WO2025199395A1 - Use of cytomegalovirus immunity against tumors - Google Patents

Use of cytomegalovirus immunity against tumors

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Publication number
WO2025199395A1
WO2025199395A1 PCT/US2025/020840 US2025020840W WO2025199395A1 WO 2025199395 A1 WO2025199395 A1 WO 2025199395A1 US 2025020840 W US2025020840 W US 2025020840W WO 2025199395 A1 WO2025199395 A1 WO 2025199395A1
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WIPO (PCT)
Prior art keywords
cancer
hcmv
cells
subject
tumor
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PCT/US2025/020840
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French (fr)
Inventor
Christopher BENEDICT
Remi MARROCCO
Tatiana HURTADO DE MENDOZA
Andrew LOWY
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La Jolla Institute for Allergy and Immunology
University of California Berkeley
University of California San Diego UCSD
Original Assignee
La Jolla Institute for Allergy and Immunology
University of California Berkeley
University of California San Diego UCSD
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Publication of WO2025199395A1 publication Critical patent/WO2025199395A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/852—Pancreas
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This document relates to methods and materials for treating cancer in a subject (e.g., a human) in need thereof.
  • a subject e.g., a human
  • methods and materials provided herein relates to harnessing human cytomegalovirus immunity to induce an immunogenic response to a cancer within a subject.
  • methods for treating a cancer in a subject in need thereof including: (a) exposing isolated PBMCs from the subject to one or more human cytomegalovirus (HCMV) peptide epitopes; (b) determining HCMV T cell reactivity within the isolated PBMCs to one or more HCMV peptide epitopes; and (c) administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells of step (b), thereby inducing an immunogenic response against the tumor.
  • methods further include (a) expanding the HCMV reactive T cells ex vivo; and (b) administering the HCMV reactive T cells to the subject.
  • the one or more human cytomegalovirus (HCMV) peptide epitopes is administered intraperitoneally, intravenously, or subcutaneously.
  • the composition is administered intraperitoneally, intravenously, or subcutaneously.
  • the composition further includes an adjuvant.
  • the adjuvant includes unmethylated cytosine-guanine dinucleotide (CpG), Polyinosinic-Poly cytidylic Acid Stabilized with Polylysine and Carboxymethylcellulose (Poly ICLC). montanide. or combinations thereof.
  • the immunogenic response includes T cell expansion, T cell activation, T cell tumor infiltration, NK cell activation, expansion of y5 T cells, and/or tumor apoptosis.
  • methods provided herein further include determining the expression level of Neutropilin-1 and/or Integrin 05 in the subject in need thereof. In some embodiments, methods provided herein further include administering to the subject a cancer therapy, where the cancer therapy is a chemotherapeutic agent or an immunotherapeutic agent.
  • the chemotherapeutic agent includes a DNA crosslinking agent, an alky lating agent, an anti-metabolite, an antimicrotubule agent, a topoisomerase inhibitor, or a cytotoxic antibiotic.
  • the immunotherapeutic agent is a checkpoint inhibitor, an immunomodulator, a cytokine, a cancer vaccine, a monoclonal antibody, an oncolytic virus, an adoptive cell therapy, a CAR T cell therapy, or combinations thereof.
  • the cancer therapy is co-administered with the composition.
  • the cancer therapy is administered before the administration of the composition.
  • the cancer therapy is administered after the administration of the composition.
  • the subject is a mammal.
  • the cancer is a solid tumor.
  • the solid tumor is a gastric cancer, a breast cancer, a lung cancer, a prostate cancer, a glioma, a melanoma, an ovarian cancer, a liver cancer, a pancreatic cancer, a kidney cancer, a bladder cancer, a thyroid cancer, a sarcoma, a stomach cancer, a head and neck cancer, a cervical cancer, an endometrial cancer, an esophageal cancer, a thymoma, a soft tissue sarcoma, a bone cancer, a testicular cancer, a penile cancer, a gallbladder cancer, a uterine sarcoma, an adrenal gland cancer, an ampullary cancer, a hepatic angiosarcoma, a nasal, or a paranasal sinus cancer.
  • the cancer is a blood cancer.
  • the blood cancer is a leukemia, a lymphoma, a myeloma, a myelodysplastic syndrome, or a myeloproliferative neoplasm.
  • FIGS. 1A-1D MCMV memory T cells can be redirected to fight pancreatic cancer.
  • FIG. 1A Protocol overview. Each treatment consists of an iRGD injection (300pg) followed by injection of 50pg of each MCMV peptide.
  • FIG. 1C H&E and immunofluorescent analysis of the tumors at endpoint looking at apoptosis by cleaved caspase 3 (CC3-Red) and T cell infiltration by CD3 staining. Necrosis score was attributed blindly by a histopathologist.
  • FIG. ID Histology showed no liver damage or immune-mediated toxicity in all the groups (top panel).
  • FIGS. 2A-2C MCMVp therapy resulted in delayed tumor growth with increased survival and can be enhanced with chemotherapy.
  • FIG. 2C B6xl29 Fl hybrid mice were injected with the KPC46 tumor cell line and treated as in B. Normalized mouse weight (middle) and tumor weight at endpoint (right).
  • FIGS. 3A and 3D Representative dot-plots of tetramer binding MCMV-specific CD4 Tconv (FIG. 3 A) and CD8 T cells (FIG. 3B).
  • FIGS. 3B and 3E Mean proportions of MCMV tetramer binding T cells among total CD4 Tconv (FIG. 3B) and CD8 T cells (FIG. 3E).
  • FIGS. 3C and 3F Absolute number of tumor-resident MCMV tetramer-binding CD4 Tconv (FIG. 3C) and CD8 T cells (FIG. 3F). Results were compared by oneway ANOVA with Tukey correction. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • FIG. 4A Representative histograms of m45- specific CD8 T cells isolated from tumors (top) and mean fluorescence intensity' (MFI) quantification (bottom).
  • FIG. 4B Tumors were subjected to CD45+ TIL magnetic separation and the purified cells were incubated with the 6 MCMV peptides for 4 hours, in the presence of GolgiPlug. The graphs show IFNy and TNF production in CD8 or CD4 T conv cells. Results were compared by one-way ANOVA with Tukey correction. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • FIG. 5A UMAP distribution.
  • FIG. 5B Boxplot of cellular proportions in the tumor.
  • FIG. 5C GSEA analysis of epithelial cells DEGs.
  • FIG. 5D CellChat analysis of potential cell-cell interactions in the tumor. Arrowheads indicate the directionality of the interactions.
  • FIGS. 5E and 5F Relative quantification of the different pathways involved in the potential cell-cell interactions in the tumor shown in FIG. 5D. Double arrows indicate bi-directional interactions; simple arrows indicate unidirectional interactions.
  • FIGS. 6A-6G MCMV therapy led to tumor infiltration of activated and cytotoxic MCMV TCR «P cells.
  • FIG. 6A T cell clustering and UMAP distribution.
  • FIG. 6B Feature plot mapping of CD4, CD8 and regulators’ T cells within these clusters and overlay of the 2 conditions (Vehicle, MCMVp).
  • FIG. 6C Graph showing the frequency of each sample in each of the predefined clusters after normalizing the amount of cells per sample FIG.
  • FIG. 6D Dimplot representing the detection of specific MCMV-associated matched CDR3 sequence for TCRa and TCR0.
  • FIG. 6E Proportion of MCMV-specific TCRaP detected per cluster.
  • FIG. 6F Proportion of MCMV-specific TCRP detected per cluster.
  • FIG. 6G Dot plots featuring selected gene-sets and expression levels among clusters. Arrow s point to clusters containing high proportion of MCMV-specific T cells (m45, m38, ml42, m25).
  • FIGS. 7A-7K Vaccine-induced T cells localize to tumors, but had no clinical benefit in the absence of MCMV infection.
  • FIG. 7A KPC1242 infected in vitro with MCMV-mCherry for 48 hours.
  • FIG. 7B IE1 DNA qPCR normalized to actin for KPC1242 tumor cells harvested from uninfected or MCMV infected animals after tumor implantation. Positive controls were spleen and liver from mice 4 post tumor cell injection (dpi).
  • FIG. 7C Protocol for peptide immunization and tumor challenge.
  • FIG. 7D Tumor volume measured by ultrasound over time (left) and end point tumor w eight (right) in the different treatment conditions after immunization (CT-Non immunized, treated with MCMV peptides.
  • CFA Complete Freund's Adjuvant
  • IFA Incomplete Freund’s Adjuvant
  • FIG. 7E Representative m45 -tetramer binding in MCMVp-immunized mice from FIG. 7D is shown.
  • FIG. 7F Representative OT-I-tetramer binding in OVAp- immunized mice from FIG. 7D is shown. Proportions of tetra-OT-I (in CD8 T cells) were quantified in draining lymph node, spleen, and tumor tissues.
  • FIG. 7G Phenotype of tumor-infiltrating m45-specific CD8 T cells compared to m45-tetramer- negative.
  • FIG. 7H Purified CD45+ cells from tumors were incubated with the 6 MCMV peptides, or the 2 OVAp for 4 hours, in the presence of GolgiPlug, and IFNy and TNF production was measured in CD8 and CD4 T conv cells.
  • FIG. 71 The level of GzrnB was measured in CD8 T cells from mice tested in FIG. 7D.
  • FIG. 7 J The level of GzrnB was measured in CD8 T cells from mice tested in FIG. 4.
  • FIG. 7K qPCR detection of UL55 mRNA in human pancreatic tumor tissue from HCMV seronegative and seropositive patients. HUVEC cells infected or not with HCMV were used as a positive control (CT+ and CT-). Results were compared by one-way ANOVA with Tukey correction. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • FIGS. 8A-8C MCMVp treatment induced a marked expansion of virus specific T cells.
  • FIG. 8A Normalized weights of mice shown in FIG. IB over the course of treatment with 50pg of each MCMV peptide epitope (top). Tumor weights in the different treatment groups at end point (18 days post tumor implantation) (bottom).
  • FIG. 8B MCMV -tetramer quantification in the spleen of the mice shown in FIG. IB.
  • FIG. 8C MCMV -tetramer quantification and phenotype of CD8 T cells in tumors of infected or uninfected mice treated with iRGD+MCMVp. Results were compared by Mann-Whitney analysis. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • FIGS. 9A-9F MCMV-specific T cells preferentially localized within tumors in the absence of iRGD.
  • FIG. 9A KPC1242 tumor growth curves.
  • FIG. 9B Growth curve of KPC1242 tumors over time (left) and tumor weight at endpoint (right). Dose B was used in combination or not with iRGD.
  • FIG. 9C Kaplan- Meier survival graph with a similar protocol as shown in FIG. 9A using Dose A.
  • FIGS. 9D-9E MCMV-specific T cell analysis in spleen, liver and tumor at endpoint from mice shown in FIG. 9B.
  • FIG. 9D Percentage of each tetramer among total Tconv (left) or CD8 T cells (right) in the tumor.
  • FIG. 9E Percentage of the 3 dominant MCMV-specific T cells (m25. m38, and m45) in the 3 tissues tested.
  • FIG. 10A Absolute number of CD4 Tconv and CD8 T cell quantification at endpoint in the 3 tissues analyzed.
  • FIGS. 10B-10C Absolute number of MCMV-specific T cells at endpoint in the spleen and liver for Tconv (FIG. 10B) and CD8 T cells (FIG. 10C).
  • FIG. 10D Proportion of MCMV-specific FoxP3+ Tregs among total Tregs in the 3 tissues analyzed. Results were compared by one-way ANOVA with Tukey correction. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • FIG. 11A Representative histograms of the m45-specific CD8 T cells in the liver stained with the indicated markers (top) and mean fluorescence intensity (MFI) quantification (bottom).
  • FIGS. 11B-11C Same analysis for m38-specific CD8 T cells in the liver (FIG. 11B) and tumor (FIG. 11C).
  • FIG. 11A Representative histograms of the m45-specific CD8 T cells in the liver stained with the indicated markers (top) and mean fluorescence intensity (MFI) quantification (bottom).
  • FIGS. 11B-11C Same analysis for m38-specific CD8 T cells in the liver (FIG. 11B) and tumor (FIG. 11C).
  • FIG. 11A Representative histograms of the m45-specific CD
  • FIGS. 12A-12D Tumor infiltration by MCMV-specific T cell was accompanied by changes in the tumor microenvironment (TME). Additional analysis from the data shown in FIG. 5.
  • FIG. 12A Cell type distribution per sample.
  • FIG. 12B GSEA analysis of DEGs in fibroblasts and macrophages.
  • FIG. 12C DEGs in epithelial cells.
  • FIG. 12D Markers used to annotate the cell types from FIG. 5.
  • FIGS. 13A-13D Analysis of tumor infiltrating T cells. Additional analysis from the data shown in FIGS. 6.
  • FIG. 13A T cell clustering and UMAP distribution split by sample conditions.
  • FIG. 13B Dimplot representing the detection of specific MCMV-associated matched CDR3 sequence for TCRp.
  • FIG. 13C Absolute number of cells in each cluster.
  • FIG. 13D Top5 DEGs per cluster.
  • FIGS. 14A and 14B MCMV infection was not necessary for tumor localization of MCMV-specific T cells.
  • FIG. 14A IE1 DNA qPCR normalized with actin in KPC46 tumors harvested from uninfected or MCMV infected B6xl29 Fl hybrid mice. Positive controls were spleen and liver from mice 4 dpi.
  • FIG. 14B Percentage quantification of MCMV and OVA tetramer binding T cells in MCMVp and OVAI (SIINFEKL) immunized mice from the draining lymph node, spleen and tumor. Results were compared by one-way ANOVA with Tukey correction. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • FIGS. 15. Patient serotyping by ELISA. CMV status of PDAC patients are shown. Four out of five sera with patient IDs (121.22. 121.23, 121.25 and 121.26) tested positive for CMV.
  • FIG. 16 Organoids generated from patient’s tumor. Organoids were passaged for up to three generations.
  • FIG. 17 Ultrasound images showing an orthotopically implanted PDAC tissue in NSG mice. The tissue was obtained from a treatment naive PDAC patient. Tumor growth was monitored post tumor implantation.
  • FIG. 18 Dot plots showing the gating strategy' for immune cell identification by flow cytometry to monitor T-cell engraftment.
  • FIGS. 19A-19F Engraftment kinetics of PBMCs from a healthy donor.
  • FIG. 19A Percentage changes in body weight relative to initial mouse weight are shown.
  • FIG. 19B Engraftment ofhCD45+ cells over the course of 6 weeks is shown.
  • FIG. 19C Bar plot showing the percentage of human CD4+ and CD8+ T cells when injected with 10 million PBMCs.
  • FIG. 19D Bar plot showing the percentage of human CD4+ and CD8+ T cells when injected with 5 million PBMCs.
  • FIG. 19E Bar plot show ing the percentage of human CD4+ and CD8+ T cells when injected with 2.5 million PBMCs.
  • FIG. 19F Bar plot showing % cell population of mCD45+ and hCD45+ in spleen from NSG mice engrafted with PBMCs from a healthy human donor.
