EP4608443A1 - Methods for sensitizing response to an immunotherapy - Google Patents

Methods for sensitizing response to an immunotherapy

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Publication number
EP4608443A1
EP4608443A1 EP23883528.4A EP23883528A EP4608443A1 EP 4608443 A1 EP4608443 A1 EP 4608443A1 EP 23883528 A EP23883528 A EP 23883528A EP 4608443 A1 EP4608443 A1 EP 4608443A1
Authority
EP
European Patent Office
Prior art keywords
versikine
protein
seq
cell
immunotherapy
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
EP23883528.4A
Other languages
German (de)
French (fr)
Inventor
Fotios Asimakopoulos
Athanasios PAPADAS
Alexander Patrick CICALA
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.)
University of California
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California
University of California Berkeley
University of California San Diego UCSD
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Publication date
Application filed by University of California, University of California Berkeley, University of California San Diego UCSD filed Critical University of California
Publication of EP4608443A1 publication Critical patent/EP4608443A1/en
Pending legal-status Critical Current

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    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001174—Proteoglycans, e.g. glypican, brevican or CSPG4
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725—Proteoglycans, e.g. aggreccan
    • 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
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3084—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • TME T-cell-inflamed tumor microenvironments
  • cDCls Tumor antigen cross-presentation and CD8+ T cell effector priming by stimulatory type 1 conventional dendritic cells (cDCls) is integral to spontaneous and therapeutic anti -tumor immunity.
  • cDCls regulate effector cell influx into the TME.
  • TME tumor microenvironment
  • cDCls are crucial for responses to vaccination strategies, immune checkpoint inhibitors, and engineered immune effector cells (e.g., chimeric antigen receptor T [CAR-T] cells). It has been shown that stimulatory cDCls are excluded from interdigitating
  • fusion proteins comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and a secretory peptide having a sequence of SEQ ID NO: 2-7.
  • fusion proteins comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and a protein selected from the group consisting of: GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, and IFNgamma.
  • a protein selected from the group consisting of GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, and IFNgamma comprises a sequence selected from the group consisting of SEQ ID NO 8-21.
  • the fusion protein further comprises a secretory peptide.
  • the secretory peptide comprises a sequence of SQ ID NO: 2-7.
  • vectors comprising any one of the fusion peptides described herein.
  • the vector is a viral vector.
  • the viral vector is a lentiviral vector.
  • Also provided herein are methods of introducing versikine to antigen-presenting cells comprising: introducing to an antigen-presenting cell any one of the fusion proteins or the vectors described herein, wherein the introducing comprises a lentiviral vector.
  • the antigen-presenting cells comprise macrophages, B cells, fibroblasts, and/or dendritic cells.
  • the antigen-presenting cell is a cell culture. In some embodiments, the antigen-presenting cell is in a subject.
  • conjugated polypeptides comprising a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 and a small molecule STING agonist.
  • the small molecule STING agonist comprises DMXAA or RVU-27065.
  • compositions comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and a STING agonist.
  • the STING agonist is DMXAA or RVU-27065
  • the versikine protein is co-administered with the STING agonist.
  • the versikine protein is administered before the STING agonist.
  • compositions comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and an anti-PDl and/or anti-PD-Ll inhibitor.
  • the anti-PDl and/or anti-PD-Ll inhibitor comprises pembrolizumab, nivolumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, or BMS-986189.
  • the versikine protein is co-administered with the anti-PDl and/or anti-PD-Ll inhibitor. In some embodiments, the versikine protein is administered before the anti-PDl and/or anti-PD-Ll inhibitor.
  • compositions comprising a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 and anti-CD40 immunotherapy.
  • the anti-CD40 immunotherapy is selected from the group consisting of selicrelumab, APX005M, JNJ-64457107, SEA-CD40, ChiLob7/4, CDX-1140H, Dacetuzumab, and ABBV-428.
  • the versikine protein is co-administered with the anti- CD40 immunotherapy.
  • the versikine protein is administered before the anti-CD40 immunotherapy.
  • TME tumor microenvironment
  • the versikine protein or versikine nucleic acid is co-administered with another pharmaceutical compound.
  • the pharmaceutical compound comprises a stimulator of interferon genes (STING) agonist immunotherapy.
  • the pharmaceutical compound comprises an anti-PDl immunotherapy.
  • the pharmaceutical compound comprises an anti-CD40 immunotherapy.
  • the administering comprises delivery of the versikine.
  • the cell is a dendritic cell. In some embodiments, the cell is an antigen-presenting cell. In some embodiments, the delivery of the proteolytic fragment comprises a virus-based delivery. In some embodiments, the virus-based delivery comprises a lentivirus.
  • the versikine is generated by proteolysis of VCAN at the Glu441-Ala442 bond.
  • the versikine protein or versikine nucleic acid is conjugated to another protein.
  • the other protein is selected from the group consisting of: GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, or IFN.
  • the other protein comprises a secretory peptide
  • the secretory peptide is selected from the group consisting of: human OSM, human immunoglobulin, human chymotrypsinogen, human trypsinogen 2, human IL-2, or human insulin.
  • the versikine protein or versikine nucleic acid is conjugated to a drug.
  • the drug comprises a small molecule STING agonist.
  • the versikine protein or versikine nucleic acid is conjugated to a small molecule.
  • the versikine protein or versikine nucleic acid is conjugated to a radiochemical.
  • the versikine protein or versikine nucleic acid is conjugated to an immunogenic component.
  • the versikine protein or versikine nucleic acid is conjugated to a label.
  • the label is a radioisotope or a fluorophore.
  • the versikine is a nucleic acid. In some embodiments, the versikine nucleic acid is fused to non-versikine nucleic acid. In some embodiments, the non- versikine nucleic acid is a DNA or RNA molecule.
  • a method provided herein further comprises administering a pharmaceutical agent.
  • the pharmaceutical agent comprises an immunotherapy agent.
  • the immunotherapy agent comprises a STING agonist, a PD-1 inhibitor, aPD-Ll inhibitor, or a CD40 agonist.
  • the subject has a tumor.
  • the tumor is a refractory tumor.
  • Also provided herein are methods of predicting a subject’s response to an immunotherapy comprising: (a) analyzing a biological sample obtained from the subject; and (b) detecting a proteolytic fragment of an extracellular matrix (ECM) proteoglycan in the biological sample, thereby predicting the subject’s response to the immunotherapy.
  • the analyzing comprises staining the biological sample.
  • the staining comprises an immunofluorescence stain.
  • the staining comprises an immunohistochemistry stain.
  • the biological sample is stained with an antibody.
  • the antibody is selected from the group consisting of: an anti-DPEAAE neo-epitope antibody, anti-HA antibody, anti-XCRl
  • the detecting comprises determining the level of the proteolytic fragment by measuring the antibody in the biological sample.
  • the immunotherapy comprises a stimulator of interferon genes (STING) agonist immunotherapy.
  • the immunotherapy comprises an anti-PDl immunotherapy.
  • the immunotherapy comprises an anti-CD40 immunotherapy.
  • the ECM proteoglycan is versican (VCAN).
  • the proteolytic fragment of the ECM proteoglycan is versikine.
  • the versikine is generated by proteolysis of VCAN at the Glu441-Ala442 bond.
  • the subject has a tumor.
  • the tumor is a refractory tumor.
  • FIGs. 1A-1J show that the VCAN pathway regulates tumor cDCls.
  • FIG. 1A shows an exemplary schematic showing versican (VCAN)-Vl functional domains and site-specific proteolysis to generate versikine (scissors represent ADAMTS proteolytic cleavage). CS, chondroitin sulphate.
  • FIG. IB shows stromal distribution of anti-DPEAAE IHC staining in human lung cancers. DPEAAE constitutes the C terminus of versikine (chromogen, DAB; counterstain, hematoxylin).
  • FIG. 1C shows triple IHC staining of human lung cancers (DPEAAE, teal; XCR1, brown; CD8, purple).
  • FIG. ID shows distribution of VCAN expression across TCGA carcinomas, ordered on the horizontal axis by median VCAN expression.
  • FIG. IE shows distribution of cDCl (BATF3-DC) score across TCGA carcinomas,
  • FIG. IF shows levels of correlation between cDCl (BATF3-DC) score and VCAN expression across TCGA carcinomas. The ranked median of VCAN expression and measured cDCl (BATF3-DC) score is shown across the x axis (1, highest; 20, lowest). Significant (q ⁇ 0.1) correlations after multiple hypothesis correction are colored red. Error bars represent the standard error of the correlation coefficient measured using Python statsmodels.
  • FIG. 1G shows generation of Vcan 1 - mice through CRISPR-Cas9-based targeting of Vcan exon 3.
  • FIG. 1H shows mass cytometry of CD45+ cells from WT (LLC implanted into WT recipients, left) and Vcan- depleted tumors (LLC VcanKD tumor cells implanted into Vcan +A recipients, right).
  • FIG. 1J shows representative flow cytometry plots showing cDCl and cDC2 frequency in WT, Vcan-depleted (LLC-EV VcanKD : Vcan +A ), and Vkine-rescued (LLC-Vkine VcanKD : Vcan +A ) tumors.
  • FIG. IK shows human tonsil immunohistochemical staining for cDCl lineage markers XCR1 and CLEC9A. Left, composite image after spectral unmixing. XCR1 (DAB), CLEC9A.
  • FIG. IL shows an exemplary schematic depiction of the deletions in the two mutant Vcan founders, 1053 (16bp deletion) and 1058 (47bp deletion). Sequence of exon 3 primer used in RT-PCR experiments is shown.
  • FIG. IM shows DNA amplification using primers flanking the targeted region. Shown are a 128bp WT amplicon and the mutated amplicons in founders Fcanl053 and Fcc?nl058.
  • FIG. IL shows an exemplary schematic depiction of the deletions in the two mutant Vcan founders, 1053 (16bp deletion) and 1058 (47bp deletion). Sequence of exon 3 primer used in RT-PCR experiments is shown.
  • FIG. IM shows DNA amplification using primers flanking the targeted region. Shown are a 128bp WT amplicon and the mutated amplicons in founders Fcanl053 and Fcc?nl058.
  • FIG. 10 shows validation of Vcan knockdown in LLC VcanKD cells. LLC cells were transfected with each of 3 hairpins (shRNA #1, 2 or 3) targeting exon 8 (encoding GAG0 domain depicted in FIG. 1A) Vcan message was assayed using exon 3 primers (left) and exon 15 primers (right).
  • shRNA #1, 2 or 3 3 hairpins targeting exon 8 (encoding GAG0 domain depicted in FIG. 1A)
  • IP shows gating strategy to delineate tumor-associated dendritic cells (TADC) per van Ginderachter schema.
  • FIG. IQ shows eDC subset frequencies in steady-state splenic tissue from WT and Vcan-/- mice.
  • FIG. 1R shows total eDC (cDCl + cDC2) absolute counts per mg tumor mass in LLC:WT, Vcan-depleted (LLC-EV VcanKD : Vcan+/-) and versikine-rescue (LLC-
  • FIG. IS shows growth rates of WT, Vcan-depleted (LLC-EV VcanKD : Vcan+/-) and versikine-rescue (LLCVkine VcanKD :Vcan+/-) tumors.
  • LLC-EV VcanKD Vcan+/-
  • LLCVkine VcanKD Vcan+/- tumors.
  • the colon separates the genoty pe of implanted tumor cells from genotype of recipient animal.
  • FIGs. 2A-2G show that the VCAN-matrikine versikine promotes cDCl abundance in vivo.
  • FIG. 2A shows an exemplary schematic of the experiment. LLC tumor cells that were engineered to express hemagglutinin (HA)-tagged versikine (LLC-Vkine) or empty vector controls (LLC-EV) and injected subcutaneously (s.c.) on the flank or intravenously using a retro-orbital approach.
  • FIG. 2B shows gross morphology of orthotopic (top) and s.c. (bottom) LLC-EV and LLC- Vkine tumors.
  • FIG. 2C shows anti -HA tag western blotting detects a 75-kDa band in LLC- Vkine tumor lysates, consistent with versikine.
  • FIG. 2D shows representative immunohistochemistry (IHC) images showing a-DPEAAE and HA tag staining of LLC-EV and LLC-Vkine tumors. Endogenous DPEAAE proteolysis is low level and similar between LLC-EV and LLC-Vkine. Anti-HA staining localizes in a membranous distribution in LLC- Vkine cells (inset, larger magnification).
  • FIG. 2E shows flow cytometric analysis of eDC subsets in s.c.
  • FIG. 2F shows comparison of immune contexture (CD45 + fraction) in LLC-EV versus LLC-Vkine tumors by 31 -marker mass cytometry.
  • FIG. 2G shows flow cytometry analysis of eDC subsets in orthotopic LLC-EV and LLC-Vkine tumors (lung metastases induced by intravenous injection). A summary of eDC and TADC subset frequencies is depicted on the right.
  • FIG. 2H shows full-length western blot for FIG. 2C.
  • FIG. 21 shows example of human lung cancer biopsy with stromal plus epithelial DPEAAE staining.
  • 10X objective scalebar 240mm
  • 40X objective scalebar 60mm.
  • FIG. 2J shows examples of negative DPEAAE staining in human lung cancer biopsies.
  • 10X objective scalebar 240mm
  • 40X objective scalebar 60mm.
  • FIG. 2K shows membranous localization of HAtagged ectopic versikine in B16 melanoma, a tumor model characterized by absence of cellautonomous Vcan expression.
  • HA-tag, chromogen BCIP/NBT; counterstain: nuclear fast red.
  • FIG. 2L shows growth rates of subcutaneous LLC- EV and LLC-Vkine tumors.
  • FIG. 2M shows absolute counts (cell count/ mg of tumor) of major intratumoral DC subsets, following optimized cell dissociation protocols (Miltenyi Dissociation Kit).
  • FIG. 2N shows flow cytometric analysis of eDC subsets in orthotopically- implanted 4T1 mammary carcinoma tumors engineered to express empty -vector (4T1-EV) or
  • FIG. 20 shows growth rates of orthotopic 4T1-EV and 4T1-Vkine tumors.
  • FIG. 2P shows flow cytometric analysis of eDC subsets from bone marrow following intracardiac injection of VQ myeloma cells, engineered to express empty-vector (VQ-EV) or versikine (VQ-Vkine). Representative flow plots (left) and frequencies (right) of eDC subsets are shown.
  • FIG. 2Q shows Kaplan-Meier curves depicting time-to-hindlimb paralysis (a clinical sequela of myeloma progression) in recipients of VQ-EV vs. VQ-Vkine myeloma tumors.
  • FIGs. 3A-3G show that versikine selectively activates cDCl in vivo.
  • FIG. 3A shows RT-PCR analysis for cDCl “signature” transcripts in bulk LLC-EV and LLC-Vkine tumor mRNA. Data are presented as mean ⁇ SEM.
  • FIG. 3B shows summary of CD40 staining intensity (MFI, mean fluorescence intensity) in DC subsets from LLC-EV and LLC-Vkine tumors (experiment 1). Examples of individual histogram plots for each DC subset are shown.
  • FIG. 3C shows a summary of PD-L1 staining intensity in DC subsets from LLC-EV and LLC-Vkine tumors. Examples of individual histogram plots for each DC subset are shown.
  • FIG. 3A shows RT-PCR analysis for cDCl “signature” transcripts in bulk LLC-EV and LLC-Vkine tumor mRNA. Data are presented as mean ⁇ SEM.
  • FIG. 3B shows summary of CD40 sta
  • FIG. 3D shows a layout of the experiment to compare trans criptomic profiles in LLC-EV versus LLC-Vkine tumor immune infiltrates.
  • FIG. 3E shows hierarchical clustering of transcriptomic profiles by RNA sequencing (RNA-seq) analysis of CD45+ tumor-infiltrating leukocytes (TILs) extracted from LLC-EV versus LLC-Vkine tumors.
  • FIG. 3F shows volcano plot highlighting key differentially expressed genes in CD45+ TILs from LLC-Vkine tumors compared with LLC- EV tumors. Genes whose overexpression has been linked to APC activation are shown in red and genes whose overexpression has been linked to T cell activation in green.
  • FIG. 31 shows Gene Ontology (GO) analysis of pathways enriched in CD45+ fractions from LLC-Vkine versus LLC-EV tumors.
  • FIG. 3H shows expression pattern of murine IrJ8, Batf3 andld2. Data from BioGPS.
  • FIG. 31 shows an exemplary schematic layout of the pre-DC adoptive transfer experiment. Pre-DC were harvested from the BM of Flt31-m vivo mobilized CD45.2+ mice and adoptively transferred into LLCEV or LLC-Vkine tumors implanted in CD45.1+ recipients. In vivo pre-DC mobilization w as achieved through implantation of Flt31-secreting B16 cells, according to standard protocols.
  • FIG. 3J shows gating strategy for flow sorting pre-DC from BM of F113I-/7? vivo mobilized donors, per the schema of van Ginderachter.
  • FIG. 3K shows representative flow plots of CD45.1+ endogenous eDC subsets (left) and frequencies (right).
  • FIG. 3L shows representative flow plots of CD45.2+ adoptively -transferred eDC subsets (left) and frequencies
  • FIGs. 4A-4K show cDCl activation by versikme is cell autonomous.
  • FIG. 4A shows an exemplary schematic layout of the experiment. MutuDC1940-EV or -Vkine cells were stimulated for 4 h with vehicle (PBS) or the TLR4 agonist lipopolysaccharide (LPS) (100 ng/mL) before RNA extraction.
  • FIG. 4B shows gross morphology of MutuDC 1940 cells engineered to express versikine (Vkine) or empty vector (EV). Phase contrast, 1003magnification; scale bar, 220 mm.
  • FIG. 4C shows hierarchical clustering of MutuDC1940 transcriptomic profiles expressing EV or versikine (Vkine) and stimulated with the TLR4 agonist LPS or vehicle (PBS).
  • FIG. 4D shows volcano plot highlighting key differentially expressed genes in MutuDC1940-Vkine versus -EV cells (without LPS).
  • FIG. 4E shows gene set enrichment analysis (GSEA) of significantly upregulated (left and center) and downregulated (right) pathways in MutuDC 1940- Vkine versus -EV cells (without LPS).
  • FIG. 4F shows Ccl7 RT-PCR using LLC-EV and LLC-Vkine tumor bulk mRNA (left) and CD1 lc+ magnetically separated fraction mRNA (right).
  • FIG. 4G shows Cxcl9 and CxcllO RT-PCR using CD1 lc+ magnetically separated fractions from LLC-EV or LLC-Vkine tumors.
  • FIG. 4H shows and exemplary schematic of the antigen presentation experiment.
  • FIG. 41 shows flow cytometry for endogenous IFN-g and IL-2 of OT-I CD8+ T cells co-cultured with SIINFEKL peptide-loaded MutuDC1940 cells, EV- or Vkine-expressing, with or without LPS.
  • FIG. 4J shows quantitation of OT-I flow cytometry analysis of the antigen presentation assay.
  • FIG. 4K shows IFN-g by ELISA in supernatants from OT-I and MutuDC1940:SIINFEKL co-cultures in the antigen presentation assay.
  • FIG. 4L shows gene ontology (GO) pathway analysis of differentially expressed genes between MutuDC 1940-Vkine vs. -EV. Versikine’s proapoptotic program (“positive regulation of programmed cell death/ “positive regulation of apoptotic process”) is reminiscent of versikine’s proapoptotic activities during development.
  • FIG. 4M shows RT-PCR of Cxcl9/10 in MutuDC1940-EV vs. MutuDC 1940-Vkine stimulated with TLR4 agonist LPS or vehicle (PBS).
  • FIG. 4N shows ELISA detection of secreted Cxcl9 by MutuDC 1940-EV- and MutuDC 1940-Vkine stimulated with LPS or vehicle (PBS) plotted against time (hours).
  • FIG. 40 shows RT-PCR for I127p28 and Ebi3 message in MutuDC 1940- EV-vs. -Vkine stimulated with LPS or vehicle (PBS).
  • FIG. 4P shows ELISA detection of secreted I127p28 by MutuDC 1940-EV- and MutuDC 1940-Vkine stimulated with LPS or vehicle (PBS) plotted against time (hours).
  • FIG. 4Q shows RT-PCR for selected versikine- signature genes using RNA from MutuDC 1940 cells (unmanipulated) exposed to supernatant from versikine-secreting HEK293 cells (Vkine sup) vs.
  • FIG. 4R shows flow cytometry for endogenous IFNg and IL2 of OT-I CD8+ T cells at baseline (left) and PMA-stimulated, prior to addition of DC (right).
  • FIG. 4S shows FMO controls shown for IFNg (left) and IL2 (right) under conditions of maximal stimulation (Vkine +LPS, compare to FIG. 41).
  • FIG. 4T shows IFNy by ELISA in supernatants from OT-I+ MutuDC1940:SIINFEKL co-cultures in the antigen presentation assay.
  • FIGs. 5A-5G show cDCl accumulation requires innate lymphoid support.
  • FIG. 5A shows RT- PCR for NK cell-activating cytokine transcripts expressed by ex vivo magnetically separated CDl lc+ cells from LLC-EV and LLC-Vkine tumors.
  • FIG. 5B shows RT-PCR profile of NKp46+ NK1. 1+ cells flow-sorted from LLC-EV and LLC-Vkine tumors.
  • FIG. 5C shows an exemplary schematic of the NK cell depletion experiment.
  • FIG. 5D shows summary of eDC subset frequency by flow cytometric analysis in LLC-EV versus LLC-Vkine tumors after treatment with NK cell-depleting antibody (anti-ASGMl) or vehicle (PBS).
  • FIG. 5A shows RT- PCR for NK cell-activating cytokine transcripts expressed by ex vivo magnetically separated CDl lc+ cells from LLC-EV and LLC-Vkine tumors.
  • FIG. 5E shows Csf2 (GM-CSF) RT-PCR of RNA extracted from NKp46+ NK1.1+ cells flow-sorted from LLC-EV and LLC-Vkine tumors growing in WT or Batf3_/_ hosts.
  • FIG. 5F shows stromal localization of NCR1+ (NKp46+) cells in human lung cancers (chromogen, DAB; counterstain, hematoxylin).
  • FIG. 5G shows annexin V/7-AAD apoptosis assay of MutuDC1940-EV or - Vkine dendritic cells exposed to graded staurosporine concentrations with or without murine GMCSF.
  • FIG. 5H shows validation of intratumoral NK (NK1.
  • FIG. 51 shows flow cytometric analysis of eDC subsets in LLC-EV vs. LLC-Vkine tumors following treatment with NK-depleting antibody (anti-ASGMl) or vehicle (PBS).
  • FIG. 5J shows flow cytometric analysis of intratumoral basophils (defined as CD45inlCD49b+FceRI+lgE+c-Kit cells, gating per.
  • FIG. 5K shows absolute counts/ mg tumor tissue of intratumoral basophils compared to intratumoral NK1.1+CD3- cells, in LLC- EV vs. LLC-Vkine tumors.
  • FIG. 51 shows flow cytometric analysis of eDC subsets in LLC-EV vs. LLC-Vkine tumors following treatment with NK-depleting antibody (anti-ASGMl) or vehicle (PBS).
  • FIG. 5J shows flow cytometric analysis of intratumoral basophils (defined as CD45inlCD49b+FceRI+lgE+c-Kit cells
  • FIG. 5L shows absence of intratumoral cDCl in Batf3-I- recipients by multiparametric flow cytometry.
  • FIG. 5M shows flow cytometric analysis of eDC subsets in LLC-EV vs. LLC-Vkine tumors implanted in WT or Tlr2-/- recipients.
  • FIG. 5N shows summary of eDC subset frequency by flow cytometric analysis in LLC-EV vs. LLC-Vkine tumors implanted in WT or Tlr2-/- recipients.
  • FIG. 50 shows growth rates of LLC-EV and LLC-Vkine tumors in WT vs. Tlr2-i- background.
  • FIG. 5P shows flow cytometric analysis of eDC subsets in LLC-EV vs. LLC-Vkine tumors implanted in WT or Cd44-/- recipients.
  • FIG. 5Q shows summary of eDC subset frequency by flow cytometric analysis in LLC-EV vs. LLC-
  • FIG. 5R shows growth rates of LLC-EV and LLC-Vkine tumors in WT vs. Cd44-I- genetic background.
  • FIGs. 6A-6L show stroma-licensed cDCls are "‘poised” and hypersensitive to nucleic acid sensing in vivo.
  • FIG. 6A shows an exemplary schematic of the experiment.
  • FIG. 6B shows growth curves of LLC-EV and LLC-Vkine tumors challenged with a single subtherapeutic dose (200 mg) of intratumoral (IT) DMXAA (DMXAA200) or vehicle (NaHCO3) on day 0.
  • FIG. 6C shows Kaplan-Meier survival curves for the experiment in (B); **p ⁇ 0.01 by log rank test.
  • FIG. 6D shows representative images showing development of hemorrhagic necrosis and a necrotic eschar in LLC-Vkine but not LLC-EV tumors 24 h after IT DMXAA200 administration.
  • FIG. 6E shows transcriptomic profile of LLC-EV and LLC-Vkine tumors harvested 2 h after IT DMXAA200.
  • FIG. 6F shows versikine -DMXAA synergy generates an abscopal effect in LLC tumors that produces a survival advantage. **p ⁇ 0.01 by log rank test.
  • FIG. 6G shows growth curves of treatment-side LLC-EV and LLC-Vkine tumors challenged with a single subtherapeutic dose (200 mg) of IT DMXAA (DMXAA200) or vehicle (NaHCO3) on day 0.
  • FIG. 6H shows growth curves of contralateral side unmanipulated LLC tumors; treated side as in FIG. 6G.
  • FIG. 61 shows response to DMXAA200 is lost in Batf3' /_ recipients. Shown are growth curves of LLC-EV and LLC-Vkine tumors challenged with a single subtherapeutic dose (200 mg) of IT DMXAA (DMXAA200) or vehicle (NaHCO3) on day 0 in Bat 3 /_ recipients.
  • FIG. 6J shows Batf3 loss abrogates the survival advantage seen in the WT (FIG. 6C).
  • FIG. 6K shows efficacy of DMXAA200 in LLC-Vkine tumors implanted into BatfT' recipients is restored after adoptive transfer of 1CD103.
  • FIG. 6L shows adoptive transfer of iCD103 in LLC-Vkine tumors implanted into Batf3 /_ recipients restores the survival advantage of mice treated with DMXAA200.
  • FIG. 6M shows Versikme-DMXAA synergy generates an abscopal effect in 4T1 mammary carcinomas. Growth curves of treatment-side 4T1-EV and 4T1-Vkine tumors challenged with a single sub-therapeutic dose (200 mcg) of IT DMXAA on Day 0 (DMXAA200) or vehicle (NaHCO3).
  • FIG. 6N shows growth curves of contralateral side unmanipulated 4T1 tumors, according to corresponding treatment side configuration (treatment as in FIG. 6M).
  • FIG. 6P shows an exemplary schematic layout of iCD103 cell adoptive transfer experiments.
  • FIG. 6Q shows flow-cytometric validation of the iCD103 cells, generated as described in the protocol by Merad, Sparwasser and colleagues, using standard cDCl markers.
  • FIG. 6R shows growth curves of B16-EV and B16-Vkine tumors challenged with a single subtherapeutic dose (200 mcg) of IT DMXAA on Day 0 (DMXAA200) or vehicle (NaHCO3).
  • FIG. 6S shows Kaplan-
  • FIG. 6T shows response to DMXAA200 is lost in B16-Vkine tumors implanted in Batf3-I- recipients.
