is cleavable by a cleaving agent (e.g., an enzyme). Preferably, the linker contains protease cleavage sites. In some cases, the linker contains one or more cleavage sites. The linker may contain a single protease cleavage site. Alternatively, the linker may contain two or more protease cleavage sites. For example, two cleavage sites, three cleavage sites, four cleavage sites, five cleavage sites, or more. If the linker contains two or more protease cleavage sites, the cleavage sites may be cleaved by the same protease or different proteases. A linker containing two or more cleavage sites is referred to as a "tandem linker". Two or more cleavage sites can be aligned in any desired orientation, including, but not limited to, one cleavage site adjacent to another, one cleavage site overlapping with another, or one cleavage site followed by another with an amino acid interposed between the two cleavage sites.

[0090] Of particular interest in the present invention are disease-specific protease-cleaving linkers. Protease-cleaving linkers that are preferentially cleaved at desired locations within the body, such as the tumor microenvironment, compared to peripheral circulation are also preferred. For example, the rate at which a protease-cleaving linker is cleaved in the tumor microenvironment can be at least about 10 times, at least about 100 times, at least about 1000 times, or at least about 10,000 times faster at a desired location within the body (e.g., the tumor microenvironment) compared to peripheral circulation (e.g., in plasma).

[0091] Proteases known to be associated with affected cells or tissues include serine protease, cysteine ​​protease, aspartate protease, threonine protease, glutamate protease, metalloprotease, asparagine peptide lyase, serum protease, cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin S, cathepsin K, cathepsin L, kallikrein, hKl, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidine, bromelain, calpain, caspase, caspase-3, and Mirl - Includes, but is not limited to, 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, MMP19, MMP20, urokinase 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 the chimeric nucleic acid sequences provided herein) may be selected from the group consisting of, for example, prostate-specific antigen (PSA), matrix metalloproteinase (MMP), A disintegrin and metalloproteinase (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase.MMPs may be, for example, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 14 (MMP14), matrix metalloproteinase 19 (MMP19), or matrix metalloproteinase 20 (MMP20). Furthermore, or alternatively, the linker may be cleaved by cathepsins (such as cathepsin B, cathepsin C, cathepsin D, cathepsin S, cathepsin E, cathepsin G, cathepsin K, and / or cathepsin L). Preferably, the linker may be cleaved by MMP14 or cathepsin L.

[0092] Table 3 shows proteases useful for linker cleavage and for use in the inducible IFN alpha-prodrugs disclosed herein, and Table 4 shows exemplary proteases and their cleavage sites. Table 3. Proteases related to inflammation and cancer [Table 3-1] [Table 3-2] Table 4. Exemplary proteases and protease recognition sequences [Table 4-1] [Table 4-2] [Table 4-3]

[0093] Exemplary protease-cleaving linkers include, but are not limited to, kallikrein-cleaving linkers, thrombin-cleaving linkers, zymase-cleaving linkers, carboxypeptidase A-cleaving linkers, cathepsin-cleaving linkers, elastase-cleaving linkers, FAP-cleaving linkers, ADAM-cleaving linkers, PR-3-cleaving linkers, granzyme M-cleaving linkers, calpain-cleaving linkers, matrix metalloproteinase (MMP)-cleaving linkers, plasminogen activator-cleaving linkers, caspase-cleaving linkers, tryptase-cleaving linkers, or tumor cell surface proteases. Specifically, MMP9-cleaving linkers, ADAM-cleaving linkers, CTSL1-cleaving linkers, FAPα-cleaving linkers, and cathepsin-cleaving linkers. Some preferred protease-cleaving linkers are cleaved by MMPs and / or cathepsins.

[0094] Linker sequences disclosed herein are typically less than 100 amino acids. Such linker sequences may be of varying lengths (e.g., 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, etc.). In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acid lengths. Preferred linkers are typically between about 5 and about 30 amino acids.

[0095] Preferably, the linker length varies from 2 to 30 amino acids and is optimized for each condition so as not to impose any constraints on the conformation or interaction of the domain to which the linker is linked.

[0096] In some embodiments, the linker uses the array GPAGLYAQ (sequence number 6); GPAGMKGL (sequence number 7); PGGPAGIG (sequence number 8); ALFKSSFP (sequence number 9); ALFFSSPP (sequence number 10); LAQRLRSS (sequence number 11); LAQKLKSS (sequence number 12); GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (sequence number 13); RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (sequence number 14); KGGGPAGLYAQGPAGLYAQGPAGLYAQGPAGLYAQGSR (sequence number 15); RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (sequence number 16); KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (sequence number 17); SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (sequence number 18); RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (sequence number 19); GGGALFKSSFPLAQKLKSSPGGPAGIGGGR (array array Number 20); RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (Sequence No. 21); RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (Sequence No. 22); RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (Sequence No. 23); GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (Sequence No. 24); GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (Sequence No. 25); RGGALFKSSFPLAQKLKSS Includes GPAGLYAQGGK (sequence number 26); RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (sequence number 27); SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (sequence number 28); KGGPGGPAGIGPLAQRLRSSALFKSSFPGR (sequence number 29); KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (sequence number 30); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (sequence number 31).

[0097] Certain preferred linkers include the sequence GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9). Linkers disclosed herein may contain one or more identical or different cleavage motifs or functional variants. A linker may contain one, two, three, four, five, or more cleavage motifs or functional variants. A linker containing 30 amino acids may contain two cleavage motifs or functional variants, three cleavage motifs or functional variants, or more. A “functional variant” of a linker is capable of being cleaved with high efficiency at a target site (e.g., a tumor microenvironment expressing high levels of protease) but not cleaved or cleaved with low efficiency in peripheral areas (e.g., serum). For example, a functional variant retains at least approximately 50%, 55%, 60%, 70%, 80%, 85%, 95%, or more of the cleavage efficiency of a linker containing any one of sequence numbers 6-31 or 232-233.

[0098] Linkers containing one or more cleavage motifs may be selected from SEQ ID NOs: 6-12 or 232-233 and combinations thereof. Preferred linkers containing one or more cleavage motifs contain amino acids selected from SEQ ID NOs: 13-31.

[0099] A linker can contain both ALFKSSFP (sequence number 9) and GPAGLYAQ (sequence number 6). A linker can contain two cleavage motifs, each having the sequence GPAGLYAQ (sequence number 6). Alternatively or additionally, a linker can contain two cleavage motifs, each having the sequence ALFKSSFP (sequence number 9). A linker can contain a third cleavage motif, identical or different.

[0100] In some embodiments, the linker includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NOs. 6-31 or SEQ ID NOs. 232-233 over the entire length of SEQ ID NOs. 6-31 or SEQ ID NOs. 232-233.

[0101] Furthermore, this disclosure relates to functional variants of linkers including SEQ ID NOs. 6-31 or 232-233. Functional variants of linkers including SEQ ID NOs. 6-31 or 232-233 generally differ from SEQ ID NOs. 6-31 or 232-233 by one or more amino acids (including substitutions, deletions, insertions, or any combination thereof) and substantially retain the ability to be cleaved by proteases.

[0102] A functional variant may contain at least one or more amino acid substitutions, deletions, or insertions compared to a linker containing SEQ ID NOs. A functional variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to a linker containing SEQ ID NOs. In some preferred embodiments, a functional variant differs from a linker containing SEQ ID NOs. 6-31 by fewer than 10, 8, 5, 4, 3, 2, or 1 amino acid changes (e.g., amino acid substitutions or deletions). In other embodiments, a functional variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to a linker containing SEQ ID NOs. Amino acid substitutions may be conserved or non-conservative, but are preferably conserved.

[0103] In other embodiments, the functional variant of the linker may contain one, two, three, four, or five or more non-conservative amino acid substitutions compared to the linker containing SEQ ID NOs. 6-31 or 232-233. Non-conservative amino acid substitutions can be recognized by those skilled in the art. The functional variant of the linker preferably contains one, two, three, four, or five or fewer amino acid deletions.

[0104] The amino acid sequences disclosed in a linker can be described by their relative linear positions within the linker relative to sissile bonds. As will be well understood by those skilled in the art, a linker containing eight amino acid protease substrates (e.g., SEQ ID NOs. 6-12 or 232-233) contains amino acids at positions P4, P3, P2, P1, P1', P2', P3', and P4', where the sissile bond is located between P1 and P1'. For example, the amino acid positions for a linker containing the sequence GPAGLYAQ (SEQ ID NOs. 6) can be described as follows: [Table 10]

[0105] "GPAGLYAQ" was disclosed as Sequence ID No. 6.

[0106] The amino acid positions for the linker containing the sequence ALFKSSFP (sequence number 9) can be described as follows: [Table 11]

[0107] "ALFKSSFP" was disclosed as Sequence ID No. 9.

[0108] Preferably, the amino acids surrounding the cleavage site (for example, positions P1 and P1' for sequence numbers 6-12 or 232-233) are not substituted.

[0109] In various embodiments, the linker includes the sequence GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9) or a functional variant of SEQ ID NO: 6 or a functional variant of SEQ ID NO: 9. As described herein, the functional variants of GPAGLYAQ (SEQ ID NO: 232) or ALFKSSFP (SEQ ID NO: 9) include one or more amino acid substitutions and can substantially retain the ability to be cleaved by proteases. Specifically, the functional variant of GPAGLYAQ (SEQ ID NO: 6) is cleaved by MMP14, and the functional variant of ALFKSSFP (SEQ ID NO: 9) is cleaved by captepsin L (CTSL1). Furthermore, the functional variants retain the ability to be cleaved with high efficiency at target sites (e.g., tumor microenvironments expressing high levels of proteases). For example, functional variants of GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9) retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95%, or more of the cleavage efficiency of linkers containing the amino acid sequence GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9), respectively.

[0110] Preferably, the functional variant of GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9) contains 1, 2, 3, 4, or 5 or fewer conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9). Preferably, the amino acids at positions P1 and P1' are not substituted. In SEQ ID NO: 6, the amino acids at positions P1 and P1' are G and L, and in SEQ ID NO: 9, the amino acids at positions P1 and P1' are K and S.

[0111] A functional variant of GPAGLYAQ (SEQ ID NO: 6) may preferably include one or more of the following: a) arginine amino acid substitution at the P4 position, b) leucine, valine, asparagine, or proline amino acid substitution at the P3 position, c) asparagine amino acid substitution at the P2 position, d) histidine, asparagine, or glycine amino acid substitution at the P1 position, e) asparagine, isoleucine, or leucine amino acid substitution at the P1' position, f) tyrosine or arginine amino acid substitution at the P2' position, g) glycine, arginine, or alanine amino acid substitution at the P3' position, h) or serine, glutamine, or lysine amino acid substitution at the P4' position. The following amino acid substitutions are undesirable in functional variants of GPAGLYAQ (SEQ ID NO: 6): a) arginine or isoleucine at position P3, b) alanine at position P2, c) valine at position P1, d) arginine, glycine, asparagine, or threonine at position P1', e) aspartic acid or glutamic acid at position P2', f) isoleucine at position P3', g) valine at position P4'. In some embodiments, functional variants of GPAGLYAQ (SEQ ID NO: 6) do not contain amino acid substitutions at positions P1 and / or P1'.

