EP4680264A2 - Promédicaments d'interféron - Google Patents

Promédicaments d'interféron

Info

Publication number
EP4680264A2
EP4680264A2 EP24771700.2A EP24771700A EP4680264A2 EP 4680264 A2 EP4680264 A2 EP 4680264A2 EP 24771700 A EP24771700 A EP 24771700A EP 4680264 A2 EP4680264 A2 EP 4680264A2
Authority
EP
European Patent Office
Prior art keywords
ifnalpha
therapeutics
antibody
seq
cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24771700.2A
Other languages
German (de)
English (en)
Inventor
Jose Andres SALMERON-GARCIA
Heather R. BRODKIN
Kyriakos ECONOMIDES
Daniel J. Hicklin
Cynthia Seidel-Dugan
Philipp W. STEINER
William M. Winston
Christopher J. NIRSCHL
Robin C. HUMPHREYS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jazz Pharmaceuticals Ireland Ltd
Original Assignee
Jazz Pharmaceuticals Ireland Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jazz Pharmaceuticals Ireland Ltd filed Critical Jazz Pharmaceuticals Ireland Ltd
Publication of EP4680264A2 publication Critical patent/EP4680264A2/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • Interferons 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.
  • IFN Interferons
  • IFN’s are a potent immune antagonist and has been considered a promising therapeutic agent for oncology 7 .
  • IFN’s have shown to have a narrow therapeutic window 7 because they are highly potent and have a short serum half-life. Consequently, therapeutic administration of IFN produce undesirable systemic effects and toxicities.
  • IFN cytokines
  • 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.
  • 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.
  • the inducible IFNalpha prodrug is conditionally active.
  • the prodrug typically remains intact.
  • the intact prodrug has attenuated IFNalpha receptor agonist activity 7 .
  • 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.
  • 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. These methods 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.
  • 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.
  • the method results in the at least one effect over
  • 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.
  • 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 activity 7 in the tumor microenvironment than in other locations.
  • the method results in increased expression of at least one, two, three, four, five, six or more immune checkpoint proteins.
  • the immune checkpoint protein is PD-F1.
  • the immune checkpoint protein is PD- 1.
  • the immune checkpoint protein is TIGIT and/or PVR.
  • 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.
  • 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.
  • HSA human serum albumin
  • 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.
  • 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.
  • 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), AGEN2034
  • CTX-8371 Compass Therapeutics
  • CX-072 CytomX Therapeutics
  • CX-188 CytomX Therapeutics
  • cypalizumab Hardbin Gloria Pharmaceuticals
  • DB004 DotBio
  • EMB02 EpimAb Biotherapeutics
  • Geptanblimab / genolimzumab Apollomics
  • GS19 Suzhou Zelgen Biopharmaceuticals
  • HLX10 Shanghai Henlius Biotech
  • HX008 Tiaizhou HanZhong Pharmaceuticals
  • HY003 Jitatias Cell Therapy
  • IBB 15 / BH2950 Innovent Biologies
  • IBB 18 Innovent Biologies
  • IBB 19 Innovent Biologies
  • IMM1802 ImmuneOnco Biopharma
  • IMT200 TrusteBinding
  • Jemperli / dostarlimab AnaptysBio
  • JTX-4014 Jounce Therapeutics
  • 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).
  • 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
  • IBB 18 Innovent Biologies
  • IBI322 Innovent Biologies
  • IBI323 Innovent Biologies
  • IGM-7354 IGM Biosciences
  • IMC-001 Surrento 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 Jubilant-PD-Ll -unknown
  • KD033 Kermmon Holdings
  • KN046 Alphahamab 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 Nab Therapeutics
  • 0X001R Olford 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 Najing Sanhome Pharmaceutical
  • sugemalimab Liigand Pharmaceuticals
  • atezolizumab Roche
  • TST005 Transcenta Holding
  • TT-01 Topicmunnity Therapeutics
  • TTX-siPDLl TransCode Therapeutics
  • UniCAR-T-PD-Ll GEMoaB monoclonals
  • VXM10 Vaximm
  • YBL- 013 Y-Biologics
  • 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.
  • HSA human serum albumin
  • 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.
  • 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.
  • mIFNal murine interferon alpha 1
  • mIFNal 1 murine interferon alpha 11
  • IFNA2b human interferon alpha 2b
  • IFNA8 interferon alpha 1 1
  • IFNA8 interferon alpha 8
  • IFNA14 interferon alpha 14
  • IFNA16 interferon alpha 16
  • the IFNalpha polypeptide comprises the amino acid sequence of SEQ ID NO: 234-237.