  • FIGS. 20A-20B Engraftment of PBMCs isolated from a treatment naive PDAC patient.
  • FIG. 20A Percentage changes in body weight relative to initial weight.
  • FIG. 20B Percentage of human CD45+ cells quantified by flow cytometry.
  • FIGS. 21A-21B Addition of a-CD40 further improved the MCMV therapy.
  • B6xl29 Fl hybrid mice were injected with the KPC46 tumor cell line and treated with vehicle or Dose A (50 ,g CD4p/l pg CD8p) alone or in combination with Gemcitabine (Gem) or a-CD40 antibody and compared with Gem or a-CD40 antibody alone.
  • FIGS. 22A-22C B6 mice were injected with the KPC1242 tumor cell line and treated with vehicle or Dose A (50pg CD4p/lpg CD8p) alone or in combination with anti-ILla antibody (FIG. 22A) or gemcitabine (Gem) (FIG. 22B), and compared with either agent alone. Tumor volume was measured over time (top panels) and a Kaplan- Meir Survival curves are shown in the bottom panels.
  • FIG. 23 Schematics illustrating MCMV replication cycle.
  • Immunotherapy has been most successful in treating tumors with high mutational burdens such as melanoma and non-small cell lung cancer (1-4). These mutations result in the generation of numerous neoantigens that the immune system recognizes as “non-self eliciting an anti-tumor immune response that can often be enhanced with immune checkpoint therapy.
  • Many common cancers, such as pancreatic cancer are largely resistant to existing immunotherapies due to a highly immunosuppressive TME (8, 9) and a modest mutational burden (5-7), which results in a paucity of activated T cells that can reach and/or effectively kill cancer cells. Consequently, numerous strategies to enhance anti-tumor immunity through immune checkpoint blockade and/or by modulating the TME have been largely unsuccessful (10-12).
  • cytomegalovirus (CMV) immunity can be re-directed to control a tumor (e.g., pancreatic cancer) that is normally refractor ⁇ ’ to immunotherapy via injection of viral epitopes.
  • a tumor e.g., pancreatic cancer
  • anticancer agents e.g., a chemotherapeutic agent
  • Immunodominant CMV-specific memory' T cells have shown broad tissue residency and an effector memory phenotype in people and mice. Approximately -10% of all circulating CD4 and CD8 T cells are CMV-specific (26, 27). Methods and materials disclosed herein have the potential for development of “off-the-shelf ’ therapeutics by stimulating pre-existing antiviral T cells; and can be widely applicable due to high prevalence of CMV immunity in general population. Using subcutaneous tumor models, it was previously shown that intra-tumoral injection of antiviral T cell peptide epitopes in mice that were previously infected with the virus led to growth arrest and even complete remission (14, 15). However, direct intra-tumoral injection is not always feasible. Methods and materials described herein demonstrate that systemic delivery of epitopes has the potential to be much more applicable to a variety of tumor types.
  • the present methods demonstrate that systemic treatment of cancer with T cell peptide epitopes can efficiently recall CMV memory T cells and result in their tumor localization and induction of an anti-tumor immune response.
  • described herein are methods and materials for harnessing antiviral memory T cells to treat a cancer and/or induce an immunogenic response to a cancer.
  • an immunogenic response to a cancer can be enhanced T cell responses, increased T cell infiltration into the tumor or around the tumor margin, activation of immune cells, T cell expansion, T cell activation, T cell tumor infiltration, NK cell (e.g., NKG2C+ NK cells) expansion, and/or tumor apoptosis, thereby reducing or eliminating (e.g., completely or partially) the tumor and potentially restricting recurrence and/or metastases.
  • activation of immune cells include activation of T cells, natural killer (NK) cells (e.g., NKG2C+ NK cells), dendritic cells, macrophages, B cells, tumor-infiltrating lymphocytes (TILs), neutrophils, eosinophils, basophils, and/or innate lymphoid cells.
  • T cell is a cytotoxic T cell (e.g., CD8+ T cells), a helper T cell (e.g., CD4+ T cell), a regulatory T cell (Tregs), a memory T cell, a gamma delta T cell (y5 T cell), and/or a natural killer T cell (NKT cell).
  • CMV infection induces a huge expansion of T cells, y5 T cells, and/or NK cells (e.g., NKG2C+ NK cells).
  • the subject has pre-existing CMV immunity. In some embodiments, the subject does not have pre-existing CMV immunity.
  • the method can include administering a composition containing one or more human cytomegalovirus (HCMV) peptide epitopes (e.g., a CD4 T cell epitope and/or a CD8 T cell epitope) to the subject.
  • HCMV human cytomegalovirus
  • the method can include inducing cytomegalovirus immunity by vaccinating a subject in need thereof with individual/pooled viral (e.g., CMV) polypeptides or mRNA encoding the CMV polypeptides (e.g., mRNA encoding the whole CMV viral protein or mRNA encoding one or more CMV peptide epitopes described herein).
  • individual/pooled viral e.g., CMV
  • mRNA encoding the CMV polypeptides e.g., mRNA encoding the whole CMV viral protein or mRNA encoding one or more CMV peptide epitopes described herein.
  • the method can include inducing cytomegalovirus immunity by administering a therapeutically effective amount of a composition comprising one or more HCMV peptide epitopes and an adjuvant.
  • one or more refers to one or more copies of the same item.
  • one or more HCMV peptide epitopes can be multiple copies of the same HCMV peptide epitope (e.g., one copy, two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or more copies of the same HCMV peptide epitope).
  • one or more refers to one or more variations of an item.
  • one or more HCMV peptide epitopes can be HCMV peptide epitopes of different sequences (e.g., one, two, three, four, five, six, seven, eight, nine, ten. more different HCMV peptide epitope sequences).
  • CMV immunity can be induced in a subject with no pre-existing CMV immunity by administering to the subject one or more HCMV (e.g.. an attenuated virus) or one or more HCMV peptide epitopes (e.g., a CD4 and/or CD8 T cell epitope).
  • HCMV e.g.. an attenuated virus
  • HCMV peptide epitopes e.g., a CD4 and/or CD8 T cell epitope.
  • methods and materials described herein include determining the expression level of Neutropilin-1 and/or Integrin 05 in the subject in need thereof.
  • the expression level of Neutropilin-1 and/or Integrin 05 is determined before administration of a composition containing one or more HCMV peptide epitopes.
  • one or more HCMV peptide epitopes are a CD4 T cell epitope and/or a CD8 T cell epitope.
  • a CD4 T cell epitope and/or a CD8 T cell epitope can be a 9-15 mer peptide epitope.
  • a 9-15 mer peptide epitope can include 9-15 amino acids.
  • the 9-15 mer peptide epitope includes a core epitope that binds to MHC-I of CD8 T cells.
  • the 9-15 mer peptide epitope binds to MHC-II, thereby- stimulating CD 4 T cells.
  • the 9-15 mer peptide epitope includes a 9 mer core epitope that binds to MHC-I of CD8 T cells and a 10-15 mer that binds to MHC-II to stimulate CD4 T cells.
  • one or more HCMV peptide epitopes can include, consist essentially of, or consist of amino acid sequences set forth in SEQ ID NOS: 1 -422 (Tables 2 and 3).
  • a composition containing one or more HCMV peptide epitopes include a CD4 T cell epitope, a CD8 T cell epitope, or a 15mer peptide.
  • a 15mer peptide includes a CD8 epitope and a CD4 epitope.
  • the composition containing one or more HCMV peptide epitopes can include, consist essentially of, or consist of amino acid sequences set forth in SEQ ID NOS: 1-422 (Tables 2 and 3).
  • the immunogenic response against the tumor can be induced by exposing isolated PBMCs to one or more HCMV peptide epitopes, determining which HCMV T cells of the isolated PBMCs are reactive to one or more HCMV peptide epitopes, and administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells.
  • HCMV peptide epitopes that are reactive to one or more HCMV peptide epitopes can induce a positive reaction or activation of T cells.
  • the immunogenic response against the tumor can be induced by isolating peripheral blood mononuclear cells (PBMCs) from the subject, exposing the isolated PBMCs to one or more HCMV peptide epitopes, determining an HCMV T cell peptide of the isolated PBMCs reactive to one or more HCMV peptide epitopes, and administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells.
  • PBMCs peripheral blood mononuclear cells
  • the immunogenic response against the tumor can be induced by isolating PBMCs from the subject, exposing the isolated PBMCs to one or more HCMV peptide epitopes, determining which HCMV T cells of the isolated PBMCs are reactive to one or more HCMV peptide epitopes, expanding the HCMV reactive T cell ex vivo, administering the HCMV reactive T cells back to the subject; and administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells (e.g.. one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response).
  • the immunogenic response against the tumor can be induced by exposing isolated PBMCs to one or more HCMV peptide epitopes, determining an HCMV T cell of the isolated PBMCs reactive to one or more HCMV peptide epitopes, expanding the HCMV reactive T cell ex vivo, administering the HCMV reactive T cells back to the subject, and administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells (e.g., one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response).
  • determining which HCMV T cells of the isolated PBMCs are reactive to one or more HCMV peptide epitopes described herein include dividing the one or more HCMV peptide epitopes into several pools and then subjecting these pools to T cells (e.g., T cells isolated from a subject in need thereof).
  • T cells e.g., T cells isolated from a subject in need thereof.
  • the HCMV peptide pools described herein can be further divided into subpools based on the reactivity of T cells to the HCMV peptide epitopes described herein until one or more HCMV peptide epitopes are identified that elicit a strong HCMV-specific T cell response.
  • the HCMV reactive T cells are expanded in vitro in the presence of cytokines (e.g., interleukin-2 (IL-2), IL-7, IL- 15, IL-21, IL-17, IL-12, IFNy, TGFp, or any other appropriate cytokines known in the art to induce T cells with specific phenotypes of interest) and one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response.
  • the HCMV reactive T cells are administered back to the subj ect before administering one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response.
  • the HCMV reactive T cells are administered back to the subject after administering one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response. In some embodiments, the HCMV reactive T cells are co-administered with one or more HCMV peptide epitopes that were identified to elicit a strong HCMV- specific T cell response.
  • the immunogenic response against the tumor can be induced by isolating PBMCs from the subject, exposing the isolated PBMCs to one or more human cytomegalovirus (HCMV) peptide epitopes, determining which HCMV T cells of the isolated PBMCs encode specific HLAs reactive to one or more HCMV peptide epitopes, genetically modifying the T cells to express a chimeric antigen receptor (CAR) that targets one or more HCMV peptide epitopes (e.g., one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response), administering the genetically modified T cells back to the subject, and administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells (e.g., one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response).
  • CAR chimeric antigen receptor
  • T cell receptors (TCRs) of the HCMV reactive T cells can be sequenced to generate the genetically modified T cells (e.g., a genetically modified T cell expressing CAR that targets one or more HCMV peptide/HLA complexes that can elicit a strong HCMV- specific T cell response).
  • the genetically modified T cells are administered back to the subject before administering one or more HCMV peptide epitopes recognized by the HCMV reactive T cells (e.g., one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response).
  • the genetically modified T cells are administered back to the subject after administering one or more HCMV peptide epitopes recognized by the HCMV reactive T cells. In some embodiments, the genetically modified T cells are co-administered with one or more HCMV peptide recognized by the HCMV reactive T cells.
  • a CAR (e.g., HCMV peptide-specific CAR) can include an antigen-binding domain (e.g.. HCMV peptide-binding domain), a hinge, a transmembrane domain, and/or one or more signaling domains.
  • antigen-binding include, without limitation, an antigen-binding fragment (Fab), a variable region of an antibody heavy (VH) chain, a variable region of a light (VL) chain, and/or a single chain variable fragment (scFv).
  • an antigen-binding domain of a CAR can target (e.g., can target and bind to) HCMV peptide in a subject (e.g., a human).
  • a CAR can include an optional signal peptide, an antigen binding domain, an optional hinge, a transmembrane domain, and one or more intracellular signaling domains.
  • a CAR can include a hinge region.
  • a hinge region can be located between an antigen-binding domain and a transmembrane domain of a CAR.
  • a hinge region can provide a CAR with increased flexibility for the antigen-binding domain.
  • a hinge region can reduce spatial limitations of an antigen-binding domain of a CAR and its target antigen. Examples of hinge regions that can be used as described herein include, without limitation, a membrane-proximal region from an IgG, a membrane-proximal region from CD8, and a membrane-proximal region from CD28.
  • a CAR can include any appropriate transmembrane domain.
  • a transmembrane domain can be located between an antigen-binding domain and a signaling domain of a CAR and/or located between a hinge and a signaling domain of a CAR.
  • a transmembrane domain can provide structural stability for the CAR (e.g., HCMV-specific CAR).
  • a transmembrane domain can include a structure (e.g., a hydrophobic alpha helix structure) that can span a cell membrane and can anchor the CAR to the plasma membrane.
  • transmembrane domains examples include, without limitation, CD3 ⁇ transmembrane domains, CD4 transmembrane domains, CD8 (e.g., a CD8a) transmembrane domains, CD28 transmembrane domains, CD 16 transmembrane domains, and erythropoietin receptor transmembrane domains.
  • a CAR can include any appropriate signaling domain or combination of signaling domains (e.g., a combination of two, three, or four signaling domains).
  • a signaling domain of a CAR can be an intracellular signaling domain normally found within T cells or NK cells. Examples of signaling domains that can be used as described herein include, without limitation. BB signaling domains, 28 ⁇ signaling domains, CD2 signaling domains, CD3 ⁇ signaling domains, CD28 signaling domains, Toll-like receptor (TLR) signaling domains (e g., TLR3 or TLR4 signaling domains), CD27 intracellular signaling domains. 0X40 (CD134) intracellular signaling domains.
  • TLR Toll-like receptor
  • a CAR for use as described herein can be designed to be a first-generation CAR having a CD3 ⁇ intracellular signaling domain.
  • a CAR for use as described herein can be designed to be a second-generation CAR having a CD28 intracellular signaling domain followed by a CD3 ⁇ intracellular signaling domain.
  • a CAR for use as described herein can be designed to be a third generation CAR having (a) a CD28 intracellular signaling domain followed by (b) a CD27 intracellular signaling domain, an 0X40 intracellular signaling domains, or a 4- IBB intracellular signaling domain followed by (c) a CD3 intracellular signaling domain.
  • the CAR T cell described herein includes one or more suicide gene.
  • a suicide gene can be a nucleic acid sequence introduced to a CAR T cell by standard methods known in the art that, when activated, causes death of the CAR T cell.
  • Suicide genes may facilitate effective tracking and elimination of the CAR T cell in vivo in case of severe toxicities.
  • suitable suicide gene systems include inducible caspase 9 protein or herpes simplex virus thymidine kinase (HSV-tk/ganciclovir (GCV) suicide gene therapy system. Any appropriate method can be used to express a CAR on aT cell.
  • nucleic acid encoding an antigen receptor can be introduced into one or more T cells.
  • viral transduction can be used to introduce nucleic acid encoding an antigen receptor into a non-dividing a cell.