[0112] The amino acid substitutions of the functional variant of GPAGLYAQ (SEQ ID NO: 6) preferably include amino acid substitutions at the P4 and / or P4' positions. For example, the functional variant of GPAGLYAQ (SEQ ID NO: 6) may contain leucine at the P4 position, or serine, glutamine, lysine, or phenylalanine at the P4 position. Alternatively or additionally, the functional variant of GPAGLYAQ (SEQ ID NO: 6) may contain glycine, phenylalanine, or proline at the P4' position.

[0113] In some embodiments, amino acid substitutions at the P2 or P2' position of GPAGLYAQ (SEQ ID NO: 6) are undesirable.

[0114] In some embodiments, functional variants of GPAGLYAQ (SEQ ID NO: 6) include amino acid sequences selected from SEQ ID NOs: 32-106. Specific functional variants of GPAGLYAQ (SEQ ID NO: 6) include GPAGLLYAQ (SEQ ID NO: 70) and GPAGLKGA (SEQ ID NO: 60). Table 5. Sequence of functional variants [Table 5-1] [Table 5-2]

[0115] Functional variants of LFKSSFP (SEQ ID NO: 233) preferably include hydrophobic amino acid substitutions. Functional variants of LFKSSFP (SEQ ID NO: 233) preferably include one or more of the following: (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; (e) (f) P1' position histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine; (g) P2' position phenylalanine, leucine, isoleucine, lysine, alanine, glutamine, or proline; (g) P3' position phenylalanine, leucine, glycine, serine, valine, histidine, alanine, or asparagine; and phenylalanine, histidine, glycine, alanine, serine, valine, glutamine, lysine, or leucine.

[0116] The inclusion of aspartic acid and / or glutamic acid in the functional variant of SEQ ID NO: 233 is generally undesirable and should be avoided. Additionally, the following amino acid substitutions are undesirable in the functional variant of LFKSSFP (SEQ ID NO: 233): (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; (g) aspartic acid at the P4' position.

[0117] The amino acid substitutions of the functional variant of LFKSSFP (SEQ ID NO: 233) preferably include amino acid substitutions at the P4 position and / or P1. In some embodiments, amino acid substitutions of the functional variant of LFKSSFP (SEQ ID NO: 233) at the P4' position are undesirable.

[0118] In some embodiments, a functional variant of LFKSSFP (SEQ ID NO: 233) includes an amino acid sequence selected from SEQ ID NOs: 107-185. Specific functional variants of LFKSSFP (SEQ ID NO: 233) include ALFFSSPP (SEQ ID NO: 10), ALFKSFPP (SEQ ID NO: 157), ALFKSLPP (SEQ ID NO: 158); ALFKHSPP (SEQ ID NO: 146); ALFKSIPP (SEQ ID NO: 159); ALFKSSLP (SEQ ID NO: 167); or SPFRSSRQ (SEQ ID NO: 108). Table 6. Sequence of functional variants [Table 6]

[0119] The linkers disclosed herein, together with the amino acid sequences (e.g., domains) to which they link, can form a physiologically stable prodrug while also being cleavable by proteases. For example, the linkers are stable in circulation (e.g., not cleaved or cleaved with low efficiency) and cleaved with higher efficiency at a target site (i.e., the tumor microenvironment). Thus, an inducible IFN alpha-prodrug containing the linkers disclosed herein has, if desired, a longer circulating half-life and / or lower biological activity in circulation compared to the components of the inducible IFN alpha-prodrug as separate molecular entities. However, when at a desired location (e.g., the tumor microenvironment), the linkers can be efficiently cleaved to release the components linked with the linkers, restoring, or nearly restoring, the half-life and biological activity of the components as separate molecular entities.

[0120] The linker preferably maintains stability in circulation for a period of 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.

[0121] In some embodiments, the linker is cleaved by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or less than 1% in circulation compared to the target site. Furthermore, the linker is stable in the absence of an enzyme capable of cleaving it. However, upon exposure to a suitable enzyme (i.e., a protease), the linker is cleaved, and the linked domains are separated.

[0122] E. Pharmaceutical Compositions Also provided herein are a pharmaceutical composition comprising an inducible IFN alpha-prodrug described herein, a vector comprising a polynucleotide encoding the inducible IFN alpha-prodrug, or a host cell transformed by the vector, and at least one pharmaceutically acceptable carrier.

[0123] This specification provides pharmaceutical formulations or compositions comprising the inducible IFN alpha-prodrug described herein and a pharmaceutically acceptable carrier. Compositions comprising the inducible IFN alpha-prodrug described herein are suitable for in vitro or in vivo administration. The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the efficacy of the biological activity of the component and is not toxic to the subject to which it is administered. Examples of suitable pharmaceutically acceptable carriers, well known in the art, 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 appropriate doses. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antimicrobial and antifungal agents.

[0124] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make it isotonic, although the formulation may be hypertonic or hypotonic if desired. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffer solutions 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, e.g., films, liposomes, or microparticles. For example, certain carriers may be more preferred depending on the route of administration and the concentration of the composition being administered. The carrier is suitable for administering IFN alpha or an inducible IFN alpha prodrug, or a nucleic acid sequence encoding an inducible IFN alpha prodrug, to humans or other subjects.

[0125] In some embodiments of the pharmaceutical composition, the inducible IFN alpha-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 inducible IFN alpha-prodrug is attached to liposomes. In some examples, the inducible IFN alpha-prodrug is conjugated on the surface of liposomes. In some examples, the inducible IFN alpha-prodrug is encapsulated within the shell of liposomes. In some examples, the liposomes are cationic liposomes.

[0126] The inducible IFN alpha-prodrugs described herein are intended for pharmaceutically active use. Administration is carried out by various methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of treatment 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 any 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 treatment, the patient's overall health, and other drugs administered in conjunction. "Effective dose" refers to the amount of active ingredient sufficient to affect the course and severity of the disease and thereby bring about relief or remission of such condition, and can be determined using known methods.

[0127] If necessary, an inducible IFN alpha prodrug or a nucleic acid sequence encoding an inducible IFN alpha prodrug is administered by a vector. Several 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 an expression vector. These methods and compositions can be broadly divided into two classes: virus-based delivery systems and non-virus-based delivery systems. Such methods are well known in the art and are readily adaptable for use in 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 encoded chimeric polypeptides, or to therapeutically deliver nucleic acids to a subject. The components of the IFN alpha polypeptides disclosed herein are typically operably linked in frame to encode a fusion protein.

[0128] Where used herein, plasmid vectors or viral vectors are activators that include a promoter that delivers the nucleic acid of the disclosure to a cell without degradation, resulting in the expression of nucleic acid molecules and / or polypeptides in the delivered cell. Viral vectors are, for example, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, polioviruses, Sindobis viruses, and other RNA viruses, including these viruses having an HIV skeleton. Any viral family that shares properties of these viruses suitable for use as vectors is also preferred. Retroviral vectors and methods for constructing them are generally described in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). Construction of replication-deficient adenoviruses has been described (Berkner et al., J.Virol.61:1213-20(1987); Massie et al., Mol.Cell.Biol.6:2872-83(1986); Haj-Ahmad et al., J.Virol.57:267-74(1986); Davidson et al., J.Virol.61:1226-39(1987); Zhang et al., BioTechniques 15:868-72(1993)). The advantage and use of these viruses as vectors is that while they can replicate within the initial infected cell, they cannot form novel infectious viral particles, thus limiting their range of 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, replication and host-limited non-replicating vaccinia virus vectors.

[0129] The inducible IFN alpha-prodrug and / or nucleic acid molecule provided may be delivered via virus-like particles. Virus-like particles (VLPs) consist of viral proteins(s) derived from the structural proteins of a virus. Methods for the preparation and use of virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0130] The inducible IFN alpha-prodrugs disclosed herein may be delivered by subviral dense bodies (DBs). DBs transport proteins to 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 provided polypeptides may be delivered by tegument aggregates. Methods for constructing and using tegument aggregates are described in International Publication WO2006 / 110728.

[0131] Non-viral-based delivery methods may include expression vectors containing nucleic acid molecules and nucleic acid sequences encoding polypeptides, with the nucleic acids operably ligated to expression regulatory sequences. Suitable vector backbones include, for example, plasmids, artificial chromosomes, BACs, YACs, or PACs, which are routinely used in the field. 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, induction elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription start sites, termination sequences, polyadenylation sequences, and introns. Furthermore, such vectors can be used to produce inducible IFN alpha-prodrugs by expression in suitable host cells (e.g., CHO cells).

[0132] Preferred promoters for controlling transcription from vectors in mammalian hosts can be obtained from a variety of sources, for example, from the genomes of viruses such as polyomas, 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). Of course, promoters derived from host cells or related species are also useful herein.

[0133] Enhancers generally refer to DNA sequences that can reside either 5' or 3' relative to the transcription unit, rather than functioning at a fixed distance from the transcription start site. Furthermore, enhancers can reside not only within introns but also within the coding sequence itself. They are typically between 10 and 300 base pairs (bp) in length and function in cis. Enhancers usually function to increase transcription from neighboring promoters. Enhancers can also contain response elements that mediate transcriptional regulation. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), but typically, enhancers derived 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.

[0134] Promoter and / or enhancer regions may be inducible (e.g., chemically or physically modulated). Chemically modulated promoters and / or enhancers may be modulated by, for example, alcohol, tetracycline, steroids, or metals. Physically modulated promoters and / or enhancers may be modulated by, for example, environmental factors such as temperature and light. If necessary, 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. If necessary, 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-term repeats (LTRs).

[0135] Furthermore, vectors may include, for example, origins of replication and / or markers. Marker genes can confer a selectable phenotype to cells, such as antibiotic resistance. The marker product is used to determine whether the vector is delivered to cells and whether it is expressed at the time of delivery. Examples of selective markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. If such selective markers are successfully transferred to mammalian host cells, the transformed mammalian host cells can survive under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, expression vectors may include tag sequences designed to facilitate the manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG® tag (Kodak; New Haven, Conn.) sequences 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.

[0136] F. Applications in treatment Methods and uses for treating diseases, disorders, or conditions related to a target antigen, comprising administering the inducible IFN alpha-prodrug described herein to a subject in need thereof. Diseases, disorders, or conditions include, but are not limited to, cancer, inflammatory diseases, immune disorders, autoimmune diseases, and infectious diseases (i.e., bacterial, viral, or parasitic diseases). Preferably, the disease, disorder, or condition is cancer.

[0137] In one embodiment, the present disclosure provides a method for treating cancer, comprising administering to a subject in need of such treatment an inducible interferon alpha (IFN alpha) prodrug comprising a checkpoint inhibitor and a fusion polypeptide having the formula:[D]-[L1]-[A]-[L2']-[H], where, [A] is interferon alpha (IFNa) polypeptide, its mutain, or active fragment. [D] is the blocking part, [H] is the half-life extension portion, [L1] is a protease-cleaving polypeptide linker containing the amino acid sequence of SEQ ID NO: 6, 9, or 12. [L2'] is a protease-cleaving polypeptide linker containing the amino acid sequence of SEQ ID NOs: 6, 9, or 12. Here, the blocking portion and the half-life extension portion each independently contain human serum albumin (HSA) or an antibody or antibody fragment that binds to HSA.