  • each of the blocking moiety and the half-life extension moiety comprises HSA.
  • each of the blocking moiety and the half-life extension moiety comprises the antibody or antibody fragment that binds HSA.
  • 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.
  • 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.
  • each of [LI] and [L2’] comprises SEQ ID NO: 6, 9 or 12.
  • the fusion polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 1-5 and 238-257.
  • 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.
  • NSCLC-Ad non-small cell lung cancer- adenocarcinoma
  • NSCLC-Sq non-small cell lung cancer- squamous prodrug
  • 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).
  • 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.
  • 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.
  • PMN-MDSCs FIG. 3C
  • G-MDSCs FIG. 3D
  • Tregs FIG. 3E
  • 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.
  • data are represented as the mean + SD, and P values are derived from t tests (**, p ⁇ 0.01; ***, p ⁇ 0.001; ****, p ⁇ 0.00001).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • FIGs. 9A B-cell MCH class I
  • FIG. 9B CD1 lb+ dendritic cells MHC class I
  • FIG. 9C CD 103+ dendritic cells MHC class I
  • B-cell MHC class II FIG. 9D
  • CDl lb+ DC: MHC class II FIG. 9E
  • CD103+ DC MHC class II FIG. 9
  • FIG. 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+.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. 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
  • 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.
  • 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.
  • 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.
  • 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).
  • 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. 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.
  • 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.
  • 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-4 antibody 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.
  • 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.
  • FIG. 23 shows Compound 5 activation by human tumor samples compared to human healthy cells.
  • 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).
  • OSCC oral squamous cell carcinoma
  • 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.
  • 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.
  • IFNg interferon gamma
  • 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. 27A shows 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.
  • 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).
  • 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.
  • the disclosure relates to inducible IFNalpha prodrugs that contain at least one polypeptide chain, and can contain two or more polypeptide chains, if desired.
  • 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.
  • 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.
  • 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.
  • a form of IFNalpha is released that is active (e.g., more active than the inducible IFNalpha prodrug).
  • the released IFNalpha is at least 10 x more active than the inducible IFNalpha prodrug.
  • 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.
  • 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 activity 7 (e.g., tumor microenvironment).
  • 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],
  • 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.
  • HSA human serum albumin
  • 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.
  • 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.
  • 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).
  • half-life extension elements e.g., an antibody fragment that binds HSA.
  • the inducible IFNalpha prodrug can have the Formula XII: [D] -[L 1 ] - [A]-[L1 ’]-[D’].
  • [A] is an IFNalpha polypeptide.
  • [LI] is a protease-cleavable polypeptide linker
  • [LT] is a protease-cleavable polypeptide linker
  • [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.
  • 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.
  • 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.
  • the IFNalpha polypeptide comprises the amino acid sequence of SEQ ID NOs: 234-237.
  • the IFNalpha polypeptide and the blocking element and/or the half-life extension element can be operably linked by the protease-cleavable polypeptide.
  • the inducible IFNalpha prodrug can be of any of 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
  • [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.
  • each of [LI] and [L2’] comprises SEQ ID NO: 6, 9 or 12.
  • 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.
  • 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.
  • VL+CL antibody light chain
  • VL light chain variable domain
  • VH + CHI antibody heavy chain Fab fragment
  • VH heavy chain variable domain
  • 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],
  • [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
  • [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).
  • 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 a half-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.
  • IFNalpha prodrugs comprising two or more polypeptide chains have been described in International Application No.: PCT/US2022/040564.
  • the half-life extension element increases the in vivo half-life and provides altered pharmacodynamics and pharmacokinetics of the inducible IFNalpha prodrugs.
  • the half-life extension element alters pharmacodynamics properties including alteration of tissue distribution, penetration, and diffusion of the inducible IFNalpha prodrug.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) and a variable domain of camelid-type nanobody (VHH), a dAb and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • HSA human serum albumin
  • 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.
  • 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.
  • Fab antigen-binding fragment
  • scFv single chain variable fragment
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VHH camelid-type nanobody
  • 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.
  • suitable blocking polypeptides include polypeptides that sterically inhibit or block binding of IFNalpha to its cognate receptor.
  • such moieties can also function as half-life extending elements.
  • 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.
  • IFNalpha blocking elements that are suitable are single chain variable fragments (scFv) or Fab fragments.
  • 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.
  • VL+CL antibody light chain
  • VL light chain variable domain
  • VH + CHI antibody heavy chain Fab fragment
  • VH heavy chain variable domain
  • 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.