  • FIG. 6U shows efficacy of sub-therapeutic DMXAA200 in B16-Vkine tumors implanted in Batf3-I- recipients is restored following adoptive transfer of iCD103 cells. A subset of B16- bearing tumors did not “take” iCD103 cells, likely attributable to the pauci-immune environment of B16 tumors.
  • FIGs. 7A-7F show versikine promotes CD8+ responses and overcomes resistance to anti-PDl inhibitors in vivo.
  • FIG. 7A shows an exemplary schematic of the experiment.
  • FIG. 7B shows frequency of MHCLSIINFEKL tetramer+ CD8+ splenocytes in mice bearing LLC-EV versus LLC-Vkine tumors 5 days after challenge with a therapeutic dose of a STING agonist (DMXAA500).
  • FIG. 7C shows correlation between in vitro versikine signature and CD8+ T cell scores across TCGA human lung cancers. Significance was measured using a linear model while accounting for total immune infiltration.
  • FIG. 7D shows DPEAAE staining in human lung cancers and associated CD8+ infiltration.
  • FIG. 7E shows distribution of DPEAAE stromal staining intensity across lung cancer prognostic subgroups (pauci-immune [poor prognosis] and immune-rich [favorable prognosis] at cutoff 3 CD8+ TILs/HPF). p ⁇ 0.001 by two-tailed Mann-Whitney test.
  • FIG. 7F shows Top: schematic of the experiment. Bottom: tumor growth rates and survival curves of LLC-EV and LLC-Vkine-bearing animals treated with 3 doses of anti-PDl antibody or isotype control.
  • FIG. 7G shows CD8+ T cell subset frequency in the spleen of mice treated as in the schema depicted in FIG. 7A.
  • FIG. 7H shows correlation between in vitro versikine response signature and corrected CD8+ T cell scores across TCGA lung cancers. CD8+ T cell scores corrected for immune infiltration to remove variation associated with immune state.
  • FIG. 7J shows growth response curves of individual tumors in the anti-PDl experiment depicted in
  • FIG. 8 shows an exemplary schematic of stromal remodeling regulating dendritic cell abundance and activity in the tumor microenvironment.
  • T-cell-inflamed tumor microenvironments are a prerequisite for immunotherapy efficacy. However, why some tumors are inflamed, and others are not remains poorly understood.
  • Tumor antigen cross-presentation and CD8+ T cell effector priming by stimulatory type 1 conventional dendritic cells (cDCls) is integral to spontaneous and therapeutic antitumor immunity.
  • cDCls regulate effector cell influx into the TME. From a translational perspective, cDCls are crucial for responses to vaccination strategies, immune checkpoint inhibitors, and engineered immune effector cells (e.g., chimeric antigen receptor T [CAR-T] cells).
  • CAR-T chimeric antigen receptor T
  • V CAN The large aggregating extracellular matrix proteoglycan versican (V CAN) is a central component of the embryonic provisional matrix, playing key non-redundant roles in development of the cardiovascular system and limbs.
  • Vcan-null mice die in utero by embryonic day 10.5 because of defects along the anterior-posterior cardiac axis.
  • VC AN proteolysis by AD AMTS a disintegrin and metalloproteinase with thrombospondin motifs
  • AD AMTS a disintegrin and metalloproteinase with thrombospondin motifs
  • proteases at the Glu441-Ala442 bond VI isofonn enumeration
  • AD AMTS a disintegrin and metalloproteinase with thrombospondin motifs
  • VI isofonn enumeration is an essential requirement that acts in part through the specific neoactivities of the released bioactive N-terminal fragment (matrikme) versikine, wherein disruption of the Glu441-Ala442 proteolytic site that generates versikine leads to developmental abnormalities
  • VC AN proteolysis at the versikine C-terminal Glu441-Ala442 bond correlates with CD8+ T cell infiltration in solid and hematopoietic human tumors.
  • the present disclosure describes methods for increasing T cell activation in a tumor microenvironment (TME) in a subject that includes administering a proteolytic fragment of an extracellular matrix (ECM) proteoglycan. Also provided herein are methods for sensitizing a response to an immunotherapy in a subject in need thereof that includes administering a proteolytic fragment of an extracellular matrix (ECM) proteoglycan. Also provided herein are methods of predicting a subject’s response to an immunotherapy that include (a) analyzing a biological sample obtained from the subject; and (b) detecting a proteolytic fragment of an extracellular matrix (ECM) proteoglycan in the biological sample, thereby predicting the subject’s response to the immunotherapy.
  • ECM extracellular matrix
  • biological sample can refer to a sample generally including cells and/or other biological material.
  • a biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX).
  • Biological samples can be derived from a homogeneous culture or population of organisms or alternatively from a collection of several different organisms, for example, in a community or ecosystem.
  • the biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei).
  • the biological sample can be a nucleic acid sample and/or protein sample.
  • the biological sample can be a carbohydrate sample or a lipid sample.
  • the biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate.
  • the sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample.
  • the sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions.
  • a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
  • precancerous e.g., benign
  • malignant pre-metastatic
  • metastatic metastatic
  • a relevant cancer may be characterized by a metastatic solid tumor.
  • SUBSTITUTE SHEET (RULE 26 ) be characterized by a hematologic tumor.
  • examples of different types of cancers known in the art include, for example, a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, Hodgkin lymphoma, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer.
  • hematopoietic cancers can include leukemias, lymphomas (Hodgkin’s and nonHodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, precancerous pathology such as myelodysplastic syndromes, acquired aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobin
  • a “cell” can refer to either a prokaryotic or eukary otic cell, optionally obtained from a subject or a commercially available source.
  • delivering can refer to the introduction of an exogenous polynucleotide into a host cell, irrespective of the method used for the introduction.
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., viral infection/transfection, or vanous other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (e.g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • the introduced polynucleotide may be stably or transiently maintained in the host cell.
  • Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • an extrachromosomal replicon e.g., a plasmid
  • a nuclear or mitochondrial chromosome e.g., a nuclear or mitochondrial chromosome.
  • a polynucleotide can be inserted into a host cell by a gene delivery molecule.
  • gene delivery molecules can include, but are not limited to, liposomes, micelle biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral
  • SUBSTITUTE SHEET (RULE 26 ) envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • nucleic acid is used to include any compound and/or substance that comprise a polymer of nucleotides.
  • a polymer of nucleotides is referred to as polynucleotides.
  • Exemplary nucleic acids or polynucleotides can include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2’-amino-LNA having a 2’-amino functionalization, and 2’-amino-a-LNA having a 2’-amino functionalization) or hybrids thereof.
  • RNAs ribonucleic acids
  • DNAs
  • Naturally- occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e g., found in ribonucleic acid (RNA)).
  • a deoxyribose sugar e.g., found in deoxyribonucleic acid (DNA)
  • RNA ribonucleic acid
  • a nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art.
  • a deoxyribonucleic acid (DNA) can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid (RNA) can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G).
  • nucleic acid refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is DNA. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is RNA
  • a subject refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease,
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • a “proteoglycan” refers a family of charged molecules containing a core protein and one or more covalently attached glycosaminoglycan side chains. Proteoglycans are important components of extracellular matrices (ECM) and have multiple functions that depend on both their protein and carbohydrate constituents.
  • ECM proteoglycan is versican (VCAN).
  • Versican is a large extracellular matrix proteoglycan that is present in a variety of tissues and plays key roles in multiple facets of cancer development, ranging from proliferative signaling, evasion of growth-suppressor pathways, regulation of cell death, promotion of neoangiogenesis, and tissue invasion and metastasis. Multiple lines of evidence implicate versican and its bioactive proteolytic fragments (matrikines) in the regulation of cancer inflammation and antitumor immune responses.
  • Matrikines can be defined as ECM-derived fragments that regulate cell activity, often in a manner distinct from that of their parent macromolecule. Proteolytic processing of versican at Glu441-Ala442 generates a bioactive N-terminal matnkine, versikine. In some embodiments, the proteolytic fragment of the ECM proteoglycan is versikine. In some embodiments, the versikine is generated by proteolysis of VCAN at the Glu441-Ala442 bond.
  • compositions of the disclosure contain a versikine ammo acid sequence.
  • the versikine amino acid sequence is the sequence of SEQ ID NO: 1.
  • the versikine amino acid comprises a sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%>, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 1.
  • the versikine is a nucleic acid molecule. In some embodiments, the versikine comprises a nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the versikine nucleic acid is at least 90% identical (e.g., at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 23).
  • the versikine nucleic acid molecule is fused to a non-versikine nucleic acid molecule. In some embodiments, the versikine nucleic acid molecule is fused to a non-versikine nucleic acid molecule via a linker. In some embodiments, the non-versikine nucleic acid molecule encodes a secretory peptide. In some embodiments, the non-versikine nucleic acid molecule encodes an immunomodulator. In some embodiments, the non-versikine nucleic acid molecule is a DNA or RNA molecule.
  • the versikine protein is conjugated to another protein. In some embodiments, the versikine protein is conjugated to another protein via a linker.
  • the linker comprises the amino acid sequence MFINIKSILWMCSTLIVTHA (SEQ ID NO: 22). In some embodiments, the linker comprises the amino acid sequence GGGS (SEQ ID NO: 24), GGGSGGGSGGGS (SEQ ID NO: 25), GGGGGGGG (SEQ ID NO: 26), EAAAKEAAAKEAAAK (SEQ ID NO: 27), PAPAP (SEQ ID NO: 28), or AEAAAKEAAAKA (SEQ ID NO: 29).
  • the linker comprises the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 30), EGKSSGSGSESKST (SEQ ID NO: 31), or GSAGSAAGSGEF (SEQ ID NO: 32). Additional examples of linkers are described for example in Chen et al., Adv Drug Deliv Rev. 2013 Oct;65(10): 1357-69, the entire contents of which are incorporated herein by reference.
  • the versikine protein is conjugated to another protein at the N-terminal. In some embodiments, the versikine protein is conjugated to another protein at the C-terminal.
  • the other protein compnses a secretory peptide.
  • the versikine protein is conjugated to the secretory peptide via a linker.
  • the versikine protein is conjugated to the secretory peptide at the N-terminal.
  • the secretory peptide is selected from the group consisting of: human OSM, human immunoglobulin, human chymotrypsinogen, human trypsinogen 2, human IL-2, or human insulin.
  • the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 2, wherein SEQ ID NO: 2 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 2 is fused to the C terminal of SEQ ID NO: 1,
  • the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 3, wherein SEQ ID NO: 3 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 3 is fused to the C terminal of SEQ ID NO: 1 , optionally including a linker.
  • the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 4, wherein SEQ ID NO: 4 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 4 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 5, wherein SEQ ID NO: 5 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 5 is fused to the C terminal of SEQ ID NO: 1 , optionally including a linker.
  • the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 6, wherein SEQ ID NO: 6 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 6 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 7, wherein SEQ ID NO: 7 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 7 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the other protein comprises an immunomodulator.
  • the versikine protein is conjugated to the immunomodulator via a linker.
  • the versikine protein is conjugated to the immunomodulator at the N-terminal.
  • the versikine protein is conjugated to the immunomodulator at the C- terminal.
  • the other protein is selected from the group consisting of: GM- CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL- 2, IFN01, IFNa2, or IFNy.
  • the other protein can comprise an IFN-a subtype (e.g, IFN-al, IFN-a2, IFN-a4, IFN-a5, IFN-a6, IFN-a7, IFN-a8, IFN-alO, IFN- al3, IFN-al4, IFN-al7, or IFN-a21).
  • IFN-a subtype e.g, IFN-al, IFN-a2, IFN-a4, IFN-a5, IFN-a6, IFN-a7, IFN-a8, IFN-alO, IFN- al3, IFN-al4, IFN-al7, or IFN-a21.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 8, wherein SEQ ID NO: 8 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 8 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 9, wherein SEQ ID NO: 9 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 9 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 10, wherein SEQ ID NO: 10 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 10 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 11, wherein SEQ ID NO: 11 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 11 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 12, wherein SEQ ID NO: 12 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 12 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 13, wherein SEQ ID NO: 13 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 13 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 14, wherein SEQ ID NO: 14 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 14 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • SEQ ID NO: 14 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 14 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 15, wherein SEQ ID NO: 15 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 15 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 16, wherein SEQ ID NO: 16 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 16 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 17, wherein SEQ ID NO: 17 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 17 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 18 wherein SEQ ID NO: 18 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 18 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator compnses SEQ ID NO: 1 and SEQ ID NO: 19, wherein SEQ ID NO: 19 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 19 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 20, wherein SEQ ID NO: 20 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 20 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 21, wherein SEQ ID NO: 21 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 21 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 33, wherein SEQ ID NO: 33 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 33 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 34, wherein SEQ ID NO: 34 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 34 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
  • the secretory peptides fused to versikine may optionally be fused to the immunomodulators of the disclosure.
  • the fusion proteins of the disclosure can be represented by the formula A - L - V - B, wherein A is selected from the group consisting of SEQ ID NOs: 2-7, 8-21, or 33-34, L comprises a linker sequence, V comprises an amino acid having at least 90% identity to SEQ ID NO: 1, and B is selected from the group consisting of SEQ ID NOs: 8-21, and 33-34.
  • the versikine nucleic acid molecule is fused to a non-versikine nucleic acid molecule. In some embodiments, the versikine nucleic acid molecule is fused to a non-versikine nucleic acid molecule via a linker. In some embodiments, the non-versikine nucleic acid molecule encodes a secretory peptide. In some embodiments, the non-versikine nucleic acid molecule encodes an immunomodulator. In some embodiments, the non-versikine nucleic acid molecule is a DNA or RNA molecule.
  • the versikine protein is conjugated to a drug.
  • the drug comprises a small molecule STING agonist.
  • the versikine protein is conjugated to a small molecule.
  • the versikine protein is conjugated to a radiochemical.
  • the versikine protein is conjugated to an immunogenic component.
  • the versikine protein is conjugated to a label.
  • the label is a radioisotope or a fluorophore.
  • the versikine protein is conjugated to a small molecule immunomodulator.
  • a small molecule immunomodulator comprises an indoleamine 2,3 -dioxygenase 1 (IDO1) inhibitor.
  • the indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor comprises Indoximod, Epacadostat, BMS-986205, Navoximod (GDC-0919, NLG-919), PF- 06840003, KHK2455, or LY3381916.
  • compositions containing versikine DNA and/or versikine fusion proteins are pharmaceutical compositions comprising versikine DNA and/or versikine fusion proteins described herein.
  • pharmaceutical composition refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers.
  • the composition is suitable for administration to a human or animal subject.
  • the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • vectors comprising any one of the nucleic acids described herein.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses).
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • the vector is a lentivirus (such as an integration-deficient lentiviral vector) or adeno-associated viral (AAV) vector.
  • lentivirus refers to a genus of the Retroviridae family.
  • Lentiviruses are unique among the retroviruses in being able to infect non-dividmg cells; they can deliver a significant amount of genetic information into the DNA of a host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
  • Exemplary viral vectors include polyoma, SV40, adenovirus, vaccinia virus, adeno- associated virus, herpes viruses including HSV and EBV, lentivirus, Sindbis viruses, alphaviruses and retroviruses of avian, murine, and human origin.
  • Baculovirus Autographa califomica multinuclear polyhedrosis virus; AcMNPV
  • AcMNPV Autographa califomica multinuclear polyhedrosis virus
  • Suitable vectors include retrovirus vectors, orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, adenoviral vectors, herpes virus vectors, alpha virus vectors, baculovirus vectors, Smdbis virus vectors, vaccinia virus vectors
  • SUBSTITUTE SHEET (RULE 26 ) and poliovirus vectors.
  • Specific exemplary vectors are poxvirus vectors such as vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus (MV A), adenovirus, baculovirus and the like.
  • Pox viruses of use include orthopox, suipox, avipox, and capripox virus.
  • Orthopox include vaccinia, ectromeha, and raccoon pox.
  • One example of an orthopox of use is vaccinia.
  • Avipox includes fowlpox, canary pox and pigeon pox.
  • Capripox include goatpox and sheeppox.
  • the suipox is swinepox.
  • Other viral vectors that can be used include other DNA viruses such as herpes simplex virus and adenoviruses, and RNA viruses such
  • vectors are capable of directing the expression of genes to which they are operatively -linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid provided herein in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively -linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described.
  • Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection).
  • Enzy matic reactions and purification techniques may be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2 nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • the administering of a proteolytic fragment of an ECM proteoglycan comprises delivery of the proteolytic fragment into a cell, wherein the cell is not currently integrated into a multi-cellular organism (e.g., a living subject).
  • the cell is part of a cell culture.
  • the cell is part of an organoid.
  • a cell is a primary hematopoietic, a non-hematopoietic human cell, or an iPS (inducible pluripotent stem cell).
  • the cell is an antigen-presenting cell (e.g., a macrophage, B cell, or dendritic cell).
  • the cell is a non-hematopoietic antigen-presenting cell (e.g., antigen-presenting fibroblast, lymph node stromal cell, or endothelial cell).
  • the versikine can be delivered into a cell by a variety of well-known techniques.
  • the delivery method can include a vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes).
  • the delivery method can include methods facilitating the delivery of “naked” polynucleotides (e.g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • the delivering comprises transfection, electroporation, or a virus-based delivery.
  • the delivery of the proteolytic fragment comprises a virus-based delivery.
  • the virus-based delivery comprises an adeno-associated virus or a lentivirus.
  • the virus-based delivery comprises a lentivirus.
  • the versikine nucleic acid can be introduced into/uptaken by dendritic or other antigen-presenting cells (e.g., macrophage, B cell, and/or dendritic cell) to generate cell-based vaccines.
  • dendritic or other antigen-presenting cells e.g., macrophage, B cell, and/or dendritic cell
  • the versikine nucleic acid can be introduced into a dendritic cell and enhancing antigen-presentation capacity of the dendritic cell, thereby generating a DC cellular vaccine.
  • cell lines are grown, and the versikine nucleic acid (SEQ ID NO: 23) is introduced to the cell via a viral vector.
  • the virus strain replicates in the mammalian cells and is then extracted from the cells and purified.
  • a versikine nucleic acid can be stably introduced into a cell for vaccine production.
  • a versikine nucleic acid can be delivered into a cell via standard transfection or using lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • a versikine protein can be expressed ex vivo and introduced into a subject.
  • in vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • the administering of a proteolytic fragment of an ECM proteoglycan comprises delivery of the proteolytic fragment into a cell, wherein the cell is integrated into a subject.
  • the cell of the subject is an antigen-presenting cell (e.g., a macrophage, B cell, or dendritic cell).
  • the cell of the subject is anon-hematopoietic antigen- presenting cell (e.g. , antigen-presenting fibroblast, lymph node stromal cell, or endothelial cell).
  • the versikine can be delivered into a cell by a variety of well-known techniques.
  • the delivery method can include a vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes).
  • the delivering comprises transfection or a virusbased delivery.
  • the delivery of the proteolytic fragment comprises a virus-based delivery.
  • the virus-based delivery comprises an adeno- associated virus or a lentivirus.
  • the virus-based delivery comprises a lentivirus.
  • the versikine composition administered as described herein treats cancer by increasing T cell activation in a tumor microenvironment, and/or potentiating the effects of other cancer treatment.
  • compositions described herein can be used to treat cancer.
  • cancer malignancy
  • neoplasm tumor
  • tumor tumor
  • cancer tumor
  • carcinoma cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation.
  • a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
  • the present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant.
  • a relevant cancer may be characterized by a solid tumor.
  • a relevant cancer may be characterized by a hematologic tumor.
  • examples of different types of cancers know n in the art include, for example, hematopoietic
  • SUBSTITUTE SHEET (RULE 26 ) cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like,
  • the versikine DNA, fusion proteins, and/or the compositions described herein may be administered alone or with an immunotherapy agent and/or a pharmaceutical agent
  • the pharmaceutical agent comprises a protein selected from the group consisting of: GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, or IFN.
  • the immunotherapy agent comprises a STING agonist, a PD-1 inhibitor, a PD-L1 inhibitor, or a CD40 agonist.
  • the versikine and the pharmaceutical agent can be administered simultaneously.
  • the versikine can be administered before administering the pharmaceutical agent.
  • the pharmaceutical agent can be administered after administering the versikine composition.
  • administration typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition.
  • agents that are, or is included in, the composition.
  • routes may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
  • administration may be ocular, oral, parenteral, topical, etc.
  • administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc.
  • administration may involve only a single dose.
  • administration may involve application of a fixed number of doses.
  • administration may involve dosing that is intermittent (e.g., a plurality of doses
  • SUBSTITUTE SHEET ( RULE 26 ) separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing.
  • administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • a therapeutically effective amount means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition.
  • a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
  • a therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual.
  • a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
  • term “therapeutically effective amount”, refers to an amount which, when administered to an individual in need thereof in the context of inventive therapy, will block, stabilize, attenuate, or reverse a cancer-supportive process occurring in said individual, or will enhance or increase a cancer-suppressive process in said individual.
  • a “therapeutically effective amount” is an amount which, when administered to an individual diagnosed with a cancer, will prevent, stabilize, inhibit, or reduce the further development of cancer in the individual.
  • a particularly preferred “therapeutically effective amount” of a composition described herein reverses (in a therapeutic treatment) the development of a malignancy such as a pancreatic carcinoma or helps achieve or prolong remission of a malignancy.
  • a therapeutically effective amount administered to an individual to treat a cancer in that individual may be the same or different from a therapeutically effective amount administered to promote remission or inhibit metastasis.
  • the therapeutic methods described herein are not to be interpreted as, restricted to, or otherwise limited to a “cure” for cancer; rather the methods of treatment are directed to the use of the described compositions to “treat” a cancer, i.e., to effect a desirable or beneficial change in the health of an individual who has cancer.
  • Such benefits are recognized by skilled healthcare providers in the field of oncology and include, but are not limited to, a stabilization of patient condition, a decrease in tumor size (tumor regression), an improvement in vital functions (e.g., improved function of cancerous tissues or organs), a decrease or inhibition of further metastasis, a decrease in opportunistic infections, an increased survivability, a decrease in pain, improved motor function, improved cognitive
  • SUBSTITUTE SHEET ( RULE 26 ) function, improved feeling of energy (vitality, decreased malaise), improved feeling of wellbeing, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof.
  • regression of a particular tumor in an individual may also be assessed by taking samples of cancer cells from the site of a tumor (e.g., over the course of treatment) and testing the cancer cells for the level of metabolic and signaling markers to monitor the status of the cancer cells to verify at the molecular level the regression of the cancer cells to a less malignant phenotype.
  • a therapeutically effective amount may be formulated and/or administered in a single dose.
  • a therapeutically effective amount may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
  • an immunotherapy refers to a treatment of disease (e.g., cancer) by activating or suppressing the immune system.
  • cancer immunotherapy uses the immune system and its components to mount an anti-tumor response through immune activation.
  • an immunotherapy can include an immune checkpoint inhibitor, an oncolytic virus therapy, a cell-based therapy, a CAR-T cell therapy, or a cancer vaccine.
  • an immunotherapy can include immune checkpoint blockade (ICB), wherein an immune checkpoint inhibitor is administered.
  • Immune checkpoint blockade (ICB) is a treatment that uses immune checkpoint inhibitors to address a disease (e.g., cancer), wherein the immune checkpoint inhibitor blocks a checkpoint protein from binding to its partner protein or receptor.
  • the versikine compositions of the disclosure may be used in methods of treating cancer and/or in conjunction with other immunotherapy.
  • the immunotherapy includes administration of an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor. Examples of a PD-1 inhibitor that can be used in conjunction with versikine compositions of the disclosure can include, but are not limited to,
  • the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • a PD-L1 inhibitor can include, but are not limited to, atezolizumab, avelumab, durvalumab, KN035, CK.-301, AUNP12, CA-170, and BMS- 986189.
  • the immune checkpoint inhibitor can be any checkpoint inhibitor, e.g., as described in Mazzarella et al., Eur J Cancer (2019) 117:14-31, hereby incorporated by reference.
  • the immunotherapy comprises an anti-PDl immunotherapy.
  • the immunotherapy comprises a stimulator of interferon genes (STING) agonist immunotherapy.
  • the immunotherapy includes activation of the cGAS-STING signaling pathway (e.g., STING agonist immunotherapy).
  • cGAS cyclic GMP-AMP synthase
  • STING interferon genes
  • Activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway induces the expression of type I interferons and proinflammatory cytokines, promoting a robust adaptive antitumor immunity.
  • STING is ubiquitously expressed in immune and nonimmune cells, wherein STING activation has been demonstrated to propagate the cancer immunity cycle, remodel the tumor microenvironment, and ultimately eliminate tumor cells.
  • Non-limiting examples of STING agonists that can be used in conjunction with versikine compositions of the disclosure include ENPP1 inhibitors (e.g., MV-626, SR-8314, SR-8291, SR8541A), bacterial vectors (e.g., SYNB1891 and STACT-TREX-1), CDN compounds (ADU-S100, BI-STING, BMS-986301, GSK532, JNJ-4412, MK-1454, SB11285, 3’3’cyclic AIMP), non-CDN small molecules (e.g., ALG-031048, ASA404, DMXAA, E7766, JNP6196, MK-2118, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-100001), nanovaccines (e.g., PC7A NP, cGAMP-NP, ONM-500), antibody-drug conjugates (e.g., XMT-2056 and CRD-5500
  • the immunotherapy includes CD40 agonist immunotherapy.
  • CD40 is a cell-surface member of the TNF (tumor necrosis factor) receptor superfamily, and upon activation, CD40 can license dendritic cells (DCs) to promote antitumor T cell activation and re-educate macrophages to destroy tumor stroma.
  • DCs dendritic cells
  • CD40 activation can play a role in driving antitumor immunity, whereby CD40-activated DCs are poised to prime or activate tumor-specific T cells.
  • CD40 agonists can be used as an immunotherapy for patients with cancer, wherein CD40 agonist immunotherapy can include agonistic anti- CD40 monoclonal antibodies (mAbs), trimeric CD40 ligand (CD40L), or ectopic expression of CD40L using gene therapy of transferred tumor or other cells.
  • mAbs agonistic anti- CD40 monoclonal antibodies
  • CD40L trimeric CD40 ligand
  • ectopic expression of CD40L using gene therapy of transferred tumor or other cells.
  • SUBSTITUTE SHEET ( RULE 26 ) immunotherapy comprises an anti-CD40 immunotherapy.
  • CD40 agonists that can be used in conjunction with versikine compositions of the disclosure include antibodies (e g., selicrelumab (CP-870,893 or R07009789), APX005M, JNJ-64457107, SEA- CD40, ChiLob7/4, CDX-1140H, Dacetuzumab (SGN-40), ABBV-428).
  • TME tumor microenvironment
  • ECM extracellular matrix
  • a tumor microenvironment refers to a highly complex and dynamic structure of cells of which a variety of immune cells are a major component.
  • the TME contains cells of the immune system (e.g., T cells, B cells, dendritic cells, myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (T AMs)), a complicated network of fibroblasts, blood vessels, lymphatics, and the cells of the cancer itself.
  • TME elements are surrounded by a dense meshwork of collagen and elastin fibers that comprise the extracellular matrix (ECM), wherein the TME is also composed of a complicated network of cytokines, chemokines, growth factors, and inflammatory, as well as matrix remodeling enzymes.
  • ECM extracellular matrix
  • the TME is a critical facilitator of immune escape and cancer progression, and the interaction between cancer cells and the diverse cell population within the TME influences tumor resistance, progression, and metastasis.