[0138] Any suitable cancer may be treated with the inducible IFN alpha-prodrugs provided herein. Exemplary suitable cancers, particularly solid tumors, such as sarcomas and carcinomas. For example, the methods and compositions disclosed herein may be used for 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, ductal carcinoma, germ cell carcinoma, endometrial cancer, ependymoma, esophageal cancer, sensory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, stomach 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, carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Mercke Sulfur cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary origin, median ductal carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, cancers of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, chromaffin cell tumor, pituitary gland It may be used to treat tumors, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, cancer of the renal pelvis and ureter, 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, teratoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0139] In certain embodiments, the methods and compositions disclosed herein are used for adrenocortical carcinoma, anal carcinoma, appendiceal carcinoma, 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, sensory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, Germ cell tumors, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gastrointestinal trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytic hyperplasia, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, carcinoma in situ, lung cancer, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, primary cancer of unknown origin Metastatic cervical squamous cell carcinoma, median ductal carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, cancers of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, chromaffin cell tumor, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma It may be used to treat cancers of the renal pelvis and ureter, 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, teratoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, non-Hodgkin lymphoma, squamous cell carcinoma of the head and neck, malignant pleural mesothelioma, and Wilms' tumor.

[0140] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), oral squamous cell carcinoma (OSCC), classical Hodgkin lymphoma (cHL), primary 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 mutation burden cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), urothelial carcinoma, colorectal cancer, or esophageal cancer.

[0141] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat Merkel cell carcinoma (MCC), urothelial carcinoma (UC), renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), triple-negative breast cancer (TNBC), endometrial cancer, cutaneous squamous cell carcinoma (CSCC), basal cell carcinoma (BCC), melanoma, malignant pleural mesothelioma, classical Hodgkin lymphoma (cHL), squamous cell carcinoma of the head and neck (SCCHN), hepatocellular carcinoma (HCC), esophageal squamous cell carcinoma (ESCC), non-squamous non-small cell lung cancer, or nasopharyngeal carcinoma (NPC).

[0142] Preferably, the methods and compositions disclosed herein are used to treat colon cancer, lung cancer, melanoma, renal cell carcinoma, or breast cancer.

[0143] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat melanoma. For example, the methods and compositions disclosed herein may be used to treat melanoma in subjects having unresectable or metastatic melanoma. In another example, the methods and compositions disclosed herein may be used for adjuvant treatment of subjects having melanoma with lymph node involvement after complete excision.

[0144] In some embodiments, methods are provided herein for enhancing the immune response in subjects requiring enhancement of the immune response by administering an effective amount of an inducible IFN alpha-prodrug provided herein to the subject. The enhanced immune response may prevent, delay, or treat the development of cancer, tumors, or viral diseases. While not bound by theory, inducible IFN alpha-prodrugs enhance the immune response by activating innate and adaptive immunity. In some embodiments, the methods described herein increase the activity of natural killer cells and T lymphocytes. In some embodiments, the inducible IFN alpha-prodrugs provided herein can induce IFNγ release from natural killer cells as well as CD4+ and CD8+ T cells.

[0145] The method may further include the administration of one or more additional agents for treating cancer, such as chemomastic agents (e.g., Adriamycin, Seruvidine, Bleomycin, Alkeran, Verban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisanthren, Noantrone, Tiguanine, Cytaribine, Procalabidine), immunotumor agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-LAG3, anti-CD47, anti-GD2), cell therapies (e.g., CAR-T, T-cell therapy), and oncolytic viruses. Non-limiting examples of anticancer drugs that may be used include: Asibicin; Akurarubicin; Acodazole hydrochloride; Acronin; Adzelesin; Aldesleukin; Altretamine; Ambomycin; Amethantrone acetate; Aminoglutethimide; Amsacrin; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisanthren hydrochloride; Bisnafido dimesylate; Biseresin; Bleomycin sulfate; Brequinal sodium; Bropyrimin; Busulfan; Kakutinomycin; Carsterone; Calasemide; Carvethymer; Carboplatin; Carmustine; Carbicin hydrochloride; Carzelesin; Sedefingol; Chlorambucil; Cyloremycin; Cisplatin; Cladribine; Crisnator mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Da Cutinomycin; Daunorubicin hydrochloride; Decitabine; Dexormaplatin; Desaguanine; Desaguanine mesylate; Diadicon; Docetaxel; Doxorubicin; Doxorubicin hydrochloride; Doroxifene; Doroxifene citrate; Dromostanolone propionate; Duazomycin; Edatrexate; Eflornithine hydrochloride; Ersamitrusine; Enloplatin; Empromate; Epipropidine; Epirubicin hydrochloride N; Elbrozol; Esolubicin hydrochloride; Estramustine; Estramustine sodium phosphate; Etanidazole; Etoposide; Etoposide phosphate; Etoprine; Fadrozol hydrochloride; Fazarabine; Fenretinide; Furoxuridine; Fludarabine phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostoliesin 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-I; Interferon Gamma-Ib; Iproplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprolide acetate; Rialozol hydrochloride; Lometrexol sodium; Lomustine; Rosoxantrone hydrochloride; Masopropyl; Maytansine; Mechlore hydrochloride Tamin; Megestrol acetate; Melengestrol acetate; Melphalan; Menogalyl; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprin; Metsuredepa; Mitindomide; Mitocalcin; Mitochromin; Mitogiline; Mitomarcin; Mitomycin; Mitospel; Mitotan; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole; Nogaramycin; Ormaplatin; Oxythran; Paclitaxel; Pegaspargase; Periomycin; Pentamustine; Peplomycin sulfate; Perphosphamide; Pipobroman; Piposul Fan; Pyroxantrone hydrochloride; Plicamycin; Promethane; Porfimer sodium; Porphyromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofulin; Ribopurine; Logretimide; Safingol; Safingol hydrochloride; Semustine; Simtrazene; Sparphosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Slofenul; Talisomycin; Tecogalan sodium; Tegafur; Teloxa hydrochloride Tetolone; temoporfin; teniposide; teroxylone; testolactone; thiamiprine; thioguanine; thiotepa; thiazofrine; tirapazamine; toremifene citrate; trestron acetate; trisilibine phosphate; trimethrexate; trimethrexate glucuronide; triptrelin; tubrosol hydrochloride; uracil mustard; uredepa; bapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine sulfate; vindesine sulfate; pinepidine sulfate; vinricinate sulfate; vinoleulosine sulfate; vinorelbine tartrate;Contains binzolidine sulfate; binzolidine sulfate; borozol; zeniplatin; dinostatin; and zolbicin hydrochloride.

[0146] In some embodiments of the methods described herein, an inducible IFN alpha-prodrug or inducible IFN alpha-prodrug is administered in combination with an agent for the treatment of a particular disease, disorder, or condition. The agents include, but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutic agents), hormones (such as steroids and peptides), radiotherapy (such as direct delivery of gamma rays, C rays, and / or radioisotopes, microwaves, and UV irradiation), gene therapy (e.g., antisense and retroviral therapies), and other immunotherapies. In some embodiments, the inducible IFN alpha-prodrug is administered in combination with an antidiarrheal, antiemetic, analgesic, and / or nonsteroidal anti-inflammatory drug.

[0147] This disclosure relates to one or more further agents for treating cancer (e.g., lymphoma), such as chemotherapeutic agents (e.g., cyclophosphamide, mechloretamine, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomid, temozolomid, thiotepa, mitomycin, diazicon (AZQ), cisplatin, carboplatin, oxaliplatin, procarbazine, hexamethylmelamine, methotrexate, pemetrexed, fluorouracil (e.g., 5-fluorouracil), capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, and more. This relates to therapeutic combinations of any of the inducible IFN alpha-prodrugs disclosed herein in combination with immunotumor agents and immune checkpoint inhibitors (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-LAG3, anti-CD47, anti-GD2), as well as oncolytic viruses).

[0148] The inducible IFN alpha-prodrugs disclosed herein can be combined with any desired further anticancer agent. The inducible IFN alpha-prodrugs disclosed herein can be combined with any desired anti-PD-1 antibody or any desired anti-PD-L1 antibody.

[0149] Exemplary anti-PD-1 antibodies that can be combined with inducible IFN alpha-prodrugs include AMP-224 (AstraZenica), 609A (3SBio), 704 (3SBio), 705 (3SBio), ABBV-181 (AbbVie), ADU-1503 / bion-004 (Chinook Therapeutics), AGEN2034 / valstilimab (Agenus), AK103 (Akeso), AK104 (Akeso), AK112 (Akeso), AK123 (Akeso), AMG256 (Amgen), AMG404 (Amgen), ANB030 (AnaptysBio), ANKEBIO anti-PD1 formulation (Anhui Anke Biotechnology), anti-PD-1 / anti-CD47 (DiNonA), and ASKG915 (Ask Gene Pharmaceuticals), AV-MEL-1 (Aivita Biomedical), BCD-100 (Biocad CJSC), BI754091 (Boehringer Ingelheim), BiCKI-IL-7 (OSE Immunotherapeutics), Boehringer-PD-1-Unknown (Boehringer Ingelheim), BSK-050K01 (Biosion), Camrelizumab (Jiangsu Hengrui) Medicine), CB201 (Crescendo Biologics), CB213 (Crescendo Biologics), CC-90006 (AnaptsBio), Setrelimab (J&J), chPD1 (Kiromic Biopharma), CMAB819 (Mabpharm), CS1003 (CStone Pharmaceuticals), CS17938 (Shenzhen Chipscreen Biosciences), CTX-8371 (Compass Therapeutics), CX-072 (CytomX Therapeutics), CX-188 (CytomX Therapeutics), Ciparizumab (Harbin Gloria Pharmaceuticals), DB004 (DotBio), EMB02 (EpimAb Biotherapeutics), Geptambrimab / Genolimuzumab (Apollomics), GS19 (Suzhou Zelgen)Biopharmaceuticals, HLX10 (Shanghai Henlius Biotech), HX008 (Taizhou HanZhong Pharmaceuticals), HY003 (Juventus Cell Therapy), IBI315 / BH2950 (Innovent Biologics), IBI318 (Innovent Biologics, IBI319 (Innovent Biologics), IMM1802 (ImmuneOnco Biopharma), IMT200 (TrueBinding), Jemperli / AnaptysBio (AnaptysBio), JTX-4014 (Jounce Therapeutics, Keytruda / Basin (Merck), LBL-006 (Nanjing Leads Biolabs), Libtayo / Nanjing-rwlc (Regeneron Pharmaceuticals), LVGN3616 (Lyvgen). Biopharma, LXF821 (Novartis), LY01015 (Luye Pharma Group), LY3462817 (Eli Lilly), MCLA-134 (Merus). NV) MEDI5752 (AstraZenica) NIR178 (Novartis) ONCR-177 (Oncorus) ONO-4685 (Ono Pharmaceutical) Pharmaceutical, MGD019 (MacroGenius), PD1-GDT CAR-T (Kiromic Biopharma). Biotherapeutics) PT-001 (Merck) PT627 (Merck) RB-M1 (Refuge). Biotechnologies) RG6139 (Roche), RG6279 (Roche), RTX-002 (RubrYc). Therapeutics), Schervier (Pfizer), Servier-PD1xLAG3-Download (Servier), SL-279252 / TAK-252 (Shattuck Labs), Sofusa PD1 (Sorrento).This list includes, but is not limited to, the following: Therapeutics, spartalizumab (Novartis), SSI-361 (Lyvgen Biopharma), Sym021 (Servier), teboterimab (MacroGenics), tislerizumab (BeiGene), TSR-075 (AnaptsBio), Tuhura-DO / PD-1-unknown (Tuhura Biopharma), tripalimab (Shanghai Junshi Biosciences), cintilimab (Innovent Biologics), Unicar-CAR-T&PD-1-unknown (Shanghai Unicar-Therapy Bio-Medicine Technology), Xdivane (Xbrane Biopharma), XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y-Biologics), and zimbererimab (Arcus Biosciences).