  • 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.
  • the desired protease is enriched or selectively expressed at the desired target site of IFNalpha activity (e.g., the tumor microenvironment).
  • the inducible IFNalpha prodrug is preferentially or selectively cleaved at the target site of desired IFNalpha activity.
  • 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.
  • 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.
  • 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.
  • the linker is cleavable by a cleaving agent, e.g., an enzyme.
  • the linker comprises a protease cleavage site.
  • 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.
  • protease-cleavable linkers that are preferentially cleaved at a desired location in the body, such as the tumor microenvironment, relative to the peripheral circulation.
  • 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).
  • 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
  • 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.
  • MMP matrix metalloproteases
  • Proteases capable of cleaving linker amino acid sequences 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).
  • 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.
  • the linker can be cleaved by MMP14 or Cathepsin L.
  • 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.
  • Some preferred protease-cleavable linkers are cleaved by a MMP and/or a cathepsin.
  • 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.
  • 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.
  • 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.
  • 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); GGPLAQKLKSSA
  • 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).
  • 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.
  • 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.
  • 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).
  • the linker can comprise two cleavage 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • amino acid positions for the linker comprising the sequence GPAGLYAQ SEQ ID NO: 6
  • sequence GPAGLYAQ SEQ ID NO: 6
  • AFKSSFP disclosed as SEQ ID NO: 9.
  • amino acids surrounding the cleavage site e.g., positions Pl and Pl ’for SEQ ID NOs: 6-12 or 232-233
  • the amino acids surrounding the cleavage site are not substituted.
  • 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.
  • 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.
  • the functional variants of GPAGLYAQ (SEQ ID NO: 6) is cleaved by MMP14.
  • ALFKSSFP (SEQ ID NO: 9) is cleaved by Capthepsin L (CTSL1).
  • CTSL1 Capthepsin L
  • 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).
  • a target site e.g., a tumor microenvironment that expresses high levels of the protease.
  • 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.
  • 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).
  • 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
  • the amino acids at positions Pl and Pl ’ in SEQ ID NO: 9 are K and S.
  • the functional variant of GPAGLYAQ 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‘.
  • GPAGLYAQ 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’.
  • the functional variant of GPAGLYAQ (SEQ ID NO: 6) does not comprise an amino acid substitution at position Pl and/or Pl’.
  • the amino acid substitution of the functional variant of GPAGLYAQ preferably comprises an amino acid substitution at position P4 and/or P4’.
  • 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.
  • the functional variant of GPAGLYAQ (SEQ ID NO: 6) can comprise a glycine, phenylalanine, or a proline at position P4’.
  • amino acid substitutions at position P2 or P2’ of GPAGLYAQ are not preferred.
  • the functional variant of GPAGLYAQ comprises the amino acid sequence selected from SEQ ID NOs: 32-106.
  • Specific functional variants of GPAGLYAQ include GPLGLYAQ (SEQ ID NO: 70). and GPAGLKGA (SEQ ID NO: 60).
  • the functional variants of LFKSSFP preferably comprises hydrophobic amino acid substitutions.
  • the functional variant of LFKSSFP 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’,
  • aspartic acid and/or glutamic acid 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’.
  • the amino acid substitution of the functional variant of LFKSSFP preferably comprises an amino acid substitution at position P4 and/or PL In some embodiments, an amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 233) at position P4 ? is not preferred.
  • the functional variant of LFKSSFP comprises the amino acid sequence selected from SEQ ID NOs: 107-185.
  • Specific functional variants of LFKSSFP 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).
  • 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.
  • 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).
  • inducible IFNalpha prodrugs that include the linkers disclosed herein can, if desired, have a prolonged circulation half-life and/or lower biological activity in the circulation in comparison to the components of the inducible IFNalpha prodrugs as separate molecular entities.
  • the linkers 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.
  • 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.
  • 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.
  • 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.
  • compositions comprising the inducible IFNalpha prodrugs as described herein are suitable for administration in vitro or in vivo.
  • 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.
  • suitable pharmaceutical carriers 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.
  • 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.
  • 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).
  • 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.
  • the inducible IFNalpha prodrug described herein is encapsulated in nanoparticles.
  • the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods.
  • the inducible IFNalpha prodrug is attached to liposomes.
  • the inducible IFNalpha prodrugs are conjugated to the surface of liposomes.
  • the inducible IFNalpha prodrug are encapsulated within the shell of a liposome.
  • the liposome is a cationic liposome.
  • 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.
  • 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.