  • Nucleic acid encoding an antigen receptor can be introduced in a T cell using any appropriate method.
  • nucleic acid encoding an antigen can be introduced into aT cell by transduction (e.g., viral transduction using a retroviral vector such as a lentiviral vector) or transfection.
  • nucleic acid encoding an antigen receptor can be introduced ex vivo into one or more T cells.
  • ex vivo engineering of T cells expressing an antigen receptor can include transducing isolated T cells with a lentiviral vector encoding an antigen receptor.
  • the T cells can be obtained from any appropriate source (e.g., a mammal such as the mammal to be treated or a donor mammal, or a cell line).
  • the mammal is a human.
  • the T cell is an allogenic T cell.
  • methods and materials provided herein can be used to reduce the size of a tumor within a subject having a cancer.
  • a subject in need thereof can be administered a therapeutically effective amount of a composition comprising one or more HCMV peptide epitopes, HCMV reactive T cells, T cellrecognizing HCMV peptide epitopes, or a genetically modified CAR-T cell (e.g.. HCMV-specific CAR-T cell) to reduce the size of the cancer in the subject.
  • methods and materials provided herein can be used as described herein to reduce the number of cancer cells in the subject by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • the methods and materials provided herein can be used as described herein to reduce the volume of one or more solid tumors in the subject by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • methods and materials provided herein can be used to improve survival of a subject (e.g., a human) having a cancer described herein.
  • a subject in need thereof can be administered a therapeutically effective amount of a composition comprising one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell (e.g., HCMV-specific CAR-T cell) to improve survival of the subject.
  • methods and materials described herein can be used to improve the survival of a subject having a cancer described herein by, for example. 10. 20. 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • methods and materials described herein can be used to improve the survival of a subject having a cancer described herein by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).
  • at least 6 months e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more.
  • methods and materials provided herein can be used to improve the immunogenic response of a subject (e.g., a human having a cancer) to a cancer described herein.
  • a subject in need thereof can be administered a therapeutically effective amount of a composition comprising one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell (e.g., HCMV-specific CAR-T cell) to improve the immunogenic response of a subject to a cancer described herein.
  • methods and materials described herein can be used to improve the immunogenic response of a subject to a cancer described herein by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell (e.g., HCMV-specific CAR-T cell) described herein can be used as the sole active agent(s) to treat a subject having a cancer described herein.
  • a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein can be administered to a subject having a cancer together with one or more (e.g., one, two, three, four, or more) additional agents used to treat cancer as described herein.
  • additional agents that can be used to treat cancer includes, without limitation, an adjuvant, a tumor targeting agent, or a cancer therapy.
  • an adjuvant is unmethylated cytosine-guanine dinucleotide (CpG), Polyinosinic-Poly cytidylic Acid Stabilized with Polylysine and Carboxymethylcellulose (Poly ICLC), montanide, or combinations thereof.
  • the tumor targeting agent is certepetide.
  • a tumor targeting agent is a polypeptide, a nanoparticle, or a viral particle, each containing the HCMV peptide epitopes described herein.
  • one or more HCMV peptide epitopes is conjugated to a tumor targeting agent (e.g., certepetide) or a cancer therapy.
  • the tumor targeting agent or a cancer therapy can be conjugated to an HMCV peptide via a linker such as a peptide linker.
  • a linker can be a cleavable linker such as an acid cleavable linker, a GSH cleavable linker, a cathepsin cleavable linker, a glycosidase cleavable linker, or a phosphatase cleavable linker.
  • a cancer therapy is a chemotherapeutic agent, an immunotherapeutic agent, surgery, radiation therapy, carbon ion therapy, or proton therapy.
  • a chemotherapy can include a DNA crosslinking agent, an alkylating agent, an anti -metabolite, an anti-microtubule agent, a topoisomerase inhibitor, or a cytotoxic antibiotic.
  • an immunotherapeutic agent can include a checkpoint inhibitor, an immunomodulator, a cytokine, a cancer vaccine, a monoclonal antibody, an oncolytic virus, an adoptive cell therapy, a CAR T cell therapy, or combinations thereof.
  • a cancer therapy can include, without limitation, trametinib, dabrafenib, binimetinib, selumntinib, vemurafenib, encorafenib, cobimetinib, busulfan, cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), liposomal doxorubicin, melphalan, pemetrexed, topotecan.
  • vinorelbine goserelin. leuprolide. tamoxifen, letrozole, anastrozole, exemestane, bevacizumab, olaparib, rucaparib, niraparib, ipilimumab (e.g., YERVOY®), pembrolizumab (e.g., KEYTRUDA®), nivolumab (e.g., OPDIVO®) and any combinations thereof.
  • tamoxifen letrozole
  • anastrozole exemestane
  • bevacizumab olaparib
  • rucaparib e.g., niraparib
  • ipilimumab e.g., YERVOY®
  • pembrolizumab e.g., KEYTRUDA®
  • nivolumab e.g., OPDIVO®
  • methods and materials provided herein can be used to improve the efficacy of additional agents described herein in a subject having a cancer.
  • a subject in need thereof can be administered a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein to improve the efficacy of additional agents in the subject.
  • methods and materials provided herein can be used to improve the efficacy of additional agents by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • methods and materials provided herein for treating a cancer or inducing an immunogenic response in a subject in need thereof can include administering a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cellrecognizing HCMV peptide epitopes, or a genetically modified CAR-T cell to a subject having cancer together with an additional agent/therapy.
  • a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein are used in combination with one or more additional agents used to treat cancer or induce an immunogenic response in a subject in need thereof (e.g., a subject having cancer)
  • the one or more additional agents can be administered at the same time (e.g., in a single composition described herein and the one or more additional agents) or independently.
  • T cellrecognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein can be administered first, and the one or more additional agents administered second, or vice versa.
  • a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein are used in combination with one or more additional agents used to treat cancer or induce an immunogenic response in a subject in need thereof (e.g., a subject having cancer), the one or more additional agents can be performed at the same time or independently of the administration of the composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein.
  • the composition described herein can be administered before, during, or after the one or more additional agents are administered, or in any order.
  • Table 1 Exemplary sequences of HCMV peptide epitopes
  • One or more HCMV peptide epitopes, HCMV reactive T cells, T cellrecognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein can be formulated into a composition (e.g., a pharmaceutical composition) for administration to a subject in need thereof (e.g., a subject having a cancer).
  • a composition e.g., a pharmaceutical composition
  • a subject in need thereof e.g., a subject having a cancer
  • a therapeutically effective amount of one or more HCMV peptide epitopes e.g., one or more HCMV peptide epitopes or a composition comprising a HCMV peptide
  • HCMV reactive T cells e.g., T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein
  • a pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sterile aqueous or non-aqueous solutions, suspensions, emulsions, and the like, compatible with pharmaceutical administration.
  • non-aqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and organic esters.
  • Aqueous carriers include, without limitation, water, alcohol, saline, and buffered solutions. Acceptable carriers also can include physiologically acceptable aqueous vehicles (e.g., physiological saline) or other known carriers for oral administration.
  • Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration.
  • routes of administration can include oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or inhaled administration.
  • administering includes oral administration, injection, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intratumoral injection, inhalation, or any combinations thereof.
  • the pharmaceutical composition provided herein suitable for parenteral administration can include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
  • the pharmaceutical composition provided herein can be administered locally or systemically.
  • a composition provided herein can be administered locally by intratumoral administration (e.g., injection into tumors) or by administration into biological spaces infiltrated by tumors (e.g. intraspinal administration, intracerebellar administration, intraperitoneal administration and/or pleural administration).
  • the pharmaceutical composition provided herein can be administered systemically by intravenous administration (e.g., injection or infusion) to a mammal (e.g., a human).
  • one or more HCMV peptide epitopes e.g., one or more HCMV peptide epitopes or a composition comprising a HCMV peptide
  • HCMV reactive T cells e.g., T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein when formulated as a pharmaceutical composition
  • the pharmaceutical composition can be in the form of a liquid, solution, suspension, tablet, powder, granule, pill, capsule, gel, cream, mist, atomized vapor, aerosol, soft gelatin capsule, or hard gelatin capsule.
  • tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents.
  • the tablets can be coated by methods known in the art.
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspension, or they can be presented as a dry product for constitution with saline or other suitable liquid vehicle before use.
  • Liquid preparations also can contain pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles, preservatives, buffer salts, flavoring agents, coloring agents, and sweetening agents as appropriate.
  • Preparations for oral administration can be suitably formulated to give controlled release of the compound.
  • the pharmaceutical composition e.g., an antigen-binding molecule and/or a bispecific-binding molecule
  • Treating cancer or inducing an immunogenic response in a subject in need thereof include administering a therapeutically effective amount of the pharmaceutical composition described herein.
  • a “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results.
  • an effective amount is one that achieves a desired therapeutic effect, e.g., an amount necessary to treat a disease, or to reduce risk of development of disease or disease symptoms (also referred to as a prophylactically effective amount).
  • An effective amount can be administered in one or more administrations, applications, or dosages.
  • a therapeutically effective amount of a therapeutic compound i.e., an effective dosage) depends on the therapeutic compounds selected.
  • a therapeutically effective amount of a pharmaceutical composition as provided herein can be effective to reduce tumor size (e.g., size of a primary 7 tumor or a metastatic tumor), improve survival, or improve immunogenic response.
  • a therapeutically effective amount of a pharmaceutical composition as provided herein can be effective to improve at least one symptom of cancer or complications related to cancer.
  • the pharmaceutical composition provided herein can be administered one or more times per year (e.g., one time per year, two times per year, three times per year, four times per year, or five times per year) to one or more times per month (e.g., one time per month, two times per month, three times per month, four times per month, five times per month), including once every other month, once every 7 three months, or twice a month.
  • the pharmaceutical composition provided herein can be administered one or more times per week (e.g., one time per week, two times per week, three times per week, four times per week, five times per week, six times per week, seven times per w eek, or more than seven times per week).
  • treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.
  • Various factors can influence the actual amount used for a particular application. For example, the frequency of administration. duration of treatment, combination of other agents, site of administration, stage of disease (if present), and the anatomical configuration of the treated area may require an increase or decrease in the actual amount administered.
  • An effective duration for administering a pharmaceutical composition provided herein can be any duration that reduces the severity 7 of cancer, reduce tumor size (e.g., size of a primary tumor or a metastatic tumor), improve survival, or improve immunogenic response in a subject without producing significant toxicity to the subject.
  • the effective duration can vary from several days to several weeks, to several months, or longer. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the effective amount, frequency of administration, use of route of administration, and severity of the subject’s condition.
  • Example 1 Redirecting cytomegalovirus (CMV) immunity against pancreatic cancer
  • This Example demonstrates that repurposing CMV-specific T cells can combat pancreatic cancer.
  • Wild-type (WT) C57BL/6J (B6) mice and C57BL/6J x 129Sl/SVlmJ (129) Fl hybrids (B6129SF1/J, Strain #: 101043) were purchased from Jackson laboratories at 4-5 weeks of age, and infected or not with MCMV at 5-6 weeks of age. This study was carried out in strict accordance with the guidelines of Association for assessment and Accreditation of laboratory Animal Care (AAALAC) and the National Institutes of Health (NIH).
  • MCMV smith strain
  • MCMV infected mice (2-3 months post infection) and age matched uninfected controls were orthotopically injected with KPC1242 (syngeneic with B6 mice) or KPC46 (syngeneic with B6/129 Fl hybrid mice) pancreatic cancer cells. These cells were cultured in vitro in RPMI with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. Once confluent, they were trypsimzed. washed, and resuspended in Matrigel® (Coming Matrigel Matrix) for injection (5,000 KPC1242 or KPC46 tumor cells in 20pLof matrigel, per mouse).
  • FBS Fetal Bovine Serum
  • Matrigel® Coming Matrigel Matrix
  • mice were subjected to ultrasound (Convex L20 HD3. Clarius) twice a week (under isofluorane) for tumor growth monitoring. Tumor volume was calculated using the ellipsoid volume calculation formula. In addition, mice were weighed twice a week together with health checks. Mice were enrolled in treatment studies and randomly assigned to different treatment conditions once most tumors were visible by ultrasound, usually around 9 to 11 days post tumor cell injection. For survival studies, the euthanasia criteria included mice loosing 20% or more of their initial weight or having tumors reaching 2000mm 3 or more plus any evident signs of distress.
  • MCMV peptides (CD4T: m09133-147; m25409-423; ml4224-38; CD8T: IE3416-423; m38316-323; m45985-993) were purchased from Mimotopes (Australia), and ovalbumin peptides (OVA257-264; OVA323-339) were from Eurogentec-Anaspec, CA, USA. Table 3 lists the amino acid sequences of the MCMV peptides described herein.
  • mice were treated twice a week by retro-orbital (RO) injections of the indicated peptides (50pg of m09133-147; m25409-423; ml4224-38 or OVA323-339; Ipg of IE3416-423; m3831 -323; m45985-993 or OVA257-264, unless indicated otherwise) and iRGD (300pg).
  • RO retro-orbital
  • mice were first injected with iRGD in one eye followed by the mix of MCMV peptides 5 minutes later in the other eye.
  • mice were intraperitoneally injected with anti-PDl (lOmg/kg twice a week. Bioxcell) or anti-ILlOR (200pg/qL, once a week, Bioxcell) antibodies, or gemcitabine (5mg/kg, twice a week, Selleck Chem).
  • Control mice were retro-orbitally injected with vehicle (PBS/DMSO equivalent to the amount in the MCMV peptide mix) and saline, or intraperitoneally injected with rat IgG controls for anti-PDl and anti-ILlOR blocking antibodies.
  • mice 8-week-old mice were immunized subcutaneously at the base of the tail with 200pL of an emulsion containing 50pg of each indicated peptide (MCMVp or OVAp) and CFA (0.5mg/mL final; Sigma).
  • Booster immunizations containing peptides + IFA were performed 3 weeks later. Mice were tumor challenged 3 weeks later as described herein.
  • FFPE formalin-fixed paraffin-embedded
  • Histological and immunofluorescence imaging were performed using a ZEISS AxioScan Z1 automated slide scanning microscope equipped with a 20x objective (NA 0.8).
  • an LED illumination system was employed in conjunction with a Hitachi HV-FL202SCL camera.
  • Immunofluorescence imaging was conducted using a Colibri7 LED illumination system and ZEISS single-band filter sets (filter sets 49, 38, 43, and 50). Fluorescence images were captured using a Hamamatsu Orca Flash 4.0 v2 camera operating in 16-bit mode (Microscopy core, LJI). Staining was quantified using the Qpath software. Hematoxilyn and Eosin staining was performed and necrosis scores w ere assessed by a histopathologist blinded to the different treatment conditions.
  • Tumors were minced into small pieces and incubated at 37°C with rotation for 30 minutes in 5 mL of digestion buffer consisting of DMEM high glucose, 10% Gentle Collagenase/Hyaluronidase (GCH, STEM CELL), 10% FBS, and 10% DNasel (1 mg/mL stock, Roche). Samples were then filtered using a 70-pm filter and smashed with the back of a syringe while adding 5 mL of 2% FBS in PBS solution. Flow through was spun at 300g for 10 minutes and subjected to RBC lysis buffer(Pharmalyse, BD. Biosciences).