[0150] Anti-PD-1 antibodies that can be combined with inducible cytokine prodrugs are typically approved anti-PD-1 antibodies. Approved anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA), dostallimab (JEMPERLI), semiprimab-rwlc (LIBATYO), nivolumab (OPDIVO), camrelizumab, tisrelizumab, tripalimab, and cintilimab (TYVYT).

[0151] Exemplary anti-PD-L1 antibodies that can be combined with inducible cytokine prodrugs include: A167 (Sichuan Kelun), ABL501 (ABL Bio), ABL503 (ABL Bio), ABSK041 (Abbisko Therapeutics), ACE1708 (Acepodia), ACE-NK-PDL1 (Acepodia), ADG104 (Adagene), AK106 (Akeso), ALPN-202 (Alpine Immune Sciences), AN4005 (Adlai Nortye Biopharma), BMS-936559 / MDX-1105 (BMS), APL-502 / TQB2450 (Apollomics), Arbutus-PD-L1-Unknown (Arbutus Biopharma), ASC22 (Ascletis Pharma), ATG-101 (Antengene), AVA-004 (Avacta Group), AVA021 (Avacta Group), AVA027 (Avacta Group), AVA-040-100 (Avacta Group), AVA04-Vbp (Avacta Group), Bavencio / Avelumab (Merck), BCD-135 (Biocad CJSC), BGB-A333 (BeiGene), Bintrafusp alfa / GSK4045154 (Merck), CA-170 / aupm-170 (Dr.Reddy's Laboratories), CCX559 (ChemoCentryx), CDR101 (CDR-Life), cosibelimab (Checkpoint Therapeutics), CTX-8371 (Compass Therapeutics), DiNonA-Solid Tumor-Unknown (DiNonA), DR30207 (Zhejiang Doer Biologics), DuoBody-PD-L1x4-1BB (Ligand Pharmaceuticals), Embafolimab (Alphamab Oncology), EPIM-001 (Elpis Biopharmaceuticals), ES101 (Elpiscience Biopharma), INBRX-105 (Inhibrx), FAZ053 (Novartis), FS118 (F-starTherapeutics), GB262 (Genor Biopharma), GS-4224 (Gilead), GT900008 (Kintor Pharmaceuticals), GX-P2 (Genexine), Hamni-PS-L1 / CD47-inhibitor (Hanmi Pharmaceutical), HBM7015 (HBM). Holdings, HBM9167 (HBM Holdings), HLX20 (Shanghai Henlius Biotech), HTI-1088 (Jiangsu Hengrui Medicine), IBI318 (Innovent Biologics), IBI322 (Innovent Biologics), IBI323 (Innovent Biologics, IGM-7354 (IGM Biosciences), IMC-001 (Sorrento Therapeutics), Imfinzi / Marine (AstraZenica), IMM25 (ImmuneOnco Biopharma), IMM2502 (ImmuneOnco). Biopharma, IMM2503 (ImmuneOnco Biopharma), IMM2504 (ImmuneOnco Biopharma), INCB86550 (Incyte), IO103 (IO Biotech), JS003 (Shanghai Junshi Biosciences), Jubilant-PD-L1-derived (Jubilant Therapeutics, KD033 (Kadmon Holdings), KN046 (Alphamab Oncology), KY1003 (Sanofi), KY1043 (Sanofi), LY3300054 (Eli Lilly), LY3415244 (Eli Lilly)、MRNA-6981(Modern)、MSB2311(Transcenta Holding)、MT-6035(Molecular Templates)、ND021 / NM21-1480(Numab Therapeutics)、OX001R(Oxford). BioTherapeutics) PD-L1ベーシ's BsAbs(I-Mab), PD-L1 Boltbody ISAC(Bolt Biotherapeutics), PDL-GEX(GlycotopeGmbH), PMC-122 (PharmAbcine), PMI06 (D&D Pharmatech), Protheragen-RV-scFv-PDL1-Unknown (Protheragen), PRS-344 (Pieris Pharmaceuticals), Q-1802 (Merck), RC98 (Yantai Rongchang Pharmaceutical), RV-scFv-PDL1 (Protheragen), SenI_TAAx22P (Hebei Senlang Biotechnology), SHC020 (Nanjing Sanhome Pharmaceutical), Sugemalimab (Ligand Pharmaceuticals), Atezolizumab (Roche), TST005 (Transcenta Holding), TT-01 (Topmunnity Therapeutics), TTX-siPDL1 (TransCode Therapeutics), UniCAR-T-PD-L1(GEMoaB This includes, but is not limited to, monoclonals, Vaximm (VXM10), and YBL-013 (Y-Biologics).

[0152] Anti-PD-L1 antibodies that can be combined with inducible IFN alpha-prodrugs are typically approved anti-PD-L1 antibodies. Approved anti-PD-1 antibodies include, but are not limited to, avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ).

[0153] Anti-CTLA4 antibodies that can be combined with inducible IFN alpha-prodrugs are typically approved anti-CTLA4 antibodies. Approved anti-CTLA4 antibodies include, but are not limited to, ipilimumab (YERVOY) and tremelimumab (IMJUDO).

[0154] Anti-LAG3 antibodies that can be combined with inducible INF alpha-prodrugs are typically approved anti-LAG3 antibodies. Approved anti-LAG3 antibodies include relatrimab (OPDUALAG).

[0155] G. Definition Throughout this specification and the claims, various terms relating to the manner of description are used. Unless otherwise indicated, such terms should be given their ordinary meanings in the art. Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein. The techniques and procedures described or referenced herein are generally well understood and commonly employed by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methods described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Procedures, including the use of commercially available kits and reagents where necessary, are generally carried out according to the protocols and conditions defined by the manufacturers unless otherwise specified.

[0156] Where used herein, the singular forms "a," "an," and "the" include the plural form unless explicitly indicated otherwise in the context. The terms "includes" and "such as" are intended to express unrestricted inclusion unless otherwise specified.

[0157] Unless otherwise indicated, the terms “at least,” “less than,” and “about,” or similar terms preceding a set of elements or scope should be understood to refer to any element within that set or scope. Those skilled in the art can identify or confirm numerous equivalents to the specific embodiments of the invention described herein by means of routine experimentation alone. Such equivalents are intended to be encompassed within the following claims.

[0158] As used herein, the terms “activatable,” “activate,” “inducible,” and “inducible” refer to inducible IFN alpha-prodrugs having a reduced active form (e.g., reduced receptor binding and / or agonist activity) and an active form. Inducible IFN alpha-prodrugs are activated by protease cleavage of a linker that dissociates the blocking element and the half-life-extending element from the inducible IFN alpha-prodrug. The induced / activated IFN alpha-prodrug can bind to the IFN alpha receptor with increased affinity / avidity.

[0159] The terms “antibody” and “immunoglobulin” are used interchangeably herein. Where used herein, “antibody” or “immunoglobulin” is intended to refer to an immunoglobulin molecule consisting of two heavy (H) chains. Typically, mammalian antibodies (e.g., human, rodent, and monkey) contain four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds). Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CHI, CH2, and CH3. Each light chain consists of a light chain variable region (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), interposed by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs aligned in the following order from the amino-terminus to the carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Antibodies can include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific 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., camel antibodies and shark antibodies).

[0160] As used herein, the term “attenuated” refers to an IFN alpha receptor agonist in which receptor agonist activity is reduced compared to the naturally occurring agonist of the IFN alpha receptor. Attenuated IFN alpha agonists may have agonist activity that is 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, or at least about 1000 times lower than the naturally occurring agonist of the receptor. When an inducible IFN alpha prodrug containing IFN alpha described herein is described as “attenuated” or having “attenuated activity”, it means that the inducible IFN alpha prodrug is an attenuated IFN alpha receptor agonist.

[0161] The term "cancer" refers to a physiological condition in mammals characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and / or specific morphological features. Often, cancer can take the form of a tumor or mass, but it can exist alone within a subject or circulate in the bloodstream as independent cells such as leukemia cells 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, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatome, breast cancer (e.g., triple-negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck 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.

[0162] Where used herein, “conservative” amino acid substitution generally refers to the substitution of one amino acid residue from a recognized group with another amino acid residue, which can alter the structure of a peptide but substantially preserve the peptide’s 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 following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. For example, those skilled in the art will reasonably predict that a single substitution of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions between structurally related amino acids will not significantly affect the biological activity of the resulting molecule.

[0163] As used herein, the term “half-life extension element” in the context of the inducible IFN alpha-prodrugs disclosed herein means a chemical element, preferably a polypeptide, that increases the serum half-life and improves the pK by altering its size (e.g., above the renal filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and excretion.

[0164] As used herein, the term “operably linked” in the context of inducible IFN alpha prodrugs refers to the orientation of the components of the inducible IFN alpha prodrug that enables the components to function in the intended manner. For example, a polypeptide comprising an IFN alpha subunit and an IFN alpha blocking element is operably linked by the protease-cleaving linker in the inducible IFN alpha prodrug if, for example, the IFN alpha blocking element can inhibit the IFN alpha receptor activating activity of the IFN alpha polypeptide, but the inhibition of the IFN alpha receptor activating activity of the IFN alpha polypeptide by the IFN alpha blocking element is reduced or eliminated because, upon cleavage of the protease-cleaving linker, for example, the IFN alpha blocking element can diffuse away from IFN alpha.

[0165] Where used herein, the terms “peptide,” “polypeptide,” or “protein” are used broadly to mean two or more amino acids linked by peptide bonds. Furthermore, protein, peptide, and polypeptide are used interchangeably herein to refer to an amino acid sequence. The term polypeptide is not used herein to suggest a specific size or number of amino acids constituting a molecule, and it should be recognized that the peptides of the present invention may contain up to several or more amino acid residues.