  • the administering of a proteolytic fragment of an ECM proteoglycan compnses delivery of the proteolytic fragment into a cell, thereby increasing T cell activation in a tumor microenvironment.
  • the cell is an antigen-presenting cell (e.g., a macrophage, B cell, or dendritic cell).
  • the cell is a non-hematopoietic antigen-presenting cell (e.g., antigen-presenting fibroblast, lymph node stromal cell, or endothelial cell).
  • the proteolytic fragment can be delivered into a cell by a variety of well-known techniques.
  • the delivery method can include a vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes).
  • the delivery method can include methods facilitating the delivery of “naked” polynucleotides (e g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • the delivering compnses transfection, electroporation, or a virus-based delivery.
  • the delivery of the proteolytic fragment comprises a virus-based delivery.
  • the virus-based delivery comprises an adeno-associated virus or a lentivirus.
  • the virus-based delivery comprises a lentivirus.
  • SUBSTITUTE SHEET ( RULE 26 ) as described herein increases T cell activation in a tumor microenvironment, thereby treating cancer and/or potentiating the effects of other cancer treatment.
  • a proteolytic fragment of an extracellular matrix (ECM) proteoglycan e.g., versikine
  • ECM extracellular matrix
  • the term “sensitizing” or “improve sensitivity” can refer to increasing response rates to a specific treatment (e g., immunotherapy).
  • improving sensitivity to ICB treatment can refer to inhibiting negative regulatory immune checkpoints or stimulating activating immune checkpoints.
  • the subj ect has refractory cancer (e. g. , a refractory' tumor).
  • the subject has undergone previous treatments for cancer.
  • the subject has not shown improvement in cancer symptoms and/or cancer markers after previous treatments for cancer.
  • administration of the versikine compositions sensitizes a response to immunotherapy in a subject, such that the subject shows improvement in cancer symptoms and/or cancer markers over similar immunotherapy without administration of versikine compositions.
  • methods of predicting a subject’s response to an immunotherapy include (a) analyzing a biological sample obtained from the subject; and (b) detecting a proteolytic fragment of an extracellular matrix (ECM) proteoglycan in the biological sample, thereby predicting the subject’s response to the immunotherapy.
  • ECM extracellular matrix
  • the analyzing comprises staining the biological sample.
  • stains can include histological stains (e.g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains).
  • the staining includes the use of hematoxylin and eosin.
  • a biological sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin.
  • biological stains including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osm
  • the biological sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxy lin and eosin (H&E), Jenner’s, Leishman, Masson’s trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright’s, and/or Periodic Acid Schiff (PAS) staining techniques.
  • PAS staining is typically performed after
  • the staining can include the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
  • the staining comprises an immunofluorescence stain. In some embodiments, the staining comprises an immunohistochemistry stain. In some embodiments, the biological sample is stained with an antibody. In some embodiments, the antibody is selected from the group consisting of: an anti-DPEAAE neo-epitope antibody, anti-HA antibody, anti-XCRl antibody, anti-CLEC9A antibody, anti-NCRl antibody, and anti-CD8 antibody.
  • the detecting can include imaging of the biological sample (e.g., a fixed and/or stained biological sample).
  • the biological sample can be visualized or imaged using bright field microscopy.
  • the biological sample can be visualized or imaged using fluorescence microscopy.
  • non-limiting examples of visualization and imaging can include expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy.
  • the detecting comprises determining the level of the proteolytic fragment by measuring the antibody in the biological sample.
  • HEK293T cells were transfected with a mixture of ps-PAX2 (packaging plasmid) and pVSV- G (envelope plasmid), and transfer plasmids encoding respective open reading frames or empty control.
  • pseudotype virus-containing culture medium was harvested, filtered, supplemented with 7.5 mg/mL polybrene (Sigma-Aldrich), and immediately applied to target cells for spinfection (120min, 2500xg at 32C). After spinfection, the medium was exchanged for fresh complete RPMI1640 medium. Target cells were passaged at least three times after retroviral transduction
  • MutuDC1940 cells were transduced with HA tagged versikine (Vkine) - or empty vector (EV)- containing lentivirus as detailed above.
  • Vkine HA tagged versikine
  • EV empty vector
  • SUBSTITUTE SHEET (RULE 26 ) were selected with lOmg/mL blasticidin for 2 weeks. HA-tagged versikine expression was confirmed by western blotting using anti-HA antibody (clone: C29F4, Cell Signaling). LLC- EV or -Vkine cell lines were transduced with pHIV-Luc-OVA-ZsGreen lentivirus. LLC-OVA expressing cells were FACS-sorted based on ZsGreen expression to ensure comparable transduction rates between different cell lines. shRNA mediated VCAN knockdown
  • the lentiviral shRNA vector set targeting mouse Vcan (NM 019389.2) and scrambled control were purchased from GeneCopoeia (#MSH080253-LVRU6H and #CSHCTR001-LVRU6H).
  • 2> ⁇ 10’ LLC cells were plated per well in a 6-well plate and incubated overnight.
  • 2mL freshly harvested lentiviral supernatant (expressing either shambled control, Vcan shRNA#l, #2 or #3), ImL of culture medium and 7.5mg/mL polybrene was added per well.
  • the plate was centrifuged at 800g for 2h at 37°C and returned to CO2 incubator. After 72h, 200mg/mL Hygromycin B was added and the cells were under antibiotic selection for 2 weeks.
  • Vcan knockdow n was confirmed by RT-PCR.
  • Intratumoral injections were performed using a 28G insulin syringe, when tumors had reached 100-150 mm 3 , using surgical forceps to hold the tumor constantly.
  • a retro-orbital approach was adopted. Mice were anesthetized using inhaled isoflurane in a chamber. The eyeball was partially protruded from the socket by applying downward pressure to the skin dorsal and ventral to the eye. Injections were performed by placing the needle, bevel face-down, in order to decrease the likelihood of damaging the eyeball. Once the injection was complete, the needle was slowly and smoothly withdrawal. Triple antibiotic ophthalmic ointment was then applied to the eye.
  • Intraperitoneal inj ection was performed using a 28.5G insulin-syringe with the head tilted down. The needle was inserted at a 30° angle in the lower left or right quadrant. Transplantation of 37
  • SUBSTITUTE SHEET ( RULE 26 ) myeloma VQ4935 cells was performed via intracardiac injection after the 6-8 week old C57BL/6J recipient mice were sub-1 ethally irradiated at 6.0 Gy using an X-RAD 320 Irradiator. Intracardiac injection was performed by placement of needle in the fourth intercostal space and into the left ventricle. The needle was inserted at a 90° angle in the middle of the imaginary line connecting the sternal notch and xyphoid process serving as anatomical landmarks, and the needle was inserted slightly left of the sternum.
  • Recipient syngeneic mice (10 per arm) were injected with LLC-EV or LLC-Vkine cells (5 x 10 5 cells per inoculum).
  • Antibody treatments were with 100 ug of antibody in 100 uL of volume each (aPDl: Bio X Cell InVivoPlus, rat IgG2a, clone RPM1-14, Cat# BP0146, Lot# 806321 J2B;
  • Isotype control Bio X Cell InVivoPlus, rat IgG2a, clone 2A3, Cat# BE0089, Lot# 796721M2)
  • Treatments were administered on days 7, 10, and 14 post-inoculation.
  • Tumor burden was tracked by measuring tumors with an electronic caliper every two days, beginning with Day 5 post-inoculation. Mice reached endpoint when they were found dead, were in clear distress, or when tumors reached 20 mm in any dimension. Animals found dead were considered to have reached endpoint on the off-day of measurements.
  • tumors were excised 21 days after transplantation.
  • tumors were cut into pieces and digested with either Collagenase la (Img/mL) C2674 Sigma Aldrich and Hyaluronidase V (O.lmg/mL) H6254 Sigma Aldrich for 40min at 37°C or with a mouse tumor dissociation kit (Miltenyi Biotec #130-096-730) using gentle MACS dissociator.
  • Tissue was passed through a 70mm cell strainer (Falcon) and washed with FACS buffer (PBS with 1% FCS) before proceeding with antibody staining.
  • FACS buffer PBS with 1% FCS
  • homogenization was performed in RLT buffer (QIAGEN) facilitated by a closed tissue grinder system (Fisher brand #02-542-09, 15mL).
  • Tumor tissue was harvested and processed for mass cytometry analyses using the protocol described above for flow cytometry. After single cell suspensions were acquired, cells were washed with PBS, centrifuged at 300-400g for 5 minutes and supernatant was discarded by aspiration. Cells were resuspended in PBS and Cell-ID Cisplatin (Fl ui digm, #201064) was added to a concentration of 5uM. After rigorous mixing, cells were incubated at room 38
  • SUBSTITUTE SHEET ( RULE 26 ) temperature for 5 minutes. Cells were then quench stained with MaxPar Cell Staining Buffer (Fluidigm, #201068) using 5* the volume of the cell suspension, centrifuged and supernatant was discarded by aspiration. The process was continued with surface staining. 50ul of the antibody cocktail was added to each tube so the total staining volume was lOOul (50ul of cell suspension-!- 50ul antibody cocktail). Cells were stained for an hour at room temperature. All antibodies used for staining were either bought pre-conjugated to metal isotopes or were conjugated using the Maxpar Antibody Labelling Kit (Fluidigm, 201160B).
  • the immune milieu of the tumor (CD45+) was enriched by manual gating among single events, equally subsampled to 6,000 events, then run through a Barnes Hut implementation of the t-SNE algorithm, viSNE, in the R package ‘Rtsne’, using optimized parameters (iterations: 1000, perplexity:30, learning rate:455).
  • viSNE t-SNE algorithm
  • Rtsne R package ‘Rtsne’
  • Flow cytometnc analyses were performed using an LSR II and/or LSR Fortessa X20. Data were analyzed using FlowJo (Tree Star). DAPI (0.5 mg/mL, Sigma-Aldrich) or a Live/Dead fixable cell stain (Ghost 780 Tonbo Biosciences) was used to exclude dead cells in all experiments, and anti-CD16/CD32 antibody (2.4G2) was used to block non-specific binding of antibodies via Fc-receptors. Quantification of total cell numbers by flow cytometry was done using fluorescent beads
  • SUBSTITUTE SHEET ( RULE 26 ) analyzed by flow cytometry. Sorting of tumor cells after retroviral transduction was done using a BD FACSAria or a BD FACS Aria Fusion. Purity of cell populations was determined by reanalysis of a fraction of sorted cell samples.
  • 1.5xl0 6 BM cells were cultured in lOmL RPMI1640 medium supplemented with 10% heat- inactivated FCS (Biochrom), penicillin/streptomycin and 50mM b-mercaptoethanol.
  • Recombinant human FLT3L 300-19, Peprotech
  • recombinant murine GM-CSF 315-03, Peprotech
  • 5mL complete medium was added between day 5 and day 6 to minimize apoptosis.
  • Non-adherent cells were harvested on day 9, counted and re-plated at 3x10 6 cells in lOmL complete medium supplemented with FLT3L and GMCSF as on day 0.
  • Non-adherent iCD103 were harvested on days 15-16. Cells were then validated by assaying for CD103, CD24, Clec9A, and CD11c by flow cytometry.
  • MutuDC1940 cells were left unstimulated or were in vitro stimulated with LPS for 8 or 24 hours at 37°.
  • Cell-free supernatant was assessed for CXCL9 (R&D Quantikine mouse CXCL9 #MCX900) and IL27p28 (R&D Quantikine mouse IL27p28 #M2728) protein levels by ELISA according to the manufacturer’s instructions (R&D).
  • CXCL9 R&D Quantikine mouse CXCL9 #MCX900
  • IL27p28 R&D Quantikine mouse IL27p28 #M2728 protein levels by ELISA according to the manufacturer’s instructions (R&D).
  • R&D antigen-presentation assay, cell- free supernatants were collected and assessed for IFNg levels (R&D Quantikine mouse IFNg P233156).
  • SUBSTITUTE SHEET ( RULE 26 ) Paraffin-embedded murine tumor sections and unstained 4-5 mm-thick human lung carcinoma TMA (US Biomax Inc., BC041115e) sections were deparaffinized and rehydrated using standard methods. Antigen retrieval was earned out in citrate buffer, pH 6.0 (Vector Laboratories, #H-3300) for DPEAAE and HA; and pH 8.0 for XCR1 and CD8 (Abeam, ab93680).
  • aDPEAAE Primary antibodies included aDPEAAE (PA1-1748A, Thermo Fisher), anti-HA (C29F4, Cell Signaling Technology), anti-XCRl (D2F8T, Cell Signaling Technology), anti- CLEC9A (ab223188, Abeam), anti-NCRl (NKp46) (MAB1850, R&D) and anti-CD8 (C8/144B, Ebioscience).
  • the aDPEAAE neoepitope antibody has been previously validated. Stained slides were examined using an Echo Revolve microscope with attached digital camera. aDPEAAE immunostaining score was assessed by scoring staining intensity (0 for no staining, 1 for low/weak staining, 2 for moderate staining and 3 for strong/intense staining).
  • Dual staining XCR1/CLEC9A on human tonsil was performed by the UW TRIP lab as follows: The experiment was run on Roche Ventana Medical System’s Discovery Ultra Automated platform. Deparaffinization was carried out on the instrument, as was heat induced epitope retrieval in the form of “cell conditioning” with CC1 buffer (Ventana #950-500), a Tris based buffer pH 8.4 for approximately 56 minutes at 95°C. Slide was incubated with the first primary antibody XCR1 diluted 1:40 in DaVinci Green antibody diluent (BioCare Medical #PD900H) for 60 min at 37°. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration.
  • Discovery OmniMap anti-Rabbit HRP (Ventana #760-4311) was applied for 16 min at 37°. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery ChromoMap DAB detection (Ventana #760-159) was applied for the preset time. Denaturing agent in the form of Discovery Inhibitor (Ventana #760-4840) was applied for the preset time. Slide was incubated with the second primary antibody CLEC9A diluted 1:50 in Ventana antibody diluent with casein (Ventana #760-219) for 60 min at 37°. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration.
  • Discovery OmniMap anti-Rabbit HRP (Ventana #760-4311) was applied for 16 min at 37°. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery Teal HRP detection kit (Ventana #760-247) was applied for 32 minutes. Slide was removed from the instrument and rinsed with dawn dish soap and warm tap water followed by rinsing with dH2O. Slide was counterstained with Harris hematoxylin (1:5 diluted in dH2O) for 45 seconds. Slide was rinsed with dH2O. Slide was dehydrated in the oven (60 degrees C) followed by dipping in Xylene. Slide was cover slipped.
  • SUBSTITUTE SHEET ( RULE 26 ) 20X images were acquired for analysis.
  • a customized spectral library algorithm for all chromogens and counterstain was created using Nuance Software version 3.0.2 (PerkinElmer).
  • the inForm software version 2.4.7 was used to segment tissue subcellular compartments (nucleus, cytoplasm or membrane), and to measure biomarker expression.
  • the double positivity algorithm was used to measure colocalization of cells expressing XCR1 and CLEC9A as double-positive percentage rate.
  • RT2 Profiler PCR Array (QIAGEN, Cat. no. PAMM-021Z) was used.
  • RNA was isolated from tumors and was reverse transcribed using kits mentioned above.
  • cDNA was mixed with RT 2 SYBR®Green qPCR Mastermix (Cat. no. 330529). The mixture was aliquoted across the RT2 Profiler PCR Array (in 96-well format) and was run on the Real Time PCR machine. Data analysis was performed using the manufacturer’s online platform for RT2 Profiler Data analysis software.
  • SUBSTITUTE SHEET ( RULE 26 ) well were seeded in 12 well plate. The following day, 10% supernatant was added to the plate media. Cells were then incubated for 72 hours. After incubation, total RNA was extracted from MutuDC1940 cells.
  • RNA sample preparations A total amount of 1 mg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X) First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H.
  • NEBNext® UltraTM RNA Library Prep Kit for Illumina® NEB, USA
  • index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic
  • PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR pnmers and Index (X) Primer.
  • PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
  • the clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.
  • Raw data (raw reads) of FASTQ format were firstly processed through fastp.
  • clean data clean reads
  • Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
  • Reference genome and gene model annotation files (GRCm38) were downloaded from genome website browser (NCBI/UCSC/Ensembl) directly Paired-end clean reads were 44
  • SUBSTITUTE SHEET (RULE 26 ) aligned to the reference genome using the Spliced Transcripts Alignment to a Reference (STAR) software (v2.6.1d).
  • FeatureCounts (vl.5.0-p3) was used to count the read numbers mapped of each gene RPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
  • Differential expression analysis between two conditions/groups (three biological replicates per condition) was performed using DESeq2 R package (v 1.20.0).
  • DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting p values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted p value ⁇ 0.05 found by DESeq2 were assigned as differentially expressed.
  • Gene Set Enrichment Analysis was performed by comparing MutuDC1940-Vkine (treated with PBS, 4h) RNA-seq data to the corresponding MutuDC1940-EV sample. 4736 differentially expressed gene features for each condition were ranked by the signal to noise metric of GSEA and the analysis was performed using the standard weighted enrichment statistic against human gene sets contained in the Molecular Signatures Database (MSigDB v7.4) that included all (H) Hallmark gene sets, (C2) curated gene sets, and (C3) motif gene sets. The normalized enrichment score (NES) was calculated using 1000 gene set permutations.
  • MSigDB v7.4 Molecular Signatures Database
  • mice were injected i.p. with 50 ug of anti-asialoGMl (Wako Pure Chemical Industries, lOOmL/mouse) on days -1, 0, 7, 14 around tumor inoculation.
  • anti-asialoGMl Wang Chemical Industries, lOOmL/mouse
  • MutuDC1940 were cultured and treated with LPS or PBS control respectively overnight. Next day, the cells were harvested and plated on 96-well round bottom plates at a density of 100,000 cells per plate. DC were then loaded with OVA peptide 257-264 (SIINFEKL) (3ng/mL) and incubated for 4 hours at 37°C. MutuDC1940 were then washed with 0.1% PBS-BSA and centrifuged at 800 x g and were fixed with 50 mL per well of freshly made PBS-glutaraldehyde (GT A) 0.008% (vol/vol) and incubated for 5 minutes on ice.
  • SIINFEKL OVA peptide 257-264
  • GT A PBS-glutaraldehyde
  • PBS-glycine 0.4M 50mL was added to the PBS-GTA 0.008% solution and cells were centrifuged at 800 * g for 2 minutes at 4°C. Plates were subsequently flicked. Finally, 100 mL of PBS-glycine 0.2M was added to each well and centrifugation of the plates at 800 x g for 2 min at 4°C followed.
  • T cell culture medium RPMI 1640 containing 10% heat inactivated FBS, 100 lU/mL penicillin, lOOmg/mL streptomycin, 2mM glutamax, 50mM P-mercaptoethanol, IxMEM nonessential ammo acids, lx sodium pyruvate
  • 100,000 OT-I T cells per well in lOOmL T cell culture medium were added (to a final volume of 200mL).
  • the co-cultured OT-I T cells with the cross-fixed DCs were incubated for 18 h at 37°C.
  • SUBSTITUTE SHEET (RULE 26 ) Level 4 gene expression data were downloaded from the TCGA Data Portal and fdtered to retain only cancer types of known epithelial origin for a total of 7591 samples across 20 different cancer types
  • Single-sample Gene Set Enrichment Analysis (ssGSEA) was performed to measure the signature of gene sets designed to measure overall immune infiltration, cDCl density and CD8+ T cell density.
  • TCGA samples were grouped by cancer type and sorted based on median expression of versican (VC AN) and median cDCl signature.
  • ssGSEA was then used to measure the signature of these two gene sets in the 1017 lung samples in the TCGA cohort and overall versikine response level was summarized as the difference between versikine-up and versikine-down signature levels.
  • This versikine response signature was then compared to the CD8 effector T cell signature using an ordinary least squares linear model including the overall immune infiltration signature as a covariate, p-values of less than 0.05 were considered significant.
  • Example 1 Conventional type dendritic cells (cDCls) localize in peritumoral stroma and are regulated by the VCAN pathway
  • VCAN Versican
  • hyaluronan binding partner hyaluronan are cardinal components of the provisional extracellular matrix in development, wound healing, and cancer.
  • stromal provisional matrix is thought to coordinate critical pro-tumor functions (e.g., angiogenesis) and prime conversion toward collagen-rich, desmoplastic stroma.
  • VCAN is sourced from stromal mesenchymal cells, immune infiltrating cells (particularly myeloid cells, such as macrophages), and, in some cases (such as in lung 47
  • VCAN proteolytic processing is located primarily in stroma because of the local activity of stromal fibroblast-derived ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs; a family of multidomain extracellular protease enzymes) VCANases (enzymes which cleave versican).
  • ADAMTS a disintegrin and metalloproteinase with thrombospondin motifs; a family of multidomain extracellular protease enzymes
  • VCANases enzyme which cleave versican.
  • VCAN proteolysis signal was observed in approximately 83% of human lung cancer cases in a stromal distribution (FIG. IB).
  • multiplex IHC was performed with antibodies detecting the cDCl lineage marker XCR1 and CD8.
  • XCR1+ cells were confined within stromal sheets recurrently undergoing VCAN proteolysis (FIG. 1C).
  • the antibody against XCR1 has been previously validated, and close correlation between XCR1 staining and the signal for the cDCl lineage marker CLEC9A was confirmed in human tonsils (FIG. IK).
  • Matrikines such as versikine have been defined as ‘‘peptides liberated by partial proteolysis of extracellular matrix macromolecules which are able to regulate cell activities not triggered by their full-size parent macromolecules”. Notwithstanding its distinct neo-activity, versikine ultimately derives from parental VCAN through ADAMTS proteolysis (FIG. 1A); therefore, it was hypothesized that VCAN expression (the substrate for versikine) and cDCl abundance correlate inhuman cancer. VCAN gene expression and cDCl signature scores were compared across 7,591 samples from 20 The Cancer Genome Atlas (TCGA) cancer types (FIGs. ID and IE). A significantly positive correlation between VCAN expression and cDCl signature scores was observed in several human carcinomas (FIG. IF), suggesting that the VCAN pathway broadly regulates cDCls.
  • TCGA Cancer Genome Atlas
  • Vcan exons 2-6 novel Vcan -targeted models were generated that disrupt exons coding for Vcan’s N terminus (Vcan exons 2-6).
  • the widely used Vcan h ⁇ null mutant (hdf, heart defect) targeting exon 7 is embryonic lethal in homozygosity.
  • Vcan hd f hemizygosity demonstrates functional haploinsufficiency for CD8+ -mediated control of viral infection.
  • CRISPRCas9-mediated mutagenesis was used to disrupt exon 3 sequences, abolishing transcription of all Vcan isoforms (and, consequently, generation of versikine) (FIG. 1G).
  • Vcanl053 and Vcanl058 bearing Vcan exon 3 deletions were found (16 bp and 47 bp, respectively) (FIGs. IL and IM).
  • Defective Vcan message induction was confirmed after stimulation of BM-derived macrophages (BMDM) with the Toll -like receptor (TLR)-4 agonist lipopolysaccharide (LPS) (FIG. IN).
  • Vcan +I ' SUBSTITUTE SHEET
  • LLC VcanKD Lewis lung carcinoma
  • ShRNA short hairpin RNA
  • GAG glycosaminoglycan
  • FIG. 10 The intratumoral immune contexture was characterized in LLC VcanKD tumors implanted in Vcan +/ ⁇ mice through mass cytometry and compared it with wild-type (WT) controls (FIG.
  • Vcan depletion resulted in expansion of CD8+ T cells, consistent with the known role of non-proteolyzed VCAN in T cell exclusion.
  • cDCl loss was observed in Vcan-depleted tumors (FIG. 1H).
  • intratumoral DCs were delineated through 9-color flow cytometry (FIG. IP) cDCls were depleted in LLC VcanKD tumors implanted into Vca V 1 ' mice (FIGs. II and 1J).
  • steady-state splenic cDCls were not reduced (in fact, they were mildly increased) in Vcan+I- mice (FIG. IQ).
  • Example 2 VCAN matrikine versikine promotes cDCl abundance in vivo
  • VCAN proteolysis is a composite event that produces two simultaneous, coupled consequences: first, parental VCAN clearance, and second, the novel activities of the released matrikine versikine.
  • LLC cells stably expressing hemagglutinin (HA)-tagged versikine in the WT background were generated (FIG. 2A).
  • HA hemagglutinin
  • FIG. 2B Ectopically expressed versikine was readily detectable by anti-HA tag western blotting in murine tumor lysates at approximately 75 kDa (FIG. 2C and 2H).
  • Murine implantable tumor models do not recapitulate the human architecture of epithelial nests and stromal sheets: this limitation has been attributed to acquisition of mesenchymal features through successive passaging (FIG. 2D).
  • the LLC model does retain physiological relevance because of its tumor-intrinsic production of Vcan that regulates myeloid cells in the TME.
  • LLC mimics a subset of human lung cancers with detectable VCAN production and processing in stromal and epithelial compartments (FIG. 2I-2J). Ectopically expressed versikine was detected in a membranous distribution consistent with its accumulation in the pericellular glycocalyx (FIG.
  • FIG. 2N Versikine promoted cDCls in the BALB/c-derived, orthotopic 4T1 mammary carcinoma model. Growth rates of 4T1 versikine-replete tumors did not differ from their EV counterparts (FIG. 20). Earlier a role of VCAN proteolysis was reported in shaping the human BM myeloma immune microenvironment. More recently, the first Ras-driven immunocompetent myeloma model was developed, VQ. Versikine-replete VQ myeloma tumors demonstrated enhanced cDCls (FIG. 2P) Myeloma clinical progression was 50
  • the Balf3 transcript increase corroborates versikine-induced cDCl abundance because the Batf3 expression range is very narrow (FIG. 3H) Id2 transcripts did not differ between versikine- replete and control tumors, but Id2 is expressed more broadly and not highly expressed in cDCls (FIG. 3H).
  • CD45.2+ pre-DC precursors were sorted from the BM of Flt31 in-vivo-mobilized donor mice (FIGs. 31-3 J). Donor mice were implanted with Flt31-secreting B16 tumor cells to provide a continuous source of circulating Flt31.
  • CD45.2+ pre-DCs were adoptively transferred intratumorally into subcutaneous LLC-EV and LLC- Vkine tumors implanted into CD45.1+ recipients. 72 h after adoptive transfer, tumors were dissociated, and CD45.2+ as well as endogenous CD45.1+ DC fractions were enumerated by flow cytometry.
  • the CD45.1+ endogenous eDC composition served as an internal control.
  • CD45.1+ endogenous cDCls were increased in LLC-Vkine tumors (FIG. 3K).
  • CD45.2+ cDCls and cDC2s did not differ between LLC-Vkine and -EV controls (FIG. 3L)
  • stromal signals did not affect pre-DC differentiation into cDCls versus cDC2s within the time frame of the differentiation assay and given the assay’s limitations.
  • TILs tumor-infiltrating leukocytes
  • FIGs. 3F and 3G show that I12rb, Cd38, Light, and Gitr were detected in T cellspecific transcripts (CD3e and T cell receptor [TCR] genes), consistent with the CD8+ T cell expansion seen by mass cytometry (FIG. 2F).
  • Hallmarks of antigen-presenting cell (APC) activation were detected (upregulation of MHCII, Ccr7, Ifnbl, Irf7, and several interferonresponsive genes) (FIG. 3F).
  • Example 5 - cDCl activation by versikine is cell autonomous
  • MutuDC1940-Vkine cell lines were generated through lentiviral transduction (FIG. 4A). MutuDC1940-Vkine cells had a slightly more developed dendritic appearance compared with EV (FIG. 4B).
