Interferon prodrugs

Abstract

This disclosure relates to methods and compositions for treating cancer using an inducible IFNalpha prodrug.

Description

INTERFERON PRODRUGSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 490.240, filed March 14, 2023, and U.S. Provisional Patent Application No. 63 / 493,802, filed April 3, 2023, the disclosures of which are incorporated herein by reference in entirety7.1. BACKGROUND

[0002] Cancer immunotherapy has rapidly established itself as the fourth pillar of cancer treatment largely owing to the clinical success of checkpoint inhibitors. Despite the durable responses achieved by some patients using these new therapies, the proportion of responders is still relatively low and restricted to only some cancer types. Tumor mutational burden, the presence or absence of T cell infiltration in tumors, and the overall immunosuppressive microenvironment of tumors greatly influences the response to immunotherapies. Although immune checkpoint blockade can prevent the physiological stop-signal that arises in response to immune activation, other approaches can be used to positively stimulate the anti-tumor immune response. One approach involves the use of immune-activating cytokines. Numerous preclinical and clinical studies have demonstrated the promise of cytokine therapy to increase anti-tumor immunity7. In fact, these were some of the first cancer immunotherapies approved for clinical use. However, systemic toxicity and poor pharmacokinetic profiles have limited their clinical application.

[0003] Interferons (“IFNs”) are a family of related signal proteins grouped in three major types, alpha, beta and gamma. Upon binding to specific receptors they lead to the activation of a signal transduction pathway that activates a broad range of genes, that are now known involved not only in antiviral but also in immunomodulatory and antiproliferative activities.

[0004] IFN’s are a potent immune antagonist and has been considered a promising therapeutic agent for oncology7. However, IFN’s have shown to have a narrow therapeutic window7because they are highly potent and have a short serum half-life. Consequently, therapeutic administration of IFN produce undesirable systemic effects and toxicities. This is exacerbated by the need to administer large quantities of cytokines (i.e., IFN) in order to achieve the desired levels of cytokine at the intended site of cytokine action (e.g., a tumor microenvironment). Unfortunately, due to the biology7of cytokine and the inability7to effectively target and control their activity, cytokines have not achieved the hoped for clinical advantages in the treatment in tumors.

[0005] Inducible IFNalpha prodrug constructs have been described in International Application Nos. PCT / US2019 / 032320, PCT / US2020 / 060624, and PCT / US2022 / 040564 to overcome the toxicity and short half-life problems that have limited clinical use of IFNalpha in oncology. The previously described inducible IFNalpha prodrug constructs comprise a polypeptide chain containing IFN and a human serum albumin or an antigen binding polypeptide that binds human serum albumin that also is capable of extending the half-life.2. SUMMARY

[0006] This disclosure relates to compositions and methods for treating cancer using an inducible IFNalpha prodrug. The method generally comprises administering to a subject in need thereof an effective amount of an inducible IFNalpha prodrug. The inducible IFNalpha prodrug can be Compound 1, Compound 2, Compound 3, Compound 4, or Compound 5. The inducible IFNalpha prodrug comprises an IFNalpha polypeptide, an IFNalpha blocking element (e.g., a steric blocking element), and a protease cleavable polypeptide linker. The IFNalpha prodrug can further comprise a half-life extension element, if desired.

[0007] The inducible IFNalpha prodrug is conditionally active. When the inducible IFNalpha prodrug is not in a site of interest (e.g., a tumor microenvironment), the prodrug typically remains intact. The intact prodrug has attenuated IFNalpha receptor agonist activity7. When the inducible IFNalpha prodrug is in a site of interest (such as a tumor microenvironment), the protease cleavable linker is cleaved by a protease active in the site of interest, releasing an unattenuated form of IFNalpha. This conditional activity preserves the immune stimulatory effects of while limiting the systemic toxicity associated with non-inducible IFNalpha therapy. The intact inducible IFNalpha prodrug can. if desired, contain an element that extends its half-life, but the post-cleavage unattenuated form of IFNalpha does not. As a result, the short half-life of IFNalpha effectively limits toxicity outside of the site of interest.

[0008] This disclosure relates to methods for selectively activating effector CD8+ T cells in the tumor microenvironment, and to a method for selectively activating tumor infdtrating lymphocytes. These methods comprising administering to a subject in need thereof and effective amount of an inducible IFNalpha prodrug. The inducible IFNalpha prodrug is ty pically administered systemically and is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations. The method results a significantly higher frequency of CD8+ T cells that produce granzyme B+ and / or IFN gamma within the tumor in comparison to peripheral tissue. These methods can result in a significant increase in the tumor reactive CD8+ / Treg ratio in the tumor microenvironment. Thesemethods can result in a decrease in the frequency of myeloid-derived suppressor cells and / or Treg cells. The methods can result in upregulation of MHC class I and MCH class II expression. The methods can result in prolonged natural killer cell activation.

[0009] Another general aspect of the application relates to a method of regulating tumor microenvironment, comprising administering to a subject in need thereof an effective amount of an inducible interferon alpha (IFNalpha) prodrug, wherein the inducible IFNalpha prodrug is administered systemically and is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, wherein the method results in at least one effect selected from the group consisting of selectively activating effector CD8+ T cells in the tumor microenvironment, selectively activating tumor infiltrating lymphocytes, increasing in the tumor reactive CD8+ / Treg ratio within the tumor microenvironment, decreasing in the frequency of myeloid-derived suppressor cells and / or Treg cells within the tumor microenvironment, increasing expression of an immune checkpoint protein in the tumor microenvironment, increasing expression of MHC class I and MCH class II expression, and / or prolonging natural killer cell activation. In certain embodiments, the method results in the at least one effect over at least about 7 days, such as at least about 7, 8, 9, 10, 11, 12, 13, 14 or 15 days, after the final administration of the inducible IFNalpha prodrug.

[0010] In embodiments of the methods of this disclosure the inducible IFNalpha prodrug can be administered about twice a week or less frequently, once a week or less frequently or about once every two weeks or less frequently. In certain embodiments, the inducible IFNalpha prodrug can be administered about once every two weeks.

[0011] The disclosure also generally relates to methods of increasing the expression of an immune checkpoint protein, the method comprising administering to a subject in need thereof an effective amount of an inducible interferon alpha (IFNalpha) prodrug, wherein the inducible IFNalpha prodrug is administered systemically and is activated by cleavage by a protease that has higher activity7in the tumor microenvironment than in other locations. In certain embodiments, the method results in increased expression of at least one, two, three, four, five, six or more immune checkpoint proteins. In certain embodiments, the immune checkpoint protein is PD-F1. 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 results in increased expression of the immune checkpoint protein in a tumor microenvironment.

[0012] In certain embodiments, the inducible interferon alpha (IFNalpha) prodrug comprises a fusion polypeptide having the formula of: [D]-[L1]-[A]-[L2?]-[H], wherein,[A] is an interferon alpha (IFNalpha) polypeptide, a mutein, or an active fragment thereof,[D] is a blocking moiety,[H] is a half-life extension moiety,[LI] is a protease-cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 6, 9, or 12, and[L2’] is a protease-cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 6, 9, or 12. wherein the blocking moiety and the half-life extension moiety each independently comprise human serum albumin (HSA) or an antibody or antibody fragment that binds the HSA.

[0013] In certain embodiments, inducible IFNalpha prodrugs for use in the methods of this disclosure consist essentially 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 an amino acid sequence variant of any of the foregoing. Preferred, inducible IFNalpha prodrugs for use in the methods of this disclosure are 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 foregoing.

[0014] The disclosure further relates to methods for treating cancer by administering to a subject in need thereof a combination therapy. The combination therapy can 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 an amino acid sequence variant of any of the foregoing and an anti-PD-1 antibody, any anti-PD-Ll antibody, or an anti-CTL-4 antibody, anti-TIGIT antibody, anti-PVR antibody, anti-LAG3 antibody, or an antigen binding fragment of any of the foregoing, or any other check point inhibitor. The methods comprise administering an effective amount of the combination therapy to the subject.

[0015] In embodiments, an anti-PD-1 antibody can be administered. The anti-PD-1 antibody can be selected from the group consisting of AMP-224 (AstraZenica), 609A (3SBio), 704 (3SBio), 705 (3SBio), ABBV-181 (AbbVie), ADU-1503 I bion-004 (Chinook Therapeutics), AGEN2034 I balstilimab (Agenus), AK103 (Akeso), AK104 (Akeso), AK112 (Akeso), AK123 (Akeso), AMG 256 (Amgen), AMG 404 (Amgen), ANB030 (AnaptysBio), ANKEBIO Anti-PDl product (Anhui Anke Biotechnology), Anti PD-1 / Anti-CD47(DiNonA), ASKG915 (Ask Gene Pharmaceuticals), AV -MEL-1 (Aivita Biomedical), BCD- 100 (Biocad CJSC), BI 754091 (Boehringer Ingelheim), BiCKI-IL-7 (OSE Immunotherapeutics), Boehringer-PD-1 -unknown (Boehringer Ingelheim), BSK-050K01 (Biosion), Camrelizumab (Jiangsu Hengrui Medicine), CB201 (Crescendo Biologies), CB213 (Crescendo Biologies), CC-90006 (AnaptsBio), cetrelimab (J&J), chPDl (Kiromic Biopharma), CMAB819 (Mabpharm), CS1003 (CStone Pharmaceuticals), CS17938 (Shenzhen Chipscreen Biosciences). CTX-8371 (Compass Therapeutics), CX-072 (CytomX Therapeutics), CX-188 (CytomX Therapeutics), cypalizumab (Harbin Gloria Pharmaceuticals), DB004 (DotBio), EMB02 (EpimAb Biotherapeutics), Geptanblimab / genolimzumab (Apollomics), GS19 (Suzhou Zelgen Biopharmaceuticals), HLX10 (Shanghai Henlius Biotech), HX008 (Taizhou HanZhong Pharmaceuticals), HY003 (Juventas Cell Therapy), IBB 15 / BH2950 (Innovent Biologies), IBB 18 (Innovent Biologies), IBB 19 (Innovent Biologies), IMM1802 (ImmuneOnco Biopharma), IMT200 (TrueBinding), Jemperli / dostarlimab (AnaptysBio), JTX-4014 (Jounce Therapeutics), Keytruda / pembrolizumab (Merck), LBL-006 (Nanjing Leads Biolabs), Libtayo / cemiplimab-rwlc (Regeneron Pharmaceuticals), LVGN3616 (Lyvgen Biopharma), LXF821 (Novartis), LY01015 (Luye Pharma Group), LY3462817 (Eh Lilly), MCLA-134 (Merus N.V.), MEDI5752 (AstraZenica), NIR178 (Novartis), ONCR-177 (Oncorus), ONO-4685 (Ono Pharmaceutical), Opdivo / nivolumab (Ono Pharmaceutical), MGD019 (MacroGenius), PD1- GDT CAR-T (Kiromic Biopharma), penpulimab (Akeso), PSB205 (Qilu Puget Sound Biotherapeutics), PT-001 (Merck), PT627 (Merck), RB-M1 (Refuge Biotechnologies), Retifanlimab (MacroGenics), RG6139 (Roche), RG6279 (Roche), RTX-002 (RubrYc Therapeutics), sasanlimab (Pfizer), Servier-PDlxLAG3-unknown (Servier), SL-279252 I TAK-252 (Shattuck Labs). Sofusa anti-PDl (Sorrento Therapeutics), spartalizumab (Novartis), SSI-361 (Lyvgen Biopharma), Sym021 (Servier), Tebotelimab (MacroGenics), tislelizumab (BeiGene), TSR-075 (AnaptsBio), Tuhura-DO / PD-1 -unknown (Tuhura Biopharma), toripalimab (Shanghai Junshi Biosciences), sintilimab (Innovent Biologies), Unicar-CAR-T&PD-l -unknown (Shanghai Unicar-Therapy Bio-Medicine Technology), Xdivane (Xbrane Biopharma). XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y - Biologies), zimberelimab (Arcus Biosciences).

[0016] In embodiments, an anti-PD-Ll antibody is administered and can be chosen from the group consisting of Al 67 (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-Ll -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 Tumors-unknown (DiNonA), DR30207 (Zhejiang Doer Biologies), DuoBody-PD-Llx4-lBB (Ligand Pharmaceuticals), envafblimab (Alphamab Oncology ), EPIM-001 (Elpis Biopharmaceuticals), ES101 (Elpiscience Biopharma), INBRX-105 (Inhibrx), FAZ053 (Novartis), FS118 (F-star Therapeutics), GB262 (Genor Biopharma), GS-4224 (Gilead), GT900008 (Kintor Pharmaceuticals), GX-P2 (Genexine), Hamni-PS-Ll / CD47-Unknoxvn (Hanmi Pharmaceutical), HBM7015 (HBM Holdings), HBM9167 (HBM Holdings), HLX20 (Shanghai Henlius Biotech), HTI-1088 (Jiangsu Hengrui Medicine). IBB 18 (Innovent Biologies), IBI322 (Innovent Biologies), IBI323 (Innovent Biologies), IGM-7354 (IGM Biosciences), IMC-001 (Sorrento Therapeutics), Imfinzi / durvalumab (AstraZenica), IMM25 (ImmuneOnco Biopharma), IMM2502 (ImmuneOnco Biopharma), IMM2503 (ImmuneOnco Biopharma), IMM2504 (ImmuneOnco Biopharma), INCB86550 (Incyte), 10103 (10 Biotech), JS003 (Shanghai Junshi Biosciences), Jubilant-PD-Ll -unknown (Jubilant Therapeutics). KD033 (Kadmon Holdings), KN046 (Alphamab Oncology), KYI 003 (Sanofi), KYI 043 (Sanofi), LY3300054 (Eli Lilly), LY3415244 (Eli Lilly), MRNA-6981 (Modema), MSB2311 (Transcenta Holding), MT-6035 (Molecular Templates), ND021 / NM21-1480 (Numab Therapeutics), 0X001R (Oxford BioTherapeutics). PD-L1 based BsAbs (I-Mab), PD-L1 Boltbody IS AC (Bolt Biotherapeutics), PDL-GEX (Glycotope GmbH), PMC- 122 (PharmAbcine), PMI06 (D&D Pharmatech), Protheragen-RV-scFv-PDLl -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-siPDLl (TransCode Therapeutics), UniCAR-T-PD-Ll (GEMoaB monoclonals), Vaximm (VXM10), and YBL- 013 (Y-Biologics).