  • NIH-provided biotinylated monomers were tetramerized using streptavidin-APC or -PE, and subsequently used to stain the cells. Briefly, tetramer staining was performed at room temperature for 1.5 to 2 hours. Live/dead and surface/intracellular staining were done subsequently. Data was analyzed using the FlowJo software.
  • CD45+ cells from the tumors were incubated for 4 hours at 37°C and 5% CO2 in 96- well plates in the presence of the indicated peptides (5pg/mL for each peptide) and GolgiPlug (Greeneldin A; eBioscience). Control cells were incubated in the presence of GolgiPlug alone. Cells were then stained as described herein.
  • CellChat (v 2.1.2) (Jin et al., 2024) was used to perform CellChat analysis. Filtering was further performed to only include communications found in at least 10 cells and for pathways that were expressed in a minimum of 2 samples.
  • T cell receptor (TCR) repertoire analysis was integrated with transcriptomic data using the SCrepertoire R package.
  • the Seurat object was employed to organize both transcriptomic and clonotype metadata.
  • TCR diversity’ was characterized through clonotype expansion analysis, V(D)J gene usage patterns, and CDR3 length distributions. Functions highlightClonesQ and combineExpressionQ were employed to map clonotype distributions onto UMAP projections.
  • Results MCM V memory T cells can be redirected to fight pancreatic cancer
  • mice To induce an MCMV memory T cell pool, 4-5 weeks old C57bl6/J (B6) mice were infected and waited a minimum of 2 months to allow the establishment of latency, defined by maintenance of the viral genome without detectable lytic replication. MCMV -infected mice and non-infected control mice were then surgically implanted with KPC1242 tumor cells in their pancreas.
  • iRGD or MCMVp alone had no beneficial effect, but surprisingly MCMVp alone had the same effect on T cell infiltration, tumor growth, and survival despite the omission of iRGD (FIGS. 9B-9D).
  • MCMV T cells expanded following treatment, and preferentially localize to the tumor
  • MCMV-specific T cells were identified using both MHC-I and -II tetramers labeled in both APC and PE, and only double-positive cells were analyzed (FIGS. 3 A and D).
  • MCMV-specific CD4 Tconv cells were detected exclusively in tumors from infected mice treated the MCMVp therapy.
  • the spleen contained a low proportion of MCMV T cells, while the liver and tumor had higher and more comparable levels.
  • the dominant epitope in the liver was ml42, while tumors were more enriched with m25 specific T cells (FIGS. 3A and 3B). Quantification of the absolute number of m09, m25 and ml 42 specific Tconv cells in tumors (FIG.
  • FIG. 10B livers and spleens
  • FIG. 10D shows that in vivo expansion of MCMV-specific CD4 T cells resulted in the differentiation of a bonified antiviral effector CD4 T cell rather than an immunoregulatory one.
  • m38 CD8 T cells (and to a lesser extent IE3) were detectable in tumors from infected mice, even in the absence of MCMVp therapy while m45 remained low, as compared to uninfected mice where virtually no MCMV T cells were detected (FIG. 3E). However, this did not translate to a significantly higher absolute number of these T cells in the tumor (FIG. 3F). This slight tumor infiltration of IE3 and m38 T cells in the absence of MCMVp therapy may be due to the effector memory phenotype and “inflationary ” nature of these memory' T cells that increase in numbers over time, while m45 cells show a central memory phenotype and do not inflate.
  • M45-specific CD8 T cells also showed a very similar phenotype in the liver, suggesting a conserved phenoty pe in multiple tissues (FIG. 11A). While PD1, LAG3 and TIM3 are often called “exhaustion markers”, they don’t define functional exhaustion as they are also upregulated early during T cell activation to dampen excessive inflammation (31). Consistently, a strong expression of activation markers and cytotoxic molecules in T cells expressing these immune checkpoint molecules was observed, arguing against an exhausted state.
  • MCMV -specific T cells also established tumor residence in infected mice that did not receive MCMVp therapy, as shown by tetramer binding (FIG. 3E) and cytokine production (FIG. 4B). MCMVp therapy further increased both IFNy and TNF production by these TILs. This enhanced IFNy was most evident in the MCMVp+Gem group, correlating with greater tumor growth control, and the same was true for IFNy production by CD4 Tconv in MCMVp+Gem treated mice (FIG. 4B).
  • m45-specific CD8 T cells showed conventional early expansion/contraction kinetics and displayed a central memory T cell phenotype. Conversely, m38- specific CD8 T cells displayed unique kinetics. They underwent early expansion paralleling m45, but only weakly contracted and continued to “inflate” in numbers over time displaying an effector- memory T cell phenotype.
  • tumor resident m38 T cells downregulated GzmA expression after MCMVp therapy, but upregulated GzmB (FIG. 11C).
  • This downregulation of GzmA may 7 result from the TME, but, importantly, did not prevent the beneficial effect of MCMVp therapy.
  • scRNAseq was performed on whole tumors from MCMV infected mice treated with vehicle or MCMVp (FIG. 5A).
  • the major tumor cell populations were epithelial cells, macrophages, T cells, fibroblasts, and dendritic cells.
  • FIGS. 5A, B, and 12A the most significant change in the MCMVp therapy group was a marked increase in T cells (0.27%Veh vs 5.7%MCMVp).
  • the KRAS signaling pathway was also decreased in all three cell types, a pathway which is highly active in KPC tumor cells. In epithelial cells, a notable difference was decreased collagen and increased MFIC-I expression (FIG. 12C), two factors that would be consistent with an enhanced anti-tumor immune response.
  • TGF- P dependent signaling netw orks also decreased betw een T cells, epithelial cells and fibroblasts (FIGS. 5E and 5F).
  • MCMVp therapy increased potential FasLg interactions between T cells and fibroblasts, but not between T cells and epithelial cells.
  • fibroblast-epithelial cell interactions involving grow th factors (FGF, VEGF, EGF) were reduced following MCMVp therapy, which would be consistent with a better prognosis (FIG. 5F).
  • cluster 1 Up to 50% of cluster 1 was composed of MCMV-specific CD8 T cells, half of that population expressed m45-specific CDR3, and the other half expressed CDR3 ascribed to both m45/m38 (FIG. 6F). Since cluster 1 was the second most abundant cluster (FIG. 13C), it was concluded that MCMV-specific CD8 T cells constituted the majority of the tumor infiltrating T cells.
  • All 20 clusters were analyzed for the expression of key transcription factors, cytotoxic molecules, cytokines and co-signaling receptors that regulate T cell effector function (FIG. 6G). All clusters induced by the MCMVp therapy expressed high levels of Tbx21 (T-bet) and 7/hy. Cluster 1 expressed very high levels oiPrfl, Gzmh. Gzmk and Fasl, indicative of high cytotoxic potential. As seen by flow cytometry (FIGS. 4 and 11), elevated expression of co-inhibitory receptors was observed in cluster 1 (Pdcdl. Haver 2. Lag3), as well as Ctla4 and Tigit. However, enhanced levels of several costimulatory receptors were also observed (Cd226.
  • cluster 7 which contained the highest numbers of m25 and ml 42 CD4 Tconv, had the most IJhy expression of any cluster, and also showed high expression of costimulatory receptors Cd28, Tnfrsf4 (0X40), Tnfrsfl8 (GITR), and Cd4()lg. which was consistent with a strong effector phenotype.
  • the top 5 differentially expressed genes in cluster 1 were Haver 2 (TIM3), Pdcdl (PD1), Rgsl6 ⁇ I.agS, and Cxcr .
  • Vaccine-induced T cells localized to tumors, but had no clinical benefit in the absence of MCMV infection
  • Example 2 Testing the effects of CMV peptide epitopes in humanized pancreatic cancer mouse models
  • GvHD Graft Versus Host Disease
  • NSG Node B scid gamma mice
  • NSG-MHC I/II DKO mice were retro-orbitally injected with a patient’s PBMCs for T-cell engraftment.
  • GvHD can kill the animal within 3-5 weeks post PBMC inoculation, particularly in NSG mice.
  • NSG MHCI/II DKO mice show delayed GvHD and may be more suitable for these experiments.
  • mice were then engrafted with patient’s human pancreatic tumor tissue or cells, and w ere challenged with IV injection of a human CMV peptide pool or specific HCMV peptide epitopes (previously selected based on blood reactivity) to elicit a CMV- specific T-cell killing of the tumor.
  • Tumor models were generated using pancreatic cancer tissues obtained from both CMV-positive and -negative patients with available PBMC samples or PBMCs obtained in serial collections. Tumor characteristics and grow th kinetics w ere evaluated to optimize timing and determine inter-patient tumor variability.
  • CMV+/- tumors were orthotopically implanted into corresponding humanized mice generated using the patient's PBMC. Mice were treated twice a week with CMV-peptide epitopes to elicit CMV-specific T-cell killing of the tumor.
  • PBMCs were isolated using two protocols, SepMate and Easy Sep.
  • the PBMCs were isolated from whole blood obtained from a healthy donor. Table 5 show s the cell counts and viabilities obtained from the tw o protocols.
  • the Easy Sep protocol yielded the highest live cell count, about 9 million more cells in the same total volume of cells, and a viability of about 77 %.
  • the SepMate had a lower cell count with a 10 % decrease in cell viability. Therefore, the Easy Sep protocol was used all future PBMC isolations.
  • the Cry oStor and the Cell- Vive storage solutions yielded 20 million total cells with 57 % and 69 % viabilities, respectively.
  • the thawed PBMCs were mixed and injected into MHC I/II DKO mice for engraftment.
  • the DKO mice were monitored for T-cell engraftment.
  • ELISA was performed on serum samples obtained from the blood of PDAC patients. The analysis showed a high prevalence of CMV IgG present in the patient population (FIG. 15).
  • PBMC has been isolated from the same patient (Patient ID 121.26) for engraftment in both NSG and NSG-MHC I/II DKO mice.
  • Tumor sample was obtained from the patient with ID 121.26. Organoids were generated and expanded from the tumor tissues (Figure 16). These organoids can be orthotopically injected into the pancreas of an NSG mouse to develop into tumors.
  • PBMCs isolated from the CMV+ patient were engrafted in NSG mice and NSG MHCI/II DKO mice (FIGS. 20A and 20B).
  • the patient’s PBMCs were much slower to engraft, with the highest engraftment being 4 % hCD45+ cells.
  • T-cell engraftment kinetics in both mice models were monitored as well as the time it took for the mice to develop GvHD.
  • Virus-specific memory T cells populate tumors and can be repurposed for tumor immunotherapy. Nat Commun 10: 567. Quburu, N., L. Bialkowski. S. M. Pontejo. S. K. Sethi. A. T. F. Bell, R. Kim, C. D. Thompson, D. R. Lowy, and J. T. Schiller. 2022.
  • CD8 + exhausted T cells promotes an immunosuppressive environment in gastric metastatic cancer. J Transl Med 22: 158.
  • Cytomegalovirus promotes murine glioblastoma growth via pericyte recruitment and angiogenesis. Journal of Clinical Investigation 129: 1671-1683.
  • Macropinocytosis the Achilles’ heel of pancreatic cancer? Scandinavian Journal of Gastroenterology 56: 177-179.

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Abstract

Provided herein are methods and materials for treating cancer in a subject (e.g., a human) in need thereof. For example, provided herein are methods and materials for harnessing human cytomegalovirus immunity to induce an immunogenic response to a cancer within a subject.

Description

USE OF CYTOMEGALOVIRUS IMMUNITY AGAINST TUMORS
CLAIM OF PRIORITY
This application claims the benefit of U.S. Provisional Application Serial No. 63/640,255, filed on April 30, 2024, and U.S. Provisional Application Serial No. 63/568,619, filed on March 22, 2024. The entire contents of the foregoing are incorporated herein by reference.
SEQUENCE LISTING
This instant application contains a Sequence Listing that has been submitted electronically as an XML file named 15670-0401W01_SL_ST26.xml. The XML file, created on March 19, 2025, is 374,719 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. CA286198, AI139749, and AI101423 awarded by the National Institutes of Health. The Government has certain rights in the invention.
ADDITIONAL FUNDING
Research for this invention was supported by funding from Foundation For A Better World under Grant No. FFBW#001.
TECHNICAL FIELD
This document relates to methods and materials for treating cancer in a subject (e.g., a human) in need thereof. For example, methods and materials provided herein relates to harnessing human cytomegalovirus immunity to induce an immunogenic response to a cancer within a subject.
BACKGROUND
Immunotherapy has become the standard of care treatment for malignancies such as lung cancer and melanoma. These cancer types have high mutational burden and T-cell infiltration, culminating in better success rates for immunotherapy. However, many common cancers such as pancreatic cancer are characterized by low mutational burden and a high immunosuppressive microenvironment, making immunotherapy very challenging. Numerous strategies to enhance immune responses by using immune checkpoint inhibitors and/or by modulating the tumor microenvironment have been unsuccessful in these cancers. Personalized therapies where tumor biopsies are sequenced for the emergence of potential neoantigens followed by vaccination are currently being tested. However, the process of neoantigen prediction is complex, very expensive, and time consuming. Thus, there is need for additional therapies for treating cancer.
SUMMARY
This document provides methods and materials for treating cancer in a subject in need thereof. For example, this document provides methods and materials for harnessing human cytomegalovirus immunity to induce an immunogenic response to a cancer within a subj ect.
Provided herein are methods for treating a cancer in a subject or inducing an immunogenic response to a cancer in a subject having pre-existing cytomegalovirus (CMV) immunity, the method including administering a therapeutically effective amount of a composition including one or more of human cytomegalovirus (HCMV) peptide epitopes to the subject having pre-existing cytomegalovirus immunity. Also provided herein are methods for treating a cancer in a subject or inducing an immunogenic response to a cancer in a subject with no pre-existing cytomegalovirus (CMV) immunity, the method including: (a) inducing cytomegalovirus immunity in the subject with no pre-existing cytomegalovirus immunity; (b) administering a therapeutically effective amount of a composition including one or more human cytomegalovirus (HCMV) peptide epitopes to the subject, thereby treating the cancer in the subject.
Also provided herein are methods for treating a cancer in a subject in need thereof, the method including: (a) exposing isolated PBMCs from the subject to one or more human cytomegalovirus (HCMV) peptide epitopes; (b) determining HCMV T cell reactivity within the isolated PBMCs to one or more HCMV peptide epitopes; and (c) administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells of step (b), thereby inducing an immunogenic response against the tumor. In some embodiments, methods further include (a) expanding the HCMV reactive T cells ex vivo; and (b) administering the HCMV reactive T cells to the subject.