[0166] The term “subject” as used herein refers to any animal, including but not limited to any mammal, including humans, non-human primates, and rodents. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0167] As used herein, the term “therapeutic dose” refers to the amount of a compound described herein (i.e., an inducible IFN alpha-prodrug) sufficient to achieve the desired pharmacological or physiological effect under the conditions of administration. For example, “therapeutic dose” may be sufficient to alleviate the signs or symptoms of a disease or condition (e.g., a tumor). Those skilled in the art will recognize that the therapeutic effect does not need to be complete or curative, as long as it is somewhat beneficial 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, an appropriate dose to administer to achieve the desired therapeutic benefit.

[0168] 5. Equals Other suitable modifications and adaptations of the methods of the present invention described herein are obvious and can be readily apparent to those skilled in the art, using suitable equivalents without departing from the scope of this disclosure or embodiments. Having described in detail certain compounds and methods up to this point, they will be more clearly understood by referring to the following examples. The following examples are presented for illustrative purposes only and are not intended to limit the scope.

[0169] 6. Disclosure of the prodrug sequence [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7] [Table 12-8] [Examples]

[0170] 7. Examples The following are examples of methods and compositions of the present invention. Considering the general description provided herein, it will be understood that various other embodiments may be implemented. Example 1. In vivo administration and tumor treatment for TIL analysis. All in vivo animal experiments were performed at Charles River Laboratories (Worcester, MA) following their standard operating procedures. Briefly, MC38 cells (1 × 10⁶) in 50% Matrigel were introduced into the flanks of 6-8 week old female C57Bl / 6 mice. 5 (Number of cells) were subcutaneously transplanted, and tumor volume was measured twice a week throughout the experiment. The average tumor volume was within a predetermined limit (approximately 100 mm). 3When present, mice were randomized to the treatment group (day 0). Mice were intraperitoneally administered either vehicle or compound 1 (100 μg / dose) twice weekly for 2 weeks. Peripheral blood and tumors were collected from n=5 mice per group on days 5, 7, 11, 14, 19, 21, and 25. Tumors treated with vehicle were not collected on days 21 and 25 because all tumors in that group had reached the maximum size limit according to the IACUC protocol on earlier days, and the animals were excluded from the study. On the day of collection, tumors were collected, weighed, and placed in ice-cold RPMI-1640 + 10% heat-inactivated fetal bovine serum. Similarly, whole blood was collected in K2-EDTA tubes. Both tumor and blood samples were transported to Werewolf Therapeutics by same-day delivery for processing. Upon arrival, the tumor was immersed in 3 mL of Hanks' Equilibrium Salts (HBSS) containing 1.25 mg / mL collagenase type IV, 0.025 mg / mL hyaluronidase, and 0.01 mg / mL DNASE I, measuring 5 mm. 3 The tumor was shredded into pieces smaller than 10¹³. The tumor was then placed in a C-tube and dissociated using Miltenyi Octomax instrument program 37C_m_TDK_1. After the digestion program was complete, the single-cell suspension was washed with complete medium to quench the enzymatic reaction, passed through a MACS SmartStrainer (70 μM), and spun down at 1500 rpm for 5 minutes. The supernatant was decanted, the sample was washed two more times in complete RPMI-1640, counted, and then refrozen in complete medium to 50 × 10¹⁶ cells. 6 The samples were resuspended to the final cell / mL concentration. Tumor samples were then analyzed by flow cytometry or NanoString analysis. Peripheral blood samples were spun at 2500 rpm for 15 minutes, then plasma was collected and frozen for subsequent pharmacokinetic analysis. This experimental protocol corresponds to the data shown in Figures 1–19C.

[0171] Example 2. Pharmacokinetic Analysis Pharmacokinetic analysis was performed using the Verikine-HS mouse interferon alfa all subtype ELISA kit (PBL Assay Science) according to the manufacturer's protocol. Mouse plasma was diluted to either 1:50,000 or 1:150,000 in the sample diluent, and then analyzed for the presence of compound 1. The results are shown in Figure 2.

[0172] Example 3. Cell staining and flow cytometry To detect intracellular cytokines, some samples were re-stimulated at 37°C for 4 hours in complete RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 50 ng / mL PMA, 1 μg / mL ionomycin, and 1 × brefelzin A, prior to staining. Unstimulated samples were also incubated at 37°C for 4 hours in complete medium (RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum) without any additional additives. For both stimulated and unstimulated samples, 5 × 10⁶ cells per well were incubated in a 96-well round-bottom plate. 6 Individual cells were plated and stained. All cell staining was performed in a 96-well round-bottom plate, and all centrifugation steps were performed at 1600 rpm for 3 minutes unless otherwise specified. Initially, 5 × 10⁶ cells per well were used. 6The cells were plated in 200 μL of FACS buffer (AutoMACS rinse buffer (Miltenyi) + 0.5% bovine serum albumin (Miltenyi)). The cells were first spun down at 1500 rpm for 5 minutes. Then, the cells were resuspended in 100 μL of FACS buffer containing FC Block reagent at the concentrations detailed in the table below at 4°C for 15 minutes. Next, the cells were spun down, the supernatant was decanted, and the cells were washed once with 200 μL of FACS buffer. The cells were resuspended in 100 μL of FACS buffer containing only tetramer at the concentrations detailed in the table below, in the dark at room temperature for 25 minutes. After tetramer staining, the cells were spun down, the supernatant was decanted, and the cells were washed twice with 200 μL of FACS buffer. The cells were resuspended in 100 μL of FACs buffer containing extracellular staining antibody at the concentrations detailed in the table below. Extracellular staining was performed at 4°C for 20 minutes. After spinning down the cells, they were washed with 200 μL of FACS buffer. This step was repeated for a total of two washes. After extracellular staining, the cells were resuspended in 100 μL of diluted Fix / Perm buffer (eBioscience) and fixed at 4°C for 30 minutes. Both Fix / Perm buffer and Perm / wash buffer were diluted according to the manufacturer's instructions. After fixation / permeabilization, the cells were spun down, washed with 200 μL of Perm buffer, spun down again, and resuspended in 100 μL of Perm buffer containing intracellular staining antibodies at the concentrations detailed in the table below. Intracellular staining was performed at 4°C for 20 minutes. After spinning down the cells, they were washed with 200 μL of Perm buffer. This step was repeated for a total of two washes. Finally, the cells were spun down and resuspended in 130 μL of FACs buffer for analysis. For single-stain controls, both the eComp control beads and cells were stained with appropriate monochromatic stains using the same protocol as detailed above, except that the eComp control beads (Thermofisher Scientific) were not treated with FC blocks, but instead incubated in a single FACs buffer during that step. All flow cytometry samples were run on a Cytek Aurora system running SpectroFlo (version 2.2.0 (10212019)).After data collection, FlowJo software (v10.5.3) was used for data analysis. A gate was defined for each stain using a fluorescence minus one (FMO) control. The results are shown in Figures 3A-3G, 4A-4B, 5A-5D, 6, 7A-7C, 8A-8E, 9A-9F, and 10A-10B. Table 7. Materials: Flow cytometry panel for TIL analysis [Table 7-1] [Table 7-2]

[0173] Example 4. RNA isolation and nanostring analysis From each tumor sample, 5 × 10 5The cells were spun down (1600 rpm for 5 minutes) and resuspended in 100 μL of RLT lysis buffer. These samples were then rapidly frozen using dry ice and an ethanol bath and stored at -80°C until transportable to Canopy Biosciences for RNA extraction and NanoString analysis. All RNA extraction and NanoString analysis were performed by Canopy Biosciences using their standard protocols. Briefly, samples were thawed, and RNA was isolated using the RNEasy microkit (Qiagen) according to the manufacturer's protocol. RNA was quantified, and 100 ng was loaded into a NanoString cartridge. Nanostring analysis was performed using the Murine Pancancer Immune Profiling Panel and N Counter system, and the raw data was sent to Werewolf Therapeutics for in-house analysis. Nanostring analysis was performed using Nsolver software (v4.0.70) with the advanced analysis module installed. All graphs were created using Graphpad Prism (v8.4.3). Using Partek software (v10.0.22.0428), pathway analysis was performed based on transcripts showing significantly different expression after treatment with compound 1, under the conditions of a p-value of less than 0.05 and an FDR step-up of less than 0.05. The results are shown in Figures 11A-11D, 12, 13, 14A-14B, 15A-15F, 16A-16C, 17A-17C, 18A-18C, and 19A-19C.

[0174] Example 5. Antitumor activity of Compound 1 in combination with immune checkpoint blockade in a CT26 tumor model All in vivo animal experiments were performed at Charles River Laboratories (Morrisville, NC) following their standard operating procedures. Briefly, CT26 cells (3 × 10⁶) in 0% Matrigel were introduced into the flanks of 6-8 week old female Balb / C mice. 5Subcutaneous transplantation of () was performed, and tumor volume was measured twice a week throughout the experiment. When the average tumor volume was within a preset limit (approximately 100 - 150 mm 3 ), the mice were randomized into treatment groups (day 0). The mice were intraperitoneally administered either vehicle or compound 1 at the doses specified in the figure legend twice a week for 2 weeks. Similarly, the mice were administered individual checkpoint inhibitors at the doses specified in the figure on the same schedule (twice a week for 2 weeks). The results are shown in Figures 20A - 20E. Table 8. Materials: Checkpoint Inhibitors

Table 8

[0175] Example 6. Antitumor Activity of Compound 1 in Combination with Immune Checkpoint Blockade in the MC38 Tumor Model All in vivo animal experiments were conducted at Charles River Laboratories (Morrisville, NC) according to its standard operating procedures. Briefly, MC38 cells (5 × 10 5 ) in 50% Matrigel were subcutaneously transplanted into the flanks of 6 - 8 - week - old female C57Bl / 6 mice, and tumor volume was measured twice a week throughout the study. When the average tumor volume was within a preset limit (approximately 100 - 150 mm 3 ), the mice were randomized into treatment groups (day 0). The mice were intraperitoneally administered either vehicle or compound 1 at the doses specified in the figure legend twice a week for 2 weeks. Similarly, the mice were administered individual checkpoint inhibitors at the doses specified in the figure on the same schedule (twice a week for 2 weeks). The results are shown in Figures 21A - 21E.

[0176] Example 7. In Vivo Administration in EMT - 6, B16 - F10, A20, and EG.7 Tumor Models All in vivo animal experiments were performed at either Charles River Laboratories (Worcester, MA) (B16-F10 and EMT-6) or Covance Laboratories (A20 and EG.7), following their standard operating procedures. Briefly, for the A20 model, 5 × 10⁶ units in 0% Matrigel were used. 5 A20 cells were subcutaneously transplanted into 6-8 week old Balb / C mice. For the EG.7 model, 1 × 10⁶ cells in 0% Matrigel were used. 6 EG.7 cells were subcutaneously transplanted into 6-8 week old C57Bl / 6 mice. For the B16-F10 model, 1 × 10⁶ cells in 50% Matrigel were used. 5 B16-F10 cells were subcutaneously transplanted into 6-8 week old C57Bl / 6 mice. Finally, for the EMT6 model, 1 × 10¹⁶ cells in 50% Matrigel were used. 5 EMT6 cells were subcutaneously transplanted into 6-8 week old Balb / C mice. Tumor volume and body weight were measured every 2-3 days throughout the experiment for all models. The average tumor volume was set to a predetermined limit (approximately 100 mm for all tested models). 3 When present, mice were randomized to the treatment group (day 0). Mice were administered intraperitoneally twice a week for two weeks either the vehicle or compound 1 at the specific doses described in the legend in the figure.