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

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

[0019] In certain embodiments, wherein the IFNalpha polypeptide comprises a murine interferon alpha 1 (mIFNal), murine interferon alpha 11 (mIFNal 1), human interferon alpha 2b (IFNA2b), murine interferon alpha 1 1 (mIFNal 1), interferon alpha 8 (IFNA8), interferon alpha 14 (IFNA14), interferon alpha 16 (IFNA16), or a mutein thereof.

[0020] In certain embodiments, the IFNalpha polypeptide comprises the amino acid sequence of SEQ ID NO: 234-237.

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

[0022] In certain embodiments, each of the blocking moiety and the half-life extension moiety comprises the antibody or antibody fragment that binds HSA.

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

[0024] In certain embodiments, at least one of the blocking moiety and the half-life extension moiety comprises the antibody or antibody fragment that binds HSA. and the antibody or antibody fragment has the amino acid sequence of residues 1 to 1 16 of SEQ ID NO: 5.In certain embodiments, each of [LI] and [L2’] comprises SEQ ID NO: 6, 9 or 12.

[0025] In certain embodiments, the fusion polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 1-5 and 238-257.In certain embodiments, the inducible interferon alpha (IFNalpha) prodrug is activated in a tumor microenvironment of a bladder cancer, a 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 (NSCLC-Sq), ovarian cancer, or uterine cancer.3. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is a graph showing anti-tumor activity of Compound 1 in the murine MC38 model. Compound 1 was dosed intraperitonially twice a week for two weeks at 100 pg / dose and a vehicle (control).

[0027] FIG. 2 is a graph showing the presence of Compound 1 or vehicle over time in the peripheral from MC38 tumor-bearing mice treated with Compound 1 or vehicle.

[0028] FIGs. 3A-3B are graphs showing that CD8+ T cells (CD8+ T-cells (FIG. 3A) and Tetramer CD8+ T cells (FIG. 3B)) make up about 50% of infiltrating immune cells. Dosing with Compound 1 results in long term infiltration of CD8+ T cells. FIGs. 3C-3E are graphs showing that treatment with Compound 1 decreases the frequency of immunosuppressive cell populations (PMN-MDSCs (FIG. 3C). G-MDSCs (FIG. 3D)), Tregs (FIG. 3E) among TILs paired with a large increase in the frequency of CD8+T cells. FIGs. 3F-3G shows that Compound 1 increases CD8+ to Treg ratio (FIG. 3F) and tetramer CD8+to Treg ratio over time in the MC38 syngeneic tumor model. Unless otherwise stated, data are represented as the mean + SD, and P values are derived from t tests (**, p <0.01; ***, p < 0.001; ****, p <0.00001).

[0029] FIG. 4A-4B are graphs showing that treatment with Compound 1 decreases the frequency of Tregs among total CD4+ T cells in tumors. FIG. 4A is a graph showing the % of CD4+T conventional cells over time in the MC38 syngeneic tumor model treated with Compound I and vehicle. FIG. 4B is a graph showing the percentage of FoxP3+ T cells in the population of CD4+T cells. Unless otherwise stated, data are represented as the mean + SD, and P values are derived from t tests (**, p <0.01; ***, p < 0.001; ****, p <0.00001).

[0030] FIG.s 5A-5D are graphs showing that treatment with Compound 1 activates tumor infiltrating tetramer+ CD8+ T cells. FIGs. 5A-5D show the frequency of IFN gamma and / or granzyme in tetramer CD8+ T cells and total CD8+ T cells.

[0031] FIG. 6 are pie chart graphs showing the frequency of intratumoral polyfunctional tetramer positive CD8+ T cells for MC38 tumor-bearing mice treated with Compound 1 or vehicle.

[0032] FIGs. 7A-7C are graphs showing that PD-1 is expressed by tumor infiltrating T cells (Treg (%) PD-1 (FIG. 7A), total CD8+ % PD-1+ (FIG. 7B), CD4+ T conventional % PD-1+ (FIG. 7C)) with treatment of Compound 1 in the MC38 syngeneic tumor model.

[0033] FIGs. 8A-8E are graphs showing increased expression of PD-L1 on various intratumoral immune cell populations, B-cells (FIG. 8A), CD1 lb+ dendritic cells (FIG. 8B), CD103+ dendritic cells (FIG. 8C), Ml macrophages (FIG. 8D), and M2 macrophages (FIG. 8E) with treatment of Compound 1 in the MC38 syngeneic tumor model.

[0034] FIGs. 9A-9F are graphs showing treatment with Compound 1 in the MC38 syngeneic tumor model increases upregulation of MHC class I and MHC class II on intratumoral antigen presenting cell populations, B-cell MCH class I (FIG. 9A), CD1 lb+ dendritic cells MHC class I (FIG. 9B), CD 103+ dendritic cells MHC class I (FIG. 9C), B-cell MHC class II (FIG. 9D), CDl lb+ DC: MHC class II (FIG. 9E), and CD103+ DC MHC class II (FIG. 9F).

[0035] FIGs. 10A-10B are graphs showing that treatment with Compound 1 in the MC38 syngeneic tumor model increases and prolongs intratumoral NK cell activation. FIG. 10A shows the percentage of tumor infiltrating NK cells producing IFNy and FIG. 1 OB shows the percentage of tumor infiltrating NK cells producing granzyme B+.

[0036] FIGs. 11A-1 ID are heatmaps of transcripts over time in mice treated with vehicle. The pathway analysis shows that the transcriptional profile of vehicle treated animals progresses away from immune activation and towards cancer progression over time in the MC38 model.

[0037] FIG. 12 is a graph showing that treatment of Compound 1 results in the accumulation of persistent transcriptional differences in the tumor microenvironment over about a week (e.g., day 7) after the final dose, and plateauing throughout the remainder of treatment.

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

[0039] FIGs. 14A-14B is a heat map and pathway analysis showing that treatment of Compound 1 enriches transcripts associated with IFNalpha signaling as early as day 5 of treatment and at subsequent time points.

[0040] FIGs. 15A-15F are a series of graphs showing that treatment with Compound 1 increases immune activation and drives cytotoxic cell infiltration and activation via the interferon pathway.

[0041] FIG. 15 A shows that treatment with Compound 1 provides a robust and persistent IFNalpha signaling in the tumor microenvironment. FIGs. 15B-15F are graphs showing pathway analysis scores of increased infiltration by activated cytotoxic cells (FIG. 15 A), adoptive immunity (FIG. 15B), innate immunity (FIG. 15C), apoptosis (FIG. 15D), NK cell function (FIG. 15E), and T cell function (FIG. 15F).

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

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

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

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

[0046] FIGs. 20A-20D are graphs showing combination activity of Compound 1 with several CPI in the CT26 syngeneic tumor model. Compound 1 in combination with an anti-PDl inhibitor (FIG. 20A), an anti-PD-Ll antibody, an anti-CTLA-4 antibody (FIG. 20C), or an anti-LAG3 antibody (FIG. 20D). FIG. 20A shows tumor volume over time in mice treated with Compound 1 at 50 pg / animal, Compound 1 at 200 pg / animal. Compound 1 at 50 pg / animal in combination with an anti-PDl inhibitor, Compound 1 at 200 pg / animal in combination with an anti-PDl inhibitor, an anti-PDl inhibitor at 200 pg / animal, and vehicle. FIG. 20B shows tumor volume over time in mice treated with Compound 1 at 50 pg / animal, Compound 1 at 200 pg / animal, Compound 1 at 50 pg / animal in combination with an anti- PD-Ll inhibitor, Compound 1 at 200 pg / animal in combination with an anti-PD-Ll inhibitor.an anti-PD-Ll inhibitor at 200 pg / animal, and vehicle. FIG. 20C shows tumor volume over time in mice treated with Compound 1 at 50 pg / animal. Compound 1 at 200 pg / animal, Compound 1 at 50 pg / animal in combination with an anti-CTLA-4 antibody, Compound 1 at 200 pg / animal in combination with an anti-CTLA-4 antibody, an anti-PDl inhibitor at 200 pg / animal, and vehicle in the CT26 murine model. FIG. 20D shows tumor volume over time in mice treated with Compound 1 at 50 pg / animal, Compound 1 at 200 pg / animal, Compound 1 at 50 pg / animal in combination with an anti-LAG3 antibody. Compound 1 at 200 pg / animal in combination with an anti-LAG3 antibody, an anti-PDl inhibitor at 200 pg / animal, and vehicle in the CT26 murine model. FIG. 20E shows data from individual mice. Combination therapy using Compound 1 and an anti-PD-1 antibody and / or an anti- CTLA-4 antibody showed improved tumor control than either Compound 1 or an anti-PD-1 antibody and / or an anti-CTLA-4 antibody monotherapy.

[0047] FIGs. 21A-21D are graphs showing combination activity of Compound 1 with several CPI in the MC38 syngeneic tumor model. Compound 1 in combination with an anti-PDl inhibitor (FIG. 21 A), an anti-PD-Ll inhibitor (FIG. 2 IB), an anti-CTLA-4 antibody (FIG. 21C), or an anti-LAG3 antibody (FIG. 2 ID).

[0048] FIG. 21 A shows tumor volume overtime in mice treated with Compound 1 at 50 pg / animal, Compound 1 at 200 pg / animal, Compound 1 at 50 pg / animal in combination with an anti-PDl inhibitor, Compound 1 at 200 pg / animal in combination with an anti-PDl inhibitor, an anti-PDl inhibitor at 200 pg / animal, and vehicle. FIG. 21B shows tumor volume over time in mice treated with Compound 1 at 50 pg / animal, Compound 1 at 200 pg / animal, Compound 1 at 50 pg / animal in combination with an anti-PD-Ll inhibitor, Compound 1 at 200 pg / animal in combination with an anti-PD-Ll inhibitor, an anti-PD-Ll inhibitor at 200 pg / animal, and vehicle. FIG. 21C shows tumor volume over time in mice treated with Compound 1 at 50 pg / animal, Compound 1 at 200 pg / animal. Compound 1 at 50 pg / animal in combination with an anti-CTLA-4 antibody, Compound 1 at 200 pg / animal in combination with an anti-CTLA-4 antibody, an anti-PDl inhibitor at 200 pg / animal, and vehicle in the CT26 murine model. FIG. 2 ID shows tumor volume over time in mice treated with Compound 1 at 50 pg / animal, Compound 1 at 200 pg / animal, Compound 1 at 50 pg / ammal in combination with an anti-LAG3 antibody. Compound 1 at 200 pg / animal in combination with an anti-LAG3 antibody, an anti-PDl inhibitor at 200 pg / animal, and vehicle in the CT26 murine model. FIG. 21E shows data from individual mice. Combination therapy using Compound 1 and an anti-PD-1 antibody and / or an anti-CTLA-4 antibody showed improved tumor control than either Compound 1 or an anti-PD-1 antibody and / or an anti-CTLA-4antibody monotherapy. Combination therapy using Compound 1 at 50 pg and an anti-PD-1 antibody showed significant improvement over Compound 1 treatment at 50 pg alone. Combination therapy using 200 pg Compound 1 and an anti-PD-1 antibody and combination therapy using 200 pg Compound 1 and an anti-CTLA4 antibody both showed significant improvement over Compound 1 treatment alone.

[0049] FIGs. 22A-E are graphs showing tumor volume over time in mice treated with Compound 1 at 10 pg / dose, 50 pg / dose. 200 pg / dose. or vehicle in various tumor cell models. FIG. 22A shows tumor growth over time in the MC38 murine model. FIG. 22B shows tumor grow th over time in the EMT6 murine model. FIG. 22C shows tumor growth over time in the B16-F10 murine model. FIG. 22D shows tumor growth over time in the A20 murine model. FIG. 22E shows tumor growth over time in the EG7 murine model.

[0050] FIG. 23 shows Compound 5 activation by human tumor samples compared to human healthy cells.

[0051] FIG. 24 is a graph showing tumor volume over time in mice treated with Compound 1 (WW0610) at 100 pg / dose, 400 pg / dose, vehicle (PBS) dosed twice per week for two weeks, or free IFNa (WW0126) dosed at 35 pg i.p. BID for five days with two days off for two weeks in a syngeneic murine tumor model of HPV-driven oral squamous cell carcinoma (OSCC) (mEER model).

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

[0053] FIGs. 26A-B are graphs showing that treatment with Compound 1 (WW0610) activates tumor infiltrating NK cells in a syngeneic mEER tumor model. FIG. 26A shows quantification of the percent of granzyme B (GRZB)-positive NK cells out the total NK cells collected. FIG. 26A shows quantification of the percent of interferon gamma (IFNg)-positive NK cells out the total NK cells collected.

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

[0055] FIGs. 28A-F are graphs showing that treatment with Compound 1 (WW0610) dose- dependently upregulates cytokine expression in a syngeneic mEER tumor model. FIG. 28A shows quantification of plasma interferon gamma (IFNy). FIG. 28B shows quantification of plasma tumor necrosis factor alpha (TNFa). FIG. 28C shows quantification of plasma CXCL10. FIG. 28C shows quantification of plasma CXCL10. FIG. 28D shows quantification of plasma interleukin-10 (IL-10). FIG. 28E shows quantification of serum interleukin-6 (IL-6). FIG. 28F shows quantification of serum interleukin-5 (IL-5).4. DETAILED DESCRIPTIONA. Inducible Interferon Pro-Drugs

[0056] The disclosure relates to inducible IFNalpha prodrugs that contain an attenuated IFNalpha and that have a long half-life in comparison to naturally occurring IFNalpha.

[0057] The disclosure relates to inducible IFNalpha prodrugs that contain at least one polypeptide chain, and can contain two or more polypeptide chains, if desired.

[0058] The inducible IFNalpha prodrugs comprises a IFNalpha, an IFNalpha blocking element, a protease cleavable linker, and optionally a half-life extension element. The IFNalpha can be a include human IFN-alphal, human IFN-alpha2, human IFN-alpha4. human IFN-alpha5, human IFN-alpha6, human IFN-alpha7, human IFN-alpha8, human IFN- alphal 0, human IFN-alphal 3, human IFN-alphal 4, human IFN-alphal 6, human IFN- alphal 7, human IFN-alpha2.