Also provided herein are methods for treating a cancer in a subject in need thereof, the method including: (a) exposing the isolated PBMCs from the subject to one or more human cytomegalovirus (HCMV) peptide epitopes; (b) determining which HCMV T cells of the isolated PBMCs are reactive to one or more HCMV peptide epitopes; (c) genetically modifying T cells to express a chimeric antigen receptor (CAR) that targets one or more HCMV peptide epitopes of step (b); (d) administering the genetically modified T cells of step (c) to the subject; and (e) administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells of step (b), thereby inducing an immunogenic response against the tumor.
In some embodiments, inducing cytomegalovirus immunity to a subject with no pre-existing cytomegalovirus immunity includes administering to the subject one or more human cytomegalovirus, one or more human cytomegalovirus (HCMV) peptide epitopes, or one or more mRNA molecules encoding one or more HCMV polypeptides. In some embodiments, the one or more human cytomegalovirus includes an attenuated virus.
In some embodiments, the one or more human cytomegalovirus (HCMV) peptide epitopes include a 9-15mer peptide. In some embodiments, the composition including one or more human cytomegalovirus (HCMV) peptide epitopes includes a 9-15mer peptide. In some embodiments, the 9-15mer peptide includes a CD8 T cell epitope and/or a CD4 T cell epitope. In some embodiments, the one or more human cytomegalovirus (HCMV) peptide epitopes include one or more amino acid sequences set forth in SEQ ID NOS: 1-422. In some embodiments, the composition including one or more HCMV peptide epitopes include one or more amino acid sequences set forth in SEQ ID NOS: 1-422.
In some embodiments, the one or more human cytomegalovirus (HCMV) peptide epitopes is administered intraperitoneally, intravenously, or subcutaneously. In some embodiments, the composition is administered intraperitoneally, intravenously, or subcutaneously.
In some embodiments, the composition further includes an adjuvant. In some embodiments, the adjuvant includes unmethylated cytosine-guanine dinucleotide (CpG), Polyinosinic-Poly cytidylic Acid Stabilized with Polylysine and Carboxymethylcellulose (Poly ICLC). montanide. or combinations thereof.
In some embodiments, methods include administering a tumor targeting agent. In some embodiments, the tumor targeting agent includes certepetide. In some embodiments, the tumor targeting agent includes a polypeptide, a nanoparticle, or a viral particle. In some embodiments, the tumor targeting agent is co-administered with the composition. In some embodiments, tumor targeting agent is administered before the administration of the composition. In some embodiments, the composition is conjugated to a tumor targeting agent or a cancer therapy.
In some embodiments, the immunogenic response includes T cell expansion, T cell activation, T cell tumor infiltration, NK cell activation, expansion of y5 T cells, and/or tumor apoptosis.
In some embodiments, methods provided herein further include determining the expression level of Neutropilin-1 and/or Integrin 05 in the subject in need thereof. In some embodiments, methods provided herein further include administering to the subject a cancer therapy, where the cancer therapy is a chemotherapeutic agent or an immunotherapeutic agent. In some embodiments, the chemotherapeutic agent includes a DNA crosslinking agent, an alky lating agent, an anti-metabolite, an antimicrotubule agent, a topoisomerase inhibitor, or a cytotoxic antibiotic. In some embodiments, the immunotherapeutic agent is a checkpoint inhibitor, an immunomodulator, a cytokine, a cancer vaccine, a monoclonal antibody, an oncolytic virus, an adoptive cell therapy, a CAR T cell therapy, or combinations thereof. In some embodiments, the cancer therapy is co-administered with the composition. In some embodiments, the cancer therapy is administered before the administration of the composition. In some embodiments, the cancer therapy is administered after the administration of the composition.
In some embodiments, the subject is a mammal.
In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is a gastric cancer, a breast cancer, a lung cancer, a prostate cancer, a glioma, a melanoma, an ovarian cancer, a liver cancer, a pancreatic cancer, a kidney cancer, a bladder cancer, a thyroid cancer, a sarcoma, a stomach cancer, a head and neck cancer, a cervical cancer, an endometrial cancer, an esophageal cancer, a thymoma, a soft tissue sarcoma, a bone cancer, a testicular cancer, a penile cancer, a gallbladder cancer, a uterine sarcoma, an adrenal gland cancer, an ampullary cancer, a hepatic angiosarcoma, a nasal, or a paranasal sinus cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is a leukemia, a lymphoma, a myeloma, a myelodysplastic syndrome, or a myeloproliferative neoplasm.
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.
DESCRIPTION OF DRAWINGS
FIGS. 1A-1D. MCMV memory T cells can be redirected to fight pancreatic cancer. FIG. 1A: Protocol overview. Each treatment consists of an iRGD injection (300pg) followed by injection of 50pg of each MCMV peptide. FIG. IB: KPC1242 tumor growth over time, monitored by ultrasound. (n=4-5/group). FIG. 1C: H&E and immunofluorescent analysis of the tumors at endpoint looking at apoptosis by cleaved caspase 3 (CC3-Red) and T cell infiltration by CD3 staining. Necrosis score was attributed blindly by a histopathologist. FIG. ID: Histology showed no liver damage or immune-mediated toxicity in all the groups (top panel). Graphs showing the quantification of immunofluorescent staining of liver sections with CD3 and cleaved caspase 3 to measure T cell infiltration and apoptosis (bottom panel). Results were compared by one-way ANOVA with Tukey correction, and repeated measures by two-way ANOVA with Sidak correction. *P < 0.05; **P < 0.01; ***p < 0.001; ****P < 0.0001.
FIGS. 2A-2C. MCMVp therapy resulted in delayed tumor growth with increased survival and can be enhanced with chemotherapy. FIG. 2A: KPC1242 tumor growth curves using Dose A (50pg CD4p/lpg CD8p). Normalized mouse weight (middle) and Kaplan-Meier survival graph (right). (n=5-8/group). FIG. 2B: Dose A was used in combination or not with gemcitabine (Gem). Normalized mouse weight (middle) and tumor weight at endpoint (right). (n=5-10/group). FIG. 2C: B6xl29 Fl hybrid mice were injected with the KPC46 tumor cell line and treated as in B. Normalized mouse weight (middle) and tumor weight at endpoint (right). (n=5- 7/group). Results were compared by one-way ANOVA with Tukey correction, and repeated measures by two-way ANOVA with Sidak correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****p < 0.0001.
FIGS. 3A-3F. MCMV T cells expanded after MCMVp therapy and preferentially localize to the tumor. Mice were implanted with KPC 1242 cells and treated as described in FIG. 2B. At day 25 post injection, spleen, liver, and tumors tissues were collected to analyze TILs by flow cytometry. (n=5/group). FIGS. 3A and 3D: Representative dot-plots of tetramer binding MCMV-specific CD4 Tconv (FIG. 3 A) and CD8 T cells (FIG. 3B). FIGS. 3B and 3E: Mean proportions of MCMV tetramer binding T cells among total CD4 Tconv (FIG. 3B) and CD8 T cells (FIG. 3E). FIGS. 3C and 3F: Absolute number of tumor-resident MCMV tetramer-binding CD4 Tconv (FIG. 3C) and CD8 T cells (FIG. 3F). Results were compared by oneway ANOVA with Tukey correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
FIGS. 4A and 4B. MCMV T cells were highly activated and cytotoxic within tumors. Immunophenotyping and cytokine production analysis of the TILs described in FIG. 3. (n=5/group). FIG. 4A: Representative histograms of m45- specific CD8 T cells isolated from tumors (top) and mean fluorescence intensity' (MFI) quantification (bottom). FIG. 4B: Tumors were subjected to CD45+ TIL magnetic separation and the purified cells were incubated with the 6 MCMV peptides for 4 hours, in the presence of GolgiPlug. The graphs show IFNy and TNF production in CD8 or CD4 T conv cells. Results were compared by one-way ANOVA with Tukey correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
FIGS. 5A-5F. MCMVp treatment induced profound changes in the tumor microenvironment. Infected mice bearing tumors were treated with vehicle or MCMVp as previously described, tumors were collected and used for 5’scRNAseq (n=3/group, each sample being the pool of 1-2 mice). FIG. 5A: UMAP distribution. FIG. 5B: Boxplot of cellular proportions in the tumor. FIG. 5C: GSEA analysis of epithelial cells DEGs. FIG. 5D: CellChat analysis of potential cell-cell interactions in the tumor. Arrowheads indicate the directionality of the interactions. FIGS. 5E and 5F: Relative quantification of the different pathways involved in the potential cell-cell interactions in the tumor shown in FIG. 5D. Double arrows indicate bi-directional interactions; simple arrows indicate unidirectional interactions.
FIGS. 6A-6G. MCMV therapy led to tumor infiltration of activated and cytotoxic MCMV TCR«P cells. A different cohort of mice treated as in FIG. 5 was used to purify T cells by FACS sorting for CD3, CD4 and CD8 (n=3/group, each sample being the pool of 1-2 mice). The purified T cells were used for 5’scRNAseq and TCR sequencing. FIG. 6A: T cell clustering and UMAP distribution. FIG. 6B: Feature plot mapping of CD4, CD8 and regulators’ T cells within these clusters and overlay of the 2 conditions (Vehicle, MCMVp). FIG. 6C: Graph showing the frequency of each sample in each of the predefined clusters after normalizing the amount of cells per sample FIG. 6D: Dimplot representing the detection of specific MCMV-associated matched CDR3 sequence for TCRa and TCR0. FIG. 6E: Proportion of MCMV-specific TCRaP detected per cluster. FIG. 6F: Proportion of MCMV-specific TCRP detected per cluster. FIG. 6G: Dot plots featuring selected gene-sets and expression levels among clusters. Arrow s point to clusters containing high proportion of MCMV-specific T cells (m45, m38, ml42, m25).
FIGS. 7A-7K. Vaccine-induced T cells localize to tumors, but had no clinical benefit in the absence of MCMV infection. FIG. 7A: KPC1242 infected in vitro with MCMV-mCherry for 48 hours. FIG. 7B: IE1 DNA qPCR normalized to actin for KPC1242 tumor cells harvested from uninfected or MCMV infected animals after tumor implantation. Positive controls were spleen and liver from mice 4 post tumor cell injection (dpi). FIG. 7C: Protocol for peptide immunization and tumor challenge. FIG. 7D: Tumor volume measured by ultrasound over time (left) and end point tumor w eight (right) in the different treatment conditions after immunization (CT-Non immunized, treated with MCMV peptides. PBS- Immunized with Complete Freund's Adjuvant (CFA) / Incomplete Freund’s Adjuvant (IFA) no peptides, treated with MCMV peptides, OVAp-Immunized and treated with OVA-I and OVA-II and MCMVp-Immunized and treated with MCMV peptides). FIG. 7E: Representative m45 -tetramer binding in MCMVp-immunized mice from FIG. 7D is shown. Proportions of m45-tetramer (in CD8 T cells) were quantified in draining lymph node, spleen, and tumor tissues. FIG. 7F: Representative OT-I-tetramer binding in OVAp- immunized mice from FIG. 7D is shown. Proportions of tetra-OT-I (in CD8 T cells) were quantified in draining lymph node, spleen, and tumor tissues. FIG. 7G: Phenotype of tumor-infiltrating m45-specific CD8 T cells compared to m45-tetramer- negative. FIG. 7H: Purified CD45+ cells from tumors were incubated with the 6 MCMV peptides, or the 2 OVAp for 4 hours, in the presence of GolgiPlug, and IFNy and TNF production was measured in CD8 and CD4 T conv cells. FIG. 71: The level of GzrnB was measured in CD8 T cells from mice tested in FIG. 7D. FIG. 7 J: The level of GzrnB was measured in CD8 T cells from mice tested in FIG. 4. FIG. 7K: qPCR detection of UL55 mRNA in human pancreatic tumor tissue from HCMV seronegative and seropositive patients. HUVEC cells infected or not with HCMV were used as a positive control (CT+ and CT-). Results were compared by one-way ANOVA with Tukey correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
FIGS. 8A-8C. MCMVp treatment induced a marked expansion of virus specific T cells. FIG. 8A: Normalized weights of mice shown in FIG. IB over the course of treatment with 50pg of each MCMV peptide epitope (top). Tumor weights in the different treatment groups at end point (18 days post tumor implantation) (bottom). FIG. 8B: MCMV -tetramer quantification in the spleen of the mice shown in FIG. IB. FIG. 8C: MCMV -tetramer quantification and phenotype of CD8 T cells in tumors of infected or uninfected mice treated with iRGD+MCMVp. Results were compared by Mann-Whitney analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
FIGS. 9A-9F. MCMV-specific T cells preferentially localized within tumors in the absence of iRGD. FIG. 9A: KPC1242 tumor growth curves. Various doses of CD8 and CD4 T peptide epitopes were tested (Dose A 50pg CD4p/lpg CD8p, Dose B 50pg CD4p/0.1pg CD8p, Dose C lOpg CD4p/3pg CD8p, Dose D lOpg CD4p/lpg CD8p), iRGD was always used at 300pg per injection. Normalized mouse weight (middle) and survival (right). (n=5-8/group). FIG. 9B: Growth curve of KPC1242 tumors over time (left) and tumor weight at endpoint (right). Dose B was used in combination or not with iRGD. FIG. 9C: Kaplan- Meier survival graph with a similar protocol as shown in FIG. 9A using Dose A. FIGS. 9D-9E: MCMV-specific T cell analysis in spleen, liver and tumor at endpoint from mice shown in FIG. 9B. FIG. 9D: Percentage of each tetramer among total Tconv (left) or CD8 T cells (right) in the tumor. FIG. 9E: Percentage of the 3 dominant MCMV-specific T cells (m25. m38, and m45) in the 3 tissues tested. FIG. 9F: Assessment of combination therapy to boost MCMVp treatment with anti-ILlOR and/or anti-PDl. Tumor growth over time (left) and Kaplan-Meier survival graph (right). Results were compared by one-way ANOVA with Tukey correction, and repeated measures by two-way A NOVA with Sidak correction. *P < 0.05; ***P < 0.001; ****P < 0.0001.
FIGS. 10A-10D. MCMV-specific T cells expanded following treatment, and preferentially localize to the tumor. Mice were tumor challenged and treated as described in FIGS. 2B, to analyze spleen, liver, and tumor T cell infiltrates. (n=5/group). FIG. 10A: Absolute number of CD4 Tconv and CD8 T cell quantification at endpoint in the 3 tissues analyzed. FIGS. 10B-10C: Absolute number of MCMV-specific T cells at endpoint in the spleen and liver for Tconv (FIG. 10B) and CD8 T cells (FIG. 10C). FIG. 10D: Proportion of MCMV-specific FoxP3+ Tregs among total Tregs in the 3 tissues analyzed. Results were compared by one-way ANOVA with Tukey correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
FIGS. 11A-11D. MCMV-specific T cells were highly activated, cytotoxic and cytokine producers. Immunophenotyping and cytokine production analysis of the TILs described in FIG. 3. (n=5/group). FIG. 11A: Representative histograms of the m45-specific CD8 T cells in the liver stained with the indicated markers (top) and mean fluorescence intensity (MFI) quantification (bottom). FIGS. 11B-11C: Same analysis for m38-specific CD8 T cells in the liver (FIG. 11B) and tumor (FIG. 11C). FIG. 11D: Total liver cells were incubated with the 6 MCMV peptides for 4 hours, in the presence of GolgiPlug, and then stained for analysis of cytokine production and cytotoxicity. Results were compared by one-way ANOVA with Tukey correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
FIGS. 12A-12D. Tumor infiltration by MCMV-specific T cell was accompanied by changes in the tumor microenvironment (TME). Additional analysis from the data shown in FIG. 5. FIG. 12A: Cell type distribution per sample. FIG. 12B: GSEA analysis of DEGs in fibroblasts and macrophages. FIG. 12C: DEGs in epithelial cells. FIG. 12D: Markers used to annotate the cell types from FIG. 5. FIGS. 13A-13D. Analysis of tumor infiltrating T cells. Additional analysis from the data shown in FIGS. 6. FIG. 13A: T cell clustering and UMAP distribution split by sample conditions. FIG. 13B: Dimplot representing the detection of specific MCMV-associated matched CDR3 sequence for TCRp. FIG. 13C: Absolute number of cells in each cluster. FIG. 13D: Top5 DEGs per cluster.