  • Versikine-upregulated genes involved in DC maturation interferon- stimulated genes such as Ifi209 and Ifi204), chemokines (Ccl7, Ccl2, Cxcl9, and CxcllO), and co-stimulatory signals (Cd80 and Cd40) (FIG. 4D).
  • Downregulated genes included components of transforming growth factor b (TGF-b) and Wnt pathways, both associated with immunosuppression.
  • Gene set enrichment analysis confirmed upregulation of immune activation gene sets (e.g., interferon [IFN]-a response, IFN-g signaling, nuclear factor kB [NF- kB] -induced tumor necrosis factor [TNF] signaling, and inflammation) and downregulation of immunosuppressive Wntb-catenin and TGF-b signaling (FIG. 4E).
  • IFN interferon
  • NF- kB nuclear factor kB
  • TGF-b signaling e.g., NF- kB
  • TGF-b signaling e.g., IL- kB
  • TGF-b signaling e.g., interferon [IFN]-a response, IFN-g signaling, nuclear factor kB [NF- kB] -induced tumor necrosis factor [TNF] signaling, and inflammation
  • Example 6 - cDCl accumulation requires atypical innate lymphoid support
  • cDCl activation by versikine was cell autonomous, cDCl accumulation might still require supporting actors, such as natural killer (NK) cells.
  • NK natural killer
  • CDl lc+ DCs from primary versikine- replete versus EV tumors were characterized Freshly explanted CDllc+ cells from LLC- Vkine tumors expressed higher levels of the NK regulators IL-23 (a subunit), IL-27 (p28 and EBB subunits), and IL-15 (FIG. 5A). Therefore, versikine’s cDCl activation program incorporated an NK cell -activating module.
  • NK cell-derived differentiation/survival mediator FLT3L as well as the chemo-attractants XCL1 and CCL5 in cDCl support.
  • NK cell-derived IFN-g has been shown recently to induce the cDCl “terminal selector” IRF8.
  • NKp46+ NK1.1+ cells from LLC-Vkine tumors were potent expressors of Csf2 (granulocyte-macrophage colony stimulating factor [GM-CSF]) and relatively weak expressors of IFN-g compared with the LLC-EV-derived counterparts (FIG. 5B), whereas expression of Xcll,
  • an antiasialo-GMl antibody (anti-ASGMl) was used for in vivo NK cell depletion (FIG. 5C).
  • Anti-ASGMl antibody treatment (FIG. 5H) completely abrogated versikine-mediated enhancement of cDCls in the TME (FIG. 5D and 51).
  • Example 7 Versikine regulates cDCl-NK cell cross-talk through cDCls
  • MutuDC1940 cells are sensitive to apoptotic signals and that this sensitivity is mitigated/rescued by exogenous murine GM-CSF (FIG. 5G).
  • NK cells are dominant GM-CSF producers compared with sparser GM-CSF expressors, such as basophils (FIGs. 5J and 5K).
  • Example 8 - TLR2 and CD44 are dispensable for cDCl accumulation in response to versikine
  • Example 9 Stroma-licensed cDCls are “poised” and hypersensitive to nucleic acid sensing in vivo
  • mice bearing tumors implanted in both flanks were examined.
  • the treated side was inoculated with EV- or versikine-expressing LLC cells; the contralateral, nontreated side was inoculated with unmanipulated (untransduced) LLC cells.
  • Ectopic versikine is bound in the pericellular halo (glycocalyx) (FIG. 2D) and probably does not circulate to an appreciable degree.
  • a consistent abscopal effect was observed when versikine-replete tumors were injected with 200 mg DMXAA (FIGs. 6F-6H).
  • EV tumors treated with the same subtherapeutic dose failed to elicit any response on the treatment or contralateral side.
  • STING agonist hypersensitivity produced consistent primary tumor and abscopal effects across genetic backgrounds; e.g., in the orthotopic 4T1 mammary carcinoma model (FIGs. 6M-6O).
  • MutuDC1940 cells cannot be used for adoptive transfer experiments because of their immunogenicity. A consistent cDCl -like phenotype was confirmed in iCD103 cells (FIG. 6Q). Adoptive transfer of iCD103 cells restored subtherapeutic STING agonist efficacy (FIG. 6K) and survival benefits (FIG. 6L). To confirm the findings in a different C57BL6/J model, the B 16 melanoma model was chosen. B 16 tumors responded to subtherapeutic doses of DMXAA in the presence of versikine but not EV (FIGs. 6R and 6S). Efficacy was lost in the Batf3-null background (FIG.
  • the OVA system was employed as an in vivo model antigen (FIG. 7A).
  • EV- and versikine-expressing LLC cells were additionally engineered to express full-length OVA (LLC-OVA).
  • LLC-OVA full-length OVA
  • EV- or versikine-replete LLC-OVA tumors were challenged with therapeutic DMXAA doses (500 mg).
  • DMXAA doses 500 mg.
  • spleens were harvested and analyzed by flow cytometry for antigen-specific effector responses using an antigen-specific tetramer assay.
  • Example 11 The stroma-licensed cDCl signature correlates with CD8+ T cell scores in human lung cancer
  • Example 12 Stromal VCAN proteolysis correlates with CD8+ infiltration in human lung cancer
  • CD8+ T cell infiltration has prognostic significance in human lung cancer as well as predictive significance for efficacy of checkpoint inhibitor-based immunotherapy.
  • a cutoff of 3-5 CD8+ T cells/HPF has been used in some studies to designate CD8+ TIL-rich versus -poor tumors bearing a favorable and unfavorable prognosis, respectively.
  • VCAN matrikine versikine overcomes resistance to anti-PDl checkpoint inhibition immunotherapy in vivo cDCls are critical for responses to checkpoint inhibitors.
  • the effect of versikine on anti-PDl responses in the refractory LLC model was tested (FIG. 7F and 7 J).

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Abstract

Provided herein are versikine fusions, including versikine fusions with GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, IFN, as well as secretory peptides. Additionally, provided herein are methods for introducing versikine and versikine fusion proteins to cells, in vitro and in vivo. Additionally, provided herein are methods of treating cancer, increasing T cell activation in a tumor microenvironment (TME), or sensitizing a response to an immunotherapy in a subject in need thereof, the method comprising administering a proteolytic fragment of an extracellular matrix (ECM) proteoglycan.

Description

METHODS FOR SENSITIZING RESPONSE TO AN IMMUNOTHERAPY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims pnonty to U.S. Provisional Patent Application No. 63/419,194, filed on October 25, 2022, and U.S. Provisional Patent Application No. 63/480,173, filed on January 17, 2023. The disclosure of the prior applications is considered part of the disclosure of this application and is incorporated herein by reference in its entirety.
SEQUENCE LISTING
This application contains a Sequence Listing that has been submitted electronically as an XML file named “15670-0367WO1.XML.” The XML file, created on August 14, 2023, is 34,725 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under Grant No. CA252937 awarded by the National Institutes of Health. The Government has certain rights in the invention.
BACKGROUND
T-cell-inflamed tumor microenvironments (TME) are prerequisite for immunotherapy efficacy; however, the drivers for promoting T cell priming and infiltration within the TME remain incompletely understood. Furthermore, conventional type I dendritic cells (cDCl) are indispensable for anti-tumor immunity, wherein several studies have documented the paradoxical localization of cDCl within tumor stroma.
Tumor antigen cross-presentation and CD8+ T cell effector priming by stimulatory type 1 conventional dendritic cells (cDCls) is integral to spontaneous and therapeutic anti -tumor immunity. In addition to effector priming in the lymph node and in the tumor microenvironment (TME), cDCls regulate effector cell influx into the TME. From a translational perspective, cDCls are crucial for responses to vaccination strategies, immune checkpoint inhibitors, and engineered immune effector cells (e.g., chimeric antigen receptor T [CAR-T] cells). It has been shown that stimulatory cDCls are excluded from interdigitating
1
SUBSTITUTE SHEET ( RULE 26 ) tumor nestlets and locate in peritumoral stroma. However, the mechanisms that retain cDCls at the tumor periphery remain poorly understood.
SUMMARY
Provided herein are fusion proteins comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and a secretory peptide having a sequence of SEQ ID NO: 2-7.
Also provided herein are fusion proteins comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and a protein selected from the group consisting of: GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, and IFNgamma. In some embodiments, a protein selected from the group consisting of GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, and IFNgamma comprises a sequence selected from the group consisting of SEQ ID NO 8-21. In some embodiments, the fusion protein further comprises a secretory peptide. In some embodiments, the secretory peptide comprises a sequence of SQ ID NO: 2-7.
Also provided herein are vectors comprising any one of the fusion peptides described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector.
Also provided herein are methods of introducing versikine to antigen-presenting cells, the method comprising: introducing to an antigen-presenting cell any one of the fusion proteins or the vectors described herein, wherein the introducing comprises a lentiviral vector. In some embodiments, the antigen-presenting cells comprise macrophages, B cells, fibroblasts, and/or dendritic cells. In some embodiments, the antigen-presenting cell is a cell culture. In some embodiments, the antigen-presenting cell is in a subject.
Also provided herein are conjugated polypeptides comprising a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 and a small molecule STING agonist. In some embodiments, the small molecule STING agonist comprises DMXAA or RVU-27065.
Also provided herein are pharmaceutical compositions comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and a STING agonist. In some embodiments, the STING agonist is DMXAA or RVU-27065 In some embodiments, the versikine protein is co-administered with the STING agonist. In some embodiments, the versikine protein is administered before the STING agonist.
Also provided herein are pharmaceutical compositions comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and an anti-PDl and/or anti-PD-Ll inhibitor. In
2
SUBSTITUTE SHEET ( RULE 26 ) some embodiments, the anti-PDl and/or anti-PD-Ll inhibitor comprises pembrolizumab, nivolumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, or BMS-986189. In some embodiments, the versikine protein is co-administered with the anti-PDl and/or anti-PD-Ll inhibitor. In some embodiments, the versikine protein is administered before the anti-PDl and/or anti-PD-Ll inhibitor.
Also provided herein are pharmaceutical compositions comprising a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 and anti-CD40 immunotherapy. In some embodiments, the anti-CD40 immunotherapy is selected from the group consisting of selicrelumab, APX005M, JNJ-64457107, SEA-CD40, ChiLob7/4, CDX-1140H, Dacetuzumab, and ABBV-428. In some embodiments, the versikine protein is co-administered with the anti- CD40 immunotherapy. In some embodiments, the versikine protein is administered before the anti-CD40 immunotherapy.
Also provided herein are methods of treating cancer, the method composing administering a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 or a versikine nucleic acid having at least 95% sequence identity to SEQ ID NO: 23.
Also provided herein are methods for increasing T cell activation in a tumor microenvironment (TME) in a subject, the method comprising: administering a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 or a versikine nucleic acid having at least 95% sequence identity to SEQ ID NO: 23.
Also provided herein are methods for sensitizing a response to an immunotherapy in a subject in need thereof, the method comprising: administering a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 or a versikine nucleic acid having at least 95% sequence identity to SEQ ID NO: 23.
In some embodiments, the versikine protein or versikine nucleic acid is co-administered with another pharmaceutical compound. In some embodiments, the pharmaceutical compound comprises a stimulator of interferon genes (STING) agonist immunotherapy. In some embodiments, the pharmaceutical compound comprises an anti-PDl immunotherapy. In some embodiments, the pharmaceutical compound comprises an anti-CD40 immunotherapy. In some embodiments, the administering comprises delivery of the versikine.
In some embodiments, the cell is a dendritic cell. In some embodiments, the cell is an antigen-presenting cell. In some embodiments, the delivery of the proteolytic fragment comprises a virus-based delivery. In some embodiments, the virus-based delivery comprises a lentivirus.
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SUBSTITUTE SHEET ( RULE 26 ) In some embodiments, the versikine is generated by proteolysis of VCAN at the Glu441-Ala442 bond. In some embodiments, the versikine protein or versikine nucleic acid is conjugated to another protein. In some embodiments, the other protein is selected from the group consisting of: GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, or IFN. In some embodiments, the other protein comprises a secretory peptide In some embodiments, the secretory peptide is selected from the group consisting of: human OSM, human immunoglobulin, human chymotrypsinogen, human trypsinogen 2, human IL-2, or human insulin.
In some embodiments, the versikine protein or versikine nucleic acid is conjugated to a drug. In some embodiments, the drug comprises a small molecule STING agonist. In some embodiments, the versikine protein or versikine nucleic acid is conjugated to a small molecule. In some embodiments, the versikine protein or versikine nucleic acid is conjugated to a radiochemical. In some embodiments, the versikine protein or versikine nucleic acid is conjugated to an immunogenic component. In some embodiments, the versikine protein or versikine nucleic acid is conjugated to a label. In some embodiments, the label is a radioisotope or a fluorophore.
In some embodiments, the versikine is a nucleic acid. In some embodiments, the versikine nucleic acid is fused to non-versikine nucleic acid. In some embodiments, the non- versikine nucleic acid is a DNA or RNA molecule.
In some embodiments, a method provided herein further comprises administering a pharmaceutical agent. In some embodiments, the pharmaceutical agent comprises an immunotherapy agent. In some embodiments, the immunotherapy agent comprises a STING agonist, a PD-1 inhibitor, aPD-Ll inhibitor, or a CD40 agonist.
In some embodiments, the subject has a tumor. In some embodiments, the tumor is a refractory tumor.
Also provided herein are methods of predicting a subject’s response to an immunotherapy, the method comprising: (a) analyzing a biological sample obtained from the subject; and (b) detecting a proteolytic fragment of an extracellular matrix (ECM) proteoglycan in the biological sample, thereby predicting the subject’s response to the immunotherapy. In some embodiments, the analyzing comprises staining the biological sample. In some embodiments, the staining comprises an immunofluorescence stain. In some embodiments, the staining comprises an immunohistochemistry stain. In some embodiments, the biological sample is stained with an antibody. In some embodiments, the antibody is selected from the group consisting of: an anti-DPEAAE neo-epitope antibody, anti-HA antibody, anti-XCRl
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SUBSTITUTE SHEET ( RULE 26 ) antibody, anti-CLEC9A antibody, anti-NCRl antibody, and anti-CD8 antibody. In some embodiments, the detecting comprises determining the level of the proteolytic fragment by measuring the antibody in the biological sample.
In some embodiments, the immunotherapy comprises a stimulator of interferon genes (STING) agonist immunotherapy. In some embodiments, the immunotherapy comprises an anti-PDl immunotherapy In some embodiments, the immunotherapy comprises an anti-CD40 immunotherapy.
In some embodiments, the ECM proteoglycan is versican (VCAN). In some embodiments, the proteolytic fragment of the ECM proteoglycan is versikine. In some embodiments, the versikine is generated by proteolysis of VCAN at the Glu441-Ala442 bond.
In some embodiments, the subject has a tumor. In some embodiments, the tumor is a refractory tumor.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
FIGs. 1A-1J show that the VCAN pathway regulates tumor cDCls. FIG. 1A shows an exemplary schematic showing versican (VCAN)-Vl functional domains and site-specific proteolysis to generate versikine (scissors represent ADAMTS proteolytic cleavage). CS, chondroitin sulphate. FIG. IB shows stromal distribution of anti-DPEAAE IHC staining in human lung cancers. DPEAAE constitutes the C terminus of versikine (chromogen, DAB; counterstain, hematoxylin). FIG. 1C shows triple IHC staining of human lung cancers (DPEAAE, teal; XCR1, brown; CD8, purple). FIG. ID shows distribution of VCAN expression across TCGA carcinomas, ordered on the horizontal axis by median VCAN expression. FIG. IE shows distribution of cDCl (BATF3-DC) score across TCGA carcinomas,
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SUBSTITUTE SHEET ( RULE 26 ) ordered on the horizontal axis by median measured cDCl score. FIG. IF shows levels of correlation between cDCl (BATF3-DC) score and VCAN expression across TCGA carcinomas. The ranked median of VCAN expression and measured cDCl (BATF3-DC) score is shown across the x axis (1, highest; 20, lowest). Significant (q < 0.1) correlations after multiple hypothesis correction are colored red. Error bars represent the standard error of the correlation coefficient measured using Python statsmodels. FIG. 1G shows generation of Vcan1 - mice through CRISPR-Cas9-based targeting of Vcan exon 3. FIG. 1H shows mass cytometry of CD45+ cells from WT (LLC implanted into WT recipients, left) and Vcan- depleted tumors (LLCVcanKD tumor cells implanted into Vcan+A recipients, right). FIG. II shows quantification of frequency (left) and absolute count ratios (cDCl/cDCl+cDC2 and cDC2/cDCl+cDC2) in WT, Vcan-depleted (LLC-EVVcanKD: Vcan+/ ), and versikine (Vkine)- rescued (LLC-VkmeVcanKD: Vcan+A) tumors. Data are presented as mean ± SEM. n = 5 for each group. *p < 0.05, **p < 0.01, ***p < 0.001. FIG. 1J shows representative flow cytometry plots showing cDCl and cDC2 frequency in WT, Vcan-depleted (LLC-EVVcanKD: Vcan+A), and Vkine-rescued (LLC-VkineVcanKD: Vcan+A) tumors. FIG. IK shows human tonsil immunohistochemical staining for cDCl lineage markers XCR1 and CLEC9A. Left, composite image after spectral unmixing. XCR1 (DAB), CLEC9A. Right, scoring map: double-negative; single-positive XCR1; single-positive CLEC9A; double-positive XCR1/CLEC9 A. 23 different areas were imaged, each containing approximately 4173 cells. About 80% of cells had no expression of markers and -14% had dual staining. FIG. IL shows an exemplary schematic depiction of the deletions in the two mutant Vcan founders, 1053 (16bp deletion) and 1058 (47bp deletion). Sequence of exon 3 primer used in RT-PCR experiments is shown. FIG. IM shows DNA amplification using primers flanking the targeted region. Shown are a 128bp WT amplicon and the mutated amplicons in founders Fcanl053 and Fcc?nl058. FIG. IN shows bone marrow-derived macrophage (BMDM) Vcan locus RT-PCR using exon 3 primers at baseline or after stimulation with TLR4 agonist, lipopolysaccharide (LPS). VcanI053 demonstrates a more severe defect in Vcan message induction/stability than Vcanl058. FIG. 10 shows validation of Vcan knockdown in LLCVcanKD cells. LLC cells were transfected with each of 3 hairpins (shRNA #1, 2 or 3) targeting exon 8 (encoding GAG0 domain depicted in FIG. 1A) Vcan message was assayed using exon 3 primers (left) and exon 15 primers (right). FIG. IP shows gating strategy to delineate tumor-associated dendritic cells (TADC) per van Ginderachter schema. FIG. IQ shows eDC subset frequencies in steady-state splenic tissue from WT and Vcan-/- mice. FIG. 1R shows total eDC (cDCl + cDC2) absolute counts per mg tumor mass in LLC:WT, Vcan-depleted (LLC-EVVcanKD: Vcan+/-) and versikine-rescue (LLC-
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SUBSTITUTE SHEET ( RULE 26 ) VkineVcanKD: Vcan+/-) tumors, using the Collagenase/Hyaluronidase tumor cell dissociation protocol. FIG. IS shows growth rates of WT, Vcan-depleted (LLC-EV VcanKD: Vcan+/-) and versikine-rescue (LLCVkineVcanKD:Vcan+/-) tumors. In FIG. 1R and IS, the colon separates the genoty pe of implanted tumor cells from genotype of recipient animal.
FIGs. 2A-2G show that the VCAN-matrikine versikine promotes cDCl abundance in vivo. FIG. 2A shows an exemplary schematic of the experiment. LLC tumor cells that were engineered to express hemagglutinin (HA)-tagged versikine (LLC-Vkine) or empty vector controls (LLC-EV) and injected subcutaneously (s.c.) on the flank or intravenously using a retro-orbital approach. FIG. 2B shows gross morphology of orthotopic (top) and s.c. (bottom) LLC-EV and LLC- Vkine tumors. FIG. 2C shows anti -HA tag western blotting detects a 75-kDa band in LLC- Vkine tumor lysates, consistent with versikine. FIG. 2D shows representative immunohistochemistry (IHC) images showing a-DPEAAE and HA tag staining of LLC-EV and LLC-Vkine tumors. Endogenous DPEAAE proteolysis is low level and similar between LLC-EV and LLC-Vkine. Anti-HA staining localizes in a membranous distribution in LLC- Vkine cells (inset, larger magnification). FIG. 2E shows flow cytometric analysis of eDC subsets in s.c. LLC-EV and LLC-Vkine tumors and quantification of eDC and tumor- associated DC (TADC) frequency (top) and absolute count ratios (cDCl/cDCl+cDC2 and cDC2/cDCl+cDC2) (bottom). FIG. 2F shows comparison of immune contexture (CD45+ fraction) in LLC-EV versus LLC-Vkine tumors by 31 -marker mass cytometry. FIG. 2G shows flow cytometry analysis of eDC subsets in orthotopic LLC-EV and LLC-Vkine tumors (lung metastases induced by intravenous injection). A summary of eDC and TADC subset frequencies is depicted on the right. FIG. 2H shows full-length western blot for FIG. 2C. Arrow, versikine band migrating at 75KDa. FIG. 21 shows example of human lung cancer biopsy with stromal plus epithelial DPEAAE staining. 10X objective: scalebar 240mm, 40X objective: scalebar 60mm. FIG. 2J shows examples of negative DPEAAE staining in human lung cancer biopsies. 10X objective: scalebar 240mm, 40X objective: scalebar 60mm. FIG. 2K shows membranous localization of HAtagged ectopic versikine in B16 melanoma, a tumor model characterized by absence of cellautonomous Vcan expression. HA-tag, chromogen: BCIP/NBT; counterstain: nuclear fast red. FIG. 2L shows growth rates of subcutaneous LLC- EV and LLC-Vkine tumors. FIG. 2M shows absolute counts (cell count/ mg of tumor) of major intratumoral DC subsets, following optimized cell dissociation protocols (Miltenyi Dissociation Kit). FIG. 2N shows flow cytometric analysis of eDC subsets in orthotopically- implanted 4T1 mammary carcinoma tumors engineered to express empty -vector (4T1-EV) or
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SUBSTITUTE SHEET ( RULE 26 ) versikine (4T1-Vkine). Representative flow plots (left) and frequencies (right) of eDC subsets are shown. FIG. 20 shows growth rates of orthotopic 4T1-EV and 4T1-Vkine tumors. FIG. 2P shows flow cytometric analysis of eDC subsets from bone marrow following intracardiac injection of VQ myeloma cells, engineered to express empty-vector (VQ-EV) or versikine (VQ-Vkine). Representative flow plots (left) and frequencies (right) of eDC subsets are shown. FIG. 2Q shows Kaplan-Meier curves depicting time-to-hindlimb paralysis (a clinical sequela of myeloma progression) in recipients of VQ-EV vs. VQ-Vkine myeloma tumors.
FIGs. 3A-3G show that versikine selectively activates cDCl in vivo. FIG. 3A shows RT-PCR analysis for cDCl “signature” transcripts in bulk LLC-EV and LLC-Vkine tumor mRNA. Data are presented as mean ± SEM. FIG. 3B shows summary of CD40 staining intensity (MFI, mean fluorescence intensity) in DC subsets from LLC-EV and LLC-Vkine tumors (experiment 1). Examples of individual histogram plots for each DC subset are shown. FIG. 3C shows a summary of PD-L1 staining intensity in DC subsets from LLC-EV and LLC-Vkine tumors. Examples of individual histogram plots for each DC subset are shown. FIG. 3D shows a layout of the experiment to compare trans criptomic profiles in LLC-EV versus LLC-Vkine tumor immune infiltrates. FIG. 3E shows hierarchical clustering of transcriptomic profiles by RNA sequencing (RNA-seq) analysis of CD45+ tumor-infiltrating leukocytes (TILs) extracted from LLC-EV versus LLC-Vkine tumors. FIG. 3F shows volcano plot highlighting key differentially expressed genes in CD45+ TILs from LLC-Vkine tumors compared with LLC- EV tumors. Genes whose overexpression has been linked to APC activation are shown in red and genes whose overexpression has been linked to T cell activation in green. FIG. 3G shows Gene Ontology (GO) analysis of pathways enriched in CD45+ fractions from LLC-Vkine versus LLC-EV tumors. FIG. 3H shows expression pattern of murine IrJ8, Batf3 andld2. Data from BioGPS. FIG. 31 shows an exemplary schematic layout of the pre-DC adoptive transfer experiment. Pre-DC were harvested from the BM of Flt31-m vivo mobilized CD45.2+ mice and adoptively transferred into LLCEV or LLC-Vkine tumors implanted in CD45.1+ recipients. In vivo pre-DC mobilization w as achieved through implantation of Flt31-secreting B16 cells, according to standard protocols. 72 hours post-adoptive transfer, tumors were harvested, processed and eDC subsets were analyzed by flow cytometry. FIG. 3J shows gating strategy for flow sorting pre-DC from BM of F113I-/7? vivo mobilized donors, per the schema of van Ginderachter. FIG. 3K shows representative flow plots of CD45.1+ endogenous eDC subsets (left) and frequencies (right). FIG. 3L shows representative flow plots of CD45.2+ adoptively -transferred eDC subsets (left) and frequencies
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SUBSTITUTE SHEET ( RULE 26 ) (right). FIG. 3M shows summary of CD40 staining intensity (MFI= mean fluorescence intensity ) in DC subsets from LLC-EV and LLC-Vkme tumors.
FIGs. 4A-4K show cDCl activation by versikme is cell autonomous. FIG. 4A shows an exemplary schematic layout of the experiment. MutuDC1940-EV or -Vkine cells were stimulated for 4 h with vehicle (PBS) or the TLR4 agonist lipopolysaccharide (LPS) (100 ng/mL) before RNA extraction. FIG. 4B shows gross morphology of MutuDC 1940 cells engineered to express versikine (Vkine) or empty vector (EV). Phase contrast, 1003magnification; scale bar, 220 mm. FIG. 4C shows hierarchical clustering of MutuDC1940 transcriptomic profiles expressing EV or versikine (Vkine) and stimulated with the TLR4 agonist LPS or vehicle (PBS). FIG. 4D shows volcano plot highlighting key differentially expressed genes in MutuDC1940-Vkine versus -EV cells (without LPS). FIG. 4E shows gene set enrichment analysis (GSEA) of significantly upregulated (left and center) and downregulated (right) pathways in MutuDC 1940- Vkine versus -EV cells (without LPS). FIG. 4F shows Ccl7 RT-PCR using LLC-EV and LLC-Vkine tumor bulk mRNA (left) and CD1 lc+ magnetically separated fraction mRNA (right). FIG. 4G shows Cxcl9 and CxcllO RT-PCR using CD1 lc+ magnetically separated fractions from LLC-EV or LLC-Vkine tumors. FIG. 4H shows and exemplary schematic of the antigen presentation experiment. FIG. 41 shows flow cytometry for endogenous IFN-g and IL-2 of OT-I CD8+ T cells co-cultured with SIINFEKL peptide-loaded MutuDC1940 cells, EV- or Vkine-expressing, with or without LPS. FIG. 4J shows quantitation of OT-I flow cytometry analysis of the antigen presentation assay. FIG. 4K shows IFN-g by ELISA in supernatants from OT-I and MutuDC1940:SIINFEKL co-cultures in the antigen presentation assay. FIG. 4L shows gene ontology (GO) pathway analysis of differentially expressed genes between MutuDC 1940-Vkine vs. -EV. Versikine’s proapoptotic program (“positive regulation of programmed cell death/ “positive regulation of apoptotic process”) is reminiscent of versikine’s proapoptotic activities during development. FIG. 4M shows RT-PCR of Cxcl9/10 in MutuDC1940-EV vs. MutuDC 1940-Vkine stimulated with TLR4 agonist LPS or vehicle (PBS). FIG. 4N shows ELISA detection of secreted Cxcl9 by MutuDC 1940-EV- and MutuDC 1940-Vkine stimulated with LPS or vehicle (PBS) plotted against time (hours). FIG. 40 shows RT-PCR for I127p28 and Ebi3 message in MutuDC 1940- EV-vs. -Vkine stimulated with LPS or vehicle (PBS). FIG. 4P shows ELISA detection of secreted I127p28 by MutuDC 1940-EV- and MutuDC 1940-Vkine stimulated with LPS or vehicle (PBS) plotted against time (hours). FIG. 4Q shows RT-PCR for selected versikine- signature genes using RNA from MutuDC 1940 cells (unmanipulated) exposed to supernatant from versikine-secreting HEK293 cells (Vkine sup) vs.