[0059] The inducible IFNalpha prodrugs of this disclosure have attenuated IFNalpha receptor agonist activity and the circulating half-life is extended. The IFNalpha receptor agonist activity is attenuated through the blocking element. The half-life extension element can also contribute to attenuation, for example through steric effects. The half-life extension element can also act as a blocking element that is capable of blocking all or some of the receptor agonist activity of IFNalpha. For instance, the half-life extension element can contribute to blocking when the half-life extension element is adjacent to the IFNalpha polypeptide.

[0060] The blocking element is capable of blocking all or some of the receptor agonist activity of IFNalpha by noncovalently binding to the IFNalpha and / or sterically blocking receptor binding. Upon cleavage of the protease cleavable linker a form of IFNalpha is released that is active (e.g., more active than the inducible IFNalpha prodrug). Typically, the released IFNalpha is at least 10 x more active than the inducible IFNalpha prodrug.Preferably, the released IFNalpha is at least 20 x, at least 30 x, at least 50 x, at least 100 x, at least 200 x, at least 300 x, at least 500 x, at least 1000 x, at least about 10,000X or more active than the inducible IFNalpha prodrug.

[0061] The form of IFNalpha that is released upon cleavage of the inducible IFNalpha prodrug ty pically has a short half-life, which is often substantially similar to the half-life of naturally occurring IFNalpha. Even though the half-life of the inducible IFNalpha prodrug is extended, toxicity is reduced or eliminated because the agonist activity of the circulating inducible IFNalpha prodrug is attenuated and active IFNalpha is targeted to the desired site of activity7(e.g., tumor microenvironment).

[0062] It will be appreciated by those skilled in the art, that the number of polypeptide chains, and the location of the elements, the half-life extension element, the protease cleavable linker(s), and the blocking element (and components of such elements, such as a VH or VL domain) on the polypeptide chains can vary and is often a matter of design preference. All such variations are encompassed by this disclosure.

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

[0064] The single polypeptide can comprise two or more blocking elements that also function as half-life extension elements (e.g., an antibody fragment that binds HSA). When two or more of such blocking elements are present in the inducible IFNalpha prodrug, they can block all or some of the receptor agonist activity of IFNalpha and also extend serum half-life. When two or more such a blocking elements are present in and IFNalpha prodrug, a separate half-life extension element or a separate blocking element are optional and are typically not present.

[0065] For example, the inducible IFNalpha prodrug can have the Formula XII: [D] -[L 1 ] - [A]-[L1 ’]-[D’]. In Formula XII [A] is an IFNalpha polypeptide. [LI] is a protease-cleavable polypeptide linker, [LT] is a protease-cleavable polypeptide linker, and [D] and [D’] are IFNalpha blocking elements, such as HSA or an anti-HSA antibody or fragment thereof. Preferably the blocking element functions as a half-life extension element. [D] and [D‘] can have the same or different amino acid sequence. [LI] and [LT] can have the same or different amino acid sequence and or protease-cleavage site (when L2 is protease-cleavable) as desired. The protease cleavable linker can comprise the sequence GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9), for example. Examples of preferred inducible IFNalpha prodrugs are Compounds 1-5. Additional activity regarding their activity is disclosed in International Application No.: PCT / US2020 / 060624.

[0066] In certain embodiments, each of the blocking moiety and the half-life extension moiety comprises HSA. In certain embodiments, each of the blocking moiety and the half-life extension moiety comprises the antibody or antibody fragment that binds HSA. In certain embodiments, one of the blocking moiety and the half-life extension moiety comprises HSA and the other one of the blocking moiety and the half-life extension moiety comprises the antibody or antibody fragment that binds HSA. In certain embodiments, at least one of the blocking moiety and the half-life extension moiety comprises the antibody or antibody fragment that binds 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 IFNalpha polypeptide comprises a murine interferon alpha 1 (mIFNal), murine interferon alpha 11 (mIFNal l), human interferon alpha 2b (IFNA2b), murine interferon alpha 11 (mIFNal 1), interferon alpha 8 (IFNA8), interferon alpha 14 (IFNA14), interferon alpha 16 (IFNA16), or a mutein thereof. In certain embodiments, the IFNalpha polypeptide comprises the amino acid sequence of SEQ ID NOs: 234-237.Table 1. Interferon Alpha Polypeptide SequencesTable 2. Inducible IFNalpha prodrugs

[0068] The IFNalpha polypeptide and the blocking element and / or the half-life extension element (when present) can be operably linked by the protease-cleavable polypeptide. For example, the inducible IFNalpha prodrug can be of any of Formulas (I)-(IX):[A]-[L1]-[H]-[L2]-[D] (I);[D]-[L2]-[H]-[L1]-[A] (II);[A]-[LI]-[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) - (IX), [A] is a IFNalpha polypeptide, [D] is a IFNalpha blocking element (e.g., extracellular portion of the INFalpha receptor 1 (IFNAR1) or IFNalpha receptor 2 (IFNAR2), or an antibody or antigen-binding fragment), [D’] is either the INFalpha receptor 1 (IFNAR1) or the IFNalpha receptor 2 (IFNAR2) that is not present in [D], [H] is a half-life extension element, [LI] is a protease-cleavable polypeptide linker, [L2] is an polypeptide linker that is optionally protease-cleavable, and [L2’] is a protease-cleavable polypeptide linker. [LI] and [L2] or [LI] and [L2’] can have the same or different amino acid sequence and or protease-cleavage site (when L2 is protease-cleavable) as desired. [H] can also optionally provide blocking. The protease cleavable linker can comprise the sequence GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9), for example. In certain embodiments, each of [LI] and [L2’] comprises SEQ ID NO: 6, 9 or 12.

[0070] The invention also relates to certain inducible IFNalpha prodrugs that comprise two or more polypeptide chains. Such inducible IFNalpha prodrugs comprises at least one IFNalpha polypeptide [A], a blocking element [D], a protease cleavable linker [L], and optionally a half-life extension element [H], which can be present on the same polypeptide chain or different polypeptide chains. The blocking element and half-life extension element (when present) can contain two or more components that are present on the same polypeptide chain or on different polypeptide chains. Illustrative of this, and as disclosed and exemplified herein, components of the blocking element can be present on separate polypeptide chains. For example, a first polypeptide chain can include an antibody light chain (VL+CL) or light chain variable domain (VL) and a second polypeptide can include an antibody heavy chain Fab fragment (VH + CHI) or heavy chain variable domain (VH) that is complementary to the VL+ CL or VL on the first polypeptide. In such situations, these components can associate in the peptide complex to form an antigen-binding site, such as a Fab that binds IFNalpha and attenuates IFNalpha activity.

[0071] For example, the inducible IFNalpha prodrug can have a first polypeptide of Formulas (X-XI). Formula X: [D]-[L]-[A]-[L2]-[H] or Formula XI: [H]-[L]-[A]-[L2]-[D], In Formulas (X) - (XI), [A] is a IFNalpha polypeptide, [D] is a IFNalpha antibody heavy chain Fab fragment (VH + CHI) or heavy chain variable domain (VH), [H] is a half-life extension element, [LI] is a protease-cleavable polypeptide linker, [L2] is an polypeptide linker that is optionally protease-cleavable, and [L2’] is a protease-cleavable polypeptide linker. [LI] and [L2] or [LI] and [L2’] can have the same or different amino acid sequence and or proteasecleavage site (when L2 is protease-cleavable) as desired. The inducible IFNalpha prodrug can have a second polypeptide antibody light chain (VL+CL) or light chain variable domain (VL) that is complementary to the VH + CHI or VH. The protease cleavable linker can comprise the sequence GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9).

[0072] In embodiments, the inducible IFNalpha prodrug can comprise a first polypeptide chain that comprises an IFNalpha polypeptide and an antibody heavy chain Fab fragment (VH + CHI) or heavy chain variable domain (VH) and a second polypeptide can include ahalf-life extension element and an antibody light chain (VL+CL) or light chain variable domain (VL) that is complementary to the VH+ CHI or VH on the first polypeptide.

[0073] Inducible IFNalpha prodrugs comprising two or more polypeptide chains have been described in International Application No.: PCT / US2022 / 040564.B. Half-Life Extension Element

[0074] Contemplated herein are domains which extend the half-life of the inducible IFNalpha prodrug. Increasing the in vivo half-life of therapeutic molecules with naturally short halflives allows for a more acceptable and manageable dosing regimen without sacrificing effectiveness.

[0075] The half-life extension element, increases the in vivo half-life and provides altered pharmacodynamics and pharmacokinetics of the inducible IFNalpha prodrugs. Without being bound by theory, the half-life extension element alters pharmacodynamics properties including alteration of tissue distribution, penetration, and diffusion of the inducible IFNalpha prodrug. In some embodiments, the half-life extension element can improve tissue targeting, tissue penetration, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension element. Without being bound by theory, an exemplary way to improve the pharmacokinetics of a polypeptide is by expression of an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor. Three types of proteins, e.g., human IgGs, HSA (or fragments), and transferrin, persist for much longer in human serum than would be predicted just by their size, which is a function of their ability to bind to receptors that are recycled rather than degraded in the lysosome. These proteins, or fragments retain FcRn binding and are routinely linked to other polypeptides to extend their serum half-life. HSA may also be directly bound to the pharmaceutical compositions or bound via a short linker. Fragments of HSA may also be used. HSA and fragments thereof can function as both a blocking element and a half-life extension element. Human IgGs and Fc fragments can also carry out a similar function.

[0076] The serum half-life extension element can also be an antigen-binding polypeptide that binds to a protein with a long serum half-life such as serum albumin, transferrin and the like. Examples of such polypeptides include antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), an antigen binding fragment (Fab), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) anda variable domain of camelid-type nanobody (VHH), a dAb and the like. Other suitable antigen-binding domain include non-immunoglobulin proteins that mimic antibody binding and / or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocallin and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that binds to or associates with one or more target antigens. The antibodies and fragments thereof can function as both a blocking element and a half-life extension element.

[0077] The half-life extension element can also function as both a blocking element and a half-life extension element. For instance, the half-life extension element (e.g., anti-HSA) can function as a blocking element when adjacent to the IFNalpha polypeptide.

[0078] The half-life extension element as provided herein is preferably a human serum albumin (HSA) binding domain, an antigen binding polypeptide that binds human serum albumin or an immunoglobulin Fc or fragment thereof.

[0079] The half-life extension element of a inducible IFNalpha prodrug extends the half-life of the inducible IFNalpha prodrug by at least about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about 10 days or more.C. Blocking Element

[0080] The blocking element can be any element that binds to IFNalpha and / or inhibits the abi li ty of the IFNalpha polypeptide to bind and activate its receptor. The blocking element can inhibit the ability of the IFNalpha to bind and / or activate its receptor e.g., by sterically blocking and / or by noncovalently binding to the inducible IFNalpha prodrug. Some blocking elements disclosed herein can bind to IFNalpha (e.g., human IFN-alphal, human IFN-alpha2, human IFN-alpha4, human IFN-alpha5, human IFN-alpha6, human IFN-alpha7, human IFN- alpha8, human IFN-alphal 0, human IFN-alphal 3, human IFN-alphal4, human IFN-alphal 6, human IFN-alphal 7, human IFN-alpha2).

[0081] Examples of suitable blocking elements include the full length or an IFNalpha- binding fragment or mutein of the cognate receptor of an IFNalpha. For instance, when the IFNalpha polypeptide is INFalpha2a, the blocking element can be the extracellular portion of the INF alpha receptor 1 (IFNAR1) or interferon binding portion or mutein thereof, or the extracellular portion of the IFNalpha receptor 2 (IFNAR2) or interferon binding portion or mutein thereof.

[0082] Antibodies and antigen-binding fragments thereof including, an antigen-binding fragment (Fab), a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like that bind IFNalpha can also be used. Other suitable antigen-binding domain that bind IFNalpha can also be used, include non-immunoglobulin proteins that mimic antibody binding and / or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL. fibronectin, lipocallin and CTLA4 scaffolds.

[0083] Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block binding of IFNalpha to its cognate receptor. Advantageously, such moieties can also function as half-life extending elements. For example, a peptide that is modified by conjugation to a water-soluble polymer, such as PEG, can sterically inhibit or prevent binding of the cytokine to its receptor. Polypeptides, or fragments thereof, that have long serum half-lives can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin and the like, as well as fragments and muteins of such polypeptides. Antibodies and antigen-binding domains that bind to, for example, a protein with a long serum half-life such as HSA, immunoglobulin or transferrin, or to a receptor that is recycled to the plasma membrane, such as FcRn or transferrin receptor, can also inhibit the cytokine, particularly when bound to their antigen.

[0084] IFNalpha blocking elements that are suitable are single chain variable fragments (scFv) or Fab fragments.

[0085] Also disclosed herein are inducible IFNalpha prodrugs that contains a blocking element having specificity for IFNalpha and further contains a half-life extension element.

[0086] The blocking element can contain two or more components that are present on the same polypeptide chain or on separate polypeptide chains. A first polypeptide chain can include an antibody light chain (VL+CL) or light chain variable domain (VL) and a second polypeptide can include an antibody heavy chain Fab fragment (VH + CHI) or heavy chain variable domain (VH) that is complementary to the VL+ CL or VL on the first polypeptide. In such situations, these components can associate in the peptide complex to form an antigenbinding site, such as a Fab that binds IFNalpha and attenuates IFNalpha activity.D. Protease Cleavable Linker

[0087] As disclosed herein, the inducible IFNalpha prodrug comprises one or more linker sequences. A linker sequence serves to provide flexibility between the polypeptides, such that, for example, the blocking element is capable of inhibiting the activity of IFNalpha. The linker can be located between the IFNalpha subunit, the half-life extension element, and / or the blocking element. As described herein the inducible IFNalpha prodrug comprises a protease cleavable linker. The protease cleavable linker can comprise one or more cleavage sites for one or more desired protease. Preferably, the desired protease is enriched or selectively expressed at the desired target site of IFNalpha activity (e.g., the tumor microenvironment). Thus, the inducible IFNalpha prodrug is preferentially or selectively cleaved at the target site of desired IFNalpha activity.