FIGS. 14A and 14B. MCMV infection was not necessary for tumor localization of MCMV-specific T cells. FIG. 14A: IE1 DNA qPCR normalized with actin in KPC46 tumors harvested from uninfected or MCMV infected B6xl29 Fl hybrid mice. Positive controls were spleen and liver from mice 4 dpi. FIG. 14B: Percentage quantification of MCMV and OVA tetramer binding T cells in MCMVp and OVAI (SIINFEKL) immunized mice from the draining lymph node, spleen and tumor. Results were compared by one-way ANOVA with Tukey correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
FIGS. 15. Patient serotyping by ELISA. CMV status of PDAC patients are shown. Four out of five sera with patient IDs (121.22. 121.23, 121.25 and 121.26) tested positive for CMV.
FIG. 16. Organoids generated from patient’s tumor. Organoids were passaged for up to three generations.
FIG. 17. Ultrasound images showing an orthotopically implanted PDAC tissue in NSG mice. The tissue was obtained from a treatment naive PDAC patient. Tumor growth was monitored post tumor implantation.
FIG. 18. Dot plots showing the gating strategy' for immune cell identification by flow cytometry to monitor T-cell engraftment.
FIGS. 19A-19F. Engraftment kinetics of PBMCs from a healthy donor.
FIG. 19A: Percentage changes in body weight relative to initial mouse weight are shown. FIG. 19B: Engraftment ofhCD45+ cells over the course of 6 weeks is shown. FIG. 19C: Bar plot showing the percentage of human CD4+ and CD8+ T cells when injected with 10 million PBMCs. FIG. 19D: Bar plot showing the percentage of human CD4+ and CD8+ T cells when injected with 5 million PBMCs. FIG. 19E: Bar plot show ing the percentage of human CD4+ and CD8+ T cells when injected with 2.5 million PBMCs. FIG. 19F: Bar plot showing % cell population of mCD45+ and hCD45+ in spleen from NSG mice engrafted with PBMCs from a healthy human donor. FIGS. 20A-20B. Engraftment of PBMCs isolated from a treatment naive PDAC patient. FIG. 20A: Percentage changes in body weight relative to initial weight. FIG. 20B. Percentage of human CD45+ cells quantified by flow cytometry.
FIGS. 21A-21B: Addition of a-CD40 further improved the MCMV therapy. B6xl29 Fl hybrid mice were injected with the KPC46 tumor cell line and treated with vehicle or Dose A (50 ,g CD4p/l pg CD8p) alone or in combination with Gemcitabine (Gem) or a-CD40 antibody and compared with Gem or a-CD40 antibody alone. Tumor volume was measured over time in FIG. 21A and tumor weight at endpoint in FIG. 21B. (n=5-7/group). Results were compared by one-way ANOVA with Tukey correction, and repeated measures by two-way A NOVA with Sidak correction. *P < 0.05; **P < 0.01; ***p < 0.001; ****P < 0.0001.
FIGS. 22A-22C. B6 mice were injected with the KPC1242 tumor cell line and treated with vehicle or Dose A (50pg CD4p/lpg CD8p) alone or in combination with anti-ILla antibody (FIG. 22A) or gemcitabine (Gem) (FIG. 22B), and compared with either agent alone. Tumor volume was measured over time (top panels) and a Kaplan- Meir Survival curves are shown in the bottom panels. FIG. 22C: Mice were weighed over time for all the groups shown in FIGS. 22A and 22B (n=5-8/group). Results were compared by one-way ANOVA with Tukey correction, and repeated measures by two- way ANOVA with Sidak correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
FIG. 23. Schematics illustrating MCMV replication cycle.
DETAILED DESCRIPTION
Immunotherapy has been most successful in treating tumors with high mutational burdens such as melanoma and non-small cell lung cancer (1-4). These mutations result in the generation of numerous neoantigens that the immune system recognizes as “non-self eliciting an anti-tumor immune response that can often be enhanced with immune checkpoint therapy. Many common cancers, such as pancreatic cancer, are largely resistant to existing immunotherapies due to a highly immunosuppressive TME (8, 9) and a modest mutational burden (5-7), which results in a paucity of activated T cells that can reach and/or effectively kill cancer cells. Consequently, numerous strategies to enhance anti-tumor immunity through immune checkpoint blockade and/or by modulating the TME have been largely unsuccessful (10-12). Provided herein are the foundational discoveries that harnessing antiviral memory T cells results in massive recruitment of these antiviral memory T cells to the tumor, causing substantial changes in the TME and ultimately reducing tumor growth and enhancing survival. For example, the present methods show that cytomegalovirus (CMV) immunity can be re-directed to control a tumor (e.g., pancreatic cancer) that is normally refractor}’ to immunotherapy via injection of viral epitopes. Further, disclosed herein is a mutation agnostic approach that can be enhanced by combination therapy with anticancer agents (e.g., a chemotherapeutic agent), and can benefit the majority of the population (more than 80%) that harbor latent CMV, suggesting its potential for clinical translation. Immunodominant CMV-specific memory' T cells have shown broad tissue residency and an effector memory phenotype in people and mice. Approximately -10% of all circulating CD4 and CD8 T cells are CMV-specific (26, 27). Methods and materials disclosed herein have the potential for development of “off-the-shelf ’ therapeutics by stimulating pre-existing antiviral T cells; and can be widely applicable due to high prevalence of CMV immunity in general population. Using subcutaneous tumor models, it was previously shown that intra-tumoral injection of antiviral T cell peptide epitopes in mice that were previously infected with the virus led to growth arrest and even complete remission (14, 15). However, direct intra-tumoral injection is not always feasible. Methods and materials described herein demonstrate that systemic delivery of epitopes has the potential to be much more applicable to a variety of tumor types.
In summary', the present methods demonstrate that systemic treatment of cancer with T cell peptide epitopes can efficiently recall CMV memory T cells and result in their tumor localization and induction of an anti-tumor immune response. Thus, described herein are methods and materials for harnessing antiviral memory T cells to treat a cancer and/or induce an immunogenic response to a cancer.
Methods of use
Provided herein are methods for treating a cancer in a subject (e.g., a human). Also provided herein are methods for inducing an immunogenic response to a cancer in a subject. For example, an immunogenic response to a cancer can be enhanced T cell responses, increased T cell infiltration into the tumor or around the tumor margin, activation of immune cells, T cell expansion, T cell activation, T cell tumor infiltration, NK cell (e.g., NKG2C+ NK cells) expansion, and/or tumor apoptosis, thereby reducing or eliminating (e.g., completely or partially) the tumor and potentially restricting recurrence and/or metastases. In some embodiments, activation of immune cells include activation of T cells, natural killer (NK) cells (e.g., NKG2C+ NK cells), dendritic cells, macrophages, B cells, tumor-infiltrating lymphocytes (TILs), neutrophils, eosinophils, basophils, and/or innate lymphoid cells. In some embodiments, T cell is a cytotoxic T cell (e.g., CD8+ T cells), a helper T cell (e.g., CD4+ T cell), a regulatory T cell (Tregs), a memory T cell, a gamma delta T cell (y5 T cell), and/or a natural killer T cell (NKT cell). In some embodiments, CMV infection induces a huge expansion of T cells, y5 T cells, and/or NK cells (e.g., NKG2C+ NK cells).
In some embodiments, the subject has pre-existing CMV immunity. In some embodiments, the subject does not have pre-existing CMV immunity. When treating a subject with pre-existing CMV immunity, the method can include administering a composition containing one or more human cytomegalovirus (HCMV) peptide epitopes (e.g., a CD4 T cell epitope and/or a CD8 T cell epitope) to the subject. When treating a subject with no pre-existing CMV immunity, the method can include inducing cytomegalovirus immunity by vaccinating a subject in need thereof with individual/pooled viral (e.g., CMV) polypeptides or mRNA encoding the CMV polypeptides (e.g., mRNA encoding the whole CMV viral protein or mRNA encoding one or more CMV peptide epitopes described herein). In some embodiments, when treating a subject with no pre-existing CMV immunity, the method can include inducing cytomegalovirus immunity by administering a therapeutically effective amount of a composition comprising one or more HCMV peptide epitopes and an adjuvant.
In some embodiments, one or more refers to one or more copies of the same item. For example, one or more HCMV peptide epitopes can be multiple copies of the same HCMV peptide epitope (e.g., one copy, two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or more copies of the same HCMV peptide epitope). In some embodiments, one or more refers to one or more variations of an item. For example, one or more HCMV peptide epitopes can be HCMV peptide epitopes of different sequences (e.g., one, two, three, four, five, six, seven, eight, nine, ten. more different HCMV peptide epitope sequences). CMV immunity can be induced in a subject with no pre-existing CMV immunity by administering to the subject one or more HCMV (e.g.. an attenuated virus) or one or more HCMV peptide epitopes (e.g., a CD4 and/or CD8 T cell epitope). In some embodiments, once a subject is identified as having CMV immunity, the subject is administered a therapeutically effective amount of a composition containing one or more HCMV peptide epitopes. In some embodiments, methods and materials described herein include determining the expression level of Neutropilin-1 and/or Integrin 05 in the subject in need thereof. In some embodiments, the expression level of Neutropilin-1 and/or Integrin 05 is determined before administration of a composition containing one or more HCMV peptide epitopes.
In some embodiments, one or more HCMV peptide epitopes are a CD4 T cell epitope and/or a CD8 T cell epitope. In some embodiments, a CD4 T cell epitope and/or a CD8 T cell epitope can be a 9-15 mer peptide epitope. For example, a 9-15 mer peptide epitope can include 9-15 amino acids. In some embodiments, the 9-15 mer peptide epitope includes a core epitope that binds to MHC-I of CD8 T cells. In some embodiments, the 9-15 mer peptide epitope binds to MHC-II, thereby- stimulating CD 4 T cells. In some embodiments, the 9-15 mer peptide epitope includes a 9 mer core epitope that binds to MHC-I of CD8 T cells and a 10-15 mer that binds to MHC-II to stimulate CD4 T cells. In some embodiments, one or more HCMV peptide epitopes can include, consist essentially of, or consist of amino acid sequences set forth in SEQ ID NOS: 1 -422 (Tables 2 and 3). In some embodiments, a composition containing one or more HCMV peptide epitopes include a CD4 T cell epitope, a CD8 T cell epitope, or a 15mer peptide. In some embodiments a 15mer peptide includes a CD8 epitope and a CD4 epitope. In some embodiments, the composition containing one or more HCMV peptide epitopes can include, consist essentially of, or consist of amino acid sequences set forth in SEQ ID NOS: 1-422 (Tables 2 and 3).
In some embodiments, the methods include administering a therapeutically effective amount of a composition containing one or more HCMV peptide epitopes to a subject (e.g., a subject with pre-existing CMV immunity or a subject with induced CMV immunity as described herein) having a cancer. In some embodiments, the subject having a cancer can be administered or instructed to self-administer a composition containing one or more HCMV peptide epitopes. In some embodiments, when treating a subject having a cancer as described herein, the subject can have any type of cancer. In some embodiments, a cancer treated as described herein includes one or more solid tumors. In some embodiments, a cancer treated as described herein includes one or more blood cancers. In some embodiments, a cancer treated as described herein is a primary cancer. In some embodiments, a cancer treated as described herein is a metastatic cancer. In some embodiments, a cancer treated as described herein is a relapsed cancer. In some embodiments, a cancer treated as described herein is a refractory cancer. Non-limiting examples of solid tumors include gastric cancer, breast cancer, lung cancer, prostate cancer, glioma, melanoma, an ovarian cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, thyroid cancer, sarcoma, stomach cancer, head and neck cancer, cervical cancer, an endometrial cancer, an esophageal cancer, thymoma, soft tissue sarcoma, bone cancer, testicular cancer, penile cancer, gallbladder cancer, uterine sarcoma, an adrenal gland cancer, an ampullary cancer, hepatic angiosarcoma, nasal, or paranasal sinus cancer. Non-limiting examples of blood cancers include leukemia, lymphoma, myeloma, myelodysplastic syndrome, or myeloproliferative neoplasm.
In some embodiments, the subject has an increased risk of developing cancer. For example, the subject may have a family history and/or personal history of cancer. In some embodiments, a subject is a mammal (e.g., a human, a non-human primate, a rodent, a dog, a pig, or a rabbit). Methods for treating a mammal having cancer (e g., breast cancer) can include identifying the mammal as having cancer. Any appropriate method can be used to identify a subject as having a cancer. Non-limiting examples of methods for identifying a mammal as having a cancer can include, without limitation, physical examination, laboratory tests (e.g., blood, urine, and/or circulating tumor cells (CTCs)), biopsy, imaging tests (e.g., X-ray, PET/CT, MRI, and/or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy, genetic tests, and/or other methods of identifying cancer as known in the art.
Also provided herein are methods for treating a cancer in a subject (e.g., a human) by inducing an immunogenic response against the tumor. In some embodiments, the immunogenic response against the tumor can be induced by isolating a biological sample (e.g., peripheral blood mononuclear cells (PBMCs). cord blood, bone marrow, spleen, lymph nodes, body fluids, other tissues such as skin, liver, nasal, lung, fat, brain, or tumor) from the subject. In some embodiments, the immunogenic response against the tumor can be induced by exposing isolated PBMCs to one or more HCMV peptide epitopes, determining which HCMV T cells of the isolated PBMCs are reactive to one or more HCMV peptide epitopes, and administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells. In some embodiments, HCMV peptide epitopes that are reactive to one or more HCMV peptide epitopes can induce a positive reaction or activation of T cells. In some embodiments, the immunogenic response against the tumor can be induced by isolating peripheral blood mononuclear cells (PBMCs) from the subject, exposing the isolated PBMCs to one or more HCMV peptide epitopes, determining an HCMV T cell peptide of the isolated PBMCs reactive to one or more HCMV peptide epitopes, and administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells.