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SUBSTITUTE SHEET ( RULE 26 ) control supernatant (control sup) at 72 hours. FIG. 4R shows flow cytometry for endogenous IFNg and IL2 of OT-I CD8+ T cells at baseline (left) and PMA-stimulated, prior to addition of DC (right). FIG. 4S shows FMO controls shown for IFNg (left) and IL2 (right) under conditions of maximal stimulation (Vkine +LPS, compare to FIG. 41). FIG. 4T shows IFNy by ELISA in supernatants from OT-I+ MutuDC1940:SIINFEKL co-cultures in the antigen presentation assay.
FIGs. 5A-5G show cDCl accumulation requires innate lymphoid support. FIG. 5A shows RT- PCR for NK cell-activating cytokine transcripts expressed by ex vivo magnetically separated CDl lc+ cells from LLC-EV and LLC-Vkine tumors. FIG. 5B shows RT-PCR profile of NKp46+ NK1. 1+ cells flow-sorted from LLC-EV and LLC-Vkine tumors. FIG. 5C shows an exemplary schematic of the NK cell depletion experiment. FIG. 5D shows summary of eDC subset frequency by flow cytometric analysis in LLC-EV versus LLC-Vkine tumors after treatment with NK cell-depleting antibody (anti-ASGMl) or vehicle (PBS). FIG. 5E shows Csf2 (GM-CSF) RT-PCR of RNA extracted from NKp46+ NK1.1+ cells flow-sorted from LLC-EV and LLC-Vkine tumors growing in WT or Batf3_/_ hosts. FIG. 5F shows stromal localization of NCR1+ (NKp46+) cells in human lung cancers (chromogen, DAB; counterstain, hematoxylin). FIG. 5G shows annexin V/7-AAD apoptosis assay of MutuDC1940-EV or - Vkine dendritic cells exposed to graded staurosporine concentrations with or without murine GMCSF. FIG. 5H shows validation of intratumoral NK (NK1. l+CD49b+) depletion following anti-ASGMl treatment. FIG. 51 shows flow cytometric analysis of eDC subsets in LLC-EV vs. LLC-Vkine tumors following treatment with NK-depleting antibody (anti-ASGMl) or vehicle (PBS). FIG. 5J shows flow cytometric analysis of intratumoral basophils (defined as CD45inlCD49b+FceRI+lgE+c-Kit cells, gating per. FIG. 5K shows absolute counts/ mg tumor tissue of intratumoral basophils compared to intratumoral NK1.1+CD3- cells, in LLC- EV vs. LLC-Vkine tumors. FIG. 5L shows absence of intratumoral cDCl in Batf3-I- recipients by multiparametric flow cytometry. FIG. 5M shows flow cytometric analysis of eDC subsets in LLC-EV vs. LLC-Vkine tumors implanted in WT or Tlr2-/- recipients. FIG. 5N shows summary of eDC subset frequency by flow cytometric analysis in LLC-EV vs. LLC-Vkine tumors implanted in WT or Tlr2-/- recipients. FIG. 50 shows growth rates of LLC-EV and LLC-Vkine tumors in WT vs. Tlr2-i- background. FIG. 5P shows flow cytometric analysis of eDC subsets in LLC-EV vs. LLC-Vkine tumors implanted in WT or Cd44-/- recipients. FIG. 5Q shows summary of eDC subset frequency by flow cytometric analysis in LLC-EV vs. LLC-
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SUBSTITUTE SHEET ( RULE 26 ) Vkine tumors implanted in WT or Cd44-/- recipients. FIG. 5R shows growth rates of LLC-EV and LLC-Vkine tumors in WT vs. Cd44-I- genetic background.
FIGs. 6A-6L show stroma-licensed cDCls are "‘poised” and hypersensitive to nucleic acid sensing in vivo. FIG. 6A shows an exemplary schematic of the experiment. FIG. 6B shows growth curves of LLC-EV and LLC-Vkine tumors challenged with a single subtherapeutic dose (200 mg) of intratumoral (IT) DMXAA (DMXAA200) or vehicle (NaHCO3) on day 0. FIG. 6C shows Kaplan-Meier survival curves for the experiment in (B); **p < 0.01 by log rank test. FIG. 6D shows representative images showing development of hemorrhagic necrosis and a necrotic eschar in LLC-Vkine but not LLC-EV tumors 24 h after IT DMXAA200 administration. FIG. 6E shows transcriptomic profile of LLC-EV and LLC-Vkine tumors harvested 2 h after IT DMXAA200. FIG. 6F shows versikine -DMXAA synergy generates an abscopal effect in LLC tumors that produces a survival advantage. **p < 0.01 by log rank test. FIG. 6G shows growth curves of treatment-side LLC-EV and LLC-Vkine tumors challenged with a single subtherapeutic dose (200 mg) of IT DMXAA (DMXAA200) or vehicle (NaHCO3) on day 0. FIG. 6H shows growth curves of contralateral side unmanipulated LLC tumors; treated side as in FIG. 6G. FIG. 61 shows response to DMXAA200 is lost in Batf3'/_ recipients. Shown are growth curves of LLC-EV and LLC-Vkine tumors challenged with a single subtherapeutic dose (200 mg) of IT DMXAA (DMXAA200) or vehicle (NaHCO3) on day 0 in Bat 3 /_ recipients. FIG. 6J shows Batf3 loss abrogates the survival advantage seen in the WT (FIG. 6C). FIG. 6K shows efficacy of DMXAA200 in LLC-Vkine tumors implanted into BatfT' recipients is restored after adoptive transfer of 1CD103. FIG. 6L shows adoptive transfer of iCD103 in LLC-Vkine tumors implanted into Batf3 /_ recipients restores the survival advantage of mice treated with DMXAA200. FIG. 6M shows Versikme-DMXAA synergy generates an abscopal effect in 4T1 mammary carcinomas. Growth curves of treatment-side 4T1-EV and 4T1-Vkine tumors challenged with a single sub-therapeutic dose (200 mcg) of IT DMXAA on Day 0 (DMXAA200) or vehicle (NaHCO3). FIG. 6N shows growth curves of contralateral side unmanipulated 4T1 tumors, according to corresponding treatment side configuration (treatment as in FIG. 6M). FIG. 60 shows Versikine-induced abscopal effect is accompanied by a survival advantage in 4T1 tumors. **=p<0.01 by log-rank test. FIG. 6P shows an exemplary schematic layout of iCD103 cell adoptive transfer experiments. FIG. 6Q shows flow-cytometric validation of the iCD103 cells, generated as described in the protocol by Merad, Sparwasser and colleagues, using standard cDCl markers. FIG. 6R shows growth curves of B16-EV and B16-Vkine tumors challenged with a single subtherapeutic dose (200 mcg) of IT DMXAA on Day 0 (DMXAA200) or vehicle (NaHCO3). FIG. 6S shows Kaplan-
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SUBSTITUTE SHEET ( RULE 26 ) Meier survival curves for the experiment in panel S6F, *=p<0.05 by log-rank test. FIG. 6T shows response to DMXAA200 is lost in B16-Vkine tumors implanted in Batf3-I- recipients. FIG. 6U shows efficacy of sub-therapeutic DMXAA200 in B16-Vkine tumors implanted in Batf3-I- recipients is restored following adoptive transfer of iCD103 cells. A subset of B16- bearing tumors did not “take” iCD103 cells, likely attributable to the pauci-immune environment of B16 tumors.
FIGs. 7A-7F show versikine promotes CD8+ responses and overcomes resistance to anti-PDl inhibitors in vivo. FIG. 7A shows an exemplary schematic of the experiment. FIG. 7B shows frequency of MHCLSIINFEKL tetramer+ CD8+ splenocytes in mice bearing LLC-EV versus LLC-Vkine tumors 5 days after challenge with a therapeutic dose of a STING agonist (DMXAA500). FIG. 7C shows correlation between in vitro versikine signature and CD8+ T cell scores across TCGA human lung cancers. Significance was measured using a linear model while accounting for total immune infiltration. FIG. 7D shows DPEAAE staining in human lung cancers and associated CD8+ infiltration. FIG. 7E shows distribution of DPEAAE stromal staining intensity across lung cancer prognostic subgroups (pauci-immune [poor prognosis] and immune-rich [favorable prognosis] at cutoff 3 CD8+ TILs/HPF). p < 0.001 by two-tailed Mann-Whitney test. FIG. 7F shows Top: schematic of the experiment. Bottom: tumor growth rates and survival curves of LLC-EV and LLC-Vkine-bearing animals treated with 3 doses of anti-PDl antibody or isotype control. FIG. 7G shows CD8+ T cell subset frequency in the spleen of mice treated as in the schema depicted in FIG. 7A. Naive (CD44- CD62L+), central memory [CM, (CD44+CD62L+)], effector/ effector memory [E/EM, (CD44+CD62L-)]. FIG. 7H shows correlation between in vitro versikine response signature and corrected CD8+ T cell scores across TCGA lung cancers. CD8+ T cell scores corrected for immune infiltration to remove variation associated with immune state. FIG. 71 shows distribution of DPEAAE stromal staining intensity across lung cancer prognostic subgroups [pauciimmune (poor prognosis) and immune-rich (favorable prognosis) at cutoff 5 CD8+ TIL/HPF], Low DPEAAE staining intensities are more prevalent in the pauci-immune subgroup than the immune-rich subgroup, p<0.001 by two-tailed Mann- Whitney test. FIG. 7J shows growth response curves of individual tumors in the anti-PDl experiment depicted in
FIG. 7F
FIG. 8 shows an exemplary schematic of stromal remodeling regulating dendritic cell abundance and activity in the tumor microenvironment.
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SUBSTITUTE SHEET ( RULE 26 ) DETAILED DESCRIPTION
T-cell-inflamed tumor microenvironments are a prerequisite for immunotherapy efficacy. However, why some tumors are inflamed, and others are not remains poorly understood. Tumor antigen cross-presentation and CD8+ T cell effector priming by stimulatory type 1 conventional dendritic cells (cDCls) is integral to spontaneous and therapeutic antitumor immunity. In addition to effector priming in the lymph node and in the tumor microenvironment (TME), cDCls regulate effector cell influx into the TME. From a translational perspective, cDCls are crucial for responses to vaccination strategies, immune checkpoint inhibitors, and engineered immune effector cells (e.g., chimeric antigen receptor T [CAR-T] cells). Several studies have shown that stimulatory cDCls are excluded from interdigitating tumor nestlets and locate in peritumoral stroma. However, the mechanisms that retain cDCls at the tumor periphery remain poorly understood.
The large aggregating extracellular matrix proteoglycan versican (V CAN) is a central component of the embryonic provisional matrix, playing key non-redundant roles in development of the cardiovascular system and limbs. For example, Vcan-null mice die in utero by embryonic day 10.5 because of defects along the anterior-posterior cardiac axis. VC AN proteolysis by AD AMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteases at the Glu441-Ala442 bond (VI isofonn enumeration) is an essential requirement that acts in part through the specific neoactivities of the released bioactive N-terminal fragment (matrikme) versikine, wherein disruption of the Glu441-Ala442 proteolytic site that generates versikine leads to developmental abnormalities Furthermore, it has been shown that VC AN proteolysis at the versikine C-terminal Glu441-Ala442 bond correlates with CD8+ T cell infiltration in solid and hematopoietic human tumors.
The present disclosure describes methods for increasing T cell activation in a tumor microenvironment (TME) in a subject that includes administering a proteolytic fragment of an extracellular matrix (ECM) proteoglycan. Also provided herein are methods for sensitizing a response to an immunotherapy in a subject in need thereof that includes administering a proteolytic fragment of an extracellular matrix (ECM) proteoglycan. Also provided herein are methods of predicting a subject’s response to an immunotherapy that include (a) analyzing a biological sample obtained from the subject; and (b) detecting a proteolytic fragment of an extracellular matrix (ECM) proteoglycan in the biological sample, thereby predicting the subject’s response to the immunotherapy.
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SUBSTITUTE SHEET ( RULE 26 ) Various non-limiting aspects of these methods are described herein and can be used in any combination without limitation. Additional aspects of various components of the methods described herein are known in the art
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range ofvalues that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.
As used herein, “biological sample” can refer to a sample generally including cells and/or other biological material. A biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX). Biological samples can be derived from a homogeneous culture or population of organisms or alternatively from a collection of several different organisms, for example, in a community or ecosystem.
The biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). The biological sample can be a nucleic acid sample and/or protein sample. The biological sample can be a carbohydrate sample or a lipid sample. The biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions.
As used herein, the terms “cancer”, “tumor”, and “carcinoma” refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a metastatic solid tumor. In some embodiments, a relevant cancer may
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SUBSTITUTE SHEET ( RULE 26 ) be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, Hodgkin lymphoma, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer. In some embodiments, hematopoietic cancers can include leukemias, lymphomas (Hodgkin’s and nonHodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, precancerous pathology such as myelodysplastic syndromes, acquired aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria (PNH) and 5q- syndrome and the like.
As used herein, a “cell” can refer to either a prokaryotic or eukary otic cell, optionally obtained from a subject or a commercially available source.
As used herein, “delivering”, “gene delivery”, “gene transfer”, “transducing” can refer to the introduction of an exogenous polynucleotide into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., viral infection/transfection, or vanous other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (e.g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
In some embodiments, a polynucleotide can be inserted into a host cell by a gene delivery molecule. Examples of gene delivery molecules can include, but are not limited to, liposomes, micelle biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral
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SUBSTITUTE SHEET ( RULE 26 ) envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
As used herein, “nucleic acid” is used to include any compound and/or substance that comprise a polymer of nucleotides. In some embodiments, a polymer of nucleotides is referred to as polynucleotides. Exemplary nucleic acids or polynucleotides can include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2’-amino-LNA having a 2’-amino functionalization, and 2’-amino-a-LNA having a 2’-amino functionalization) or hybrids thereof. Naturally- occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e g., found in ribonucleic acid (RNA)).
A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A deoxyribonucleic acid (DNA) can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid (RNA) can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G).
In some embodiments, the term “nucleic acid” refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is DNA. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is RNA
As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease,
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SUBSTITUTE SHEET ( RULE 26 ) disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
Versikine, versikine fusions, and versikine combinations
As used herein, a “proteoglycan” refers a family of charged molecules containing a core protein and one or more covalently attached glycosaminoglycan side chains. Proteoglycans are important components of extracellular matrices (ECM) and have multiple functions that depend on both their protein and carbohydrate constituents. In some embodiments, the ECM proteoglycan is versican (VCAN). Versican is a large extracellular matrix proteoglycan that is present in a variety of tissues and plays key roles in multiple facets of cancer development, ranging from proliferative signaling, evasion of growth-suppressor pathways, regulation of cell death, promotion of neoangiogenesis, and tissue invasion and metastasis. Multiple lines of evidence implicate versican and its bioactive proteolytic fragments (matrikines) in the regulation of cancer inflammation and antitumor immune responses.
Matrikines can be defined as ECM-derived fragments that regulate cell activity, often in a manner distinct from that of their parent macromolecule. Proteolytic processing of versican at Glu441-Ala442 generates a bioactive N-terminal matnkine, versikine. In some embodiments, the proteolytic fragment of the ECM proteoglycan is versikine. In some embodiments, the versikine is generated by proteolysis of VCAN at the Glu441-Ala442 bond.
Some compositions of the disclosure contain a versikine ammo acid sequence. In some embodiments, the versikine amino acid sequence is the sequence of SEQ ID NO: 1. In some embodiments, the versikine amino acid comprises a sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%>, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 1.
SEQ ID NO: 1 - Versikine amino acid sequence
LHKVKVGKSPPVRGSLSGKVSLPCHFSTMPTLPPSYNTSEFLRIKWSKIEVDKNGKDL KETTVLVAQNGNIKIGQDYKGRVSVPTHPEAVGDASLTWKLLASDAGLYRCDVM YGIEDTQDTVSLTVDGVVFHYRAATSRYTLNFEAAQKACLDVGAVIATPEQLFAAY EDGFEQCDAGWLADQTVRYPIRAPRVGCYGDKMGKAGVRTYGFRSPQETYDVYCY
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SUBSTITUTE SHEET ( RULE 26 ) VDHLDGDVFHLTVPSKFTFEEAAKECENQDARLATVGELQAAWRNGFDQCDYGWL SDASVRHPVTVARAQCGGGLLGVRTLYRFENQTGFPPPDSRFDAYCFKRRMSDLSVI GHPIDSESKEDEPCSEETDPVHDLMAEILPEFPDIIEIDLYHSEENEEEEEECANATDVT TTPSVQYINGKHLVTTVPKDPEAAE
In some embodiments, the versikine is a nucleic acid molecule. In some embodiments, the versikine comprises a nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the versikine nucleic acid is at least 90% identical (e.g., at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO: 23).
SEQ ID NO: 23 - Versikine nucleic acid sequence
ATGTTCATAAATATAAAGAGCATCTTATGGATGTGTTCAACCTTAATAGTAACCC ATGCGCTACATAAAGTCAAAGTGGGAAAAAGCCCACCGGTGAGGGGCTCCCTCT CTGGAAAAGTCAGCCTACCTTGTCATTTTTCAACGATGCCTACTTTGCCACCCAG TTACAACACCAGTGAATTTCTCCGCATCAAATGGTCTAAGATTGAAGTGGACAAA AATGGAAAAGATTTGAAAGAGACTACTGTCCTTGTGGCCCAAAATGGAAATATC AAGATTGGTCAGGACTACAAAGGGAGAGTGTCTGTGCCCACACATCCCGAGGCT GTGGGCGATGCCTCCCTCACTGTGGTCAAGCTGCTGGCAAGTGATGCGGGTCTTT ACCGCTGTGACGTCATGTACGGGATTGAAGACACACAAGACACGGTGTCACTGA CTGTGGATGGGGTTGTGTTTCACTACAGGGCGGCAACCAGCAGGTACACACTGA ATTTTGAGGCTGCTCAGAAGGCTTGTTTGGACGTTGGGGCAGTCATAGCAACTCC AGAGCAGCTCTTTGCTGCCTATGAAGATGGATTTGAGCAGTGTGACGCAGGCTG GCTGGCTGATCAGACTGTCAGATATCCCATCCGGGCTCCCAGAGTAGGCTGTTAT GGAGATAAGATGGGAAAGGCAGGAGTCAGGACTTATGGATTCCGTTCTCCCCAG GAAACTTACGATGTGTATTGTTATGTGGATCATCTGGATGGTGATGTGTTCCACC TCACTGTCCCCAGTAAATTCACCTTCGAGGAGGCTGCAAAAGAGTGTGAAAACC AGGATGCCAGGCTGGCAACAGTGGGGGAACTCCAGGCGGCATGGAGGAACGGC TTTGACCAGTGCGATTACGGGTGGCTGTCGGATGCCAGCGTGCGCCACCCTGTGA CTGTGGCCAGGGCCCAGTGTGGAGGTGGTCTACTTGGGGTGAGAACCCTGTATC GTTTTGAGAACCAGACAGGCTTCCCTCCCCCTGATAGCAGATTTGATGCCTACTG CTTTAAACGTCGAATGAGTGATTTGAGTGTAATTGGTCATCCAATAGATTCAGAA TCTAAAGAAGATGAACCTTGTAGTGAAGAAACAGATCCAGTGCATGATCTAATG GCTGAAATTTTACCTGAATTCCCTGACATAATTGAAATAGACCTATACCACAGTG
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SUBSTITUTE SHEET ( RULE 26 ) AAGAAAATGAAGAAGAAGAAGAAGAGTGTGCAAATGCTACTGATGTGACAACC ACCCCATCTGTGCAGTACATAAATGGGAAGCATCTCGTTACCACTGTGCCCAAGG ACCCAGAAGCTGCAGAA
In some embodiments, the versikine nucleic acid molecule is fused to a non-versikine nucleic acid molecule. In some embodiments, the versikine nucleic acid molecule is fused to a non-versikine nucleic acid molecule via a linker. In some embodiments, the non-versikine nucleic acid molecule encodes a secretory peptide. In some embodiments, the non-versikine nucleic acid molecule encodes an immunomodulator. In some embodiments, the non-versikine nucleic acid molecule is a DNA or RNA molecule.
In some embodiments, the versikine protein is conjugated to another protein. In some embodiments, the versikine protein is conjugated to another protein via a linker. In some embodiments, the linker comprises the amino acid sequence MFINIKSILWMCSTLIVTHA (SEQ ID NO: 22). In some embodiments, the linker comprises the amino acid sequence GGGS (SEQ ID NO: 24), GGGSGGGSGGGS (SEQ ID NO: 25), GGGGGGGG (SEQ ID NO: 26), EAAAKEAAAKEAAAK (SEQ ID NO: 27), PAPAP (SEQ ID NO: 28), or AEAAAKEAAAKA (SEQ ID NO: 29). In some embodiments, the linker comprises the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 30), EGKSSGSGSESKST (SEQ ID NO: 31), or GSAGSAAGSGEF (SEQ ID NO: 32). Additional examples of linkers are described for example in Chen et al., Adv Drug Deliv Rev. 2013 Oct;65(10): 1357-69, the entire contents of which are incorporated herein by reference. In some embodiments, the versikine protein is conjugated to another protein at the N-terminal. In some embodiments, the versikine protein is conjugated to another protein at the C-terminal.
Secretory peptides
In some embodiments, the other protein compnses a secretory peptide. In some embodiments, the versikine protein is conjugated to the secretory peptide via a linker. In some embodiments, the versikine protein is conjugated to the secretory peptide at the N-terminal. In some embodiments, the secretory peptide is selected from the group consisting of: human OSM, human immunoglobulin, human chymotrypsinogen, human trypsinogen 2, human IL-2, or human insulin.
In some embodiments, the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 2, wherein SEQ ID NO: 2 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 2 is fused to the C terminal of SEQ ID NO: 1,
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SUBSTITUTE SHEET ( RULE 26 ) optionally including a linker. In some embodiments, the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 3, wherein SEQ ID NO: 3 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 3 is fused to the C terminal of SEQ ID NO: 1 , optionally including a linker. In some embodiments, the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 4, wherein SEQ ID NO: 4 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 4 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 5, wherein SEQ ID NO: 5 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 5 is fused to the C terminal of SEQ ID NO: 1 , optionally including a linker. In some embodiments, the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 6, wherein SEQ ID NO: 6 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 6 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the secretory peptide comprises SEQ ID NO: 1 and SEQ ID NO: 7, wherein SEQ ID NO: 7 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 7 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
SEQ ID NO: 2 - Human OSM
MGVLLTQRTLLSLVLALLFPSMASM
SEQ ID NO: 3 - Human immunoglobulin
MDMRVPAQLLGLLLLWLRGARC
SEQ ID NO: 4 - Human chymotrypsinogen
MAFLWLLSCWALLGTTFG
SEQ ID NO: 5 - Human trypsinogen 2
MNLLLILTFVAAAVA
SEQ ID NO: 6 - Human IL-2
MYRMQLLSCIALSLALVTNS
SEQ ID NO: 7 - Human insulin
MALWMRLLPLLALLALWGPDPAAA
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SUBSTITUTE SHEET ( RULE 26 ) Immunomodulators
In some embodiments, the other protein comprises an immunomodulator. In some embodiments, the versikine protein is conjugated to the immunomodulator via a linker. In some embodiments, the versikine protein is conjugated to the immunomodulator at the N-terminal. In some embodiments, the versikine protein is conjugated to the immunomodulator at the C- terminal. In some embodiments, the other protein is selected from the group consisting of: GM- CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL- 2, IFN01, IFNa2, or IFNy. In some embodiments, the other protein can comprise an IFN-a subtype (e.g, IFN-al, IFN-a2, IFN-a4, IFN-a5, IFN-a6, IFN-a7, IFN-a8, IFN-alO, IFN- al3, IFN-al4, IFN-al7, or IFN-a21).
In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 8, wherein SEQ ID NO: 8 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 8 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 9, wherein SEQ ID NO: 9 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 9 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 10, wherein SEQ ID NO: 10 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 10 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 11, wherein SEQ ID NO: 11 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 11 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 12, wherein SEQ ID NO: 12 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 12 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 13, wherein SEQ ID NO: 13 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 13 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 14, wherein SEQ ID NO: 14 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 14 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments,
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SUBSTITUTE SHEET ( RULE 26 ) the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 15, wherein SEQ ID NO: 15 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 15 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 16, wherein SEQ ID NO: 16 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 16 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 17, wherein SEQ ID NO: 17 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 17 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 18 wherein SEQ ID NO: 18 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 18 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator compnses SEQ ID NO: 1 and SEQ ID NO: 19, wherein SEQ ID NO: 19 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 19 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 20, wherein SEQ ID NO: 20 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 20 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 21, wherein SEQ ID NO: 21 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 21 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 33, wherein SEQ ID NO: 33 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 33 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker. In some embodiments, the versikine protein conjugated with the immunomodulator comprises SEQ ID NO: 1 and SEQ ID NO: 34, wherein SEQ ID NO: 34 is fused to the N terminal of SEQ ID NO: 1 or wherein SEQ ID NO: 34 is fused to the C terminal of SEQ ID NO: 1, optionally including a linker.