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

[0089] Preferably the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked domain. In a preferred embodiment, the linker is cleavable by a cleaving agent, e.g., an enzyme. Preferably, the linker comprises a protease cleavage site. In some cases, the linker comprises one or more cleavage sites. The linker can comprise a single protease cleavage site. The linker can also comprise 2 or more protease cleavage sites. For example, 2 cleavage sites, 3 cleavage sites, 4, cleavage sites, 5 cleavage sites, or more. In cases the linker comprises 2 or more protease cleavage sites, the cleavage sites can be cleaved by the same protease or different proteases. A linker comprising two or more cleavage sites is referred to as a “tandem linker.” The two or more cleavage sites can be arranged in any desired orientation, including, but not limited tom one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, or one cleavage site following by another cleavage site with intervening amino acids between the two cleavage sites.

[0090] Of particular interest in the present invention are disease specific protease-cleavable linkers. Also preferred are protease-cleavable linkers that are preferentially cleaved at a desired location in the body, such as the tumor microenvironment, relative to the peripheral circulation. For example, the rate at which the protease-cleavable 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 in the desired location in the body, e g., the tumor microenvironment, in comparison to in the peripheral circulation (e.g.. in plasma).

[0091] Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins. Cathepsin B. Cathepsin C. Cathepsin D. Cathepsin E. Cathepsin G. Cathepsin S, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK.15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease. CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, MMP19, MMP20, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin- 10 converting enzyme, thrombin, FAP (FAPa), dipeptidyl peptidase, meprins, granzymes and dipeptidyl peptidase IV (DPPIV / CD26). Proteases capable of cleaving linker amino acid sequences (which can be encoded by the chimeric nucleic acid sequences provided herein) can, for example, be selected from the group consisting of a prostate specific antigen (PSA), a matrix metalloproteinase (MMP). an A Disintigrin and a Metalloproteinase (ADAM), a plasminogen activator, a cathepsin, a caspase, a tumor cell surface protease, and an elastase. The MMP can, for example, be matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9). matrix metalloproteinase 14 (MMP 14), matrix metalloproteinase 19 (MMP 19), or matrix metalloproteinase 20 (MMP20). In addition, or alternatively, the linker can be cleaved by a cathepsin, such as. Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin S, Cathepsin E, Cathepsin G, Cathepsin K and / or Cathepsin L. Preferably, the linker can be cleaved by MMP14 or Cathepsin L.

[0092] Proteases useful for cleavage of linkers and for use in the inducible IFNalpha prodrug disclosed herein are presented in Table 3, and exemplary proteases and their cleavage site are presented in Table 4.Table 3. Proteases relevant to inflammation and cancerTable 4. Exemplary Proteases and Protease Recognition Sequences

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

[0094] The linker sequences disclosed herein are typically less than 100 amino acids. Such linker sequences can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid 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. In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75. about 80, about 85, about 90, about 95, or about 100 amino acids in length. Preferred linkers are typically from about 5 amino acids to about 30 amino acids.

[0095] Preferably the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked domains.

[0096] In some embodiments, the linker comprises the sequence GPAGLYAQ (SEQ ID NO: 6); GPAGMKGL (SEQ ID NO: 7); PGGPAGIG (SEQ ID NO: 8); ALFKSSFP (SEQ ID NO: 9); ALFFSSPP (SEQ ID NO: 10); LAQRLRSS (SEQ ID NO: 11); LAQKLKSS (SEQ ID NO: 12); GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (SEQ ID NO: 13);RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (SEQ ID NO: 14); KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO: 15);RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO: 16); KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO: 17); SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO: 18); RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (SEQ ID NO: 19); GGGALFKSSFPLAQKLKSSPGGPAGIGGGR (SEQ ID NO: 20); RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (SEQ ID NO: 21); RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (SEQ ID NO: 22); RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO: 23); GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO: 24); GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO: 25); RGGALFKSSFPLAQKLKSSGPAGLYAQGGK (SEQ ID NO: 26); RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (SEQ ID NO: 27); SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (SEQ ID NO: 28); KGGPGGPAGIGPLAQRLRSSALFKSSFPGR (SEQ ID NO: 29); KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (SEQ ID NO: 30); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (SEQ ID NO: 31)

[0097] Certain preferred linkers comprises the sequence GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9). The linkers disclosed herein can comprise one or more cleavage motif or functional variants that are the same or different. The linkers can comprise 1, 2, 3, 4. 5, or more cleavage motifs or functional variants. Linkers comprising 30 amino acids can contain 2 cleavage motifs or functional variants, 3 cleavage motifs or functional variants or more. A “functional variant” of a linker retains the ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease) and are not cleaved or cleaved with low efficiency in the periphery (e.g., serum). For example, the functional variants retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a linker comprising any one of SEQ ID NOs: 6-31 or 232-233.

[0098] The linkers comprising more than one cleavage motif can be selected from SEQ ID NOs: 6-12 or 232-233 and combinations thereof. Preferred linkers comprising more than one cleavage motif comprise the amino acids selected from SEQ ID NO: 13-31.

[0099] The linker can comprise both ALFKSSFP (SEQ ID NO: 9) and GPAGLYAQ (SEQ ID NO: 6). The linker can comprise two cleavage motifs that each have the sequence GPAGLYAQ (SEQ ID NO: 6). Alternatively, or additionally, the linker can comprise twocleavage motifs that each have the sequence ALFKSSFP (SEQ ID NO: 9). The linker can comprise a third cleavage motif that is the same or different.

[0100] In some embodiments, the linker comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NO: 6 to SEQ ID NO: 31 or SEQ ID NOs: 232-233 over the full length of SEQ ID NOs: 6-31 or SEQ ID NOs: 232-233.

[0101] The disclosure also relates to functional variants of the linkers comprising SEQ ID NOs: 6-31 or 232-233. The functional variants of the linkers comprising SEQ ID NOs: 6-31 or 232-233 generally differ from SEQ ID NOs: 6-31 or 232-233 by one or a few amino acids (including substitutions, deletions, insertions, or any combination thereof), and substantially retain their ability to be cleaved by a protease.

[0102] The functional variants can contain at least one or more amino acid substitutions, deletions, or insertions relative to the linkers comprising SEQ ID NOs: 6-31 or 232-233. The functional variant can comprise 1, 2. 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations comparted to the linkers comprising SEQ ID NOs: 6-31 or 232-233. In some preferred embodiments, the functional variant differs from the linker comprising SEQ ID NOs: 6-31 by less than 10, less, than 8, less than 5, less than 4, less than 3, less than 2, or one amino acid alterations, e.g., amino acid substitutions or deletions. In other embodiments, the functional variant may comprise 1, 2, 3, 4. 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NOs: 6- 31 or 232-233. The amino acid substitution can be a conservative substitution or a nonconservative substitution, but preferably is a conservative substitution.

[0103] In other embodiments, the functional variants of the linkers may comprise 1, 2, 3, 4, or 5 or more non-conservative amino acid substitutions compared to the linkers comprising SEQ ID NOs: 6-31 or 232-233. Non-conservative amino acid substitutions could be recognized by one of skill in the art. The functional variant of the linker preferably contains no more than 1, 2, 3, 4, or 5 amino acid deletions.

[0104] The amino acid sequences disclosed in the linkers can be described by the relative linear position in the linker with respect to the sissile bond. As will be well-understood by persons skilled in the art, linkers comprising 8 amino acid protease substrates (e.g.. SEQ ID Nos: 6-12 or 232-233) contain amino acid at positions P4, P3, P2, Pl, Pl ’, P2’, P3’, P4’, wherein the sissile bond is between Pl and PE. For example, amino acid positions for the linker comprising the sequence GPAGLYAQ (SEQ ID NO: 6 ) can be described as follows:

[0105] "GPAGLYAQ" disclosed as SEQ ID NO: 6.

[0106] Amino acids positions for the linker comprising the sequence ALFKSSFP (SEQ IDNO: 9) can be described as follows:

[0107] "ALFKSSFP" disclosed as SEQ ID NO: 9.

[0108] Preferably, the amino acids surrounding the cleavage site (e.g., positions Pl and Pl ’for SEQ ID NOs: 6-12 or 232-233) are not substituted.

[0109] In embodiments, the linker comprises the sequence GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9) or a functional variant of SEQ ID NO: 6 or a function variant of SEQ ID NO: 9. As described herein, a functional variant of PAGLYAQ (SEQ ID NO: 232) or ALFKSSFP (SEQ ID NO: 9) can comprise one or more amino acid substitutions, and substantially retain their abi li ty to be cleaved by a protease. Specifically, the functional variants of GPAGLYAQ (SEQ ID NO: 6) is cleaved by MMP14. and the functional variant of ALFKSSFP (SEQ ID NO: 9) is cleaved by Capthepsin L (CTSL1). The functional variants also retain their ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease). For example, the 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 a linker comprising 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) comprise no more than 1, 2, 3, 4. or 5 conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 6) or ALFKSSFP (SEQ ID NO: 9). Preferably, the amino acids at position Pl and PL are not substituted. The amino acids at positions Pl and Pl ’ in SEQ ID NO: 6 are G and L, and the amino acids at positions Pl and Pl ’ in SEQ ID NO: 9 are K and S.

[0111] The functional variant of GPAGLYAQ (SEQ ID NO: 6) can preferably comprise one or more of the following: a) an arginine amino acid substitution at position P4, b) a leucine,valine, asparagine, or proline amino acid substitution at position P3, c) a asparagine amino acid substitution at position P2, d) a histidine, asparagine, or glycine amino acid substitution at position Pl, e) a asparagine, isoleucine, or leucine amino acid substitution at position Pl’, f a tyrosine or arginine amino acid substitution at position P2’, g) a glycine, arginine, or alanine amino acid substitution at position P3‘, h) or a serine, glutamine, or lysine amino acid substitution at position P4‘. The following amino acid substitutions are disfavored 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 Pl, d) arginine, glycine, asparagine, or threonine at position Pl’, e) aspartic acid or glutamic acid at position P2’, f) isoleucine at position P3’, g) valine at position P4’. In some embodiments, the functional variant of GPAGLYAQ (SEQ ID NO: 6) does not comprise an amino acid substitution at position Pl and / or Pl’.

[0112] The amino acid substitution of the functional variant of GPAGLYAQ (SEQ ID NO: 6) preferably comprises an amino acid substitution at position P4 and / or P4’. For example, the functional variant of GPAGLYAQ (SEQ ID NO: 6) can comprise a leucine at position P4, or serine, glutamine, lysine, or phenylalanine at position P4. Alternatively or additionally, the functional variant of GPAGLYAQ (SEQ ID NO: 6) can comprise a glycine, phenylalanine, or a proline at position P4’.

[0113] In some embodiments, the amino acid substitutions at position P2 or P2’ of GPAGLYAQ (SEQ ID NO: 6) are not preferred.

[0114] In some embodiments, the functional variant of GPAGLYAQ (SEQ ID NO: 6) comprises the amino acid sequence selected from SEQ ID NOs: 32-106. Specific functional variants of GPAGLYAQ (SEQ ID NO: 6) include GPLGLYAQ (SEQ ID NO: 70). and GPAGLKGA (SEQ ID NO: 60).Table 5. Functional Variant Sequences

[0115] The functional variants of LFKSSFP (SEQ ID NO: 233) preferably comprises hydrophobic amino acid substitutions. The functional variant of LFKSSFP (SEQ ID NO: 233) can preferably comprise one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, phenylalanine at position P3; (c) arginine, leucine, alanine, glutamine, or histatine at position P2; (d) phenylalanine, histidine, threonine, alanine, or glutamine at position Pl; (e) histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine at position Pl’, (f) phenylalanine, leucine, isoleucine, lysine, alanine, glutamine, or proline at position P2’; (g) phenylalanine, leucine, glycine, serine, valine, histidine, alanine, or asparagine at position P3’; and phenylalanine, histidine, glycine, alanine, serine, valine, glutamine, lysine, or leucine.

[0116] The inclusion of aspartic acid and / or glutamic acid in functional variants of SEQ ID NO: 233 are generally disfavored and avoided. The following amino acid substitutions are also disfavored in functional variants of LFKSSFP (SEQ ID NO: 233): (a) alanine, serine, or glutamic acid at position P3; (b) proline, threonine, glycine, or aspartic acid at position P2: (c) proline at position Pl; (d) proline at position Pl’; (e) glycine at position P2’; (I) lysine or glutamic acid at position P3’; (g) aspartic acid at position P4’.

[0117] The amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 233) preferably comprises an amino acid substitution at position P4 and / or PL In someembodiments, an amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 233) at position P4?is not preferred.

[0118] In some embodiments, the functional variant of LFKSSFP (SEQ ID NO: 233) comprises the 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. Functional Variant Sequences

[0119] The linkers disclosed herein can form a stable prodrug under physiological conditions with the amino acid sequences (e.g. domains) that they link, while being capable of being cleaved by a protease. For example, the linker is stable (e.g., not cleaved or cleaved with low efficiency) in the circulation and cleaved with higher efficiency at a target site (i.e. a tumor microenvironment). Accordingly, inducible IFNalpha prodrugs that include the linkers disclosed herein can, if desired, have a prolonged circulation half-life and / or lower biologicalactivity in the circulation in comparison to the components of the inducible IFNalpha prodrugs as separate molecular entities. Yeti when in the desired location (e.g.. tumor microenvironment) the linkers can be efficiently cleaved to release the components that are joined together by the linker and restoring or nearly restoring the half-life and biological activity of the components as separate molecular entities.

[0120] The linker desirably remains stable in the circulation for at least 2 hours, at least 5, hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.

[0121] In some embodiments, the linker is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%. 30%. 20%. 20%. 5%, or 1% in the circulation as compared to the target location. The linker is also stable in the absence of an enzyme capable of cleaving the linker. However, upon expose to a suitable enzy me (i.e., a protease), the linker is cleaved resulting in separation of the linked domain.E. Pharmaceutical Compositions

[0122] Also provided herein, are pharmaceutical compositions comprising an inducible IFNalpha prodrug described herein, a vector comprising the polynucleotide encoding the inducible IFNalpha prodrug or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier.

[0123] Provided herein are pharmaceutical formulations or compositions containing the inducible IFNalpha prodrugs as described herein and a pharmaceutically acceptable carrier. Compositions comprising the inducible IFNalpha prodrugs as described herein are suitable for administration in vitro or in vivo. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any earner that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the subject to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various t pes of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial 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 a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic, although the formulate can be hypertonic or hypotonic if desired. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or from about 7 to 7.5. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptides. Matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. Certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of the IFNalpha or inducible IFNalpha prodrugs or nucleic acid sequences encoding the inducible IFNalpha prodrugs to humans or other subjects.