[0177] Example 8. Treatment of compound 5 with primary dissociated human tissue samples. Cells derived from healthy human tissue were purchased from various suppliers (see table below), and dissociated tumor samples were purchased from Discovery Life Sciences. Primary cells from healthy human tissue were grown and expanded according to the manufacturer's protocol and then frozen in single-use vials. Dissociated tumor samples were generated from surgically excised primary human tumors that were enzymatically digested in situ before freezing. Therefore, these samples contain a mixture of all cell types found in primary human tumors, including immune cells, tumor cells, and other stromal cells. All purchased samples were transported to Werewolf Therapeutics on dry ice and stored at -140°C. In addition, some tumor samples were obtained fresh after surgical excision and transported overnight to Werewolf Therapeutics in RPMI-1640 medium on ice. Fresh tumor samples were processed in single-cell suspension at Werewolf Therapeutics. Upon arrival, samples were weighed and marked with a 5 mm scalpel. 3 After chopping the tissue into smaller pieces, the samples were enzymatically digested using the following enzyme cocktail in Leibovitz medium L15: collagenase I (45 U / mL), collagenase II (15 U / mL), collagenase IV (45 U / mL), DNase I (50,000 U / mL), and elastase (0.075 U / mL). The samples were digested with 25 mL of digestion medium per 500 mg of tissue, and digestion was carried out at 37°C for 45 minutes with shaking at 100 rpm. After enzymatic digestion, the samples were mechanically dissociated through a 70 μM filter, thoroughly washed, counted, and frozen in recovered cell culture freezing medium for later use. To investigate INDUKINE molecular treatment, the samples were thawed, washed, and counted. The cells were then resuspended in X-Vivo 15 medium and counted from 0.75 to 1 × 10⁶. 5 Individual viable cells were plated in each well of a 96-well round-bottom plate. INDUKINE molecules were added to each well at a final concentration of 20 nM for 48 hours, after which the cell culture supernatant was collected and frozen for later analysis. Each condition was run in series whenever possible. Protease-activating (cleaving) compound 5 was included as a positive control in all experiments. Table 9. Materials: Human cell type [Table 9]

[0178] Example 9. Measurement of compound 5 activity using human PBMCs Human buffy coat was isolated using Research Blood Components or BioIVT and transported to Werewolf Therapeutics by same-day delivery at room temperature for PBMC isolation. Upon arrival, the buffy coat was diluted 1:4 with PBS, and 25 mL of cell suspension was gently overlaid onto 25 mL of Ficoll-Paque Plus. Cells were spun at 2000 rpm for 40 minutes at room temperature with low acceleration (setting number 2) and brake off. After centrifugation, PBMCs were collected from the bilayer interface, washed three times with 50 mL of PBS, counted, and frozen in recovered cell culture freezing medium for subsequent use. To measure INDUKINE activity, PBMCs were thawed, counted, and then 1 × 10⁶ cells were placed in X-Vivo 15 medium. 6 The cells were resuspended at a concentration of cells / mL. 1 × 10⁶ cells per well were placed in a 96-well round-bottom plate. 5 100 μL of PBMCs were plated. Next, cell culture supernatant (collected from primary tumor samples incubated with INDUKINE molecules) was thawed and 100 μL was used to stimulate the PBMCs. This resulted in a 1:2 dilution of the conditioned medium with fresh medium. After 48 hours at 37°C and 5% CO2, the cell cultures were mixed three times with a multichannel pipette and spun down at 1600 rpm for 3 minutes. Supernatant was collected from the stimulated PBMCs, and IP-10 / CXCL10 production was measured using the Human IP-10 / CXCL10 AlphaLisa Kit (Perkin Elmer) according to the manufacturer's protocol, with one notable exception. Recommended concentrations of beads and antibody were used at 0.75-fold. The AlphaLisa signal was measured using a Perkin Elmer Enspire Alpha Reader with Enspire Manager software (V4.13.3005.1482). Sample measurements were fitted to a standard curve using the method described in the manufacturer's protocol. Specifically, 1 / Y2 The data was fitted using a data-weighted nonlinear four-parameter logistic regression (sigmoid dose-response curve with variable gradient). Data analysis and graphs were created using GraphPad Prism8 software (v8.4.2(679)) for Windows® (64-bit).

[0179] The dynamic range of the assay could be established by comparing the baseline activity of compound 5 incubated without primary human cells (fully intact input control, 0% of total activity) with the activity of cleaved compound 5 incubated with dissociated tumor samples (fully cleaved positive control, 100% of total activity). Normalization to the dynamic range allowed for the aggregation of results from multiple experiments into a broad dataset. Mathematically, the percentage of activity generated by exposing compound 5 to primary human samples was calculated using the following formula:

[0180]

number

[0181] Example 10. Efficacy of Compound 1 in an mEER tumor model HPV infection accounts for over 71% of oral squamous cell carcinoma (OSCC) cases in the United States. While most infections are cleared by the immune response, persistent HPV infection is a risk factor for OSCC. Persistent viral infection is partly due to the evasion of the host immune response by viral oncoplastic proteins. To test the antitumor activity of compound 1, a syngeneic mouse tumor model of HPV-OSCC (mEER) was used, featuring HPV16 E6 and E7 oncogenes and mouse pharyngeal epithelial cells transformed with H-ras. Six-to-eight-week-old female C57 / Bl6 mice (Charles River) were given 3 × 10⁶ units in 50% Matrigel on day 0. 5MEER cells were injected. Mice were intraperitoneally administered either the vehicle or 100 μg or 400 μg of compound 1 twice a week for two weeks, starting on day 0. A fourth group of mice was intraperitoneally administered 35 μg of free IFNα twice daily for five days a week for two weeks, starting on day 0. For each of the four groups, five mice were sacrificed on day 6, their spleens were collected, and the tumor-infiltrating lymphocyte (TIL) population was quantified. The study was continued with eight mice per group to determine efficacy. TILs were quantified as described in Example 3. Cytokine levels were measured using the V-Plex mouse pro-inflammatory panel 1 (Meso Scale Discovery; Rockville, MD).

[0182] The results showed that both doses of compound 1 tested had a more persistent antitumor response in the mEER model of HPV-driven OSCC compared to free IFNα (Figure 24). Furthermore, compound 1 increased the frequency of activated CD25+, IFNg+, TNFα+granzyme B+, or T-box TS factor (Tbet)+CD8+ T cells (Figure 25AE), and pluripotent cells (cells expressing more than one of IFNg, TNFα, or granzyme B; data not shown). Increased NK cell activation and upregulation of MHC class I expression were also observed in mice treated with compound 1 (Figures 26A-B and 27A-B). In addition, treatment with compound 1 resulted in dose-dependent increases in the production of IFNg, TNFα, CXCL10, and IL-10 compared to vehicle and free IFNα (Figures 28A-F).

Claims

1. A method for selectively activating effector CD8+ T cells in the tumor microenvironment, comprising administering an effective amount of an inducible interferon alpha (IFN alpha) prodrug to a subject requiring such activation, wherein the inducible IFN alpha prodrug is systemically administered and activated by cleavage by a protease having higher activity in the tumor microenvironment than in other sites, thereby significantly increasing the frequency of IFN gamma and granzyme B-producing CD8+ T cells in the tumor compared to peripheral tissue.

2. A method for selectively activating tumor-infiltrating lymphocytes, comprising administering an effective amount of an inducible interferon-alpha (IFN-alpha) prodrug to a subject requiring such activation, wherein the inducible IFN-alpha prodrug is systemically administered and activated by cleavage by a protease having higher activity in the tumor microenvironment than in other sites, thereby significantly increasing the frequency of IFN-gamma and granzyme B-producing CD8+ T cells in the tumor compared to peripheral tissues.

3. The method according to any one of claims 1 or 2, wherein the tumor-reactive CD8+ / Treg ratio in the tumor microenvironment is significantly increased by the method described above.

4. The method according to any one of the preceding claims, wherein the frequency of bone marrow-derived suppressor cells and / or Treg cells in the tumor microenvironment is reduced by the method described above.

5. The method according to any one of the preceding claims, wherein the expression of an immune checkpoint protein is increased by the method described above.

6. The method according to claim 5, wherein the immune checkpoint protein is PD-L1.

7. The method according to any one of the preceding claims, wherein the expression of MHC class I and MCH class II is increased by the method described above.

8. The method according to any one of the preceding claims, wherein the activation of natural killer cells is prolonged by the method described above.

9. The method according to any one of the preceding claims, wherein the inducible IFN alpha-prodrug is compound 1 (SEQ ID NO: 1), compound 2 (SEQ ID NO: 2), compound 3 (SEQ ID NO: 3), compound 4 (SEQ ID NO: 4), compound 5 (SEQ ID NO: 5), or an amino acid sequence variant of any of the above.

10. The method according to any one of the preceding claims, wherein the inducible IFN alpha-prodrug is administered at a frequency of about twice a week or less.

11. The method according to any one of the preceding claims, wherein the inducible IFN alpha-prodrug is administered at a frequency of about once a week or less.

12. The method according to any one of the preceding claims, wherein the inducible IFN alpha-prodrug is administered approximately once every two weeks.

13. A method for modulating the tumor microenvironment, comprising administering an effective amount of an inducible interferon alpha (IFN alpha) prodrug to a subject in need thereof, wherein the inducible IFN alpha prodrug is systemically administered and activated by cleavage by a protease having higher activity in the tumor microenvironment than at other sites, wherein the method yields at least one effect selected from the group consisting of selective activation of effector CD8+ T cells in the tumor microenvironment, selective activation of tumor-infiltrating lymphocytes, an increase in the tumor-reactive CD8+ / Treg ratio in the tumor microenvironment, a decrease in the frequency of bone marrow-derived suppressor cells and / or Treg cells in the tumor microenvironment, an increase in the expression of immune checkpoint proteins in the tumor microenvironment, an increase in the expression of MHC class I and MCH class II, and / or an extension of natural killer cell activation.

14. The method according to claim 13, wherein the expression of at least 1, 2, 3, 4, 5, 6, or more immune checkpoint proteins in the tumor microenvironment is increased by the method described above.

15. The method according to claim 13 or 14, wherein the immune checkpoint protein is PD-L1, PD-1, TIGIT, PVR, CTLA-4, or LAG-3.

16. The method according to claim 13 or 14, wherein the immune checkpoint protein is PD-L1 or PD-1.

17. The method according to any one of claims 13 to 16, wherein the inducible IFN alpha-prodrug is administered at a frequency of about twice a week or less.