SEQ ID NO: 8 - Human GM-CSF
MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLN LSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASH YKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE
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SUBSTITUTE SHEET ( RULE 26 ) SEQ ID NO: 9 - Human CCL5
MKVSAAALAVILIATALCAPASAFPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSN
PAVVFVTRKNRQVCANPEKKWVREYINSLEMS
SEQ ID NO: 10 - Human XCL1
MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIF
ITKRGLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG
SEQ ID NO: 11 - Human FLT3LG
MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVK
IRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLE
RVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQC QP
DSSTLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHWQRTRRRTPRPG
EQVPPVPSPQDLLLVEH
SEQ ID NO: 12 - Human CCL7
MKASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSH
CPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL
SEQ ID NO: 13 - Human CXCL9
MKKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCE KIEIIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRK SQRSRQKKTT
SEQ ID NO: 14 - Human CXCL 10
MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEII
ATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP
SEQ ID NO: 15 - Human IL-12
MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEED
GITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWS
TDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTC
GAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSS FFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKRE KKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS
23
SUBSTITUTE SHEET ( RULE 26 ) SEQ ID NO: 16 - Human IL-27
MGQTAGDLGWRLSLLLLPLLLVQAGVWGFPRPPGRPQLSLQELRREFTVSLHLARK
LLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQAWRRLSDPERLCFISTTLQ PFHALLGGLGTQGRWTNMERMQLWAMRLDLRDLQRHLRFQVLAAGFNLPEEEEEE EEEEEEERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSK AGHSVWPLGFPTLSPQP
SEQ ID NO: 17 - Human IL-15
MRISKPHLRSISIQCYLCLLLNSHFLTEAG1HVFILGCFSAGLPKTEANWVNVISDLKKI EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
SEQ ID NO: 18 - Human IL-18
MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFI DQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKII SFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILK KEDELGDRSIMFTVQNED
SEQ ID NO: 19 - Human IL-23
MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHM DLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGE PSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQA FVAVAARVFAHGAATLSP
SEQ ID NO: 20 - Human IL-2
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT
SEQ ID NO: 21 - Human IFNgamma (IFNy)
MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILK NWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDD FEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ
SEQ ID NO: 33 - Human IFNpi
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SUBSTITUTE SHEET ( RULE 26 ) MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDR MNFDIPEEIKQLQQFQKED AALTIYEMLQNIFAIFRQDS S STGWNETIVENLL ANVYH QINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEI LRNFYFINRLTGYLRN
SEQ ID NO: 34 - Human IFNa2
MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRH DFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQL NDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMR SFSLSTNLQESLRSKE
The secretory peptides fused to versikine may optionally be fused to the immunomodulators of the disclosure. In some embodiments, the fusion proteins of the disclosure can be represented by the formula A - L - V - B, wherein A is selected from the group consisting of SEQ ID NOs: 2-7, 8-21, or 33-34, L comprises a linker sequence, V comprises an amino acid having at least 90% identity to SEQ ID NO: 1, and B is selected from the group consisting of SEQ ID NOs: 8-21, and 33-34.
In some embodiments, the versikine nucleic acid molecule is fused to a non-versikine nucleic acid molecule. In some embodiments, the versikine nucleic acid molecule is fused to a non-versikine nucleic acid molecule via a linker. In some embodiments, the non-versikine nucleic acid molecule encodes a secretory peptide. In some embodiments, the non-versikine nucleic acid molecule encodes an immunomodulator. In some embodiments, the non-versikine nucleic acid molecule is a DNA or RNA molecule.
In some embodiments, the versikine protein is conjugated to a drug. In some embodiments, the drug comprises a small molecule STING agonist. In some embodiments, the versikine protein is conjugated to a small molecule. In some embodiments, the versikine protein is conjugated to a radiochemical. In some embodiments, the versikine protein is conjugated to an immunogenic component. In some embodiments, the versikine protein is conjugated to a label. In some embodiments, the label is a radioisotope or a fluorophore. In some embodiments, the versikine protein is conjugated to a small molecule immunomodulator. In some embodiments, a small molecule immunomodulator comprises an indoleamine 2,3 -dioxygenase 1 (IDO1) inhibitor. In some embodiments, the indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor comprises Indoximod, Epacadostat, BMS-986205, Navoximod (GDC-0919, NLG-919), PF- 06840003, KHK2455, or LY3381916.
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SUBSTITUTE SHEET ( RULE 26 ) Compositions containing versikine DNA and/or versikine fusion proteins
Provided herein are compositions containing versikine DNA and/or versikine fusion proteins. In some embodiments, the compositions of the disclosure are pharmaceutical compositions comprising versikine DNA and/or versikine fusion proteins described herein. As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
Provided herein are vectors comprising any one of the nucleic acids described herein. As used herein, “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a plasmid, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. In some embodiments, the vector is a lentivirus (such as an integration-deficient lentiviral vector) or adeno-associated viral (AAV) vector. As used herein, the term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividmg cells; they can deliver a significant amount of genetic information into the DNA of a host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
Exemplary viral vectors include polyoma, SV40, adenovirus, vaccinia virus, adeno- associated virus, herpes viruses including HSV and EBV, lentivirus, Sindbis viruses, alphaviruses and retroviruses of avian, murine, and human origin. Baculovirus (Autographa califomica multinuclear polyhedrosis virus; AcMNPV) vectors can be used and obtained from commercial sources. Other suitable vectors include retrovirus vectors, orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, adenoviral vectors, herpes virus vectors, alpha virus vectors, baculovirus vectors, Smdbis virus vectors, vaccinia virus vectors
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SUBSTITUTE SHEET ( RULE 26 ) and poliovirus vectors. Specific exemplary vectors are poxvirus vectors such as vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus (MV A), adenovirus, baculovirus and the like. Pox viruses of use include orthopox, suipox, avipox, and capripox virus. Orthopox include vaccinia, ectromeha, and raccoon pox. One example of an orthopox of use is vaccinia. Avipox includes fowlpox, canary pox and pigeon pox. Capripox include goatpox and sheeppox. In one example, the suipox is swinepox. Other viral vectors that can be used include other DNA viruses such as herpes simplex virus and adenoviruses, and RNA viruses such as retroviruses and polio.
Certain vectors are capable of directing the expression of genes to which they are operatively -linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors are often in the form of plasmids. Recombinant expression vectors can comprise a nucleic acid provided herein in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively -linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described.
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzy matic reactions and purification techniques may be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
In vitro applications of versikine compositions
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SUBSTITUTE SHEET ( RULE 26 ) The term “in vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
The veriskine nucleotides and/or the versikine pharmaceutical compositions described herein may be used in in vitro applications. In some embodiments, the administering of a proteolytic fragment of an ECM proteoglycan (e g., veriskine) comprises delivery of the proteolytic fragment into a cell, wherein the cell is not currently integrated into a multi-cellular organism (e.g., a living subject). In some embodiments, the cell is part of a cell culture. In some embodiments, the cell is part of an organoid. In some embodiments, a cell is a primary hematopoietic, a non-hematopoietic human cell, or an iPS (inducible pluripotent stem cell). In some embodiments, the cell is an antigen-presenting cell (e.g., a macrophage, B cell, or dendritic cell). In some embodiments, the cell is a non-hematopoietic antigen-presenting cell (e.g., antigen-presenting fibroblast, lymph node stromal cell, or endothelial cell). In some embodiments, the versikine can be delivered into a cell by a variety of well-known techniques. In some embodiments, the delivery method can include a vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes). In some embodiments, the delivery method can include methods facilitating the delivery of “naked” polynucleotides (e.g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). In some embodiments, the delivering comprises transfection, electroporation, or a virus-based delivery. In some embodiments, the delivery of the proteolytic fragment comprises a virus-based delivery. In some embodiments, the virus-based delivery comprises an adeno-associated virus or a lentivirus. In some embodiments, the virus-based delivery comprises a lentivirus. In some embodiments, the versikine nucleic acid can be introduced into/uptaken by dendritic or other antigen-presenting cells (e.g., macrophage, B cell, and/or dendritic cell) to generate cell-based vaccines. For example, in some embodiments, the versikine nucleic acid can be introduced into a dendritic cell and enhancing antigen-presentation capacity of the dendritic cell, thereby generating a DC cellular vaccine. For such cell-based vaccines, cell lines are grown, and the versikine nucleic acid (SEQ ID NO: 23) is introduced to the cell via a viral vector. The virus strain replicates in the mammalian cells and is then extracted from the cells and purified. In some embodiments, a versikine nucleic acid can be stably introduced into a cell for vaccine production. In some embodiments, a versikine nucleic acid can be delivered into a cell via standard transfection or using lipid nanoparticles (LNPs). In some embodiments, a versikine protein can be expressed ex vivo and introduced into a subject.
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SUBSTITUTE SHEET ( RULE 26 ) In vivo applications of versikine compositions
The term “in vivo” as used herein refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
The veriskine nucleotides and/or the versikine pharmaceutical compositions described herein may be used in in vivo applications. In some embodiments, the administering of a proteolytic fragment of an ECM proteoglycan (e.g., veriskine) comprises delivery of the proteolytic fragment into a cell, wherein the cell is integrated into a subject. In some embodiments, the cell of the subject is an antigen-presenting cell (e.g., a macrophage, B cell, or dendritic cell). In some embodiments, the cell of the subject is anon-hematopoietic antigen- presenting cell (e.g. , antigen-presenting fibroblast, lymph node stromal cell, or endothelial cell). In some embodiments, the versikine can be delivered into a cell by a variety of well-known techniques. In some embodiments, the delivery method can include a vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes). In some embodiments, the delivering comprises transfection or a virusbased delivery. In some embodiments, the delivery of the proteolytic fragment comprises a virus-based delivery. In some embodiments, the virus-based delivery comprises an adeno- associated virus or a lentivirus. In some embodiments, the virus-based delivery comprises a lentivirus. In some embodiments, the versikine composition administered as described herein treats cancer by increasing T cell activation in a tumor microenvironment, and/or potentiating the effects of other cancer treatment.
The compositions described herein can be used to treat cancer. The terms “cancer”, “malignancy”, “neoplasm”, “tumor”, and “carcinoma”, are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers know n in the art include, for example, hematopoietic
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SUBSTITUTE SHEET ( RULE 26 ) cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like,
Administration of versikine
In some embodiments, the versikine DNA, fusion proteins, and/or the compositions described herein may be administered alone or with an immunotherapy agent and/or a pharmaceutical agent In some embodiments, the pharmaceutical agent comprises a protein selected from the group consisting of: GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, or IFN. In some embodiments, the immunotherapy agent comprises a STING agonist, a PD-1 inhibitor, a PD-L1 inhibitor, or a CD40 agonist. In some embodiments, the versikine and the pharmaceutical agent can be administered simultaneously. In some embodiments, the versikine can be administered before administering the pharmaceutical agent. In some embodiments, the pharmaceutical agent can be administered after administering the versikine composition.
As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary' skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses
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SUBSTITUTE SHEET ( RULE 26 ) separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
As used herein, the term “therapeutically effective amount" means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. For example, in some embodiments, term “therapeutically effective amount”, refers to an amount which, when administered to an individual in need thereof in the context of inventive therapy, will block, stabilize, attenuate, or reverse a cancer-supportive process occurring in said individual, or will enhance or increase a cancer-suppressive process in said individual. In the context of cancer treatment, a “therapeutically effective amount” is an amount which, when administered to an individual diagnosed with a cancer, will prevent, stabilize, inhibit, or reduce the further development of cancer in the individual. A particularly preferred “therapeutically effective amount” of a composition described herein reverses (in a therapeutic treatment) the development of a malignancy such as a pancreatic carcinoma or helps achieve or prolong remission of a malignancy. A therapeutically effective amount administered to an individual to treat a cancer in that individual may be the same or different from a therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the therapeutic methods described herein are not to be interpreted as, restricted to, or otherwise limited to a “cure” for cancer; rather the methods of treatment are directed to the use of the described compositions to “treat” a cancer, i.e., to effect a desirable or beneficial change in the health of an individual who has cancer. Such benefits are recognized by skilled healthcare providers in the field of oncology and include, but are not limited to, a stabilization of patient condition, a decrease in tumor size (tumor regression), an improvement in vital functions (e.g., improved function of cancerous tissues or organs), a decrease or inhibition of further metastasis, a decrease in opportunistic infections, an increased survivability, a decrease in pain, improved motor function, improved cognitive
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SUBSTITUTE SHEET ( RULE 26 ) function, improved feeling of energy (vitality, decreased malaise), improved feeling of wellbeing, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. In addition, regression of a particular tumor in an individual (e g , as the result of treatments described herein) may also be assessed by taking samples of cancer cells from the site of a tumor (e.g., over the course of treatment) and testing the cancer cells for the level of metabolic and signaling markers to monitor the status of the cancer cells to verify at the molecular level the regression of the cancer cells to a less malignant phenotype. For example, tumor regression induced by employing the methods of this disclosure would be indicated by finding a decrease in any of the pro-angiogenic markers discussed above, an increase in anti- angiogenic markers described herein, the normalization (i.e., alteration toward a state found in normal individuals not suffering from cancer) of metabolic pathways, intercellular signaling pathways, or intracellular signaling pathways that exhibit abnormal activity in individuals diagnosed with cancer. Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
Immunotherapy / methods of treating cancer
As used herein, “immunotherapy” refers to a treatment of disease (e.g., cancer) by activating or suppressing the immune system. For example, cancer immunotherapy uses the immune system and its components to mount an anti-tumor response through immune activation. In some embodiments, an immunotherapy can include an immune checkpoint inhibitor, an oncolytic virus therapy, a cell-based therapy, a CAR-T cell therapy, or a cancer vaccine. In some embodiments, an immunotherapy can include immune checkpoint blockade (ICB), wherein an immune checkpoint inhibitor is administered. Immune checkpoint blockade (ICB) is a treatment that uses immune checkpoint inhibitors to address a disease (e.g., cancer), wherein the immune checkpoint inhibitor blocks a checkpoint protein from binding to its partner protein or receptor.
The versikine compositions of the disclosure may be used in methods of treating cancer and/or in conjunction with other immunotherapy. In some embodiments, the immunotherapy includes administration of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. Examples of a PD-1 inhibitor that can be used in conjunction with versikine compositions of the disclosure can include, but are not limited to,
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SUBSTITUTE SHEET ( RULE 26 ) pembrolizumab, nivolumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, and dostarlimab. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. Examples of a PD-L1 inhibitor can include, but are not limited to, atezolizumab, avelumab, durvalumab, KN035, CK.-301, AUNP12, CA-170, and BMS- 986189. In some embodiments, the immune checkpoint inhibitor can be any checkpoint inhibitor, e.g., as described in Mazzarella et al., Eur J Cancer (2019) 117:14-31, hereby incorporated by reference. In some embodiments, the immunotherapy comprises an anti-PDl immunotherapy.
In some embodiments, the immunotherapy comprises a stimulator of interferon genes (STING) agonist immunotherapy. In some embodiments, the immunotherapy includes activation of the cGAS-STING signaling pathway (e.g., STING agonist immunotherapy). Activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway induces the expression of type I interferons and proinflammatory cytokines, promoting a robust adaptive antitumor immunity. As part of this innate immune signaling pathway, STING is ubiquitously expressed in immune and nonimmune cells, wherein STING activation has been demonstrated to propagate the cancer immunity cycle, remodel the tumor microenvironment, and ultimately eliminate tumor cells. Non-limiting examples of STING agonists that can be used in conjunction with versikine compositions of the disclosure include ENPP1 inhibitors (e.g., MV-626, SR-8314, SR-8291, SR8541A), bacterial vectors (e.g., SYNB1891 and STACT-TREX-1), CDN compounds (ADU-S100, BI-STING, BMS-986301, GSK532, JNJ-4412, MK-1454, SB11285, 3’3’cyclic AIMP), non-CDN small molecules (e.g., ALG-031048, ASA404, DMXAA, E7766, JNP6196, MK-2118, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-100001), nanovaccines (e.g., PC7A NP, cGAMP-NP, ONM-500), antibody-drug conjugates (e.g., XMT-2056 and CRD-5500), and exoSTING. In some examples, the STING agonist is DMXAA.
In some embodiments, the immunotherapy includes CD40 agonist immunotherapy. CD40 is a cell-surface member of the TNF (tumor necrosis factor) receptor superfamily, and upon activation, CD40 can license dendritic cells (DCs) to promote antitumor T cell activation and re-educate macrophages to destroy tumor stroma. CD40 activation can play a role in driving antitumor immunity, whereby CD40-activated DCs are poised to prime or activate tumor-specific T cells. In some embodiments, CD40 agonists can be used as an immunotherapy for patients with cancer, wherein CD40 agonist immunotherapy can include agonistic anti- CD40 monoclonal antibodies (mAbs), trimeric CD40 ligand (CD40L), or ectopic expression of CD40L using gene therapy of transferred tumor or other cells. In some embodiments, the 33
SUBSTITUTE SHEET ( RULE 26 ) immunotherapy comprises an anti-CD40 immunotherapy. Non-limiting examples of CD40 agonists that can be used in conjunction with versikine compositions of the disclosure include antibodies (e g., selicrelumab (CP-870,893 or R07009789), APX005M, JNJ-64457107, SEA- CD40, ChiLob7/4, CDX-1140H, Dacetuzumab (SGN-40), ABBV-428).
Provided herein are methods for increasing T cell activation in a tumor microenvironment (TME) in a subject that include administering a proteolytic fragment of an extracellular matrix (ECM) proteoglycan (e.g., versikine).
As used herein, “a tumor microenvironment” (TME) refers to a highly complex and dynamic structure of cells of which a variety of immune cells are a major component. The TME contains cells of the immune system (e.g., T cells, B cells, dendritic cells, myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (T AMs)), a complicated network of fibroblasts, blood vessels, lymphatics, and the cells of the cancer itself. These TME elements are surrounded by a dense meshwork of collagen and elastin fibers that comprise the extracellular matrix (ECM), wherein the TME is also composed of a complicated network of cytokines, chemokines, growth factors, and inflammatory, as well as matrix remodeling enzymes. Furthermore, the TME is a critical facilitator of immune escape and cancer progression, and the interaction between cancer cells and the diverse cell population within the TME influences tumor resistance, progression, and metastasis.
In some embodiments, the administering of a proteolytic fragment of an ECM proteoglycan (e.g., venskine) compnses delivery of the proteolytic fragment into a cell, thereby increasing T cell activation in a tumor microenvironment. In some embodiments, the cell is an antigen-presenting cell (e.g., a macrophage, B cell, or dendritic cell). In some embodiments, the cell is a non-hematopoietic antigen-presenting cell (e.g., antigen-presenting fibroblast, lymph node stromal cell, or endothelial cell). In some embodiments, the proteolytic fragment can be delivered into a cell by a variety of well-known techniques. In some embodiments, the delivery method can include a vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes). In some embodiments, the delivery method can include methods facilitating the delivery of “naked” polynucleotides (e g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). In some embodiments, the delivering compnses transfection, electroporation, or a virus-based delivery. In some embodiments, the delivery of the proteolytic fragment comprises a virus-based delivery. In some embodiments, the virus-based delivery comprises an adeno-associated virus or a lentivirus. In some embodiments, the virus-based delivery comprises a lentivirus. In some embodiments, the versikine composition administered
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SUBSTITUTE SHEET ( RULE 26 ) as described herein increases T cell activation in a tumor microenvironment, thereby treating cancer and/or potentiating the effects of other cancer treatment.
Provided herein are methods for sensitizing a response to an immunotherapy in a subject in need thereof that include administering a proteolytic fragment of an extracellular matrix (ECM) proteoglycan (e.g., versikine) to a subject in need thereof. As used herein, the term “sensitizing” or “improve sensitivity” can refer to increasing response rates to a specific treatment (e g., immunotherapy). In some embodiments, improving sensitivity to ICB treatment can refer to inhibiting negative regulatory immune checkpoints or stimulating activating immune checkpoints. In some embodiments, the subj ect has refractory cancer (e. g. , a refractory' tumor). In some embodiments, the subject has undergone previous treatments for cancer. In some embodiments, the subject has not shown improvement in cancer symptoms and/or cancer markers after previous treatments for cancer. In some embodiments, administration of the versikine compositions sensitizes a response to immunotherapy in a subject, such that the subject shows improvement in cancer symptoms and/or cancer markers over similar immunotherapy without administration of versikine compositions.
Methods of predicting a subject ’s response to an immunotherapy
Provided herein are methods of predicting a subject’s response to an immunotherapy that include (a) analyzing a biological sample obtained from the subject; and (b) detecting a proteolytic fragment of an extracellular matrix (ECM) proteoglycan in the biological sample, thereby predicting the subject’s response to the immunotherapy.
In some embodiments, the analyzing comprises staining the biological sample. In some embodiments, non-limiting examples of stains can include histological stains (e.g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, the staining includes the use of hematoxylin and eosin. In some embodiments, a biological sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In some instances, the biological sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxy lin and eosin (H&E), Jenner’s, Leishman, Masson’s trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright’s, and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after
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SUBSTITUTE SHEET ( RULE 26 ) formalin or acetone fixation In some embodiments, the staining can include the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
In some embodiments, the staining comprises an immunofluorescence stain. In some embodiments, the staining comprises an immunohistochemistry stain. In some embodiments, the biological sample is stained with an antibody. In some embodiments, the antibody is selected from the group consisting of: an anti-DPEAAE neo-epitope antibody, anti-HA antibody, anti-XCRl antibody, anti-CLEC9A antibody, anti-NCRl antibody, and anti-CD8 antibody.
In some embodiments, the detecting can include imaging of the biological sample (e.g., a fixed and/or stained biological sample). In some embodiments, the biological sample can be visualized or imaged using bright field microscopy. In some embodiments, the biological sample can be visualized or imaged using fluorescence microscopy. In some embodiments, non-limiting examples of visualization and imaging can include expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy.
In some embodiments, the detecting comprises determining the level of the proteolytic fragment by measuring the antibody in the biological sample.
EXAMPLES
Lentiviral transduction
HEK293T cells were transfected with a mixture of ps-PAX2 (packaging plasmid) and pVSV- G (envelope plasmid), and transfer plasmids encoding respective open reading frames or empty control. On Day 2 post-transfection, pseudotype virus-containing culture medium was harvested, filtered, supplemented with 7.5 mg/mL polybrene (Sigma-Aldrich), and immediately applied to target cells for spinfection (120min, 2500xg at 32C). After spinfection, the medium was exchanged for fresh complete RPMI1640 medium. Target cells were passaged at least three times after retroviral transduction
Generation ofHA-lagged versikine- and OVA-ZsGreen-expressing cell lines
LLC, 4T1, B16-F10 melanoma and MutuDC1940 cells were transduced with HA tagged versikine (Vkine) - or empty vector (EV)- containing lentivirus as detailed above. The cells
36
SUBSTITUTE SHEET ( RULE 26 ) were selected with lOmg/mL blasticidin for 2 weeks. HA-tagged versikine expression was confirmed by western blotting using anti-HA antibody (clone: C29F4, Cell Signaling). LLC- EV or -Vkine cell lines were transduced with pHIV-Luc-OVA-ZsGreen lentivirus. LLC-OVA expressing cells were FACS-sorted based on ZsGreen expression to ensure comparable transduction rates between different cell lines. shRNA mediated VCAN knockdown
The lentiviral shRNA vector set targeting mouse Vcan (NM 019389.2) and scrambled control were purchased from GeneCopoeia (#MSH080253-LVRU6H and #CSHCTR001-LVRU6H). In brief, 2><10’ LLC cells were plated per well in a 6-well plate and incubated overnight. Next day, 2mL freshly harvested lentiviral supernatant (expressing either shambled control, Vcan shRNA#l, #2 or #3), ImL of culture medium and 7.5mg/mL polybrene was added per well. The plate was centrifuged at 800g for 2h at 37°C and returned to CO2 incubator. After 72h, 200mg/mL Hygromycin B was added and the cells were under antibiotic selection for 2 weeks. Vcan knockdow n was confirmed by RT-PCR.
Tumor cell inoculations and tumor growth measurement
Cells were harvested by trypsinization and washed in PBS. Mice were under isoflurane anesthesia during tumor injections. 5xl05 LLC cells were injected subcutaneously (s.c.) in lOOmL endotoxin-free PBS on the flank of recipient mice. 105 4T1 cells were injected orthotopically in the mammary fat pad of the mice. Tumor growth was measured using a digital caliper. Tumor volumes were measured biweekly and estimated by using the formula: Tumor volume = length x (width)2 divided by 2, where length represented the largest tumor diameter and width represented the perpendicular tumor diameter. Intratumoral injections were performed using a 28G insulin syringe, when tumors had reached 100-150 mm3, using surgical forceps to hold the tumor constantly. For intravenous (i.v.) inoculations, a retro-orbital approach was adopted. Mice were anesthetized using inhaled isoflurane in a chamber. The eyeball was partially protruded from the socket by applying downward pressure to the skin dorsal and ventral to the eye. Injections were performed by placing the needle, bevel face-down, in order to decrease the likelihood of damaging the eyeball. Once the injection was complete, the needle was slowly and smoothly withdrawal. Triple antibiotic ophthalmic ointment was then applied to the eye.
Intraperitoneal inj ection was performed using a 28.5G insulin-syringe with the head tilted down. The needle was inserted at a 30° angle in the lower left or right quadrant. Transplantation of 37
SUBSTITUTE SHEET ( RULE 26 ) myeloma VQ4935 cells was performed via intracardiac injection after the 6-8 week old C57BL/6J recipient mice were sub-1 ethally irradiated at 6.0 Gy using an X-RAD 320 Irradiator. Intracardiac injection was performed by placement of needle in the fourth intercostal space and into the left ventricle. The needle was inserted at a 90° angle in the middle of the imaginary line connecting the sternal notch and xyphoid process serving as anatomical landmarks, and the needle was inserted slightly left of the sternum.
Anti-PDl treatments
Recipient syngeneic mice (10 per arm) were injected with LLC-EV or LLC-Vkine cells (5 x 105 cells per inoculum). Antibody treatments were with 100 ug of antibody in 100 uL of volume each (aPDl: Bio X Cell InVivoPlus, rat IgG2a, clone RPM1-14, Cat# BP0146, Lot# 806321 J2B; Isotype control: Bio X Cell InVivoPlus, rat IgG2a, clone 2A3, Cat# BE0089, Lot# 796721M2) Treatments were administered on days 7, 10, and 14 post-inoculation. Tumor burden was tracked by measuring tumors with an electronic caliper every two days, beginning with Day 5 post-inoculation. Mice reached endpoint when they were found dead, were in clear distress, or when tumors reached 20 mm in any dimension. Animals found dead were considered to have reached endpoint on the off-day of measurements.
Processing of tumor tissue
Unless stated otherwise, tumors were excised 21 days after transplantation. For subsequent analysis by flow cytometry, tumors were cut into pieces and digested with either Collagenase la (Img/mL) C2674 Sigma Aldrich and Hyaluronidase V (O.lmg/mL) H6254 Sigma Aldrich for 40min at 37°C or with a mouse tumor dissociation kit (Miltenyi Biotec #130-096-730) using gentle MACS dissociator. Tissue was passed through a 70mm cell strainer (Falcon) and washed with FACS buffer (PBS with 1% FCS) before proceeding with antibody staining. For RNA isolation, homogenization was performed in RLT buffer (QIAGEN) facilitated by a closed tissue grinder system (Fisher brand #02-542-09, 15mL).
Mass cytometry
Tumor tissue was harvested and processed for mass cytometry analyses using the protocol described above for flow cytometry. After single cell suspensions were acquired, cells were washed with PBS, centrifuged at 300-400g for 5 minutes and supernatant was discarded by aspiration. Cells were resuspended in PBS and Cell-ID Cisplatin (Fl ui digm, #201064) was added to a concentration of 5uM. After rigorous mixing, cells were incubated at room 38
SUBSTITUTE SHEET ( RULE 26 ) temperature for 5 minutes. Cells were then quench stained with MaxPar Cell Staining Buffer (Fluidigm, #201068) using 5* the volume of the cell suspension, centrifuged and supernatant was discarded by aspiration. The process was continued with surface staining. 50ul of the antibody cocktail was added to each tube so the total staining volume was lOOul (50ul of cell suspension-!- 50ul antibody cocktail). Cells were stained for an hour at room temperature. All antibodies used for staining were either bought pre-conjugated to metal isotopes or were conjugated using the Maxpar Antibody Labelling Kit (Fluidigm, 201160B). Following incubation, cells were washed by adding 2mL Maxpar Cell Staining Buffer to each tube, then centrifuged at 300xg for 5 minutes and supernatant was removed by aspiration. This step was repeated for a total of 2 washes, and cells were resuspended in residual volume by gently vortexing after final wash/aspiration. Cells were then fixed with 1.6% FA solution and incubated at room temperature for 10 minutes. Finally, cells were labelled with Cell-ID Intercalator-Ir (Fluidigm, #201192A) at a final concentration of 125nM, incubated for an hour at room temperature and then analyzed on a Helios instrument (WB injector). All samples were resuspended in sufficient volume of 0.1 EQ beads (Fluidigm, #201078 by diluting one part beads to 9 parts Maxpar Cell Acquisition (CAS) solution.