[0125] In some embodiments of the pharmaceutical compositions, the inducible IFNalpha prodrug described herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical compositions, the inducible IFNalpha prodrug is attached to liposomes. In some instances, the inducible IFNalpha prodrugs are conjugated to the surface of liposomes. In some instances, the inducible IFNalpha prodrug are encapsulated within the shell of a liposome. In some instances, the liposome is a cationic liposome.

[0126] The inducible IFNalpha prodrugs described herein are contemplated for use as a medicament. Administration is effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient's size, body surface area, age. sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently. An "effective dose" refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.

[0127] Optionally, the inducible IFNalpha prodrug or nucleic acid sequences encoding the inducible IFNalpha prodrug are administered by a vector. There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and / or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based deliverysystems and non-viral based delivery- systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express and preferably secrete the encoded chimeric polypeptide or to therapeutically deliver nucleic acids to a subject. The components of the IFNalpha polypeptide disclosed herein are typically operably linked in frame to encode a fusion protein.

[0128] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the nucleic acid molecule and / or polypeptide in the cells into which it is delivered. Viral vectors are, for example. Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general and methods of making them are described by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). The construction of replication-defective 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 benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host- restricted non-replicating vaccinia virus vectors.

[0129] The provided inducible IFNalpha prodrugs and / or nucleic acid molecules can be delivered via virus like particles. Virus like particles (VLPs) consist of viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus likeparticles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0130] The inducible IFNalpha prodrugs disclosed herein can be delivered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003). The provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006 / 110728.

[0131] Non-viral based delivery' methods, can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding polypeptides, wherein the nucleic acids are operably linked to an expression control sequence. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations 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, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to make the inducible IFNalpha prodrugs by expression in a suitable host cell, such as CHO cells.

[0132] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40). adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g., [Lactin promoter or EFla promoter, or from hybrid or chimeric promoters (e.g., CMV promoter fused to the |3- actin promoter). Of course, promoters from the host cell or related species are also useful herein.

[0133] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' or 3' to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 base pairs (bp) in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While manyenhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0134] The promoter and / or the enhancer can be inducible (e.g., chemically or physically regulated). A chemically regulated promoter and / or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. A physically regulated promoter and / or enhancer can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region can be active in a cell type specific manner. Optionally, in certain vectors, the promoter and / or enhancer region can be active in all eukary otic cells, independent of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the [3-actin promoter, the EFla promoter, and the retroviral long terminal repeat (LTR).

[0135] The vectors also can include, for example, origins of replication and / or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.F. Therapeutic Applications

[0136] Also provided herein, are methods and uses for the treatment of a disease, disorder or condition associated with a target antigen comprising administering to a subject in needthereof a inducible IFNalpha prodrug as described herein. Diseases, disorders, or conditions include, but are not limited to, cancer, inflammatory- disease, an immunological disorder, autoimmune disease, infectious disease (i.e., bacterial, viral, or parasitic disease). Preferably, the disease, disorder, or condition is cancer.

[0137] In an aspect, the disclosure provides a method for treating cancer, comprising administering to a subject in need thereof a checkpoint inhibitor and an inducible interferon alpha (IFNalpha) prodrug comprising a fusion polypeptide having the formula of: [D] -[L 1] - [A]-[L2’]-[H], wherein,[A] is an interferon alpha (IFNa) polypeptide, a mutein, or an active fragment thereof, [D] is a blocking moiety,[H] is a half-life extension moiety.[LI] is a protease-cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 6, 9, or 12, and[L2’] is a protease-cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 6, 9, or 12. wherein the blocking moiety and the half-life extension moiety each independently comprise human serum albumin (HSA) or an antibody or antibody fragment that binds the HSA.

[0138] Any suitable cancer may be treated with the inducible IFNalpha prodrugs provided herein. Illustrative suitable cancers, in particular solid tumors, such as sarcomas and carcinomas. For examples, the methods and compositions disclosed herein can be used to treat acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer,multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary’ central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, 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 can be used to treat adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary' blastoma, primary’ central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary’ gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoidtumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer. non-Hodgkin lymphoma, squamous 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 cancer (HNSCC), oral squamous cell carcinoma (OSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability high or mismatch repair deficient cancer, microsatellite instability high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial carcinoma, tumor mutational burden high cancer, cutaneous squamous cell carcinoma (cSCC), triple negative breast cancer (TNBC), urothelial carcinoma, colorectal cancer or oesophageal carcinoma.

[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. As an example, the methods and compositions disclosed herein can be used to treat melanoma in subj ects with unresectable or metastatic melanoma. As another example, the methods and compositions disclosed herein can be used for the adjuvant treatment of subjects with melanoma with involvement of lymph node(s) following complete resection.

[0144] In some embodiments, provided herein is a method of enhancing an immune response in a subject in need thereof by administering an effective amount of an inducible IFNalpha prodrug provided herein to the subject. The enhanced immune response may prevent, delay, or treat the onset of cancer, a tumor, or a viral disease. Without being bound by theory, theinducible IFNalpha prodrug enhances the immune response by activating the innate and adaptive immunities. In some embodiments, the methods described herein increase the activity of Natural Killer Cells and T lymphocytes. In some embodiments, the inducible IFNalpha prodrug provided herein, can induce IFNy release from Natural Killer cells as well as CD4+ and CD8+ T cells.

[0145] The method can further involve the administration of one or more additional agents to treat cancer, such as chemotherapeutic agents (e.g., Adriamycin, Cerubidine. Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), immuno-oncology agents (e.g., anti-PD-Ll, anti- CTLA4, anti-PD-1, anti-LAG3, anti-CD47, anti-GD2), cellular therapies (e.g., CAR-T, T-cell therapy), oncolytic viruses and the like. Non-limiting examples of anti-cancer agents that can be used include acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa: bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine: estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a: interferon alpha-2b: interferon alpha-nl interferon alpha-n3; interferon beta-I; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin;mitosper: mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride: semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate: trestolone acetate; triciribine phosphate: trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfm; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinzolidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride.

[0146] In some embodiments of the methods described herein, the inducible IFNalpha prodrug or the inducible IFNalpha prodrug is administered in combination with an agent for the treatment of the particular disease, disorder, or condition. Agents include, but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroidal, peptide, and the like), radiotherapies (y-rays, C-rays, and / or the directed delivery of radioisotopes, microwaves, UV radiation and the like), gene therapies (e.g., antisense, retroviral therapy and the like) and other immunotherapies. In some embodiments, the inducible IFNalpha prodrug or is administered in combination with anti-diarrheal agents, anti-emetic agents, analgesics and / or non-steroidal anti-inflammatory agents.

[0147] This disclosure relates to a therapeutic combination of any of the inducible IFNalpha prodrugs disclosed herein in combination with one or more additional agents to treat cancer (such as lymphoma), such as chemotherapeutic agents (e.g., cyclophosphamide, mechlorethamine, melphalan, chlorambucil, ifosfamide, busulfan, N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU). semustine (MeCCNU), fotemustine. streptozotocin. dacarbazine, mitozolomide, temozolomide, thiotepa, mitomycin, diaziquone (AZQ), cisplatin, carboplatin, oxaliplatin, procarbazine, hexamethylmelamine, methotrexate, pemetrexed, fluorouracil (e.g. 5 -fluorouracil), capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thioguanine, mercaptopurine, vincristine, vinblastine, vinorelbine, vindesine. vinflunine, paclitaxel, docetaxel, etoposide, teniposide, doxorubicin, daunorubicin, epirubicin,idarubicin, pirarubicin. aclarubicin, mitoxantrone, actinomycin, bleomycin, bisantrene, gemcitabine, cytarabine, and the like), immuno-oncology agents and immune checkpoint inhibitors (e.g., anti-PD-Ll, anti-CTLA4, anti-PD-1, anti-LAG3, anti-CD47, anti-GD2), oncolytic viruses and the like.

[0148] The inducible IFNalpha prodrugs disclosed herein can be combined with any desired additional anti-cancer agent. The inducible IFNalpha prodrugs disclosed herein can be combined with any desired anti-PD-1 antibody or any desired anti-PD-Ll antibody.

[0149] Exemplary anti-PD-1 antibodies that can be combined with the inducible IFNalpha prodrugs include, but are not limited to, AMP-224 (AstraZenica), 609A (3SBio), 704 (3SBio), 705 (3SBio), ABBV-181 (AbbVie), ADU-1503 I bion-004 (Chinook Therapeutics), AGEN2034 I balstilimab (Agenus), AK103 (Akeso), AK104 (Akeso), AK112 (Akeso), AK123 (Akeso), AMG 256 (Amgen), AMG 404 (Amgen), ANB030 (AnaptysBio), ANKEBIO Anti-PDl product (Anhui Anke Biotechnology), Anti PD-1 / Anti-CD47 (DiNonA), ASKG915 (Ask Gene Pharmaceuticals), AV -MEL-1 (Aivita Biomedical), BCD- 100 (Biocad CJSC), BI 754091 (Boehringer Ingelheim), BiCKI-IL-7 (OSE Immunotherapeutics), Boehringer-PD-1 -unknown (Boehringer Ingelheim), BSK-050K01 (Biosion), Camrelizumab (Jiangsu Hengrui Medicine), CB201 (Crescendo Biologies), CB213 (Crescendo Biologies), CC-90006 (AnaptsBio), cetrelimab (J&J), chPDl (Kiromic Biopharma), CMAB819 (Mabpharm), CS1003 (CStone Pharmaceuticals), CS17938 (Shenzhen Chipscreen Biosciences). CTX-8371 (Compass Therapeutics), CX-072 (CytomX Therapeutics), CX-188 (CytomX Therapeutics), cypalizumab (Harbin Gloria Pharmaceuticals), DB004 (DotBio), EMB02 (EpimAb Biotherapeutics), Geptanblimab / genolimzumab (Apollomics), GS19 (Suzhou Zelgen Biopharmaceuticals), HLX10 (Shanghai Henlius Biotech), HX008 (Taizhou HanZhong Pharmaceuticals), HY003 (Juventas Cell Therapy), IBI315 / BH2950 (Innovent Biologies), IBI318 (Innovent Biologies), IBI319 (Innovent Biologies), IMM1802 (ImmuneOnco Biopharma), IMT200 (TrueBinding), Jemperli / dostarlimab (AnaptysBio). JTX-4014 (Jounce Therapeutics), Keytruda / pembrolizumab (Merck), LBL-006 (Nanjing Leads Biolabs), Libtayo / cemiplimab-rwlc (Regeneron Pharmaceuticals), LVGN3616 (Lyvgen Biopharma), LXF821 (Novartis), LY01015 (Luye Pharma Group), LY3462817 (Eli Lilly), MCLA-134 (Merus N.V.), MEDI5752 (AstraZenica), NIR178 (Novartis), ONCR-177 (Oncorus), ONO-4685 (Ono Pharmaceutical), Opdivo / nivolumab (Ono Pharmaceutical), MGD019 (MacroGenius), PD1- GDT CAR-T (Kiromic Biopharma), penpulimab (Akeso), PSB205 (Qilu Puget Sound Biotherapeutics), PT-001 (Merck), PT627 (Merck), RB-M1 (Refuge Biotechnologies),Retifanlimab (MacroGenics), RG6139 (Roche), RG6279 (Roche), RTX-002 (RubrYc Therapeutics), sasanlimab (Pfizer), Servier-PDlxLAG3-unknown (Servier), SL-279252 / TAK-252 (Shattuck Labs), Sofusa anti-PDl (Sorrento Therapeutics), spartalizumab (Novartis), SSI-361 (Lyvgen Biopharma), Sym021 (Servier), Tebotelimab (MacroGenics), tislelizumab (BeiGene), TSR-075 (AnaptsBio), Tuhura-DO / PD-1 -unknown (Tuhura Biopharma), toripalimab (Shanghai Junshi Biosciences), sintilimab (Innovent Biologies), Unicar-CAR-T&PD-l -unknown (Shanghai Unicar-Therapy Bio-Medicine Technology). Xdivane (Xbrane Biopharma), XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y - Biologies), and zimberelimab (Arcus Biosciences).

[0150] The anti-PD-1 antibody that can be combined with the inducible cytokine prodrugs is typically an approved anti-PD-1 antibody. Approved anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA), dostarlimab (JEMPERLI), cemiplimab-rwlc (LIBATYO), nivolumab (OPDIVO), camrelizumab, tislelizumab, toripalimab, and sintilimab (TYVYT).

[0151] Exemplary anti-PD-Ll antibodies that can be combined with the inducible cytokine prodrugs include, but are not limited to, A 167 (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-Ll -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 Tumors-unknown (DiNonA), DR30207 (Zhejiang Doer Biologies), DuoBody-PD-Llx4-lBB (Ligand Pharmaceuticals), envafolimab (Alphamab Oncology), EP IM-001 (Elpis Biopharmaceuticals), ESI 01 (Elpiscience Biopharma), INBRX-105 (Inhibrx), FAZ053 (Novartis), FS118 (F-star Therapeutics), GB262 (Genor Biopharma), GS-4224 (Gilead), GT900008 (Kintor Pharmaceuticals), GX-P2 (Genexine), Hamni-PS-Ll / CD47-Unknown (Hanmi Pharmaceutical), HBM7015 (HBM Holdings), HBM9167 (HBM Holdings), HLX20 (Shanghai Henlius Biotech), HTI-1088 (Jiangsu Hengrui Medicine). IBB 18 (Innovent Biologies), IBI322 (Innovent Biologies), IBI323 (Innovent Biologies), IGM-7354 (IGM Biosciences), IMC-001 (SorrentoTherapeutics), Imfinzi / durvalumab (AstraZenica), IMM25 (ImmuneOnco Biopharma), IMM2502 (ImmuneOnco Biopharma), IMM2503 (ImmuneOnco Biopharma), IMM2504 (ImmuneOnco Biopharma), INCB86550 (Incyte), 10103 (10 Biotech), JS003 (Shanghai Junshi Biosciences), Jubilant-PD-Ll -unknown (Jubilant Therapeutics), KD033 (Kadmon Holdings), KN046 (Alphamab Oncology ), KYI 003 (Sanofi), KYI 043 (Sanofi), LY3300054 (Eh Lilly), LY3415244 (Eli Lilly), MRNA-6981 (Modema), MSB2311 (Transcenta Holding), MT-6035 (Molecular Templates). ND021 / NM21-1480 (Numab Therapeutics). OXOOIR (Oxford BioTherapeutics), PD-L1 based BsAbs (LMab), PD-L1 Boltbody ISAC (Bolt Biotherapeutics), PDL-GEX (Glycotope GmbH), PMC-122 (PharmAbcine), PMI06 (D&D Pharmatech), Protheragen-RV-scFv-PDLl -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-siPDLl (TransCode Therapeutics), UniCAR-T-PD-Ll (GEMoaB monoclonals). Vaximm (VXM10), and YBL- 013 (Y-Biologics).