18. The method according to any one of claims 13 to 16, wherein the inducible IFN alpha-prodrug is administered at a frequency of about once a week or less.

19. The method according to any one of claims 13 to 16, wherein the inducible IFN alpha-prodrug is administered approximately once every two weeks.

20. The method according to any one of claims 13 to 19, wherein the method provides at least one effect over a period of at least about 7 days (such as at least about 7, 8, 9, 10, 11, 12, 13, 14, or 15 days) after the last dose of the inducible IFN alpha-prodrug.

21. The inducible interferon alpha (IFN alpha) prodrug comprises a fusion polypeptide having the formula: [D]-[L1]-[A]-[L2']-[H], where, [A] is interferon alpha (IFNa) polypeptide, its mutain, or an active fragment. [D] is the blocking part, [H] represents the half-life extension portion, [L1] is a protease-cleaving polypeptide linker containing the amino acid sequence of SEQ ID NO: 6, 9, or 12. [L2'] is a protease-cleaving polypeptide linker containing the amino acid sequence of SEQ ID NO: 6, 9, or 12. The method according to any one of claims 1 to 8 and 13 to 20, wherein the blocking portion and the half-life extension portion each independently comprise human serum albumin (HSA) or an antibody or antibody fragment that binds to HSA.

22. A method for treating cancer, comprising administering to a subject in need of such treatment a combination therapy comprising compound 1 (SEQ ID NO: 1), compound 2 (SEQ ID NO: 2), compound 3 (SEQ ID NO: 3), compound 4 (SEQ ID NO: 4), compound 5 (SEQ ID NO: 5), or any amino acid sequence variant of the above, and a checkpoint inhibitor.

23. The method according to claim 22, wherein the checkpoint inhibitor is an anti-PD-1 antibody or a fragment thereof.

24. The method according to claim 22, wherein the checkpoint inhibitor is an anti-PD-L1 antibody or a fragment thereof.

25. The method according to claim 22, wherein the checkpoint inhibitor is an anti-CTLA4 antibody or a fragment thereof.

26. The method according to claim 22, wherein the checkpoint inhibitor is an anti-LAG3 antibody or a fragment thereof.

27. The method according to claim 22, wherein an effective amount of the combination therapy is administered to the subject.

28. The anti-PD-1 antibody is AMP-224 (AstraZeneca), 609A (3SBio), 704 (3SBio), 705 (3SBio), ABBV-181 (AbbVie), ADU-1503 / bion-004 (Chinook Therapeutics), AGEN2034 / balstilimab (Agenus), AK103 (Akeso), AK104 (Akeso), AK112 (Akeso), AK123 (Akeso), AMG256 (Amgen), AMG404 (Amgen), ANB030 (AnaptysBio), ANKEBIO anti-PD1 formulation (Anhui Anke Biotechnology), anti-PD-1 / anti-CD47 (DiNonA), ASKG915 (Ask Gene Pharmaceuticals), AV-MEL-1 (Aivita Biomedical), BCD-100 (Biocad CJSC), BI754091 (Boehringer Ingelheim), BiCKI-IL-7 (OSE Immunotherapeutics), Boehringer-PD-1-unknown (Boehringer Ingelheim), BSK-050K01 (Biosion), camrelizumab (Jiangsu Hengrui Medicine), CB201 (Crescendo Biologics), CB213 (Crescendo Biologics), CC-90006 (AnaptsBio), cetrelimab (J&J), chPD1 (Kirimic Biopharma), CMBAB819 (Mabpharm), CS1003 (CStone Pharmaceuticals), CS17938 (Shenzhen Chipscreen Biosciences), CTX-8371 (Compass Therapeutics), CX-072 (CytomX Therapeutics), CX-188 (CytomX Therapeutics), siparilizumab (Harbin Gloria Pharmaceuticals), DB004 (DotBio), EMB02 (EpimAb Biotherapeutics), gepantumab / genolimuzumab (Apollomics), GS19 (Suzhou ZelgenBiopharmaceuticals), HLX10 (Shanghai Henlius Biotech), HX008 (Taizhou Hanzhong Pharmaceuticals), HY003 (Juventas Cell Therapy), IBI315 / BH2950 (Innovent Biologics), IBI318 (Innovent Biologics), IBI319 (Innovent Biologics), IMM1802 (ImmuneOnco BioPharm), IMT200 (TrueBinding), Jemperli / dostarlimab (AnaptysBio), JTX-4014 (Jounce Therapeutics), Keytruda / pembrolizumab (Merck), LBL-006 (Nanjing Leads Biolabs), Libtayo / semiplimab-rwlc (Regeneron Pharmaceuticals), LVG N3616 (Lyven BioPharm), LXF821 (Novartis), LY01015 (Luye Pharma Group), LY3462817 (Eli Lilly), MCLA-134 (Merus N.V.), MEDI5752 (AstraZeneca), NIR178 (Novartis), ONCR-177 (Oncorus), ONO-4685 (Ono Pharmaceutical), Opdivo / nivolumab (Ono Pharmaceutical), MGD019 (MacroGenius), PD1-GDT CAR-T (KiroMic BioPharm), pembrolizumab (Akeso), PSB205 (Qilu Paget Sound Biotherapeutics), PT-001 (Merck), PT627 (Merck), RB-M1 (Refuge Biotechnologies), retifanlimab (MacroGenius), RG6139 (Roche), RG6279 (Roche), RTX-002 (RubrYc Therapeutics), sasanalimab (Pfizer), Servier-PD1xLAG3-unknown (Servier), SL-279252 / TAK-252 (Shattuck Labs), Sorfusa anti-PD1 (SorrentoTherapeutics), Spartalizumab (Novartis), SSI-361 (Lyvgen Biopharma), Sym021 (Servier), Teboterimab (MacroGenics), Tithrelizumab (BeiGene), TSR-075 (AnaptsBio), Tuhura-DO / PD-1-Unknown (Tuhura Biopharma), Tripalimab (Shanghai Junshi Biosciences), Syntilimab (Innovent Biologics), Unicar-CAR-T&PD-1-Unknown (Shanghai Unicar-Therapy Bio-Medicine) The method according to claim 23, wherein a drug selected from the group consisting of Technology), Xdivane (Xbrane Biopharma), XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y-Biologicals), and zimbererimab (Arcus Biosciences).

29. The anti-PD-L1 antibody is A167 (Sichuan Kelun), ABL501 (ABL Bio), ABL503 (ABL Bio), ABSK041 (Abbisko Therapeutics), ACE1708 (Acepodia), ACE-NK-PDL1 (Acepodia), ADG104 (Adagene), AK106 (Akeso), ALPN-202 (Alpine Immune Sciences), AN4005 (Adlai Nortye Biopharma), BMS-936559 / MDX-1105 (BMS), APL-502 / TQB2450 (Apollomics), Arbutus-PD-L1-unknown (Arbutus Biopharma), ASC22 (Ascletis Pharma), ATG-101 (Antengene), AVA-004 (Avacta Group), AVA021 (Avacta Group), AVA027 (Avacta Group), AVA-040-100 (Avacta Group), AVA04-Vbp (Avacta Group), Bavencio / avelumab (Merck), BCD-135 (Biocad CJSC), BGB-A333 (BeiGene), Bintrafusp alfa / GSK4045154 (Merck), CA-170 / aupm-170 (Dr. Reddy's Laboratories), CCX559 (Chemocentryx), CDR101 (CDR-Life), cosibelimab (Checkpoint Therapeutics), CTX-8371 (Compass Therapeutics), DiNonA-solid tumor-unknown (DiNonA), DR30207 (Zhejiang Doer Biologics), DuoBody-PD-L1x4-1BB (Ligand Pharmaceuticals), enbafolimab (Alphamab Oncology), EPIM-001 (Elpis Biopharmaceuticals), ES101 (Elpiscience Biopharma), INBRX-105 (Inhibrx), FAZ053 (Novartis), FS118 (F-star Therapeutics), GB262 (GenorBioPharma), GS-4224 (Gilead), GT900008 (Kintor Pharmaceuticals), GX-P2 (Genexine), Hamni-PS-L1 / CD47 - unknown (Hanmi Pharmaceuticals), HBM7015 (HBM Holdings), HBM9167 (HBM Holdings), HLX20 (Shanghai Henlius Biotech), HTI-1088 (Jiangsu Hengrui Medicine), IBI318 (Innovent Biologics), IBI322 (Innovent Biologics), IBI323 (Innovent Biologics), IGM-7354 (IGM Biosciences), IMC-001 (Sorrento Therapeutics), Imfinzi / durvalumab (AstraZeneca), IMM25 (ImmuneOnco Biopharma), IMM2502 (ImmuneOnco Biopharma), IMM2503 (ImmuneOnco Biopharma), IMM2504 (ImmuneOnco Biopharma), INCB86550 (Incyte), IO103 (IO Biotech), JS003 (Shanghai Junshi Biosciences), Jubilant-PD-L1 - unknown (Jubilant Therapeutics), KD033 (Kadmon Holdings), KN046 (AlphaMab Oncology), KY1003 (Sanofi), KY1043 (Sanofi), LY3300054 (Eli Lilly), LY3415244 (Eli Lilly), mRNA-6981 (Moderna), MSB2311 (Transcenta Holding), MT-6035 (Molecular Templates), ND021 / NM21-1480 (Numab Therapeutics), OX001R (Oxford BioTherapeutics), PD-L1-based BsAbs (I-Mab), PD-L1 Boltbody ISAC (Bolt Biotherapeutics), PDL-GEX (GlycotopeGmbH), PMC-122 (PharmAbcine), PMI06 (D&D Pharmatech), Protheragen-RV-scFv-PDL1-unknown (Protheragen), PRS-344 (Pieris Pharmaceuticals), Q-1802 (Merck), RC98 (Yantai Rongchang Pharmaceutical), RV-scFv-PDL1 (Protheragen), SenI_TAAx22P (Hebei Senlang Biotechnology), SHC020 (Nanjing Sanhome Pharmaceutical), Sugemalimab (Ligand The method according to claim 24, wherein the drug is selected from the group consisting of Pharmaceuticals), atezolizumab (Roche), TST005 (Transcenta Holding), TT-01 (Topmunity Therapeutics), TTX-siPDL1 (TransCode Therapeutics), UniCAR-T-PD-L1 (GEMoaB monoclonals), Vaximm (VXM10), and YBL-013 (Y-Biologicals).

30. A method for treating cancer, comprising administering to a subject in need thereof an inducible interferon alpha (IFN alpha) prodrug comprising a checkpoint inhibitor and a fusion polypeptide having the formula: [D]-[L1]-[A]-[L2']-[H], where, [A] is interferon alpha (IFN alpha) polypeptide, its mutain, or an active fragment. [D] is the blocking part, [H] represents the half-life extension portion, [L1] is a protease-cleaving polypeptide linker containing the amino acid sequence of SEQ ID NO: 6, 9, or 12. [L2'] is a protease-cleaving polypeptide linker containing the amino acid sequence of SEQ ID NO: 6, 9, or 12. Herein, the blocking portion and the half-life extension portion each independently comprise human serum albumin (HSA) or an antibody or antibody fragment that binds to HSA.