  • the inducible IFNalpha prodrug or nucleic acid sequences encoding the inducible IFNalpha prodrug are administered by a vector.
  • 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.
  • compositions and methods described herein 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.
  • 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).
  • 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.
  • VLPs Virus like particles
  • Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).
  • 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.
  • 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.
  • a suitable host cell such as CHO cells.
  • 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
  • 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
  • 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 many enhancer 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.
  • 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.
  • 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.
  • the promoter and/or enhancer region can be active in a cell type specific manner.
  • 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).
  • 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.
  • 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.
  • 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 FLAGTM tag (Kodak; New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GFP glutathione S-transferase
  • GST glutathione S-transferase
  • polyhistidine polyhistidine
  • c-myc hemagglutinin
  • FLAGTM tag FLAGTM tag
  • a disease, disorder or condition associated with a target antigen comprising administering to a subject in need thereof 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).
  • the disease, disorder, or condition is cancer.
  • 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,
  • IFNa interferon alpha
  • [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.
  • HSA human serum albumin
  • any suitable cancer may be treated with the inducible IFNalpha prodrugs provided herein.
  • suitable cancers in particular solid tumors, such as sarcomas and carcinomas.
  • 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
  • ALL acute lymphoblast
  • 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, hypophary
  • 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.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • 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).
  • MCC Merkel cell carcinoma
  • UC urothelial carcinoma
  • RCC renal cell carcinoma
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • TNBC triple negative breast cancer
  • endometrial cancer cutaneous
  • the methods and compositions disclosed herein are used to treat colon cancer, lung cancer, melanoma, renal cell carcinoma, or breast cancer.
  • the methods and compositions disclosed herein are used to treat melanoma.
  • the methods and compositions disclosed herein can be used to treat melanoma in subj ects with unresectable or metastatic melanoma.
  • 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.
  • 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.
  • the inducible IFNalpha prodrug enhances the immune response by activating the innate and adaptive immunities.
  • the methods described herein increase the activity of Natural Killer Cells and T lymphocytes.
  • the inducible IFNalpha prodrug provided herein can induce IFNy release from Natural Killer cells as well as CD4+ and CD8+ T cells.
  • 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.
  • chemotherapeutic agents e.g., Adriamycin, Cerubidine. Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine
  • Non-limiting examples of anti-cancer agents 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;
  • 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.
  • the inducible IFNalpha prodrug or is administered in combination with anti-diarrheal agents, anti-emetic agents, analgesics and/or non-steroidal anti-inflammatory agents.
  • 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.
  • chemotherapeutic agents e.g., cyclophosphamide, mechlorethamine, melphalan, chlorambucil, ifosfamide, busulfan, N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU). semustine (MeCCNU), fotemustine. streptozotocin.
  • fluorouracil e.g. 5 -fluorouracil
  • capecitabine e.g. 5 -fluorouracil
  • capecitabine cytarabine
  • gemcitabine decitabine
  • immune checkpoint inhibitors e.g., anti-PD-Ll, anti-CTLA4, anti-PD-1, anti-LAG3, anti-CD47, anti-GD2
  • 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.
  • 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 In
  • CTX-8371 Compass Therapeutics
  • CX-072 CytomX Therapeutics
  • CX-188 CytomX Therapeutics
  • cypalizumab Hardbin Gloria Pharmaceuticals
  • DB004 DotBio
  • EMB02 EpimAb Biotherapeutics
  • Geptanblimab / genolimzumab Apollomics
  • GS19 Suzhou Zelgen Biopharmaceuticals
  • HLX10 Shanghai Henlius Biotech
  • HX008 Tiaizhou HanZhong Pharmaceuticals
  • HY003 Juventas Cell Therapy
  • IBI315 / BH2950 Innovent Biologies
  • IBI318 Innovent Biologies
  • IBI319 Innovent Biologies
  • IMM1802 ImmuneOnco Biopharma
  • IMT200 TrueeBinding
  • Jemperli / dostarlimab AnaptysBio.
  • JTX-4014 Jounce Therapeutics
  • LBL-006 Najing Leads Biolabs
  • Libtayo / cemiplimab-rwlc (Regeneron Pharmaceuticals)
  • LVGN3616 Livgen Biopharma
  • LXF821 Novartis
  • LY01015 Liye Pharma Group
  • LY3462817 Eli Lilly
  • MCLA-134 Merus N.V.