Analysis of mass cytometry data using viSNE
To visualize the immune contexture, the immune milieu of the tumor (CD45+) was enriched by manual gating among single events, equally subsampled to 6,000 events, then run through a Barnes Hut implementation of the t-SNE algorithm, viSNE, in the R package ‘Rtsne’, using optimized parameters (iterations: 1000, perplexity:30, learning rate:455). To characterize the myeloid and lymphoid linages markers were selected for viSNE, excluding CD45.
Flow cytometry and fluorescence-activated cell sorting
Flow cytometnc analyses were performed using an LSR II and/or LSR Fortessa X20. Data were analyzed using FlowJo (Tree Star). DAPI (0.5 mg/mL, Sigma-Aldrich) or a Live/Dead fixable cell stain (Ghost 780 Tonbo Biosciences) was used to exclude dead cells in all experiments, and anti-CD16/CD32 antibody (2.4G2) was used to block non-specific binding of antibodies via Fc-receptors. Quantification of total cell numbers by flow cytometry was done using fluorescent beads
(Biolegend Precision beads). For intracellular staining of IFNg and IL-2 in vitro, cells were treated with Golgi Plug (Brefeldin A 500*) and were collected 4h later Intracellular staining was performed in permeabilization buffer (eBioscience) for 30 min and cells were subsequently
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SUBSTITUTE SHEET ( RULE 26 ) analyzed by flow cytometry. Sorting of tumor cells after retroviral transduction was done using a BD FACSAria or a BD FACS Aria Fusion. Purity of cell populations was determined by reanalysis of a fraction of sorted cell samples.
Generation of ICD103 in vitro
1.5xl06 BM cells were cultured in lOmL RPMI1640 medium supplemented with 10% heat- inactivated FCS (Biochrom), penicillin/streptomycin and 50mM b-mercaptoethanol. Recombinant human FLT3L (300-19, Peprotech) and recombinant murine GM-CSF (315-03, Peprotech) were added at day 0 of the culture. 5mL complete medium was added between day 5 and day 6 to minimize apoptosis. Non-adherent cells were harvested on day 9, counted and re-plated at 3x106 cells in lOmL complete medium supplemented with FLT3L and GMCSF as on day 0. Non-adherent iCD103 were harvested on days 15-16. Cells were then validated by assaying for CD103, CD24, Clec9A, and CD11c by flow cytometry.
ELISA
MutuDC1940 cells were left unstimulated or were in vitro stimulated with LPS for 8 or 24 hours at 37°. Cell-free supernatant was assessed for CXCL9 (R&D Quantikine mouse CXCL9 #MCX900) and IL27p28 (R&D Quantikine mouse IL27p28 #M2728) protein levels by ELISA according to the manufacturer’s instructions (R&D). For the antigen-presentation assay, cell- free supernatants were collected and assessed for IFNg levels (R&D Quantikine mouse IFNg P233156).
Immunoblotting
Whole-cell lysates were prepared by boiling cells in Laemmli Sample Buffer (Bio-Rad) supplemented w ith lOOmM DTT for 10 min at a final concentration of 107 cells per milliliter. A total of 105 cells or 20 mg protein was resolved by SDS-PAGE and transferred to Immobilon- P PVDF membranes (Millipore). Membranes were blocked in 5% milk in TBS-T (25 mM Tris- HC1 [pH 7 4], 0.13 M NaCl, 2.7 mM KC1). Primary antibodies (anti-HA [C29F4; Cell Signaling Technologies], anti-DPEAAE [PA1-1748A; Thermo]) were diluted in 5% milk- TBS-T, and membranes were incubated overnight at 4°C. Secondary Ab-HRP conjugate, as well as anti-GAPDH-HRP conjugate (A00192; GenScript), incubations were carried out for 1 h at room temperature. Signal detection was achieved using Amersham ECL.
Immunohistochemistry
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SUBSTITUTE SHEET ( RULE 26 ) Paraffin-embedded murine tumor sections and unstained 4-5 mm-thick human lung carcinoma TMA (US Biomax Inc., BC041115e) sections were deparaffinized and rehydrated using standard methods. Antigen retrieval was earned out in citrate buffer, pH 6.0 (Vector Laboratories, #H-3300) for DPEAAE and HA; and pH 8.0 for XCR1 and CD8 (Abeam, ab93680). Primary antibodies included aDPEAAE (PA1-1748A, Thermo Fisher), anti-HA (C29F4, Cell Signaling Technology), anti-XCRl (D2F8T, Cell Signaling Technology), anti- CLEC9A (ab223188, Abeam), anti-NCRl (NKp46) (MAB1850, R&D) and anti-CD8 (C8/144B, Ebioscience). The aDPEAAE neoepitope antibody has been previously validated. Stained slides were examined using an Echo Revolve microscope with attached digital camera. aDPEAAE immunostaining score was assessed by scoring staining intensity (0 for no staining, 1 for low/weak staining, 2 for moderate staining and 3 for strong/intense staining).
Dual staining XCR1/CLEC9A on human tonsil was performed by the UW TRIP lab as follows: The experiment was run on Roche Ventana Medical System’s Discovery Ultra Automated platform. Deparaffinization was carried out on the instrument, as was heat induced epitope retrieval in the form of “cell conditioning” with CC1 buffer (Ventana #950-500), a Tris based buffer pH 8.4 for approximately 56 minutes at 95°C. Slide was incubated with the first primary antibody XCR1 diluted 1:40 in DaVinci Green antibody diluent (BioCare Medical #PD900H) for 60 min at 37°. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery OmniMap anti-Rabbit HRP (Ventana #760-4311) was applied for 16 min at 37°. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery ChromoMap DAB detection (Ventana #760-159) was applied for the preset time. Denaturing agent in the form of Discovery Inhibitor (Ventana #760-4840) was applied for the preset time. Slide was incubated with the second primary antibody CLEC9A diluted 1:50 in Ventana antibody diluent with casein (Ventana #760-219) for 60 min at 37°. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery OmniMap anti-Rabbit HRP (Ventana #760-4311) was applied for 16 min at 37°. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery Teal HRP detection kit (Ventana #760-247) was applied for 32 minutes. Slide was removed from the instrument and rinsed with dawn dish soap and warm tap water followed by rinsing with dH2O. Slide was counterstained with Harris hematoxylin (1:5 diluted in dH2O) for 45 seconds. Slide was rinsed with dH2O. Slide was dehydrated in the oven (60 degrees C) followed by dipping in Xylene. Slide was cover slipped.
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SUBSTITUTE SHEET ( RULE 26 ) Triple staining DPEAAE/XCR1/CD8+ on lung cancer TMA was performed by the UW TRIP lab as follows: The experiment was run on Roche Ventana Medical System’s Discovery Ultra Automated platform. Deparaffmization was carried out on the instrument, as was heat-induced epitope retrieval in the form of ‘’cell conditioning” with CC1 buffer (Ventana #950-500), aTris based buffer pH 8.4 for approximately 56 minutes at 95°C. Slide was incubated with the first primary antibody XCR1 diluted 1:25 in DaVinci Green antibody diluent (BioCare Medical #PD900H) for 60 min at 37°. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery OmniMap anti-Rabbit HRP (Ventana #760-4311) was applied for 16 min at 37 degrees. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery ChromoMap DAB detection (Ventana #760-159) was applied for the preset time. Denaturing agent in the form of Discovery Inhibitor (Ventana #760-4840) was applied for the preset time. Slide was incubated with the second pnmary antibody DPEAAE diluted 1:800 in Ventana antibody diluent with casein (Ventana #760-219) for 28 min at 37 degrees. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery OmniMap anti-Rabbit HRP (Ventana #760-4311) was applied for 16 min at 37 degrees. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery Teal HRP detection kit (Ventana #760-247) was applied for 32 minutes. Denaturing agent in the form of Discovery Inhibitor (Ventana #760-4840) was applied for the preset time. Slide was incubated with the third pnmary antibody CD8 (pre-diluted ready to use) for 16 min at 37 degrees. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery OmniMap anti-Rabbit HRP (Ventana #760-4311) was applied for 16 min at 37 degrees. Slide was rinsed with Reaction Buffer (Ventana #950-300) for the preset time and duration. Discovery Purple HRP detection kit (Ventana #760-229) was applied for 16 minutes. Slide was removed from the instrument and rinsed with dawn dish soap and warm tap water followed by nnsing with dH2O Slide was counterstained with Harris hematoxylin (1:5 diluted in dH2O) for 45 seconds. Slide was rinsed with dH2O. Slide was dehydrated in the oven (60 degrees C) followed by dipping in Xylene. Slide was cover slipped. An Olympus BX43 microscope with 40X objective (400X magnification) was used for data points reported as per “high power field”.
Imaging and morphometric analysis ofXCRl+CLEC9A colocalization
Imaging of a human tonsil slide was performed on a Vectra 2 multispectral scanner (Akoya Biosciences). The stained slide was then loaded onto the instrument and 24 8-bit Bright Field 42
SUBSTITUTE SHEET ( RULE 26 ) 20X images were acquired for analysis. A customized spectral library algorithm for all chromogens and counterstain was created using Nuance Software version 3.0.2 (PerkinElmer). The inForm software version 2.4.7 was used to segment tissue subcellular compartments (nucleus, cytoplasm or membrane), and to measure biomarker expression. The double positivity algorithm was used to measure colocalization of cells expressing XCR1 and CLEC9A as double-positive percentage rate.
RNA isolation and quantitative real-time PCR
RNA was isolated using QIAGEN RNeasy Mini Kit and cDNA was synthesized using the iScript Reverse Transcription Supermix (Biorad). Quantitative real-time (qRT-PCR) analysis was performed using SsoAdvanced Universal SYBR Green Supermix (Biorad #1725272) according to the manufacturer’s instructions on an CFX96 Touch Real Time PCR detection (Biorad) using the relative standard curve method. PCR conditions were 2 min at 50°C, 10 min at 95°C followed by 40 2-step cycles of 15 s at 95°C and 1 min at 60°C. Primers for the targets as well as SDHA for normalization control, were used to assess relative gene expression.
For DMXAA-response analysis, RT2 Profiler PCR Array (QIAGEN, Cat. no. PAMM-021Z) was used. In brief, RNA was isolated from tumors and was reverse transcribed using kits mentioned above. cDNA was mixed with RT2 SYBR®Green qPCR Mastermix (Cat. no. 330529). The mixture was aliquoted across the RT2 Profiler PCR Array (in 96-well format) and was run on the Real Time PCR machine. Data analysis was performed using the manufacturer’s online platform for RT2 Profiler Data analysis software.
Generation of versikine cell culture supernatant
5xl06 HEK293 and HEK-Vkine expressing cells were seeded in T-175 cell culture flasks and cultured in DMEM 10% FBS media. After 75 to 80% confluency, the cell media was changed to DMEM 1% FBS media. Subsequently, media supernatant was collected after 48 hours of incubation. The collected supernatant was centrifuged to remove debris and filtered with 0.45m filter. The filtered supernatant was then concentrated 30 times to the initial volume using Sartorius Vivaspin 20, 10,000MWCOPES concentrator (Cat. No. VS2001). Endotoxin assay was performed using Genscript ToxinSensor Gel Clot Endotoxin Assay Kit (Cat. No. L00351) according to the manufacturer’s instructions to rule out contamination. The presence of versikine in concentrated supernatant was confirmed using western blot using c-Myc Antibody (Novus Bioc-Myc Antibody (9E10) - Chimeric NBP2-52636). Concentrated supernatant containing versikine was then used to treat MutuDC1940 cells. 2 x 105 MutuDC1940 cells per
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SUBSTITUTE SHEET ( RULE 26 ) well were seeded in 12 well plate. The following day, 10% supernatant was added to the plate media. Cells were then incubated for 72 hours. After incubation, total RNA was extracted from MutuDC1940 cells.
Library preparation for RNA -sea
A total amount of 1 mg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X) First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 30 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150-200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 mL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 mm followed by 5 mm at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR pnmers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.
RNA-seq data analysis
Raw data (raw reads) of FASTQ format were firstly processed through fastp. In this step, clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Reference genome and gene model annotation files (GRCm38) were downloaded from genome website browser (NCBI/UCSC/Ensembl) directly Paired-end clean reads were 44
SUBSTITUTE SHEET ( RULE 26 ) aligned to the reference genome using the Spliced Transcripts Alignment to a Reference (STAR) software (v2.6.1d). FeatureCounts (vl.5.0-p3) was used to count the read numbers mapped of each gene RPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis between two conditions/groups (three biological replicates per condition) was performed using DESeq2 R package (v 1.20.0). DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting p values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted p value <0.05 found by DESeq2 were assigned as differentially expressed.
Gene set enrichment analysis
Gene Set Enrichment Analysis was performed by comparing MutuDC1940-Vkine (treated with PBS, 4h) RNA-seq data to the corresponding MutuDC1940-EV sample. 4736 differentially expressed gene features for each condition were ranked by the signal to noise metric of GSEA and the analysis was performed using the standard weighted enrichment statistic against human gene sets contained in the Molecular Signatures Database (MSigDB v7.4) that included all (H) Hallmark gene sets, (C2) curated gene sets, and (C3) motif gene sets. The normalized enrichment score (NES) was calculated using 1000 gene set permutations.
NK cell depletion in vivo
For depletion of NK cells, mice were injected i.p. with 50 ug of anti-asialoGMl (Wako Pure Chemical Industries, lOOmL/mouse) on days -1, 0, 7, 14 around tumor inoculation.
MutuDC1940 apoptosis assay
2xl06 MutuDC1940-EV or -Vkine cells were plated per well in a 6-well plate and allowed to attach overnight at 37°C in 5% CO2 incubator. Next day, cells were pretreated with lOng/mL murine GM-CSF (Peprotech, catalog no # 315-03) for 6 h, followed by staurosporine [(AM- 2282), Selleckchem, Catalog no. SI 421] treatment at indicated doses for 24h. Next day, for apoptosis assay, cells and medium were harvested, washed twice with cold PBS and then resuspended in Annexin-V binding buffer. The cell suspension was stained with APC Annexin V and 7-AAD viability dye according to manufacturer’s instructions (APC Annexin V Apoptosis Detection Kit
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SUBSTITUTE SHEET ( RULE 26 ) with 7-AAD, Biolegend, catalog #640930). Each sample was analyzed by flow cytometry with proper machine settings.
Antigen presentation assay
MutuDC1940 were cultured and treated with LPS or PBS control respectively overnight. Next day, the cells were harvested and plated on 96-well round bottom plates at a density of 100,000 cells per plate. DC were then loaded with OVA peptide 257-264 (SIINFEKL) (3ng/mL) and incubated for 4 hours at 37°C. MutuDC1940 were then washed with 0.1% PBS-BSA and centrifuged at 800 x g and were fixed with 50 mL per well of freshly made PBS-glutaraldehyde (GT A) 0.008% (vol/vol) and incubated for 5 minutes on ice. 50mL of PBS-glycine 0.4M was added to the PBS-GTA 0.008% solution and cells were centrifuged at 800 * g for 2 minutes at 4°C. Plates were subsequently flicked. Finally, 100 mL of PBS-glycine 0.2M was added to each well and centrifugation of the plates at 800 x g for 2 min at 4°C followed. Fixed DC were then washed twice with 200 mL/well of T cell culture medium (RPMI 1640 containing 10% heat inactivated FBS, 100 lU/mL penicillin, lOOmg/mL streptomycin, 2mM glutamax, 50mM P-mercaptoethanol, IxMEM nonessential ammo acids, lx sodium pyruvate) before being resuspended in 100 ul/well of the same medium. 100,000 OT-I T cells per well in lOOmL T cell culture medium were added (to a final volume of 200mL). The co-cultured OT-I T cells with the cross-fixed DCs were incubated for 18 h at 37°C. Cell activation cocktail with brefeldin A (PMA/Ionomycin and Brefeldin A Biolegend, #423303) was added to the wells 4 hours before harvesting. At the time of the harvest, plates were spun down at 800 x g for 2 minutes at 4°C and supernatant was kept for subsequent cytokine analysis. They were then washed with lOOmL of 0.1% PBS-BSA before proceeding with live/dead staining with fixable viability Ghost 780 dye (Tonbo Biosciences) for 30 min at 4°C in PBS. Cells were then washed with 0. 1% PBS-BSA and stained with a cocktail of 70mL/well of the surface markers (CD8a and CD3) for 40 minutes at 4°C. They were then fixed and permeabilized using the eBiosciences fixation and permeabilization buffer set (eBioscience 88-8824-00) according to the manufacturer’s instructions followed by intracellular staining of IFNy and IL-2 in permeabilization buffer. Finally, cells were washed 2 times with 0.1% PBS-BSA, centrifuged and resuspended in lOOmL/well of PBS-BSA and then analyzed by flow cytometry. cDCl and VCAN landscape analysis ofTCGA datasets
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SUBSTITUTE SHEET ( RULE 26 ) Level 4 gene expression data were downloaded from the TCGA Data Portal and fdtered to retain only cancer types of known epithelial origin for a total of 7591 samples across 20 different cancer types Single-sample Gene Set Enrichment Analysis (ssGSEA) was performed to measure the signature of gene sets designed to measure overall immune infiltration, cDCl density and CD8+ T cell density. TCGA samples were grouped by cancer type and sorted based on median expression of versican (VC AN) and median cDCl signature. To measure cancerspecific relationships between VCAN expression and the cDCl signature an ordinary least squares linear model was fit on these two variables to measure their relationship within each cancer type Nominal p values from these 20 different models were corrected for multiple hypothesis testing using the Benjamini-Hochberg method and q- values less than 0.1 were considered statistically significant.
Computational modeling of CD8+ T cell density and versikine response signature
Differential expression using DESeq2 was performed to identify genes that were differentially expressed between the PBS-EV and PBS-versikine conditions (FIGs. 4A and 4C). Genes with a q-value of less than 0.1 were considered significant. Two gene sets were defined to measure the response to versikine by selecting the 100 genes more significantly induced (versikine-up) and most significantly repressed (versikine-down). These mouse genes were then mapped to their human orthologues using the HGNC Comparison of Orthology Predictions (HCOP) tool. ssGSEA was then used to measure the signature of these two gene sets in the 1017 lung samples in the TCGA cohort and overall versikine response level was summarized as the difference between versikine-up and versikine-down signature levels. This versikine response signature was then compared to the CD8 effector T cell signature using an ordinary least squares linear model including the overall immune infiltration signature as a covariate, p-values of less than 0.05 were considered significant.
Example 1 - Conventional type dendritic cells (cDCls) localize in peritumoral stroma and are regulated by the VCAN pathway
Versican (VCAN) and its binding partner hyaluronan are cardinal components of the provisional extracellular matrix in development, wound healing, and cancer. In tumors, stromal provisional matrix is thought to coordinate critical pro-tumor functions (e.g., angiogenesis) and prime conversion toward collagen-rich, desmoplastic stroma. For deposition into stromal matrix, VCAN is sourced from stromal mesenchymal cells, immune infiltrating cells (particularly myeloid cells, such as macrophages), and, in some cases (such as in lung 47
SUBSTITUTE SHEET ( RULE 26 ) carcinomas), the tumor cells themselves. However, VCAN proteolytic processing is located primarily in stroma because of the local activity of stromal fibroblast-derived ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs; a family of multidomain extracellular protease enzymes) VCANases (enzymes which cleave versican). Using an immunohistochemistry (IHC)-validated antibody against DPEAAE, a neoepitope generated through VCAN proteolysis at Glu441-Ala442 (VI isoform) (FIG. 1 ), VCAN proteolysis signal was observed in approximately 83% of human lung cancer cases in a stromal distribution (FIG. IB). To determine the location of cDCls relative to sites of stromal VCAN proteolysis, multiplex IHC was performed with antibodies detecting the cDCl lineage marker XCR1 and CD8. Remarkably, even in T cell-inflamed tumors demonstrating intra-epithelial CD8+ T cell penetration, XCR1+ cells were confined within stromal sheets recurrently undergoing VCAN proteolysis (FIG. 1C). The antibody against XCR1 has been previously validated, and close correlation between XCR1 staining and the signal for the cDCl lineage marker CLEC9A was confirmed in human tonsils (FIG. IK).
Matrikines (such as versikine) have been defined as ‘‘peptides liberated by partial proteolysis of extracellular matrix macromolecules which are able to regulate cell activities not triggered by their full-size parent macromolecules”. Notwithstanding its distinct neo-activity, versikine ultimately derives from parental VCAN through ADAMTS proteolysis (FIG. 1A); therefore, it was hypothesized that VCAN expression (the substrate for versikine) and cDCl abundance correlate inhuman cancer. VCAN gene expression and cDCl signature scores were compared across 7,591 samples from 20 The Cancer Genome Atlas (TCGA) cancer types (FIGs. ID and IE). A significantly positive correlation between VCAN expression and cDCl signature scores was observed in several human carcinomas (FIG. IF), suggesting that the VCAN pathway broadly regulates cDCls.
To dissect the relevant mechanisms, novel Vcan -targeted models were generated that disrupt exons coding for Vcan’s N terminus (Vcan exons 2-6). The widely used Vcanh^ null mutant (hdf, heart defect) targeting exon 7 is embryonic lethal in homozygosity. Vcanhdf hemizygosity demonstrates functional haploinsufficiency for CD8+ -mediated control of viral infection. CRISPRCas9-mediated mutagenesis was used to disrupt exon 3 sequences, abolishing transcription of all Vcan isoforms (and, consequently, generation of versikine) (FIG. 1G). T 'O founders (Vcanl053 and Vcanl058) bearing Vcan exon 3 deletions were found (16 bp and 47 bp, respectively) (FIGs. IL and IM). Defective Vcan message induction was confirmed after stimulation of BM-derived macrophages (BMDM) with the Toll -like receptor (TLR)-4 agonist lipopolysaccharide (LPS) (FIG. IN). BMDMs stimulated with LPS 48
SUBSTITUTE SHEET ( RULE 26 ) preferentially transcribe the VI isoform, the precursor to versikine. The Vcanl053 transgenic line demonstrated the most severe defect in Vcan message induction (hereafter designated Vcan+I').
Lewis lung carcinoma (LLC) cells produce Vcan cell autonomously. Endogenous Vcan expression was knocked down in LLC cells using short hairpin RNA (shRNA) targeting Vcan exon 8 (encoding for the glycosaminoglycan (GAG)P domain in the VCAN- VI isoform, the major isoform produced in LLC; FIG. 1A), hereafter referred to as LLCVcanKD. The reduced transcription of Vcan was validated in LLCVcanKD cells using 5’ and -3’ Vcan primers, as shown in FIG. 10. The intratumoral immune contexture was characterized in LLCVcanKD tumors implanted in Vcan+/~ mice through mass cytometry and compared it with wild-type (WT) controls (FIG. 1H) Vcan depletion resulted in expansion of CD8+ T cells, consistent with the known role of non-proteolyzed VCAN in T cell exclusion. In keeping with the hypothesis, cDCl loss was observed in Vcan-depleted tumors (FIG. 1H). To corroborate the mass cytometry findings, intratumoral DCs were delineated through 9-color flow cytometry (FIG. IP) cDCls were depleted in LLCVcanKD tumors implanted into Vca V1' mice (FIGs. II and 1J). In contrast, steady-state splenic cDCls were not reduced (in fact, they were mildly increased) in Vcan+I- mice (FIG. IQ). Ectopic expression of versikine in LLCVcanKD cells restored near-physiological cDCl abundance (FIGs. II and 1J). Intratumoral DC absolute count ratios corroborated the cell frequency findings (FIGs. II and 1J) despite fluctuations in total eDC counts using the collagenase/hy aluromdase tumor dissociation protocol delineated in the STAR Methods (FIG. 1R). Vcan depletion and versikine add-back did not affect tumor growth rates (FIG. IS). These results demonstrate that VCAN is necessary and that its proteolytic product versikine is sufficient for eDC 1 abundance in the TME.
Example 2 - VCAN matrikine versikine promotes cDCl abundance in vivo
VCAN proteolysis is a composite event that produces two simultaneous, coupled consequences: first, parental VCAN clearance, and second, the novel activities of the released matrikine versikine. To uncouple versikine’ s activity from the effects of parental VCAN depletion, LLC cells stably expressing hemagglutinin (HA)-tagged versikine in the WT background were generated (FIG. 2A). Expression of versikine in LLC cells did not result in grossly visible increases in angiogenesis or hemorrhagic propensity (FIG. 2B). Ectopically expressed versikine was readily detectable by anti-HA tag western blotting in murine tumor lysates at approximately 75 kDa (FIG. 2C and 2H).
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SUBSTITUTE SHEET ( RULE 26 ) Murine implantable tumor models do not recapitulate the human architecture of epithelial nests and stromal sheets: this limitation has been attributed to acquisition of mesenchymal features through successive passaging (FIG. 2D). However, the LLC model does retain physiological relevance because of its tumor-intrinsic production of Vcan that regulates myeloid cells in the TME. In this regard, LLC mimics a subset of human lung cancers with detectable VCAN production and processing in stromal and epithelial compartments (FIG. 2I-2J). Ectopically expressed versikine was detected in a membranous distribution consistent with its accumulation in the pericellular glycocalyx (FIG. 2D), a physiological site of VC AN cleavage. Membranous localization of ectopic versikine was also seen in B16 melanoma tumor cells that transcribe a very low to undetectable endogenous Vcan message (FIG. 2K).
There were no differences in the growth rates between LLC-empty vector (EV) and LLC-versikine (LLC-Vkine) tumors (FIG. 2L). eDC populations were analyzed by conventional flow cytometry (FIG. IP). cDCl expansion was confirmed in LLC-Vkme tumors (FIG. 2E), notably the opposite phenotype to that of Vcan depletion (FIG. 1 J), as well as unchanged monocytic-derived dendritic cell (Mo-DC) frequency (FIG. 2E). In the later stages of the study, optimized tumor cell dissociation protocols permitted determination of absolute DC counts; only cDCls expanded in absolute terms (FIG. 2M). By mass cytometry, in addition to eDC 1 accumulation, expansion of an innate lymphoid NK1. 1+ NKp46+ population, an increase in intratumoral CD8+ T cells, as well as polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) depletion was observed (FIG. 2F), the latter being consistent with IRF8 regulation by versikine. To confirm cDCl expansion in the orthotopic lung milieu, EV and versikine (Vkine)-expressing LLC cells were injected intravenously to seed the relevant lung microenvironment and lungs bearing metastatic deposits were harvested on day 10 (FIG. 2G). LLC-Vkine tumors within the relevant lung microenvironment also showed enhanced cDCls (FIG. 2G). As with prior reports, CD 103 was found to be more consistent compared with CD24 for cDCl enumeration in native lung tissue.
Versikine promoted cDCls in the BALB/c-derived, orthotopic 4T1 mammary carcinoma model (FIG. 2N). Growth rates of 4T1 versikine-replete tumors did not differ from their EV counterparts (FIG. 20). Earlier a role of VCAN proteolysis was reported in shaping the human BM myeloma immune microenvironment. More recently, the first Ras-driven immunocompetent myeloma model was developed, VQ. Versikine-replete VQ myeloma tumors demonstrated enhanced cDCls (FIG. 2P) Myeloma clinical progression was 50
SUBSTITUTE SHEET ( RULE 26 ) unaffected by versikine (FIG. 2Q). Therefore, versikine promotes cDCl abundance in solid and hematopoietic cancers across murine genetic backgrounds.