In some embodiments, the immunogenic response against the tumor can be induced by isolating PBMCs from the subject, exposing the isolated PBMCs to one or more HCMV peptide epitopes, determining which HCMV T cells of the isolated PBMCs are reactive to one or more HCMV peptide epitopes, expanding the HCMV reactive T cell ex vivo, administering the HCMV reactive T cells back to the subject; and administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells (e.g.. one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response). In some embodiments, the immunogenic response against the tumor can be induced by exposing isolated PBMCs to one or more HCMV peptide epitopes, determining an HCMV T cell of the isolated PBMCs reactive to one or more HCMV peptide epitopes, expanding the HCMV reactive T cell ex vivo, administering the HCMV reactive T cells back to the subject, and administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells (e.g., one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response).
In some embodiments, determining which HCMV T cells of the isolated PBMCs are reactive to one or more HCMV peptide epitopes described herein include dividing the one or more HCMV peptide epitopes into several pools and then subjecting these pools to T cells (e.g., T cells isolated from a subject in need thereof). For example, the HCMV peptide pools described herein can be further divided into subpools based on the reactivity of T cells to the HCMV peptide epitopes described herein until one or more HCMV peptide epitopes are identified that elicit a strong HCMV-specific T cell response. In some embodiments, the HCMV reactive T cells are expanded in vitro in the presence of cytokines (e.g., interleukin-2 (IL-2), IL-7, IL- 15, IL-21, IL-17, IL-12, IFNy, TGFp, or any other appropriate cytokines known in the art to induce T cells with specific phenotypes of interest) and one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response. In some embodiments, the HCMV reactive T cells are administered back to the subj ect before administering one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response. In some embodiments, the HCMV reactive T cells are administered back to the subject after administering one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response. In some embodiments, the HCMV reactive T cells are co-administered with one or more HCMV peptide epitopes that were identified to elicit a strong HCMV- specific T cell response.
In some embodiments, the immunogenic response against the tumor can be induced by isolating PBMCs from the subject, exposing the isolated PBMCs to one or more human cytomegalovirus (HCMV) peptide epitopes, determining which HCMV T cells of the isolated PBMCs encode specific HLAs reactive to one or more HCMV peptide epitopes, genetically modifying the T cells to express a chimeric antigen receptor (CAR) that targets one or more HCMV peptide epitopes (e.g., one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response), administering the genetically modified T cells back to the subject, and administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells (e.g., one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response). In some cases, T cell receptors (TCRs) of the HCMV reactive T cells can be sequenced to generate the genetically modified T cells (e.g., a genetically modified T cell expressing CAR that targets one or more HCMV peptide/HLA complexes that can elicit a strong HCMV- specific T cell response). In some embodiments, the genetically modified T cells are administered back to the subject before administering one or more HCMV peptide epitopes recognized by the HCMV reactive T cells (e.g., one or more HCMV peptide epitopes that were identified to elicit a strong HCMV-specific T cell response). In some embodiments, the genetically modified T cells are administered back to the subject after administering one or more HCMV peptide epitopes recognized by the HCMV reactive T cells. In some embodiments, the genetically modified T cells are co-administered with one or more HCMV peptide recognized by the HCMV reactive T cells.
A CAR (e.g., HCMV peptide-specific CAR) can include an antigen-binding domain (e.g.. HCMV peptide-binding domain), a hinge, a transmembrane domain, and/or one or more signaling domains. Examples of antigen-binding include, without limitation, an antigen-binding fragment (Fab), a variable region of an antibody heavy (VH) chain, a variable region of a light (VL) chain, and/or a single chain variable fragment (scFv). In some cases, an antigen-binding domain of a CAR can target (e.g., can target and bind to) HCMV peptide in a subject (e.g., a human). A CAR can include an optional signal peptide, an antigen binding domain, an optional hinge, a transmembrane domain, and one or more intracellular signaling domains.
In some embodiments, a CAR can include a hinge region. In some cases, a hinge region can be located between an antigen-binding domain and a transmembrane domain of a CAR. In some cases, a hinge region can provide a CAR with increased flexibility for the antigen-binding domain. For example, a hinge region can reduce spatial limitations of an antigen-binding domain of a CAR and its target antigen. Examples of hinge regions that can be used as described herein include, without limitation, a membrane-proximal region from an IgG, a membrane-proximal region from CD8, and a membrane-proximal region from CD28.
In some embodiments, a CAR can include any appropriate transmembrane domain. A transmembrane domain can be located between an antigen-binding domain and a signaling domain of a CAR and/or located between a hinge and a signaling domain of a CAR. In some embodiments, a transmembrane domain can provide structural stability for the CAR (e.g., HCMV-specific CAR). For example, a transmembrane domain can include a structure (e.g., a hydrophobic alpha helix structure) that can span a cell membrane and can anchor the CAR to the plasma membrane. Examples of transmembrane domains that can be used as described herein include, without limitation, CD3^ transmembrane domains, CD4 transmembrane domains, CD8 (e.g., a CD8a) transmembrane domains, CD28 transmembrane domains, CD 16 transmembrane domains, and erythropoietin receptor transmembrane domains.
In some embodiments, a CAR can include any appropriate signaling domain or combination of signaling domains (e.g., a combination of two, three, or four signaling domains). In some cases, a signaling domain of a CAR can be an intracellular signaling domain normally found within T cells or NK cells. Examples of signaling domains that can be used as described herein include, without limitation. BB signaling domains, 28^ signaling domains, CD2 signaling domains, CD3^ signaling domains, CD28 signaling domains, Toll-like receptor (TLR) signaling domains (e g., TLR3 or TLR4 signaling domains), CD27 intracellular signaling domains. 0X40 (CD134) intracellular signaling domains. 4-1BB (CD137) intracellular signaling domains, CD278 intracellular signaling domains, DAP 10 intracellular signaling domains, DAP 12 intracellular signaling domains, FceRly intracellular signaling domains, CD278 intracellular signaling domains, CD 122 intracellular signaling domains, CD 132 intracellular signaling domains, CD70 intracellular signaling domains, cytokine receptor intracellular signaling domains, and CD40 intracellular signaling domains. In some cases, a CAR for use as described herein can be designed to be a first-generation CAR having a CD3^ intracellular signaling domain. In some cases, a CAR for use as described herein can be designed to be a second-generation CAR having a CD28 intracellular signaling domain followed by a CD3^ intracellular signaling domain. In some cases, a CAR for use as described herein can be designed to be a third generation CAR having (a) a CD28 intracellular signaling domain followed by (b) a CD27 intracellular signaling domain, an 0X40 intracellular signaling domains, or a 4- IBB intracellular signaling domain followed by (c) a CD3 intracellular signaling domain.
In some embodiments, the CAR T cell described herein includes one or more suicide gene. A suicide gene can be a nucleic acid sequence introduced to a CAR T cell by standard methods known in the art that, when activated, causes death of the CAR T cell. Suicide genes may facilitate effective tracking and elimination of the CAR T cell in vivo in case of severe toxicities. Non-limiting examples of suitable suicide gene systems include inducible caspase 9 protein or herpes simplex virus thymidine kinase (HSV-tk/ganciclovir (GCV) suicide gene therapy system. Any appropriate method can be used to express a CAR on aT cell. For example, nucleic acid encoding an antigen receptor (e.g., HCMV-binding domain) can be introduced into one or more T cells. In some cases, viral transduction can be used to introduce nucleic acid encoding an antigen receptor into a non-dividing a cell. Nucleic acid encoding an antigen receptor can be introduced in a T cell using any appropriate method. In some cases, nucleic acid encoding an antigen can be introduced into aT cell by transduction (e.g., viral transduction using a retroviral vector such as a lentiviral vector) or transfection. In some cases, nucleic acid encoding an antigen receptor can be introduced ex vivo into one or more T cells. For example, ex vivo engineering of T cells expressing an antigen receptor can include transducing isolated T cells with a lentiviral vector encoding an antigen receptor. In cases where T cells are engineered ex vivo to express an antigen receptor, the T cells can be obtained from any appropriate source (e.g., a mammal such as the mammal to be treated or a donor mammal, or a cell line). In some embodiments, the mammal is a human. In some embodiments, the T cell is an allogenic T cell.
In some embodiments, methods and materials provided herein can be used to reduce the size of a tumor within a subject having a cancer. For example, a subject in need thereof can be administered a therapeutically effective amount of a composition comprising one or more HCMV peptide epitopes, HCMV reactive T cells, T cellrecognizing HCMV peptide epitopes, or a genetically modified CAR-T cell (e.g.. HCMV-specific CAR-T cell) to reduce the size of the cancer in the subject. In some embodiments, methods and materials provided herein can be used as described herein to reduce the number of cancer cells in the subject by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials provided herein can be used as described herein to reduce the volume of one or more solid tumors in the subject by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some embodiments, methods and materials provided herein can be used to improve survival of a subject (e.g., a human) having a cancer described herein. For example, a subject in need thereof can be administered a therapeutically effective amount of a composition comprising one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell (e.g., HCMV-specific CAR-T cell) to improve survival of the subject. For example, methods and materials described herein can be used to improve the survival of a subject having a cancer described herein by, for example. 10. 20. 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, methods and materials described herein can be used to improve the survival of a subject having a cancer described herein by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).
In some embodiments, methods and materials provided herein can be used to improve the immunogenic response of a subject (e.g., a human having a cancer) to a cancer described herein. For example, a subject in need thereof can be administered a therapeutically effective amount of a composition comprising one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell (e.g., HCMV-specific CAR-T cell) to improve the immunogenic response of a subject to a cancer described herein. For example, methods and materials described herein can be used to improve the immunogenic response of a subject to a cancer described herein by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some embodiments, a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell (e.g., HCMV-specific CAR-T cell) described herein can be used as the sole active agent(s) to treat a subject having a cancer described herein.
In some embodiments, a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein can be administered to a subject having a cancer together with one or more (e.g., one, two, three, four, or more) additional agents used to treat cancer as described herein. For example, additional agents that can be used to treat cancer includes, without limitation, an adjuvant, a tumor targeting agent, or a cancer therapy. In some embodiments, an adjuvant is unmethylated cytosine-guanine dinucleotide (CpG), Polyinosinic-Poly cytidylic Acid Stabilized with Polylysine and Carboxymethylcellulose (Poly ICLC), montanide, or combinations thereof. In some embodiments, the tumor targeting agent is certepetide. In some embodiments, a tumor targeting agent is a polypeptide, a nanoparticle, or a viral particle, each containing the HCMV peptide epitopes described herein. In some embodiments, one or more HCMV peptide epitopes is conjugated to a tumor targeting agent (e.g., certepetide) or a cancer therapy. In some embodiments, the tumor targeting agent or a cancer therapy can be conjugated to an HMCV peptide via a linker such as a peptide linker. In some embodiments, a linker can be a cleavable linker such as an acid cleavable linker, a GSH cleavable linker, a cathepsin cleavable linker, a glycosidase cleavable linker, or a phosphatase cleavable linker.
In some embodiments, a cancer therapy is a chemotherapeutic agent, an immunotherapeutic agent, surgery, radiation therapy, carbon ion therapy, or proton therapy. Non-limiting examples of a chemotherapy can include a DNA crosslinking agent, an alkylating agent, an anti -metabolite, an anti-microtubule agent, a topoisomerase inhibitor, or a cytotoxic antibiotic. Non-limiting example of an immunotherapeutic agent can include a checkpoint inhibitor, an immunomodulator, a cytokine, a cancer vaccine, a monoclonal antibody, an oncolytic virus, an adoptive cell therapy, a CAR T cell therapy, or combinations thereof. In some embodiments, a cancer therapy can include, without limitation, trametinib, dabrafenib, binimetinib, selumntinib, vemurafenib, encorafenib, cobimetinib, busulfan, cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), liposomal doxorubicin, melphalan, pemetrexed, topotecan. vinorelbine, goserelin. leuprolide. tamoxifen, letrozole, anastrozole, exemestane, bevacizumab, olaparib, rucaparib, niraparib, ipilimumab (e.g., YERVOY®), pembrolizumab (e.g., KEYTRUDA®), nivolumab (e.g., OPDIVO®) and any combinations thereof.
In some embodiments, methods and materials provided herein can be used to improve the efficacy of additional agents described herein in a subject having a cancer. A subject in need thereof can be administered a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein to improve the efficacy of additional agents in the subject. For example, methods and materials provided herein can be used to improve the efficacy of additional agents by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some cases, a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell together with one or more (e.g., one, two, three, four, or more) additional agents described herein. For example, methods and materials provided herein for treating a cancer or inducing an immunogenic response in a subject in need thereof (e.g., a subject having cancer) can include administering a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cellrecognizing HCMV peptide epitopes, or a genetically modified CAR-T cell to a subject having cancer together with an additional agent/therapy.
In cases where a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein are used in combination with one or more additional agents used to treat cancer or induce an immunogenic response in a subject in need thereof (e.g., a subject having cancer), the one or more additional agents can be administered at the same time (e.g., in a single composition described herein and the one or more additional agents) or independently. For example, a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells. T cellrecognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein can be administered first, and the one or more additional agents administered second, or vice versa.
In some cases, a composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein are used in combination with one or more additional agents used to treat cancer or induce an immunogenic response in a subject in need thereof (e.g., a subject having cancer), the one or more additional agents can be performed at the same time or independently of the administration of the composition containing one or more HCMV peptide epitopes, HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein. For example, the composition described herein can be administered before, during, or after the one or more additional agents are administered, or in any order.
Table 1: Exemplary sequences of HCMV peptide epitopes
Table 2: Exemplary sequences of CMV-specific class II epitopes
Pharmaceutical composition
One or more HCMV peptide epitopes, HCMV reactive T cells, T cellrecognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein can be formulated into a composition (e.g., a pharmaceutical composition) for administration to a subject in need thereof (e.g., a subject having a cancer). For example, a therapeutically effective amount of one or more HCMV peptide epitopes (e.g., one or more HCMV peptide epitopes or a composition comprising a HCMV peptide), HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.
As used herein the language “pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sterile aqueous or non-aqueous solutions, suspensions, emulsions, and the like, compatible with pharmaceutical administration. Examples of non-aqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and organic esters. Aqueous carriers include, without limitation, water, alcohol, saline, and buffered solutions. Acceptable carriers also can include physiologically acceptable aqueous vehicles (e.g., physiological saline) or other known carriers for oral administration. Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration can include oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or inhaled administration. In some embodiments, administering includes oral administration, injection, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intratumoral injection, inhalation, or any combinations thereof. In some embodiments, the pharmaceutical composition provided herein suitable for parenteral administration can include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. In some embodiments, the pharmaceutical composition provided herein can be administered locally or systemically. For example, a composition provided herein can be administered locally by intratumoral administration (e.g., injection into tumors) or by administration into biological spaces infiltrated by tumors (e.g. intraspinal administration, intracerebellar administration, intraperitoneal administration and/or pleural administration). For example, the pharmaceutical composition provided herein can be administered systemically by intravenous administration (e.g., injection or infusion) to a mammal (e.g., a human).