  • MEDI5752 AstraZenica
  • NIR178 Novartis
  • ONCR-177 Oncorus
  • ONO-4685 Ono Pharmaceutical
  • MGD019 MacroGenius
  • PD1- GDT CAR-T Kiromic Biopharma
  • penpulimab Akeso
  • PSB205 Qilu Puget Sound Biotherapeutics
  • PT-001 Merck
  • Xdivane (Xbrane Biopharma), XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y - Biologies), and zimberelimab (Arcus Biosciences).
  • 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).
  • 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.
  • IBB 18 Innovent Biologies
  • IBI322 Innovent Biologies
  • IBI323 Innovent Biologies
  • IGM-7354 IGM Biosciences
  • IMC-001 Currento 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 Alphahamab Oncology
  • KYI 003 Sanofi
  • KYI 043 Sanofi
  • LY3300054 Eh Lilly
  • LY3415244 Eli Lilly
  • 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),
  • 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).
  • 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).
  • 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).
  • 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.
  • an antibody or immunoglobulin is intended to refer to immunoglobulin molecules comprised of two heavy (H) chains.
  • H heavy chain
  • mammals e.g., humans, rodents, and monkey's
  • L light 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).
  • CDR complementarity determining regions
  • FR framework regions
  • 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.
  • bispecific antibodies human antibodies
  • humanized antibodies humanized antibodies
  • chimeric antibodies immunoglobulins
  • synthetic antibodies synthetic antibodies
  • tetrameric antibodies comprising two heavy chain and two light chain molecules.
  • antibodies include camelid and shark antibodies.
  • 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.
  • 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.
  • 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.
  • 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.
  • a “conservative" amino acid substitution 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 the peptide 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.
  • 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.
  • 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.
  • 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 activity 7 of 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.
  • peptide 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 interchangeably 7 to 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.
  • the mammal is a mouse.
  • the mammal is a human.
  • 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 a desired pharmacological or physiological effect under the conditions of administration.
  • 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).
  • 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.
  • Example 1 In vivo Dosing and Tumor Processing for TIL Analysis
  • HBSS Hanks Balanced Salt Solution
  • Tumors were then placed in a C tube and dissociated on a Miltenyi Octomax instrument running program 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 xlO 6 cells 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. 19C
  • 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 the table 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.
  • RNA extraction and NanoString analysis were 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 than 0.05. Results are show n in FIGs.llA-llD, 12, 13, 14A-14B, 15A-15F, 16A-16C, 17A-17C, 18A-18C, and 19A-19C
  • mice 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 (3xl0 5 ) 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 mm 3 ), 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.
  • mice 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 (5xl0 5 ) 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 mm 3 ), 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.
  • 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.
  • A20 model 5x10 5 A20 cells were implanted in 0% Matrigel subcutaneously on 6-8 week old Balb/C mice.
  • EG.7 model 1x10 6 EG.7 cells were implanted in 0% Matrigel subcutaneously on 6-8 week old C57B1/6 mice.
  • B16-F10 model IxlO 5 B16-F 10 cells were implanted in 50% Matrigel subcutaneously on 6-8 week old C57B1/6 mice.
  • EMT6 model 1x10 5 EMT6 cells were implanted in 50% Matrigel subcutaneously on 6-8 week old Balb/C mice.
  • tumor volume and body weight were measured every 2-3 days throughout the course of the experiment.
  • mice When the average tumor volume was within a pre-set limit (approximately 100 mm 3 for 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
  • 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.
  • 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-lxl0 5 viable cells were plated in each well of a 96 well round bottom plate.
  • INDUKINE molecules were added to each well at a final concentration of 20 nM for 48 hours 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.
  • PBMCs w ere thawed and counted prior to being resuspended at IxlO 6 cells/mL in X-Vivo 15 media.
  • 1x10 s PBMCs 100 pL were plated per well in a 96 well round bottom plate.
  • the cell culture supernatants collected from primary tumor samples incubated with the INDUKINE molecules
  • the cell 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.
  • 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.
  • OSCC oral squamous cell carcinoma
  • Persistent viral infections are attributed to, in part, evasion of host immune responses by viral oncoproteins.
  • mEER syngeneic murine tumor model of HPV-OSCC
  • mEER syngeneic murine tumor model of HPV-OSCC
  • mice Six- to eight- week old female C57/B16 mice (Charles River) were inject with 3 x 10 5 MEER 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).
  • 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).

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Abstract

La présente invention concerne des procédés et des compositions pour le traitement du cancer à l'aide d'un promédicament IFNalpha inductible.
EP24771700.2A 2023-03-14 2024-03-14 Promédicaments d'interféron Pending EP4680264A2 (fr)

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