[0152] The anti-PD-Ll antibody that can be combined with the inducible IFNalpha prodrugs is ty pically an approved anti-PD-Ll antibody. Approved anti-PD-1 antibodies include, but are not limited to, avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ).

[0153] The anti-CTLA4 antibody that can be combined with the inducible IFNalpha prodrugs is typically an approved anti-CTLA4 antibody. Approved anti-CTLA4 antibodies include, but are not limited to, ipilimumab (YERVOY) and tremelimumab (IMJUDO).

[0154] The anti-LAG3 antibody that can be combined with the inducible INF alpha prodrug is ty pically an approved anti-LAG3 antibody. An approved anti-LAG3 antibody includes, relatlimab (OPDUALAG).G. Definitions

[0155] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent yvith the definitions provided herein. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as. for example, the widely utilized molecular cloning methodologies described in Sambrook et al.. Molecular Cloning: A LaboratoryManual 4th ed. (2012) Cold Spring Harbor Laboratory' Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

[0156] As used herein, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.

[0157] Unless otherwise indicated, the terms "at least," "less than," and "about," or similar terms preceding a series of elements or a range are to be understood to refer to every' element in the series or range. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0158] As used herein, the terms “activatable,” “activate,” “induce,” and “inducible” refers to a inducible IFNalpha prodrug that has an attenuated activity form (e.g.. attenuated receptor binding and / or agonist activity) and an activated form. The inducible IFNalpha prodrug is activated by protease cleavage of the linker that causes the blocking element and half-life extension element to dissociate from the inducible IFNalpha prodrug. The induced / activated IFNalpha prodrug can bind with increased affinity / avidity to the IFNalpha receptor.

[0159] The terms “antibody” and “immunoglobulin” are used interchangeably herein. An antibody or immunoglobulin, as used herein, is intended to refer to immunoglobulin molecules comprised of two heavy (H) chains. Typically, antibodies in mammals (e.g., humans, rodents, and monkey's) comprise four polypeptide chains, two heavy' (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies.monospecific antibodies, multi specific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, or tetrameric antibodies comprising two heavy chain and two light chain molecules. One of skill in the art would recognize that other forms of antibodies exist (e.g. camelid and shark antibodies).

[0160] The term “attenuated” as used herein is an IFNalpha receptor agonist that has decreased receptor agonist activity as compared to the IFNalpha receptor's naturally occurring agonist. An attenuated IFNalpha agonist can have at least about 10X, at least about 50X, at least about 100X, at least about 250X, at least about 500X, at least about 1000X or less agonist activity as compared to the receptor’s naturally occurring agonist. When a inducible IFNalpha prodrug that contains IFNalpha as described herein is described as “attenuated” or having “attenuated activity”, it is meant that the inducible IFNalpha prodrug is an attenuated IFNalpha receptor agonist.

[0161] The term "cancer" refers to the physiological condition in mammals in which a population of cells is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate and / or certain morphological features. Often cancers can be in the form of a tumor or mass, but may exist alone within the subject, or may circulate in the blood stream as independent cells, such a leukemic or lymphoma cells. The term cancer includes all types of cancers and metastases, including hematological malignancy, solid tumors, sarcomas, carcinomas and other solid and non-solid tumors. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., triple negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary’ gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancers. Triple negative breast cancer refers to breast cancer that is negative for expression of the genes for estrogen receptor (ER), progesterone receptor (PR), and Her2 / neu.

[0162] A "conservative" amino acid substitution, as used herein, generally refers to substitution of one amino acid residue with another amino acid residue from within a recognized group which can change the structure of the peptide but biological activity of thepeptide is substantially retained. Conservative substitutions of amino acids are known to those skilled in the art. Conservative substitutions of amino acids can include, but not limited to, substitutions made amongst amino acids within the following groups: (a) M, I, L, V: (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. For instance, a person of ordinary7skill in the art reasonably expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an ammo acid with a structurally related amino acid will not have a major effect on the biological activity of the resulting molecule.

[0163] As used herein, the term “half-life extension element” in the context of the inducible IFNalpha prodrug disclosed herein, refers to a chemical element, preferable a polypeptide that increases the serum half-life and improve pK. for example, by altering its size (e.g., to be above the kidney filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.

[0164] As used herein, the term “operably linked” in the context of a inducible IFNalpha prodrug refers to the orientation of the components of a inducible IFNalpha prodrug that permits the components to function in their intended manner. For example, a polypeptide comprising an IFNalpha subunit and an IFNalpha blocking element are operably linked by a protease cleavable linker in a inducible IFNalpha prodrug when the IF alpha blocking element is capable of inhibiting the IFNalpha receptor-activating activity of the IFNalpha polypeptide, but upon cleavage of the protease cleavable linker the inhibition of the IFNalpha receptor-activating activity7of the IFNalpha polypeptide by the IFNalpha blocking element is decreased or eliminated, for example because the IFNalpha blocking element can diffuse away from the IFNalpha.

[0165] As used herein, the terms “peptide”, “polypeptide”, or “protein” are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably7to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.

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

[0167] As used herein, the term “therapeutically effective amount” refers to an amount of a compound described herein (i.e., a inducible IFNalpha prodrug) that is sufficient to achieve adesired pharmacological or physiological effect under the conditions of administration. For example, a “therapeutically effective amount” can be an amount that is sufficient to reduce the signs or symptoms of a disease or condition (e.g., a tumor). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. A therapeutically effective amount of a pharmaceutical composition can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmaceutical composition to elicit a desired response in the individual. An ordinarily skilled clinician can determine appropriate amounts to administer to achieve the desired therapeutic benefit based on these and other considerations.5. EQUIVALENTS

[0168] It will be readily apparent to those skilled in the art that other suitable modifications and adaptions of the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments. Having now described certain compounds and methods in detail, the same will be more clearly understood by reference to the following examples, which are introduced for illustration only and not intended to be limiting.6. PRODRUG SEQUENCE DISCLOSURE7. EXAMPLES

[0169] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided herein.Example 1. In vivo Dosing and Tumor Processing for TIL Analysis

[0170] All in vivo animal work was performed at Charles River Laboratories (Worcester, MA) according to their standard operating procedures. Briefly, 6-8 week old female C57B1 / 6 mice were subcutaneously implanted with MC38 cells (IxlO5) in 50% Matrigel on the flank, and tumor volume was measured twice a week throughout the course of the experiment. When the average tumor volume was within a pre-set limit (approximately 100 mm3), mice were randomized into treatment groups (Day 0). Mice were dosed intraperitonially with either vehicle or Compound 1 (100 pg / dose) twice a week for two weeks. Peripheral blood and tumors were collected from n=5 mice per group on Days 5, 7, 11, 14, 19, 21, and 25. No vehicle treated tumors were collected on Days 21 and 25, as all tumors in that group had reached the maximum size limit according the IACUC protocol on earlier days and the animals were taken off study. On harvest days, tumors were collected, weighed, and placed in ice cold RPMI-1640 + 10% heat inactivated fetal bovine serum. Likewise, whole blood was collected in K2-EDTA tubes. Both tumor and blood samples were shipped by same day courier to Werewolf Therapeutics for processing. Upon arrival, tumors were chopped into <5 mm3pieces in 3 mL of Hanks Balanced Salt Solution (HBSS) containing 1.25 mg / mL Collagenase Type IV, 0.025 mg / mL Hyaluronidase, and 0.01 mg / mL DNASE I. Tumors were then placed in a C tube and dissociated on a Miltenyi Octomax instrument runningprogram 37C_m_TDK_l. After the digestion program was complete, single cell suspensions were washed with complete media to quench the enzymatic reaction, passed through a MACS SmartStrainer (70 pM), and spun down at 1500 rpm for 5 minutes. Supernatants were decanted, and samples were washed 2 additional times in complete RPMI-1640 before being counted and resuspended at a final concentration of 50 xlO6cells per rnL in complete media. Tumor samples were then analyzed by flow cytometry’ or NanoString analysis. Peripheral blood samples were spun at 2500 rpm for 15 minutes before plasma was collected and frozen for later pharmacokinetic analysis. This experimental protocol corresponds to data presented in FIG.l to FIG. 19CExample 2. Pharmacokinetic Analysis

[0171] Pharmacokinetic analysis was performed using the Verikine-HS Mouse Interferon Alpha All Subtype ELISA Kit (PBL Assay Science) according to the manufacturer’s protocol. Murine plasma was diluted either 1 :50,000 or 1:150,000 in sample diluent before being analyzed for the presence of Compound 1. Results are show n in FIG. 2.Example 3. Cell Staining and Flow Cytometry

[0172] For detection of intracellular cytokines, some samples were restimulated prior to staining for 4 hours at 37°C with complete RPMI-1640 media containing: 10% heat inactivated fetal bovine serum, 50 ng / mL PMA, 1 pg / mL lonomycin, and lx Brefeldin A. Unstimulated samples were also incubated for 4 hours in complete media (RPMI-1640 media containing 10% heat inactivated fetal bovine serum) at 37°C, but without any additional additives. For both stimulated and unstimulated samples, 5 xlO6cells were plated per well in a 96 well round bottom plate and staining was performed. All cell staining was performed in 96 well round bottom plates, and all centrifugation steps were performed at 1600 rpm for 3 minutes unless otherwise noted. To start, 5xl06cells were plated per well in 200 pL of FACS Buffer (AutoMACS Rinsing Buffer (Miltenyi) + 0.5% bovine serum albumin (Miltenyi)).Cells were initially spun down at 1500 rpm for 5 minutes. Cells were then resuspended in 100 pL of FACS buffer containing FC Block reagent at the concentration detailed in the table below for 15 minutes at 4°C. Next, the cells were spun down, and the supernatants were decanted before the cells were washed once with 200 pL of FACS buffer. The cells were resuspended in 100 pL of FACS Buffer containing only the tetramer at the concentration detailed in the table below for 25 minutes at room temperature, in the dark. After tetramer staining, the cells were spun down, and the supernatants were decanted before the cells w ere washed twice with 200 pL of FACS buffer. The cells were resuspended in 100 pl of FACs buffer containing the extracellular staining antibodies at the concentrations detailed in thetable below. Extracellular staining was performed for 20 minutes at 4°C. The cells were spun down before being washed with 200 pL of FACS Buffer. This step was repeated for a total of two washes. After extracellular staining, the cells were resuspended in 100 pL of diluted Fix / Perm Buffer (eBioscience) and fixed for 30 minutes at 4°C. Both Fix / Perm Buffer and Perm / Wash Buffer were diluted according to the manufacturer’s specifications. After fixation / permeabilization, the cells were spun down and washed with 200 pL of Perm Buffer before being spun down again and resuspended in 100 pL of Perm Buffer containing the intracellular staining antibodies at the concentrations detailed in the table below. Intracellular staining was performed for 20 minutes at 4°C. The cells were spun down before being washed with 200 pL of Perm Buffer. This step was repeated for a total of two washes. Lastly, the cells were spun down and resuspended in 130 pL of FACs Buffer for analysis. For single stain controls, both eComp control beads (Thermofisher Scientific) and cells were stained with the appropriate single-color stain using the same protocol as detailed above, except that the eComp control beads were not treated with FC block and were instead incubated in FACs buffer alone during that step. All flow cytometry samples were run on a Cytek Aurora system running SpectroFlo (Version 2.2.0 (10212019)). After collection, data analysis was performed using FlowJo software (vl0.5.3). Gates were defined using Fluorescence minus one (FMO) controls for each stain. Results are shown in FIGs.3A-3G, 4A-4B, 5A-5D, 6, 7A- 7C, 8A-8E, 9A-9F, and 10A-10BTable 7. Materials: Flow Cytometry Panel for TIL AnalysisExample 4. RNA Isolation and Nanostring Analysis

[0173] From each tumor sample, 5 xlO5cells were spun down (1600 rpm for 5 minutes) and resuspended in 100 pL of RLT Lysis buffer. These samples were then flash frozen using a dry ice and ethanol bath and stored at -80°C until they could be shipped to Canopy Biosciences for RNA extraction and NanoString analysis. All RNA extraction and NanoString analysis was performed by Canopy Biosciences using their standard protocols. Briefly, samples were thawed, and RNA was isolated using an RNEasy micro kit (Qiagen) according to the manufacturer’s protocol. RNA was quantified, and 100 ng was loaded into the NanoString cartridge. Nanostring analysis was performed using the Murine Pancancer Immune Profiling Panel and an N Counter system, and 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 generated using Graphpad Prism (v8.4.3). Pathway analysis was performed using Partek software (v 10.0.22.0428), based on transcripts with significantly different expression following Compound 1 treatment, using a p value of less than 0.05 with a FDR step-up of less than0.05. Results are show n in FIGs.llA-llD, 12, 13, 14A-14B, 15A-15F, 16A-16C, 17A-17C, 18A-18C, and 19A-19CExample 5. Anti-Tumor Activity of Compound 1 in Combination with Immune Checkpoint Blockade in the CT26 Tumor Model

[0174] All in vivo animal work was performed at Charles River Laboratories (Morrisville, NC) according to their standard operating procedures. Briefly, 6-8 week old female Balb / C mice were implanted subcutaneously with CT26 cells (3xl05) in 0% Math gel on the flank, and tumor volume was measured twice a week throughout the course of the study. When the average tumor volume was within a pre-set limit (approximately 100-150 mm3), mice were randomized into treatment groups (Day 0). Mice were dosed intraperitonially with either vehicle or Compound 1 twice a week for two weeks at the doses specified in the figure legend. Likewise, mice were dosed on the same schedule (twice a week for two weeks) with individual checkpoint inhibitors at the doses specified in the figures. Results are shown in FIGs. 20A-20E.Table 8. Materials: Checkpoint InhibitorsExample 6. Anti-Tumor Activity of Compound 1 in Combination with Immune Checkpoint Blockade in the MC38 Tumor Model