Example 3 - Pre-DC differentiation is unaffected by versikine
To explain how VCAN proteolytic products promote tumor cDCl density, it was first determined whether this occurred through uncommitted tumor-seeding pre-DC differentiation. The rationale was based on prior observation that recombinant versikine promoted cDCl generation from mouse BM treated with FLT3L in vitro. cDCl “signature” transcripts (Irf8, Batf3, Cxcl9, and CxcllO) were increased in the bulk transcriptome of versikine-replete (LLC- Vkine) tumors (FIG. 3A). Irf8 is a “terminal selector” for the cDCl lineage. The Balf3 transcript increase corroborates versikine-induced cDCl abundance because the Batf3 expression range is very narrow (FIG. 3H) Id2 transcripts did not differ between versikine- replete and control tumors, but Id2 is expressed more broadly and not highly expressed in cDCls (FIG. 3H).
To test the differentiation hypothesis, CD45.2+ pre-DC precursors were sorted from the BM of Flt31 in-vivo-mobilized donor mice (FIGs. 31-3 J). Donor mice were implanted with Flt31-secreting B16 tumor cells to provide a continuous source of circulating Flt31. CD45.2+ pre-DCs were adoptively transferred intratumorally into subcutaneous LLC-EV and LLC- Vkine tumors implanted into CD45.1+ recipients. 72 h after adoptive transfer, tumors were dissociated, and CD45.2+ as well as endogenous CD45.1+ DC fractions were enumerated by flow cytometry. The CD45.1+ endogenous eDC composition served as an internal control. As expected, CD45.1+ endogenous cDCls were increased in LLC-Vkine tumors (FIG. 3K). In contrast, CD45.2+ cDCls and cDC2s did not differ between LLC-Vkine and -EV controls (FIG. 3L) Thus, stromal signals did not affect pre-DC differentiation into cDCls versus cDC2s within the time frame of the differentiation assay and given the assay’s limitations.
Example 4 - Versikine selectively activates cDCls in vivo
Because pre-DC differentiation could not explain cDCl accumulation, the hypothesis that versikine regulates the cDCl activation-survival cycle was tested. The rationale was supported by the association between VCAN proteolysis and T cell infiltration, suggesting a role of versikine in cDCl activation. CD40 expression was specifically observed because of recent work showing that cDCls cross-prime CD4+ T cells and are licensed through CD40 back-signaling to augment CD8+ T cell priming and anti-tumor responses as well as the reported anti-apoptotic functions of CD40
51
SUBSTITUTE SHEET ( RULE 26 ) in cDCls. Flow cytometry analysis of eDC subsets from LLC-EV and LLC-Vkine tumors demonstrated that versikine selectively upregulates CD40 expression in tumor cDCls but not tumor cDC2s or Mo-DCs (FIG. 3B and 3M). CD40 expression is weakest at baseline in intratumoral cDCls. In contrast, all 3 intratumoral DC subsets upregulate PD-L1 when exposed to versikine in vivo (FIG. 3C)
CD40 induction in cDCls would be expected to promote T cell activation in the TME. To test this hypothesis, the trans criptomic profiles of CD45+ tumor-infiltrating leukocytes (TILs) isolated from versikine-replete versus control LLC tumors were compared (FIGs. 3D- 3G). A compelling T cell co-stimulation and activation signature was detected (Cd69, Ctla-4, Icos, Zap70,
I12rb, Cd38, Light, and Gitr) (FIGs. 3F and 3G) as well as a significant increase in T cellspecific transcripts (CD3e and T cell receptor [TCR] genes), consistent with the CD8+ T cell expansion seen by mass cytometry (FIG. 2F). Hallmarks of antigen-presenting cell (APC) activation were detected (upregulation of MHCII, Ccr7, Ifnbl, Irf7, and several interferonresponsive genes) (FIG. 3F).
Example 5 - cDCl activation by versikine is cell autonomous
To explore steady-state changes in the MutuDC1940 transcriptome in the presence of versikine, stable MutuDC1940-Vkine cell lines were generated through lentiviral transduction (FIG. 4A). MutuDC1940-Vkine cells had a slightly more developed dendritic appearance compared with EV (FIG. 4B).
Versikine elicited a co-stimulatory transcriptional program in MutuDC1940 cells distinct from the transcriptional program elicited by the TLR4 agonist LPS (FIG. 4C and 4L). The combination of versikine and LPS elicited a transcriptional signature distinct from either stimulus alone (FIG. 4C). Versikine-upregulated genes involved in DC maturation (interferon- stimulated genes such as Ifi209 and Ifi204), chemokines (Ccl7, Ccl2, Cxcl9, and CxcllO), and co-stimulatory signals (Cd80 and Cd40) (FIG. 4D). Downregulated genes included components of transforming growth factor b (TGF-b) and Wnt pathways, both associated with immunosuppression. Gene set enrichment analysis (GSEA) confirmed upregulation of immune activation gene sets (e.g., interferon [IFN]-a response, IFN-g signaling, nuclear factor kB [NF- kB] -induced tumor necrosis factor [TNF] signaling, and inflammation) and downregulation of immunosuppressive Wntb-catenin and TGF-b signaling (FIG. 4E). Several of the top hits were confirmed by RT-PCR and, at the protein level, by ELISA (FIGs. 4M-4P). Key hits were confirmed after exposure of MutuDC1940 cells to supernatants from HEK293 cells secreting
52
SUBSTITUTE SHEET ( RULE 26 ) versikine (FIG. 4Q) One of the top hits, Ccl7, has been shown recently to act as a cDCl chemoattractant. LLC-Vkine tumors expressed high bulk Ccl7 transcripts (FIG. 4F), as did immunomagnetically separated CD1 lc+ cells from LLC-Vkine tumors (FIG. 4G).
Then, to test versikine’ s effect on T cell priming by cDCls, antigen presentation assays were earned out using the OVA (ovalbumin) antigen system in conjunction with TCR- engineered OT-I T cells (FIG. 4H). Vkine- and EV-MutuDC1940 cells were pulsed with SIINFEKL peptides and cocultured with OT-I cells (FIGs. 4I-4K and 4R-4T). Versikine alone more than doubled the percentage of primed OT-I cells secreting IFN-g and interleukm-2 (IL- 2) by flow cytometry, confirmed through IFN-g ELISA of culture supernatants. Combining versikine with LPS further augmented T cell priming. Thus, versikine synergized with classical “danger” signals to maximize stimulatory DC antigen presentation and T cell priming.
Example 6 - cDCl accumulation requires atypical innate lymphoid support
Although cDCl activation by versikine was cell autonomous, cDCl accumulation might still require supporting actors, such as natural killer (NK) cells. To determine whether versikine regulates cDCl/NK cell cross-talk in vivo, CDl lc+ DCs from primary versikine- replete versus EV tumors were characterized Freshly explanted CDllc+ cells from LLC- Vkine tumors expressed higher levels of the NK regulators IL-23 (a subunit), IL-27 (p28 and EBB subunits), and IL-15 (FIG. 5A). Therefore, versikine’s cDCl activation program incorporated an NK cell -activating module. Versikme-exposed DCs demonstrated low IL- 12a(p35) and IL-12b(p40) subunit expression but very high IL-23a expression. It was speculated that IL-23a could combine with IL- 12b(p40) secreted by other activated myeloid cells (e.g., macrophages) to form bioactive IL-23 heterodimers, similar to extracellular IL- 12p70 generation.
It was hypothesized that versikine may trigger a distinct stroma-specific pattern of cDCl-NK cell communication. Previous studies have implicated the NK cell-derived differentiation/survival mediator FLT3L as well as the chemo-attractants XCL1 and CCL5 in cDCl support. NK cell-derived IFN-g has been shown recently to induce the cDCl “terminal selector” IRF8. NKp46+ NK1.1+ cells from LLC-Vkine tumors were potent expressors of Csf2 (granulocyte-macrophage colony stimulating factor [GM-CSF]) and relatively weak expressors of IFN-g compared with the LLC-EV-derived counterparts (FIG. 5B), whereas expression of Xcll,
Flt31, and Ccl5 remained unchanged (FIG. 5B). Versikine causes induction/expansion of an atypical NK cell subset expressing low IFN-g despite high cytotoxicity receptor (NKp46) 53
SUBSTITUTE SHEET ( RULE 26 ) expression and robust Csf2 (GM-CSF), an essential survival factor for cDCls. These results suggest that versikine engages innate lymphoid cells through a previously unreported mechanism in tumors. Versikine-induced NK cells are reminiscent of a recently reported spleen-resident ILCl-like subset that nurses cDCls and promotes CD8+ T cell pnming in viral infection.
To determine whether versikine - requires innate lymphoid cells to support cDCl abundance in the TME, an antiasialo-GMl antibody (anti-ASGMl) was used for in vivo NK cell depletion (FIG. 5C). Anti-ASGMl antibody treatment (FIG. 5H) completely abrogated versikine-mediated enhancement of cDCls in the TME (FIG. 5D and 51).
Example 7 - Versikine regulates cDCl-NK cell cross-talk through cDCls
The results so far suggested that versikine regulates cDCls and NK cells in a virtuous circle but left open the question of whether versikine’s primary target is cDCls or NK cells. To answer this question, the experiment was repeated in Batf3' ' mice, which lack intratumoral cDCls (FIG. 5L). In Batf3_ _ recipients, versikine was unable to induce Csf2 (GMCSF) in intratumoral NKp46+ NK1.1 + cells despite a modest increase in NK cell frequency (FIG. 5E). This result suggested that the primary target of versikine in the eDC 1 -NK cell cycle was eDC 1 s. This model would pre-suppose proximity of cDCls and NKp46+ cells in human tumor stroma. NCR1 (NKp46+) cells in human tumors were almost exclusively stromal in their location (FIG. 5F).
The evidence so far supports a model where versikine acts directly on stromal cDCls to activate a pro-immunogenic program. Like other known eDC 1 activators (e. g. , TLR ligands), versikine-mediated activation simultaneously triggers a homeostatic propensity for apoptosis. This pro-apoptotic propensity is mitigated/rescued by NK cell-derived GM-CSF. The end result is accumulation of activated cDCls (which can be called stroma-licensed cDCls) in stroma undergoing VCAN proteolysis. Consistent with this model, versikine’s MutuDC1940 signature incorporates a pro-apoptotic module (FIG. 4L). Experimentally, it was confirmed that versikine- expressing MutuDC1940 cells are sensitive to apoptotic signals and that this sensitivity is mitigated/rescued by exogenous murine GM-CSF (FIG. 5G). In the TME, NK cells are dominant GM-CSF producers compared with sparser GM-CSF expressors, such as basophils (FIGs. 5J and 5K).
54
SUBSTITUTE SHEET ( RULE 26 ) Example 8 - TLR2 and CD44 are dispensable for cDCl accumulation in response to versikine
The data are consistent with versikine signaling through a DC receptor whose identity is unknown. Although cDCls do not robustly express TLR2, non-proteolyzed VCAN is thought to act through TLR2 expressed broadly by DC subsets. However, Tlr2 loss had no effect on versikine- induced cDCl accumulation (FIGs. 5M-5O).
It was also considered that versikine may activate hyaluronan-dependent signaling pathways in vivo. VCAN’s N-terminal link domains bind hyaluronan (FIG. 1A) Recombinant versikine bioactivity was previously demonstrated in the absence of bound hyaluronan in vitro. Loss of Cd44, encoding the major hyaluronan receptor, did not affect versikine-induced cDCl accumulation in vivo (FIGs. 5P-5R)
Example 9 - Stroma-licensed cDCls are “poised” and hypersensitive to nucleic acid sensing in vivo
Previous studies have highlighted the paradox of immunogenic DC accumulation along the tumor nm, but no compelling mechanism has emerged. It was hypothesized that peritumoral stroma-licensed DCs are “poised” to respond to physiological maturation signals arising from necrotic tumor cells. Thus, versikine-induced CD40 expression and “stroma licensing” could serve to amplify these innate cancer-sensing signals. The cGAS/STING pathway, a sensor of exogenous double-stranded DNA, has emerged as a central mediator of innate sensing of tumors. It was hypotehsized that stroma-licensed DCs may respond to very low doses of a STING agonist (FIG. 6A). Dose-response relationships were previously established for the intratumorally administered murine STING agonist DMXAA: the maximum tolerated dose (MTD) was 500 mg; unacceptable toxicity was observed at higher doses. LLC- EV and LLC-Vkine tumors were challenged with subtherapeutic doses of the STING agonist DMXAA (150-200 mg) or vehicle (NaHCO3). Tumor response curves are shown in FIG. 6B and survival plots (Kaplan-Meier) in FIG. 6C. EV tumors did not appreciably respond to vehicle or subtherapeutic doses of DMXAA (DMXAA200). In contrast, versikine low ered the response threshold to DMXAA so that versikine-replete tumors demonstrated a consistent response to single subtherapeutic DMXAA doses. Versikine-replete tumors routinely developed necrotic eschars by 24 h after subtherapeutic DMXAA injection (FIG. 6D). In contrast, none of the control mice developed eschars within this time frame. To determine whether versikine reduced the therapeutic threshold through a classic type I IFN response to
55
SUBSTITUTE SHEET ( RULE 26 ) DMXAA, tumors for RNA extraction were harvested 2 h after DMXAA administration. Versikine-replete tumors demonstrated a several-fold increase in IFN-a transcripts (particularly IFN-a2 and IFN-a4) and, to a lesser degree, IFN-pi transcripts (FIG. 6E). These results demonstrate that versikine lowered the threshold for a classic type I IFN-mediated STING agonist response.
To determine whether low doses of the STING agonist could generate an abscopal effect, mice bearing tumors implanted in both flanks were examined. The treated side was inoculated with EV- or versikine-expressing LLC cells; the contralateral, nontreated side was inoculated with unmanipulated (untransduced) LLC cells. Ectopic versikine is bound in the pericellular halo (glycocalyx) (FIG. 2D) and probably does not circulate to an appreciable degree. A consistent abscopal effect was observed when versikine-replete tumors were injected with 200 mg DMXAA (FIGs. 6F-6H). EV tumors treated with the same subtherapeutic dose failed to elicit any response on the treatment or contralateral side. STING agonist hypersensitivity produced consistent primary tumor and abscopal effects across genetic backgrounds; e.g., in the orthotopic 4T1 mammary carcinoma model (FIGs. 6M-6O).
The hypothesis that stromal matrikines render cDCl hypersensitive to nucleic acid sensing in vivo predicts that DMXAA200 would be ineffective in the absence of cDCls. Thus, the experiment delineated in FIG. 6A was repeated in Batf3'A recipients. DMXAA200 was globally ineffective in the Batf3-null background, and all survival benefit was lost (FIGs. 61 and 6J). To confirm that the responsible actors were cDCls (rather than another BatfB- expressing lineage), it was attempted to rescue the null phenotype with intratumoral adoptive transfer of iCD103, BM-derived primary cDCl-like cells generated in culture (FIG. 6P). MutuDC1940 cells cannot be used for adoptive transfer experiments because of their immunogenicity. A consistent cDCl -like phenotype was confirmed in iCD103 cells (FIG. 6Q). Adoptive transfer of iCD103 cells restored subtherapeutic STING agonist efficacy (FIG. 6K) and survival benefits (FIG. 6L). To confirm the findings in a different C57BL6/J model, the B 16 melanoma model was chosen. B 16 tumors responded to subtherapeutic doses of DMXAA in the presence of versikine but not EV (FIGs. 6R and 6S). Efficacy was lost in the Batf3-null background (FIG. 6T), but response to subtherapeutic doses of the STING agonists was restored, at least in a subset of mice, upon iCD103 adoptive transfer (1CD103 “take” in B16, an “immune-cold” tumor, was less efficient than in LLC, an “immune-hot” tumor, FIG. 6U). Therefore, stroma-licensed cDCls are “poised” and hypersensitive to nucleic acid sensing in vivo
56
SUBSTITUTE SHEET ( RULE 26 ) Example 10 - Versikine promotes antigen-specific CD8+ T cell responses in vivo
To determine whether hypersensitivity of stromalicensed cDCls to DNA sensing translates into enhanced antigen-specific effector responses in vivo, the OVA system was employed as an in vivo model antigen (FIG. 7A). EV- and versikine-expressing LLC cells were additionally engineered to express full-length OVA (LLC-OVA). EV- or versikine-replete LLC-OVA tumors were challenged with therapeutic DMXAA doses (500 mg). Five days after challenge, spleens were harvested and analyzed by flow cytometry for antigen-specific effector responses using an antigen-specific tetramer assay. Versikine more than doubled the magnitude of the antigen-specific response in the CD8+ T cell compartment, as determined by MHCESIINFEKL-tetramer staining (FIG. 7B). Spleens contained a larger proportion of CD8+CD62L+CD44+ cells with a central memory phenotype (FIG. 7G). The results demonstrate that stromal matrikines enhance antigen-specific CD8+ T cell effector responses in vivo.
Example 11 - The stroma-licensed cDCl signature correlates with CD8+ T cell scores in human lung cancer
To correlate stromal cDCl licensing with CD8+ T cell density in human cancers, a unique cDCl response signature was generated from 200 genes whose expression was significantly altered in versikine-activated MutuDC1940 cells (FIGs. 4C and 4D). Then, this stroma-cDCl signature was correlated with CD8+ T cell scores estimated in TCGA expression data for 1,017 lung cancers (STAR Methods). The results are shown in FIG. 7C and 7H. A significant but weak correlation was observed between stroma-licensed cDCl transcriptional profiles and CD8+ T cell scores. There were obvious limitations in this analysis (application of an in vitro murine cultured cell-generated signature to primary bulk human tumor data), but the results did support a connection between stromal cDCl licensing and CD8+ T cell density in human cancer.
Example 12 - Stromal VCAN proteolysis correlates with CD8+ infiltration in human lung cancer
CD8+ T cell infiltration has prognostic significance in human lung cancer as well as predictive significance for efficacy of checkpoint inhibitor-based immunotherapy. A cutoff of 3-5 CD8+ T cells/HPF has been used in some studies to designate CD8+ TIL-rich versus -poor tumors bearing a favorable and unfavorable prognosis, respectively. To determine whether 57
SUBSTITUTE SHEET ( RULE 26 ) stromal VCAN proteolysis was associated with prognostic immune infiltration groups in humans, 98 non-small cell lung cancer (NSCLC) biopsies in our tissue microarray (TMA) were subdivided into pauci-immune (0-2 CD8+ TILs/HPF in stromal and epithelial compartments, n = 26) and immune-rich (R3 CD8+ TILs/HPF in stromal or epithelial compartments, n = 72). The distribution of stromal DPEAAE staining intensity (0, 1, 2, or 3) was compared between the groups. The results are shown in FIGs. 7D, 7E, and 71. A statistically significant association was found between stromal VCAN proteolysis intensity and CD8+ T cell infiltration in human NSCLC. Example 13 - The VCAN matrikine versikine overcomes resistance to anti-PDl checkpoint inhibition immunotherapy in vivo cDCls are critical for responses to checkpoint inhibitors. Thus, the effect of versikine on anti-PDl responses in the refractory LLC model was tested (FIG. 7F and 7 J). Versikine sensitized at least a subset of LLC tumors to a short course of anti-PDl checkpoint inhibition- based immunotherapy, resulting in enhanced animal survival.
58
SUBSTITUTE SHEET ( RULE 26 )

Claims

WHAT IS CLAIMED IS:
1. A fusion protein comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and a secretory peptide having a sequence of SEQ ID NO: 2-7.
2. A fusion protein comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and a protein selected from the group consisting of: GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, and IFNgamma.
3. The fusion protein of claims 1 or 2, wherein a protein selected from the group consisting of GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL- 27, IL- 15, IL 18, IL-23, IL-2, and IFNgamma comprises a sequence selected from the group consisting of SEQ ID NO 8-21.
4. The fusion protein of claims 1 or 2, further comprises a secretory peptide.
5. The fusion protein of any of the above claims, wherein the secretory peptide comprises a sequence of SQ ID NO: 2-7.
6. A vector comprising any of the fusion peptides of any of the above claims.
7. The vector of claim 6, wherein the vector is a viral vector.
8. The vector of claim 7, wherein the viral vector is a lentiviral vector.
9. A method of introducing versikine to antigen-presenting cells, the method comprising: introducing to an antigen-presenting cell a fusion protein of any one of claims 1-5 or a vector of any one of claims 6-8, wherein the introducing compnses a lentiviral vector.
10. The method of claim 9, wherein the antigen-presenting cells comprise macrophages, B cells, fibroblasts, and/or dendritic cells.
11. The method of claim 9, wherein the antigen-presenting cell is a cell culture.
12. The method of claim 9, wherein the antigen-presenting cell is in a subject. A conjugated polypeptide comprising a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 and a small molecule STING agonist. The conjugated polypeptide of claim 13, wherein the small molecule STING agonist comprises DMXAA or RVU-27065. A pharmaceutical composition comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and a STING agonist. The pharmaceutical composition of claim 15, wherein the STING agonist is DMXAA or RVU-27065. The pharmaceutical composition of claim 15, wherein the versikine protein is coadministered with the STING agonist. The pharmaceutical composition of claim 15, wherein the versikine protein is administered before the STING agonist. A pharmaceutical composition comprising a versikine protein having at least 95% identity to SEQ ID NO: 1 and an anti-PDl and/or anti-PD-Ll inhibitor. The pharmaceutical composition of claim 19, wherein the anti-PDl and/or anti-PD-Ll inhibitor comprises pembrolizumab, nivolumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, or BMS- 986189. The pharmaceutical composition of claim 19, wherein the versikine protein is coadministered with the anti-PDl and/or anti-PD-Ll inhibitor. The pharmaceutical composition of claim 19, wherein the versikine protein is administered before the anti-PD l and/or anti-PD-L l inhibitor. A pharmaceutical composition comprising a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 and anti-CD40 immunotherapy. The pharmaceutical composition of claim 23, wherein the anti-CD40 immunotherapy is selected from the group consisting of selicrelumab, APX005M, JNJ-64457107, SEA-CD40, ChiLob7/4, CDX-1140H, Dacetuzumab, and ABBV-428. The pharmaceutical composition of claim 23, wherein the versikine protein is coadministered with the anti-CD40 immunotherapy. The pharmaceutical composition of claim 23, wherein the versikine protein is administered before the anti-CD40 immunotherapy. A method of treating cancer, the method comprising administering a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 or a versikine nucleic acid having at least 95% sequence identity to SEQ ID NO: 23. A method for increasing T cell activation in a tumor microenvironment (TME) in a subject, the method comprising: administering a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 or a versikine nucleic acid having at least 95% sequence identity to SEQ ID NO: 23. A method for sensitizing a response to an immunotherapy in a subject in need thereof, the method comprising: administering a versikine protein having at least 95% sequence identity to SEQ ID NO: 1 or a versikine nucleic acid having at least 95% sequence identity to SEQ ID NO: 23. The method of any of the above claims, wherein the versikine protein or versikine nucleic acid is co-administered with another pharmaceutical compound. The method of claim 30, wherein the pharmaceutical compound comprises a stimulator of interferon genes (STING) agonist immunotherapy. The method of claim 30, wherein the pharmaceutical compound comprises an anti- PD1 immunotherapy. The method of claim 30, wherein the pharmaceutical compound comprises an anti- CD40 immunotherapy. The method of any one of the above claims, wherein the administering comprises delivery of the versikine. The method of any one of the above claims, wherein the cell is a dendritic cell. The method of any one of the above claims, wherein the cell is an antigen-presenting cell. The method of any one of the above claims, wherein the delivery of the proteolytic fragment comprises a virus-based delivery. The method of any one of the above claims, wherein the virus-based delivery comprises a lentivirus. The method of any one of the above claims, wherein the versikine is generated by proteolysis of VCAN at the Glu441-Ala442 bond. The method of any one of the above claims, wherein the versikine protein or versikine nucleic acid is conjugated to another protein. The method of any one of the above claims, wherein the other protein is selected from the group consisting of: GM-CSF, CCL5, XCL1, FLT3L, CCL7, CXCL9, CXCL10, IL-12, IL-27, IL-15, IL18, IL-23, IL-2, or IFN. The method of any one of the above claims, wherein the other protein comprises a secretory peptide. The method of any one of the above claims, wherein the secretory' peptide is selected from the group consisting of: human OSM, human immunoglobulin, human chymotrypsinogen, human trypsinogen 2, human IL-2, or human insulin. The method of any one of the above claims, wherein the versikine protein or versikine nucleic acid is conjugated to a drug. The method of any one of the above claims, wherein the drug comprises a small molecule STING agonist. The method of any one of the above claims, wherein the versikine protein or versikine nucleic acid is conjugated to a small molecule The method of any one of the above claims, wherein the versikine protein or versikine nucleic acid is conjugated to a radiochemical. The method of any one of the above claims, wherein the versikine protein or versikine nucleic acid is conjugated to an immunogenic component. The method of any one of the above claims, wherein the versikine protein or versikine nucleic acid is conjugated to a label. The method of any one of the above claims, wherein the label is a radioisotope or a fluorophore. The method of any one of the above claims, wherein the versikine is a nucleic acid. The method of any one of the above claims, wherein the versikine nucleic acid is fused to non-versikine nucleic acid. The method of any one of the above claims, wherein the non-versikine nucleic acid is a DNA or RNA molecule. The method of any one of the above claims, further comprising administering a pharmaceutical agent. The method of any one of the above claims, wherein the pharmaceutical agent comprises an immunotherapy agent. The method of any one of the above claims, wherein the immunotherapy agent comprises a STING agonist, a PD-1 inhibitor, a PD-L1 inhibitor, or a CD40 agonist. The method of any one of the above claims, wherein the subject has a tumor. The method of claim 57, wherein the tumor is a refractory tumor. A method of predicting a subject’s response to an immunotherapy, the method comprising:
(a) analyzing a biological sample obtained from the subject; and
(b) detecting a proteolytic fragment of an extracellular matrix (ECM) proteoglycan in the biological sample, thereby predicting the subject’s response to the immunotherapy. The method of claim 59, wherein the analyzing compnses staining the biological sample. The method of claim 60, wherein the staining comprises an immunofluorescence stain. The method of claim 60, wherein the staining comprises an immunohistochemistry stain. The method of any one of claims 60-62, wherein the biological sample is stained with an antibody. The method of claim 63, wherein the antibody is selected from the group consisting of: an anti-DPEAAE neo-epitope antibody, anti-HA antibody, anti-XCRl antibody, anti-CLEC9A antibody, anti-NCRl antibody, and anti-CD8 antibody. The method of any one of claims 59-64, wherein the detecting comprises determining the level of the proteolytic fragment by measuring the antibody in the biological sample. The method of any one of claims 59-65, wherein the immunotherapy comprises a stimulator of interferon genes (STING) agonist immunotherapy. The method of any one of claims 59-66, wherein the immunotherapy comprises an anti-PDl immunotherapy. The method of any one of claims 59-67, wherein the immunotherapy comprises an anti-CD40 immunotherapy. The method of any one of claims 59-68, wherein the ECM proteoglycan is versican (VCAN). The method of claim 59, wherein the proteolytic fragment of the ECM proteoglycan is versikine. The method of claim 70, wherein the versikine is generated by proteolysis of VCAN at the Glu441-Ala442 bond. The method of any one of claims 59-71, wherein the subject has a tumor. The method of claim 72, wherein the tumor is a refractory tumor.
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