31. The method according to claim 30, wherein the checkpoint inhibitor is an anti-PD-1 antibody or a fragment thereof.

32. The method according to claim 30, wherein the checkpoint inhibitor is an anti-PD-1 antibody or a fragment thereof.

33. The method according to claim 30, wherein the checkpoint inhibitor is an anti-CTLA4 antibody or a fragment thereof.

34. The method according to claim 30, wherein the checkpoint inhibitor is an anti-LAG3 antibody or a fragment thereof.

35. The method according to claim 22 or 30, wherein the checkpoint inhibitor is a TIGIT antibody or a fragment thereof.

36. The method according to claim 22 or 30, wherein the checkpoint inhibitor is a PVR antibody or a fragment thereof.

37. The anti-PD-1 antibody is AMP-224 (AstraZeneca), 609A (3SBio), 704 (3SBio), 705 (3SBio), ABBV-181 (AbbVie), ADU-1503 / bion-004 (Chinook Therapeutics), AGEN2034 / balstilimab (Agenus), AK103 (Akeso), AK104 (Akeso), AK112 (Akeso), AK123 (Akeso), AMG256 (Amgen), AMG404 (Amgen), ANB030 (AnaptysBio), ANKEBIO anti-PD1 formulation (Anhui Anke Biotechnology), anti-PD-1 / anti-CD47 (DiNonA), ASKG915 (Ask Gene Pharmaceuticals), AV-MEL-1 (Aivita Biomedical), BCD-100 (Biocad CJSC), BI754091 (Boehringer Ingelheim), BiCKI-IL-7 (OSE Immunotherapeutics), Boehringer-PD-1-unknown (Boehringer Ingelheim), BSK-050K01 (Biosion), camrelizumab (Jiangsu Hengrui Medicine), CB201 (Crescendo Biologics), CB213 (Crescendo Biologics), CC-90006 (AnaptsBio), cetrelimab (J&J), chPD1 (Kirimic Biopharma), CMAB819 (Mabpharm), CS1003 (CStone Pharmaceuticals), CS17938 (Shenzhen Chipscreen Biosciences), CTX-8371 (Compass Therapeutics), CX-072 (CytomX Therapeutics), CX-188 (CytomX Therapeutics), sipatrizumab (Harbin Gloria Pharmaceuticals), DB004 (DotBio), EMB02 (EpimAb Biotherapeutics), gepotidumab / genolimzumab (Apolloics), GS19 (Suzhou ZelgenBiopharmaceuticals), HLX10 (Shanghai Henlius Biotech), HX008 (Taizhou Hanzhong Pharmaceuticals), HY003 (Juventas Cell Therapy), IBI315 / BH2950 (Innovent Biologics), IBI318 (Innovent Biologics), IBI319 (Innovent Biologics), IMM1802 (ImmuneOnco Biopharm), IMT200 (TrueBinding), Jemperli / dostarlimab (AnaptysBio), JTX-4014 (Jounce Therapeutics), Keytruda / pembrolizumab (Merck), LBL-006 (Nanjing Leads Biolabs), Libtayo / semiplimab-rwlc (Regeneron Pharmaceuticals), LVGN3616 (Lyvgen Biopharm), LXF821 (Novartis), LY01015 (Luye Pharma Group), LY3462817 (Eli Lilly), MCLA-134 (Merus N.V.), MEDI5752 (AstraZeneca), NIR178 (Novartis), ONCR-177 (Oncorus), ONO-4685 (Ono Pharmaceutical), Opdivo / nivolumab (Ono Pharmaceutical), MGD019 (MacroGenics), PD1-GDT CAR-T (Kirimic Biopharm), pembrolizumab (Akeso), PSB205 (Qilu Paget Sound Biotherapeutics), PT-001 (Merck), PT627 (Merck), RB-M1 (Refuge Biotechnologies), retifanlimab (MacroGenics), RG6139 (Roche), RG6279 (Roche), RTX-002 (RubrYc Therapeutics), sasanalimab (Pfizer), Servier-PD1xLAG3-unknown (Servier), SL-279252 / TAK-252 (Shattuck Labs), Sorfusa anti-PD1 (SorrentoTherapeutics), Spartalizumab (Novartis), SSI-361 (Lyvgen Biopharma), Sym021 (Servier), Teboterimab (MacroGenics), Tithrelizumab (BeiGene), TSR-075 (AnaptsBio), Tuhura-DO / PD-1-Unknown (Tuhura Biopharma), Tripalimab (Shanghai Junshi Biosciences), Syntilimab (Innovent Biologics), Unicar-CAR-T&PD-1-Unknown (Shanghai Unicar-Therapy Bio-Medicine) The method according to claim 31, wherein a drug selected from the group consisting of Technology), Xdivane (Xbrane Biopharma), XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y-Biologicals), and zimbererimab (Arcus Biosciences).

38. The anti-PD-L1 antibody is A167 (Sichuan Kelun), ABL501 (ABL Bio), ABL503 (ABL Bio), ABSK041 (Abbisko Therapeutics), ACE1708 (Acepodia), ACE-NK-PDL1 (Acepodia), ADG104 (Adagene), AK106 (Akeso), ALPN-202 (Alpine Immune Sciences), AN4005 (Adlai Nortye Biopharma), BMS-936559 / MDX-1105 (BMS), APL-502 / TQB2450 (Apollomics), Arbutus-PD-L1-unknown (Arbutus Biopharma), ASC22 (Ascletis Pharma), ATG-101 (Antengene), AVA-004 (Avacta Group), AVA021 (Avacta Group), AVA027 (Avacta Group), AVA-040-100 (Avacta Group), AVA04-Vbp (Avacta Group), Bavencio / avelumab (Merck), BCD-135 (Biocad CJSC), BGB-A333 (BeiGene), Bintrafusp alfa / GSK4045154 (Merck), CA-170 / aupm-170 (Dr. Reddy's Laboratories), CCX559 (Chemocentryx), CDR101 (CDR-Life), cosibelimab (Checkpoint Therapeutics), CTX-8371 (Compass Therapeutics), DiNonA-solid tumor-unknown (DiNonA), DR30207 (Zhejiang Doer Biologics), DuoBody-PD-L1x4-1BB (Ligand Pharmaceuticals), enbafolimab (Alphamab Oncology), EPIM-001 (Elpis Biopharmaceuticals), ES101 (Elpiscience Biopharma), INBRX-105 (Inhibrx), FAZ053 (Novartis), FS118 (F-star Therapeutics), GB262 (GenorBioPharma), GS-4224 (Gilead), GT900008 (Kintor Pharmaceuticals), GX-P2 (Genexine), Hamni-PS-L1 / CD47 - unknown (Hanmi Pharmaceuticals), HBM7015 (HBM Holdings), HBM9167 (HBM Holdings), HLX20 (Shanghai Henlius Biotech), HTI-1088 (Jiangsu Hengrui Medicine), IBI318 (Innovent Biologics), IBI322 (Innovent Biologics), IBI323 (Innovent Biologics), IGM-7354 (IGM Biosciences), IMC-001 (Sorrento Therapeutics), Imfinzi / durvalumab (AstraZeneca), IMM25 (ImmuneOnco Biopharma), IMM2502 (ImmuneOnco Biopharma), IMM2503 (ImmuneOnco Biopharma), IMM2504 (ImmuneOnco Biopharma), INCB86550 (Incyte), IO103 (IO Biotech), JS003 (Shanghai Junshi Biosciences), Jubilant-PD-L1 - unknown (Jubilant Therapeutics), KD033 (Kadmon Holdings), KN046 (AlphaMab Oncology), KY1003 (Sanofi), KY1043 (Sanofi), LY3300054 (Eli Lilly), LY3415244 (Eli Lilly), mRNA-6981 (Moderna), MSB2311 (Transcenta Holding), MT-6035 (Molecular Templates), ND021 / NM21-1480 (Numab Therapeutics), OX001R (Oxford BioTherapeutics), PD-L1-based BsAbs (I-Mab), PD-L1 Boltbody ISAC (Bolt Biotherapeutics), PDL-GEX (GlycotopeGmbH), PMC-122 (PharmAbcine), PMI06 (D&D Pharmatech), Protheragen-RV-scFv-PDL1-unknown (Protheragen), PRS-344 (Pieris Pharmaceuticals), Q-1802 (Merck), RC98 (Yantai Rongchang Pharmaceutical), RV-scFv-PDL1 (Protheragen), SenI_TAAx22P (Hebei Senlang Biotechnology), SHC020 (Nanjing Sanhome Pharmaceutical), Sugemalimab (Ligand The method according to claim 32, wherein the drug is selected from the group consisting of Pharmaceuticals), atezolizumab (Roche), TST005 (Transcenta Holding), TT-01 (Topmunity Therapeutics), TTX-siPDL1 (TransCode Therapeutics), UniCAR-T-PD-L1 (GEMoaB monoclonals), Vaximm (VXM10), and YBL-013 (Y-Biologicals).

39. The method according to any one of claims 21 and 30 to 38, wherein the IFN alpha polypeptide comprises mouse interferon alpha 1 (mIFNa1), mouse interferon alpha 11 (mIFNa11), human interferon alpha 2b (IFNA2b), mouse interferon alpha 11 (mIFNa11), interferon alpha 8 (IFNA8), interferon alpha 14 (IFNA14), interferon alpha 16 (IFNA16), or its mutain.

40. The method according to claim 39, wherein the IFN alpha polypeptide comprises the amino acid sequences of SEQ ID NOs. 234 to 237.

41. The method according to any one of claims 21 and 30 to 40, wherein each of the blocking portion and the half-life extension portion includes an HSA.

42. The method according to any one of claims 21 and 30 to 40, wherein each of the blocking portion and the half-life extension portion comprises an antibody or antibody fragment that binds to HSA.

43. The method according to any one of claims 21 and 30 to 40, wherein one of the blocking portion and the half-life extension portion comprises an HSA, and the other of the blocking portion and the half-life extension portion comprises an antibody or antibody fragment that binds to the HSA.

44. The method according to claim 42 or 43, wherein at least one of the blocking portion and the half-life extension portion comprises an antibody or antibody fragment that binds to HSA, and the antibody or antibody fragment has the amino acid sequence of residues 1 to 116 of SEQ ID NO:

5.

45. The method according to any one of claims 21 and 30 to 44, wherein each of [L1] and [L2'] includes sequence number 6, 9, or 12.

46. The method according to claim 45, wherein the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 and 238 to 257.

47. The method according to any one of claims 1 to 46, wherein the inducible IFN alpha-prodrug is activated in the tumor microenvironment of bladder cancer, glioblastoma multiforme, head and neck cancer, gastric cancer, colorectal cancer, cervical cancer, endometrial cancer, melanoma, kidney cancer, non-small cell lung cancer-adenocarcinoma (NSCLC-Ad), non-small cell lung cancer-squamous cell (NSCLC-Sq), ovarian cancer, or uterine cancer.

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