[0175] All in vivo animal work was performed at Charles River Laboratories (Morrisville, NC) according to their standard operating procedures. Briefly, 6-8 week old female C57B1 / 6 mice were subcutaneously implanted with MC38 cells (5xl05) in 50% Matrigel on the flank, and tumor volume was measured twice a week throughout the course of the study. When the average tumor volume was within a pre-set limit (approximately 100-150 mm3), mice were randomized into treatment groups (Day 0). Mice were dosed intraperitonially with either vehicle or Compound 1 twice a week for two weeks at the doses specified in the figure legend. Likewise, mice w ere dosed on the same schedule (twice a w eek for tw o w eeks) with individual checkpoint inhibitors at the doses specified in the figures. Results are shown in FIGs. 21A-21E.Example 7. In vivo Dosing of the EMT-6, B16-F10, A20, and EG.7 Tumor Models

[0176] All in vivo animal work was performed at either Charles River Laboratories (Worcester, MA) (B16-F10 and EMT-6) or Covance Laboratories (A20 and EG.7) according to their standard operating procedures. Briefly, for the A20 model, 5x105A20 cells were implanted in 0% Matrigel subcutaneously on 6-8 week old Balb / C mice. For the EG.7 model, 1x106EG.7 cells were implanted in 0% Matrigel subcutaneously on 6-8 week old C57B1 / 6 mice. For the B16-F10 model. IxlO5B16-F 10 cells were implanted in 50% Matrigel subcutaneously on 6-8 week old C57B1 / 6 mice. Lastly, for the EMT6 model, 1x105EMT6 cells were implanted in 50% Matrigel subcutaneously on 6-8 week old Balb / C mice. For all the models, tumor volume and body weight were measured every 2-3 days throughout the course of the experiment. When the average tumor volume was within a pre-set limit (approximately 100 mm3for all the tested models), mice were randomized into treatment groups (Day 0). Mice were dosed intraperitonially with either vehicle or Compound 1 at the specific doses noted in the figure legends twice a week for two weeks.Example 8. Compound 5 Processing by Primary Dissociated Human Tissue Samples

[0177] Cells derived from healthy human tissue were purchased from various vendors (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, before being frozen into single use vials. The dissociated tumor samples were generated from surgically resected primary human tumors that were enzymatically digested on site prior to being frozen. Therefore, these samples contain a mixture of all the cell types found in a primary human tumor, including immune cells, tumor cells, and other stromal cells. All purchased samples were shipped to Werewolf Therapeutics on dry ice and were stored at -140°C. Several tumor samples were also freshly obtained after surgical resection and were shipped overnight to Werewolf Therapeutics on wet ice in RPMI- 1640 media. Fresh tumor samples w ere processed into single cell suspensions at Werewolf Therapeutics. Upon arrival, samples were weighed and minced with a scalpel into <5 mm3samples, before being enzymatically digested using the following enzyme cocktail in Leibovitz Media L 15: Collagenase I (45 U / mL). Collagenase II (15 U / mL), Collagenase IV (45 U / mL), DNase I (50,000 U / mL), Elastase (0.075 U / mL). Samples were digested in 25 mL of digestion media per 500 mg of tissue, and digestion w as performed at 37°C for 45 minutes while shaking at 100 rpm. After enzy matic digestion, samples were mechanically dissociated through a 70 pM filter, before being thoroughly washed, counted, and frozen in Recovery Cell Culture Freezing Media for later use. To examine INDUKINE molecule processing.samples were thawed, washed, and counted. Cells were then resuspended in X-Vivo 15 media, and between 0.75-lxl05viable 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 before cell culture supernatants were collected and frozen for later analysis. Each condition was run in duplicate whenever possible. In all experiments, protease activated (cleaved) Compound 5 was included as a positive control.Table 9. Materials: Human Cell TypesExample 9. Measurement of Compound 5 Activity Using Human PBMCS

[0178] Human buffy coats were isolated at Research Blood Components or BioIVT and were shipped by same day courier at room temperature to Werewolf Therapeutics for PBMC isolation. Upon arrival, buffy coats were diluted 1 :4 in PBS, and 25 mLs of the cell suspension was gently layered over 25 mLs of Ficoll-Paque Plus. Cells were spun at room temperature for 40 minutes at 2000 rpm with low acceleration (setting #2) and with the brake turned off. After centrifugation, PBMCs were collected from the interface of the two layers and washed three times with 50 mLs of PBS before being counted and frozen in Recovery Cell Culture Freezing Media for later use. To measure INDUKINE activity, PBMCs w ere thawed and counted prior to being resuspended at IxlO6cells / mL in X-Vivo 15 media. 1x10sPBMCs (100 pL) were plated per well in a 96 well round bottom plate. Next, the cell culture supernatants (collected from primary tumor samples incubated with the INDUKINE molecules) were thawed, and 100 pL was used to stimulate the PBMCs. This resulted in a 1:2 dilution of the conditioned media with fresh media. After 48 hours at 37°C and 5% CO2, thecell cultures were mixed 3 times with a multichannel pipette and spun down at 1600 rpm for 3 minutes. Supernatants from the stimulated PBMCs were collected, and IP-10 / CXCL10 production was measured with a Human IP-10 / CXCL10 AlphaLisa kit (Perkin Elmer) using the manufacturer’s protocol with one notable exception. 0.75X of the recommended concentration of beads and antibodies were used. 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 the standard curve using the method described by the manufacturer’s protocol. Specifically, a nonlinear, 4-parameter logistic regression (sigmoidal dose-response curve with variable slope) with a l / Y2data weighting was used to fit the data. Data analysis and graphs were generated in GraphPad Prism 8 Software for Windows (64- Bit) (v8.4.2 (679)).

[0179] By comparing the baseline activity of Compound 5 incubated without primary human cells (fully intact input control, 0% of full activity) to that of cleaved Compound 5 incubated with the dissociated tumor sample (fully cleaved positive control, 100% of full activity), a dynamic range for the assay could be established. By normalizing to the dynamic range, the results from multiple experiments could be compiled into a broad dataset. Mathematically, the percent of activity generated by exposing Compound 5 to the primary human samples was calculated, using the following equation:

[0180] % of Total Activity = 100 *(Sample Measurement-No Cells Control Measurement)(Cleaved WTX-613 Measurement-No Cells Control Measurement)Example 10. Efficacy of Compound 1 in mEER tumor model

[0181] 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 infections are attributed to, in part, evasion of host immune responses by viral oncoproteins. To test the anti-tumor activity of Compound 1, a syngeneic murine tumor model of HPV-OSCC (mEER) featuring murine pharyngeal epithelial cells transformed with HPV 16 E6 and E7 oncogenes and H-ras w as utilized. Six- to eight- week old female C57 / B16 mice (Charles River) were inject with 3 x 105MEER cells in 50% matrigel at day 0. Mice were dosed intrapentomally with either vehicle or Compound 1 at 100 pg or 400 pg twice a w eek for two w eeks starting at day 0. A fourth group of mice were dosed intraperitonially with 35 pg free IFNa twice daily for five days a week for tw o w eeks starting at day 0. For each of the four groups, five mice were sacrificed at day 6 to harvest spleen and quantitate populations of Tumor Infiltrating Lymphocytes(TILs), and eight mice per group continued the study to determine efficacy. TILs were quantified as described in Example 3. Cytokine levels were measured using V-Plex Mouse Proinfl ammatory Panel 1 (Meso Scale Discovery; Rockville, MD).

[0182] Results showed that Compound 1 at both doses tested had a more durable anti-tumor response in the mEER model of HPV-driven OSCC compared with free IFNa (FIG. 24). Further, Compound 1 led to increased frequency of activated CD25+. IFNg+, TNFa+ Granzyme B+,or T box TS factor (Tbet)+ CD8+ T Cells (FIGs. 25 AE) and polyfunctional cells (cells expressing more than one of IFNg, TNFa, or GranzymeB; data not shown). Increased NK cell activation and an upregulation of MHC class I expression were also observed in mice dosed with Compound 1 (FIGs. 26A-B and 27A-B). Additionally, treatment with Compound 1 led to increases in dose-dependent production of IFNg, TNFa, CXCL10 and IL-10 compared to Vehicle and free IFNa (FIGs. 28A-F).

Claims

CLAIMSIt is claimed:1 . A method for selectively activating effector CD8+ T cells in the tumor microenvironment, comprising administering to a subject in need thereof an effective amount of an inducible interferon alpha (IFNalpha) prodrug, wherein the inducible IFNalpha prodrug is administered systemically, is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, and results in significantly higher frequency of CD8+ T cells that produce IFN gamma and granzyme B within the tumor in comparison to peripheral tissue.

2. A method for selectively activating tumor infiltrating lymphocytes, comprising administering to a subject in need thereof an effective amount of an inducible interferon alpha (IFNalpha) prodrug, wherein the inducible IFNalpha prodrug is administered systemically, is activated by cleavage by a protease that has higher activity7in the tumor microenvironment than in other locations, and results in significantly higher frequency of CD8+ T cells that produce IFN gamma and granzyme B within the tumor in comparison to peripheral tissue.

3. The method of any one of claims 1 or 2, wherein the method results in a significant increase in the tumor reactive CD8+ / Treg ratio within the tumor microenvironment.

4. The method of any one of the preceding claims, wherein the method results in a decrease in the frequency of myeloid-derived suppressor cells and / or Treg cells within the tumor microenvironment.

5. The method of any one of the preceding claims, wherein the method results in increased expression of an immune checkpoint protein.

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

7. The method of any one of the preceding claims, wherein the method results in increased expression of MHC class I and MCH class II expression.

8. The method of any one of the preceding claims, wherein the method results in prolonged natural killer cell activation.

9. The method of any one of the preceding claims, wherein the inducible IFNalpha 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 foregoing.

10. The method of any one of the preceding claims, wherein the inducible IFNalpha prodrug is administered about twice a week or less frequently.

11. The method of any one of the preceding claims, wherein the inducible IFNalpha prodrug is administered about once a week or less frequently.

12. The method of any one of the preceding claims, wherein the inducible IFNalpha prodrug is administered about once every two weeks.

13. A method of regulating tumor microenvironment,, comprising administering to a subject in need thereof an effective amount of an inducible interferon alpha (IFNalpha) prodrug, wherein the inducible IFNalpha prodrug is administered systemically and is activated by cleavage by a protease that has higher activity in the tumor microenvironment than in other locations, wherein the method results in at least one effect selected from the group consisting of selectively activating effector CD8+ T cells in the tumor microenvironment, selectively activating tumor infiltrating lymphocytes, increasing in the tumor reactive CD8+ / Treg ratio within the tumor microenvironment, decreasing in the frequency of myeloid-derived suppressor cells and / or Treg cells within the tumor microenvironment, increasing expression of an immune checkpoint protein in the tumor microenvironment, increasing expression of MHC class I and MCH class II expression, and / or prolonging natural killer cell activation.

14. The method of claim 13, wherein the method results in increased expression of at least one. two, three, four, five, six or more immune checkpoint proteins in the tumor microenvironment.

15. The method of claim 13 or 14, wherein the immune checkpoint protein is PD-L1. PD-1, TIGIT, PVR, CTLA-4, or LAG-3.1 . The method of claim 13 or 14, wherein the immune checkpoint protein is PD-L1 orPD-1.

17. The method of any one of claims 13 to 16, wherein the inducible IFNalpha prodrug is administered about twice a week or less frequently.

18. The method of any one of claims 13 to 16, wherein the inducible IFNalpha prodrug is administered about once a week or less frequently.

19. The method of any one of claim 13 to 16, wherein the inducible IFNalpha prodrug is administered about once every two weeks.

20. The method of any one of claims 13-19, wherein the method results in at least one effect over at least about 7 days, such as at least about 7, 8, 9, 10, 11, 12, 13, 14 or 15 days, after the final administration of the inducible IFNalpha prodrug.

21. The method of any one of claims 1-8 and 13-20, wherein the inducible interferon alpha (IFNalpha) prodrug comprises a fusion polypeptide having the formula of: [D]-[L1]- [A]-[L2’]-[H], wherein,[A] is an interferon alpha (IFNa) polypeptide, a mutein. or an active fragment thereof[D] is a blocking moiety,[H] is a half-life extension moiety,[LI] is a protease-cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO:

6. 9, or 12, and[L2’] is a protease-cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 6, 9, or 12, wherein the blocking moiety and the half-life extension moiety each independently comprise human serum albumin (HSA) or an antibody or antibody fragment that binds the HSA.

22. A method for treating cancer, comprising administering to a subject in need thereof 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 an amino acid sequence variant of any of the foregoing and a checkpoint inhibitor.

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

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

25. The method of claim 22. wherein the checkpoint inhibitor is an anti-CTLA4 antibody or fragment thereof.

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

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

28. The method of claim 23. wherein the anti-PD-1 antibody is selected from the group consisting of AMP-224 (AstraZemca), 609A (3SBio), 704 (3SBio), 705 (3SBio), ABBV-181 (AbbVie), ADU-1503 I bion-004 (Chinook Therapeutics), AGEN2034 / balstilimab (Agenus), AK103 (Akeso), AK104 (Akeso), AK112 (Akeso), AK123 (Akeso). AMG 256 (Amgen), AMG 404 (Amgen). ANB030 (AnaptysBio), ANKEBIO Anti-PDl product (Anhui Anke Biotechnology), Anti PD-1 / Anti-CD47 (DiNonA), ASKG915 (Ask Gene Pharmaceuticals), AV-MEL-1 (Aivita Biomedical), BCD-100 (Biocad CJSC), BI 754091 (Boehringer Ingelheim), BiCKI-IL-7 (OSE Immunotherapeutics). Boehringer-PD-1 -unknown (Boehringer Ingelheim), BSK-050K01 (Biosion), Camrelizumab (Jiangsu Hengrui Medicine), CB201 (Crescendo Biologies), CB213 (Crescendo Biologies), CC-90006 (AnaptsBio), cetrelimab (J&J), chPDl (Kiromic Biopharma), CMAB819 (Mabpharm), CS1003 (CStone Pharmaceuticals), CS17938 (Shenzhen Chipscreen Biosciences), CTX-8371 (Compass Therapeutics), CX-072 (CytomX Therapeutics). CX-188 (CytomX Therapeutics), cypalizumab (Harbin Gloria Pharmaceuticals), DB004 (DotBio), EMB02 (EpimAb Biotherapeutics), Geptanblimab / genolimzumab (Apollomics), GS19 (Suzhou Zelgen Biopharmaceuticals), HLX10 (Shanghai Henlius Biotech), HX008 (Taizhou HanZhong Pharmaceuticals), HY003 (Juventas Cell Therapy), IBI315 I BH2950 (Innovent Biologies), IBB 18 (Innovent Biologies), IBB 19 (Innovent Biologies), IMM1802 (ImmuneOnco Biopharma), IMT200 (TrueBinding), Jemperli / dostarlimab (AnaptysBio), JTX-4014(Jounce Therapeutics), Keytruda / pembrolizumab (Merck), LBL-006 (Nanjing Leads Biolabs). Libtayo / cemiplimab-rwlc (Regeneron Pharmaceuticals). LVGN3616 (Lyvgen Biopharma), LXF821 (Novartis), LY01015 (Luye Pharma Group), LY3462817 (Eli Lilly), MCLA-134 (Merus N.V.), MEDI5752 (AstraZemca), NIR178 (Novartis), ONCR-177 (Oncorus), ONO-4685 (Ono Pharmaceutical), Opdivo / nivolumab (Ono Pharmaceutical), MGD019 (MacroGenius), PD1-GDT CAR-T (Kiromic Biopharma), penpulimab (Akeso), PSB205 (Qilu Puget Sound Biotherapeutics), PT-001 (Merck), PT627 (Merck), RB-M1 (Refuge Biotechnologies), Retifanlimab (MacroGenics), RG6139 (Roche), RG6279 (Roche), RTX-002 (RubrYc Therapeutics), sasanlimab (Pfizer), Servier-PD l xLAG3-unknown (Servier), SL-279252 / TAK-252 (Shattuck Labs), Sofusa anti-PDl (Sorrento Therapeutics), spartalizumab (Novartis), SSI-361 (Lyvgen Biopharma), Sym021 (Sender), Tebotelimab (MacroGenics), tislelizumab (BeiGene), TSR-075 (AnaptsBio), Tuhura-DO / PD-1 -unknown (Tuhura Biopharma), toripalimab (Shanghai Junshi Biosciences), sintilimab (Innovent Biologies), Unicar-CAR-T&PD-l -unknown (Shanghai Unicar-Therapy Bio-Medicine Technology’), Xdivane (Xbrane Biopharma), XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y-Biologics). zimberelimab (Arcus Biosciences)29. The method of claim 24, wherein the anti-PD-Ll antibody is chosen from the group consisting of A167 (Sichuan Kelun), ABL501 (ABL Bio), ABL503 (ABL Bio), ABSK041 (Abbisko Therapeutics), ACE 1708 (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-Ll -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 (ChemoC entry x), CDR101 (CDR-Life), cosibelimab (Checkpoint Therapeutics), CTX-8371 (Compass Therapeutics), DiNonA-Solid Tumors-unknown (DiNonA). DR30207 (Zhejiang Doer Biologies), DuoBody-PD-Llx4-lBB (Ligand Pharmaceuticals), envafolimab (Alphamab Oncology'), EPIM-001 (Elpis Biopharmaceuticals), ES101 (Elpiscience Biopharma), INBRX- 105 (Inhibrx), FAZ053 (Novartis). FS118 (F-star Therapeutics), GB262 (Genor Biopharma), GS-4224 (Gilead), GT900008 (Kintor Pharmaceuticals), GX-P2 (Genexine), Hamni-PS- Ll / CD47-Unknown (Hanmi Pharmaceutical), HBM7015 (HBM Holdings), HBM9167(HBM Holdings), HLX20 (Shanghai Henlius Biotech), HTI-1088 (Jiangsu Hengrui Medicine), IBI318 (Innovent Biologies), IBI322 (Innovent Biologies), IBI323 (Innovent Biologies), IGM-7354 (IGM Biosciences), IMC-001 (Sorrento Therapeutics), Imfinzi / durvalumab (AstraZenica), IMM25 (ImmuneOnco Biopharma), IMM2502 (ImmuneOnco Biopharma), IMM2503 (ImmuneOnco Biopharma), IMM2504 (ImmuneOnco Biopharma), INCB86550 (Incyte), 10103 (IO Biotech). JS003 (Shanghai Junshi Biosciences). Jubilant- PD-L I -unknown (Jubilant Therapeutics), KD033 (Kadmon Holdings), KN046 (Alphamab Oncology), KY1003 (Sanofi), KY1043 (Sanofi), LY3300054 (Eli Lilly), LY3415244 (Eli Lilly), MRNA-6981 (Modema), MSB2311 (Transcenta Holding), MT-6035 (Molecular Templates), ND021 / NM21-1480 (Numab Therapeutics), OXOOIR (Oxford BioTherapeutics), PD-L1 based BsAbs (I-Mab). PD-L1 Boltbody IS AC (Bolt Biotherapeutics), PDL-GEX (Glycotope GmbH), PMC- 122 (PharmAbcine), PMI06 (D&D Pharmatech), Protheragen-RV-scFv-PDLl -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-siPDLl (TransCode Therapeutics), UniCAR-T-PD-Ll (GEMoaB monoclonals), Vaximm (VXM10), and YBL- 013 (Y-Biologics).

30. A method for treating a cancer, comprising administering to a subject in need thereof a checkpoint inhibitor and an inducible interferon alpha (IFNalpha) prodrug comprising a fusion polypeptide having the formula of: [D]-[L1]-[A]-[L2’]-[H], wherein,[A] is an interferon alpha (IFNalpha) polypeptide, a mutein, or an active fragment thereof[D] is a blocking moiety,[H] is a half-life extension moiety,[LI] is a protease-cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO:

6. 9, or 12, and[L2’] is a protease-cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 6, 9, or 12, wherein the blocking moiety and the half-life extension moiety each independently comprise human serum albumin (HSA) or an antibody or antibody fragment that binds the HSA.

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

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

33. The method of claim 30. wherein the checkpoint inhibitor is an anti-CTLA4 antibody or fragment thereof.

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

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

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

37. The method of claim 31, wherein the anti-PD-1 antibody is selected from the group consisting of 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), AMG 256 (Amgen), AMG 404 (Amgen). ANB030 (AnaptysBio), ANKEBIO Anti-PDl product (Anhui Anke Biotechnology), Anti PD-1 1 Anti-CD47 (DiNonA), ASKG915 (Ask Gene Pharmaceuticals), AV-MEL-1 (Aivita Biomedical), BCD-100 (Biocad CJSC), BI 754091 (Boehringer Ingelheim), BiCKI-IL-7 (OSE Immunotherapeutics), Boehringer-PD-1 -unknown (Boehringer Ingelheim), BSK-050K01 (Biosion), Camrelizumab (Jiangsu Hengrui Medicine), CB201 (Crescendo Biologies). CB213 (Crescendo Biologies), CC-90006 (AnaptsBio). cetrelimab (J&J), chPDl (Kiromic Biopharma). CMAB819 (Mabpharm). CS1003 (CStone Pharmaceuticals), CS17938 (Shenzhen Chipscreen Biosciences), CTX-8371 (Compass Therapeutics), CX-072 (CytomX Therapeutics), CX-188 (CytomX Therapeutics), cypalizumab (Harbin Gloria Pharmaceuticals), DB004 (DotBio), EMB02 (EpimAb Biotherapeutics). Geptanblimab / genolimzumab (Apollomics). GS19 (Suzhou Zelgen Biopharmaceuticals), HLX10 (Shanghai Henlius Biotech), HX008 (Taizhou HanZhongPharmaceuticals), HY003 (Juventas Cell Therapy), IBI315 / BH2950 (Innovent Biologies), I Bl 318 (Innovent Biologies), IBI319 (Innovent Biologies), IMM1802 (ImmuneOnco Biopharma), IMT200 (TrueBinding), Jemperli I dostarlimab (AnaptysBio), JTX-4014 (Jounce Therapeutics), Keytruda / pembrolizumab (Merck), LBL-006 (Nanjing Leads Biolabs), Libtayo / cemiplimab-rwlc (Regeneron Pharmaceuticals), LVGN3616 (Lyvgen Biopharma), LXF821 (Novartis), LY01015 (Luye Pharma Group), LY3462817 (Eli Lilly). MCLA-134 (Merus N.V.). MEDI5752 (AstraZemca). NIR178 (Novartis), ONCR-177 (Oncorus), ONO-4685 (Ono Pharmaceutical), Opdivo / nivolumab (Ono Pharmaceutical), MGD019 (MacroGenius), PD1-GDT CAR-T (Kiromic Biopharma), penpulimab (Akeso), PSB205 (Qilu Puget Sound Biotherapeutics), PT-001 (Merck), PT627 (Merck), RB-M1 (Refuge Biotechnologies), Retifanlimab (MacroGenics), RG6139 (Roche). RG6279 (Roche), RTX-002 (RubrYc Therapeutics), sasanlimab (Pfizer), Servier-PDlxLAG3-unknown (Sender), SL-279252 / TAK-252 (Shattuck Labs), Sofusa anti-PDl (Sorrento Therapeutics), spartalizumab (Novartis), SSI-361 (Lyvgen Biopharma), Sym021 (Sender), Tebotelimab (MacroGenics), tislelizumab (BeiGene). TSR-075 (AnaptsBio). Tuhura-DO / PD-1 -unknown (Tuhura Biopharma), toripalimab (Shanghai Junshi Biosciences), sintilimab (Innovent Biologies), Unicar-CAR-T&PD-l -unknown (Shanghai Uni car-Therapy Bio-Medicine Technology ), Xdivane (Xbrane Biopharma), XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y-Biologics), zimberelimab (Arcus Biosciences)38. The method of claim 32, wherein the anti-PD-Ll antibody is chosen from the group consisting of A167 (Sichuan Kelun), ABL501 (ABL Bio), ABL503 (ABL Bio), ABSK041 (Abbisko Therapeutics), ACE 1708 (Acepodia), ACE-NK-PDL1 (Acepodia), ADG104 (Adagene). AK106 (Akeso), ALPN-202 (Alpine Immune Sciences), AN4005 (Adlai Nortye Biopharma), BMS-936559 / MDX-1 105 (BMS), APL-502 / TQB2450 (Apollomics), Arbutus-PD-Ll -unknown (Arbutus Biopharma), ASC22 (Ascletis Pharma), ATG-101 (Antengene), AVA-004 (Av acta Group), AVA021 (Av acta Group), AVA027 (Av acta Group), AVA-040-100 (Avacta Group), AVA04-Vbp (Av acta 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 Tumors-unknown (DiNonA). DR30207 (Zhejiang Doer Biologies). DuoBody-PD-Llx4-lBB (Ligand Pharmaceuticals), envafolimab (Alphamab Oncology), EPIM-001 (Elpis Biopharmaceuticals), ES101 (Elpiscience Biopharma), INBRX-105 (Inhibrx), FAZ053 (Novartis). FS118 (F-star Therapeutics), GB262 (Genor Biopharma), GS-4224 (Gilead), GT900008 (Kintor Pharmaceuticals), GX-P2 (Genexine), Hamni-PS- Ll / CD47-Unknown (Hanmi Pharmaceutical), HBM7015 (HBM Holdings), HBM9167 (HBM Holdings), HLX20 (Shanghai Henlius Biotech), HTI-1088 (Jiangsu Hengrui Medicine), IBB 18 (Innovent Biologies), IBI322 (Innovent Biologies), IBI323 (Innovent Biologies), IGM-7354 (I GM Biosciences), IMC-001 (Sorrento Therapeutics), Imfinzi / durvalumab (AstraZenica), IMM25 (ImmuneOnco Biopharma), IMM2502 (ImmuneOnco Biopharma), IMM2503 (ImmuneOnco Biopharma), IMM2504 (ImmuneOnco Biopharma), INCB86550 (Incyte), 10103 (IO Biotech), JS003 (Shanghai Junshi Biosciences), Jubilant- PD-L1 -unknown (Jubilant Therapeutics), KD033 (Kadmon Holdings), KN046 (Alphamab Oncology). KY1003 (Sanofi), KY1043 (Sanofi), LY3300054 (Eh Lilly), LY3415244 (Eli Lilly), MRNA-6981 (Modema), MSB231 1 (Transcenta Holding), MT-6035 (Molecular Templates), ND021 / NM21-1480 (Numab Therapeutics), OXOOIR (Oxford BioTherapeutics), PD-L1 based BsAbs (I-Mab), PD-L1 Boltbody IS AC (Bolt Biotherapeutics). PDL-GEX (Glycotope GmbH), PMC- 122 (PharmAbcine), PMI06 (D&D Pharmatech). Protheragen-RV-scFv-PDLl -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-siPDLl (TransCode Therapeutics), UniCAR-T-PD-Ll (GEMoaB monoclonals), Vaximm (VXM10), and YBL- 013 (Y-Biologics).

39. The method of any one of claims 21 and 30-38, wherein the IFNalpha polypeptide comprises a murine interferon alpha 1 (mIFNal), murine interferon alpha 11 (mIFNal l), human interferon alpha 2b (IFNA2b), murine interferon alpha 11 (mIFNal 1), interferon alpha 8 (IFNA8), interferon alpha 14 (IFNA14), interferon alpha 16 (IFNA16), or a mutein thereof.

40. The method of claim 39, wherein the IFNalpha polypeptide comprises the amino acid sequence of SEQ ID NOs: 234-237.

41. The method of claim any one of claims 21 and 30-40, wherein each of the blocking moiety and the half-life extension moiety comprises HSA.

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

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

44. The method of claim 42 or 43, wherein at least one of the blocking moiety' and the half-life extension moiety comprises the antibody or antibody fragment that binds 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 of any one of claims 21 and 30-44, wherein each of [LI] and [L2’] comprises SEQ ID NO:

6. 9 or 12.

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

47. The method of any one of claims 1 -46, wherein the inducible IFNalpha prodrug is activated in a tumor microenvironment of a bladder cancer, a 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), nonsmall cell lung cancer- squamous (NSCLC-Sq), ovarian cancer, or uterine cancer.

Images