In some embodiments, one or more HCMV peptide epitopes (e.g., one or more HCMV peptide epitopes or a composition comprising a HCMV peptide), HCMV reactive T cells, T cell-recognizing HCMV peptide epitopes, or a genetically modified CAR-T cell described herein when formulated as a pharmaceutical composition can be formulated in an ingestible form or a topical form. For example, the pharmaceutical composition can be in the form of a liquid, solution, suspension, tablet, powder, granule, pill, capsule, gel, cream, mist, atomized vapor, aerosol, soft gelatin capsule, or hard gelatin capsule. For oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets can be coated by methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspension, or they can be presented as a dry product for constitution with saline or other suitable liquid vehicle before use. Liquid preparations also can contain pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles, preservatives, buffer salts, flavoring agents, coloring agents, and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the compound. In some embodiments, the pharmaceutical composition (e.g., an antigen-binding molecule and/or a bispecific-binding molecule) provided herein is formulated for oral ingestion, injection, subcutaneous injection, or tissue-specific targeting.
The pharmaceutical composition (e.g., an antigen-binding molecule, a bispecific-binding molecule, and/or a CAR-T cell) provided herein can be administered by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished with nasal sprays or suppositories. Dosage
Treating cancer or inducing an immunogenic response in a subject in need thereof (e.g., a subject having cancer) according to the methods described herein include administering a therapeutically effective amount of the pharmaceutical composition described herein. A “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. For example, an effective amount is one that achieves a desired therapeutic effect, e.g., an amount necessary to treat a disease, or to reduce risk of development of disease or disease symptoms (also referred to as a prophylactically effective amount). An effective amount can be administered in one or more administrations, applications, or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. For example, a therapeutically effective amount of a pharmaceutical composition as provided herein can be effective to reduce tumor size (e.g., size of a primary7 tumor or a metastatic tumor), improve survival, or improve immunogenic response. In some embodiments, a therapeutically effective amount of a pharmaceutical composition as provided herein can be effective to improve at least one symptom of cancer or complications related to cancer.
The pharmaceutical composition provided herein can be administered one or more times per year (e.g., one time per year, two times per year, three times per year, four times per year, or five times per year) to one or more times per month (e.g., one time per month, two times per month, three times per month, four times per month, five times per month), including once every other month, once every7 three months, or twice a month. In some embodiments, the pharmaceutical composition provided herein can be administered one or more times per week (e.g., one time per week, two times per week, three times per week, four times per week, five times per week, six times per week, seven times per w eek, or more than seven times per week). The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. Various factors can influence the actual amount used for a particular application. For example, the frequency of administration. duration of treatment, combination of other agents, site of administration, stage of disease (if present), and the anatomical configuration of the treated area may require an increase or decrease in the actual amount administered.
An effective duration for administering a pharmaceutical composition provided herein can be any duration that reduces the severity7 of cancer, reduce tumor size (e.g., size of a primary tumor or a metastatic tumor), improve survival, or improve immunogenic response in a subject without producing significant toxicity to the subject. In some cases, the effective duration can vary from several days to several weeks, to several months, or longer. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the effective amount, frequency of administration, use of route of administration, and severity of the subject’s condition.
Dosage, toxicity and therapeutic efficacy of the pharmaceutical composition provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population). Compositions that exhibit high therapeutic indices are preferred. While compositions that exhibit toxic side effects may be used, care should be taken to minimize and reduce side effects. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compositions used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays or animal models.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example 1: Redirecting cytomegalovirus (CMV) immunity against pancreatic cancer
This Example demonstrates that repurposing CMV-specific T cells can combat pancreatic cancer.
Methods and Materials
Wild-type (WT) C57BL/6J (B6) mice and C57BL/6J x 129Sl/SVlmJ (129) Fl hybrids (B6129SF1/J, Strain #: 101043) were purchased from Jackson laboratories at 4-5 weeks of age, and infected or not with MCMV at 5-6 weeks of age. This study was carried out in strict accordance with the guidelines of Association for assessment and Accreditation of laboratory Animal Care (AAALAC) and the National Institutes of Health (NIH).
MCMV infection
MCMV (smith strain) was originally produced in 3T3 cells from cloned and sequenced BAC DNA (43). The strain was amplified in vivo in BALB/c mice, extracted from the salivary glands, and titrated on MEF (murine embryonic fibroblast) cells (17). Mice aged 5-6-weeks old were infected intraperitoneally with 1 x 104 pfu in lOOpL of PBS.
Pancreatic tumor models
MCMV infected mice (2-3 months post infection) and age matched uninfected controls were orthotopically injected with KPC1242 (syngeneic with B6 mice) or KPC46 (syngeneic with B6/129 Fl hybrid mice) pancreatic cancer cells. These cells were cultured in vitro in RPMI with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. Once confluent, they were trypsimzed. washed, and resuspended in Matrigel® (Coming Matrigel Matrix) for injection (5,000 KPC1242 or KPC46 tumor cells in 20pLof matrigel, per mouse). Tumor grow th was monitored initially by palpation, and after day 8 post injection, mice were subjected to ultrasound (Convex L20 HD3. Clarius) twice a week (under isofluorane) for tumor growth monitoring. Tumor volume was calculated using the ellipsoid volume calculation formula. In addition, mice were weighed twice a week together with health checks. Mice were enrolled in treatment studies and randomly assigned to different treatment conditions once most tumors were visible by ultrasound, usually around 9 to 11 days post tumor cell injection. For survival studies, the euthanasia criteria included mice loosing 20% or more of their initial weight or having tumors reaching 2000mm3 or more plus any evident signs of distress.
Treatment Studies
MCMV peptides (CD4T: m09133-147; m25409-423; ml4224-38; CD8T: IE3416-423; m38316-323; m45985-993) were purchased from Mimotopes (Australia), and ovalbumin peptides (OVA257-264; OVA323-339) were from Eurogentec-Anaspec, CA, USA. Table 3 lists the amino acid sequences of the MCMV peptides described herein. Arens (The Journal of Immunology 180f 10): 6472-6476 (2008)) and Munks (The Journal of Immunology 177(l):450-458(2006)) disclose the MCMV peptides used herein, and are incorporated herein by reference.
Table 3: Exemplary sequences of MCMV peptides iRGD (Certepetide) was acquired from LISATA Therapeutics. Mice were treated twice a week by retro-orbital (RO) injections of the indicated peptides (50pg of m09133-147; m25409-423; ml4224-38 or OVA323-339; Ipg of IE3416-423; m3831 -323; m45985-993 or OVA257-264, unless indicated otherwise) and iRGD (300pg). When iRGD was utilized, mice were first injected with iRGD in one eye followed by the mix of MCMV peptides 5 minutes later in the other eye. When indicated, mice were intraperitoneally injected with anti-PDl (lOmg/kg twice a week. Bioxcell) or anti-ILlOR (200pg/qL, once a week, Bioxcell) antibodies, or gemcitabine (5mg/kg, twice a week, Selleck Chem). Control mice were retro-orbitally injected with vehicle (PBS/DMSO equivalent to the amount in the MCMV peptide mix) and saline, or intraperitoneally injected with rat IgG controls for anti-PDl and anti-ILlOR blocking antibodies. Mice Immunization
8-week-old mice were immunized subcutaneously at the base of the tail with 200pL of an emulsion containing 50pg of each indicated peptide (MCMVp or OVAp) and CFA (0.5mg/mL final; Sigma). Booster immunizations containing peptides + IFA were performed 3 weeks later. Mice were tumor challenged 3 weeks later as described herein.
Histology’ 'Immunohistology
Upon harvesting, tumors were fixed in 10% Formalin for 24 - 48 hours and kept in 70% ethanol until paraffin embedded. Immunohistochemistry was performed. In short, formalin-fixed paraffin-embedded (FFPE) sections were de-paraffinized and subj ected to steam heat mediated antigen retrieval using a low pH buffer (eBioscience). Blocking was performed with 5% donkey and 5% goat serum. The sections were stained with cleaved caspase 3 (1:250) (Cat.no. 9579S, Cell Signaling) and CD3 (1 :250) (Cat.no. 11089, Abeam), and kept at 4 °C overnight and incubated with their respective secondary antibodies. Histological and immunofluorescence imaging were performed using a ZEISS AxioScan Z1 automated slide scanning microscope equipped with a 20x objective (NA 0.8). For brightfield imaging of H&E- stained specimens, an LED illumination system was employed in conjunction with a Hitachi HV-FL202SCL camera. Immunofluorescence imaging was conducted using a Colibri7 LED illumination system and ZEISS single-band filter sets (filter sets 49, 38, 43, and 50). Fluorescence images were captured using a Hamamatsu Orca Flash 4.0 v2 camera operating in 16-bit mode (Microscopy core, LJI). Staining was quantified using the Qpath software. Hematoxilyn and Eosin staining was performed and necrosis scores w ere assessed by a histopathologist blinded to the different treatment conditions.
FACS analysis.
Tumors were minced into small pieces and incubated at 37°C with rotation for 30 minutes in 5 mL of digestion buffer consisting of DMEM high glucose, 10% Gentle Collagenase/Hyaluronidase (GCH, STEM CELL), 10% FBS, and 10% DNasel (1 mg/mL stock, Roche). Samples were then filtered using a 70-pm filter and smashed with the back of a syringe while adding 5 mL of 2% FBS in PBS solution. Flow through was spun at 300g for 10 minutes and subjected to RBC lysis buffer(Pharmalyse, BD. Biosciences). Cells were counted and subjected to magnetic separation of tumor infiltrating lymphocytes using Easysep CD45+ TIL kit (STEM CELL). Spleen and liver cells were harvested and processed, and analyzed on an LSR-II Fortessa X20 (BD) or a Cytek Aurora. Dead cells were excluded by staining with LD blue (Invitrogen). Surface staining was performed by diluting antibodies in Brilliant Stain buffer (Invitrogen). Fixation/permeabilization was done with Foxp3/Transcription Factor Staining Buffer Set (eBioscience). For antibodies used see Table 4.
For tetramer-staining, NIH-provided biotinylated monomers were tetramerized using streptavidin-APC or -PE, and subsequently used to stain the cells. Briefly, tetramer staining was performed at room temperature for 1.5 to 2 hours. Live/dead and surface/intracellular staining were done subsequently. Data was analyzed using the FlowJo software.
Cytokine stimulation.
Total liver or purified CD45+ cells from the tumors were incubated for 4 hours at 37°C and 5% CO2 in 96- well plates in the presence of the indicated peptides (5pg/mL for each peptide) and GolgiPlug (Brefeldin A; eBioscience). Control cells were incubated in the presence of GolgiPlug alone. Cells were then stained as described herein.
Single-cell RNA-seq
Total live-tumor cells (n=3 per group) or total tumor-infiltrating T cells (n=3 per group) (some samples were a combination of 2 tumors) were sorted from the indicated group, counted, and processed for subsequent 5 'scRNAseq using Chromium GEM-X single cell 5’ v3 gene expression kit.
Data analysis
Data are presented as means ± SEM. The GraphPad Prism statistical package was used for statistical analyses (Graph- Pad Software, Inc.). Results were compared by one-way ANOVA with Tukey correction, and repeated measures by two-way A NOVA with Sidak correction. Results were considered statistically significant when P < 0.05 and are indicated in the figures as follows: *P <0.05; **P < 0.01; ***P < 0.001; ****p < 0.0001.
Whole Tumor scRNA-seq QC
FASTQ files were processed using cellranger multi (cellranger- 8.0.1) and aligned to cellranger-vdj-GRCM38. The resulting output files were then further filtered by cellbender (v0.3.2)(Fleming et al., 2023) using the default settings. Afterward, droplets that had at least a probability of 0.5 of being cells were retained for further quality filtering. Cells were then filtered for outliers by counts using the median absolute deviation. Additionally, cells were also excluded if they had a mitochondrial RNA content of greater than 5%, or if they had less than 600 UMI counts. Doublets were then filtered out using scDblFinder (v 1.16.0) (Germain et al., 2022)
Whole Tumor scRNA-seq annotation
Batch correction was performed using scvi (v 1.1.2) (Gayoso et al., 2022). To determine ideal hyperparameters, a hyperparameter sweep was performed for 100 samples from the sample space. The model with the lowest validation loss was selected, and used for training the scvi model for 114 epochs. Using the computed latent space, nearest neighbors were calculated as well as a UMAP, using 30 nearest neighbors and a minimum distance of 0.1 for the UMAP. Cells w ere clustered using the Leiden algorithm implemented in scanpy (version 1.10. 1) (Wolf et al., 2018), at a resolution of 0.6. Additionally, the igraph flavor was used, and it was run for 2 iterations. Clusters w ere then manually annotated using known marker genes (FIG. S7D)
Whole Tumor scRNA Differential Gene Expression and GSEA
For differential gene expression, cells were pseudobulked by cell type and sample. Afterward, differential gene expression was performed in EdgeR (v 4.0.16) (Robinson et al., 2010). GSEA w as then performed using ClusterProfiler (v 4.15.0.003) (Xu et aL, 2024). Whole Tumor CellChat Analysis
CellChat (v 2.1.2) (Jin et al., 2024) was used to perform CellChat analysis. Filtering was further performed to only include communications found in at least 10 cells and for pathways that were expressed in a minimum of 2 samples.
TILs single-cell Transcriptome and TCR Repertoire
Analysis of single-cell RNA sequencing (scRNA-seq) data were processed using the Cell Ranger v6. 1.2 count pipeline for library mapping, followed by aggregation with the aggr pipeline. The aggregated data were imported into the R environment, and Seurat (v4.1.1) was utilized for quality control, normalization, and clustering. Cells expressing fewer than 200 or more than 2,500 genes, or with mitochondrial gene content exceeding 5%, were excluded. Genes detected in fewer than three cells were filtered out. The gene expression matrix was normalized and scaled prior to principal component analysis (PCA). Principal components were selected based on the elbow plot, and clustering was performed at a resolution of 0.8. Differentially expressed genes within clusters were identified using Seurat’s FindAllMarkers function with the MAST statistical framework. T cell receptor (TCR) repertoire analysis was integrated with transcriptomic data using the SCrepertoire R package. The Seurat object was employed to organize both transcriptomic and clonotype metadata. TCR diversity’ was characterized through clonotype expansion analysis, V(D)J gene usage patterns, and CDR3 length distributions. Functions highlightClonesQ and combineExpressionQ were employed to map clonotype distributions onto UMAP projections. Antigen-specific TCR clonotypes for IE1, m38, and m45 were identified using the VDJ database (vdjdb.cdr3.net/) as well as an inhouse TCR sequence library derived from tetramer-sorted cells. Cells identified with dual antigen specificity' were manually reviewed to ensure proper annotation or reclassified as “other cells” when appropriate. Clonotype-specific transcriptional profiles were analyzed and visualized using UMAP embeddings, facilitating the identification of functionally distinct T cell subsets. Statistical analyses were performed to correlate clonotype features with transcriptional states. Data visualization was conducted using custom scripts and ggplot2, leveraging the capabilities of both Seurat and SCrepertoire for integrative analysis. Table 4: Antibodies and reagents used for flow cytometry staining
Results MCM V memory T cells can be redirected to fight pancreatic cancer
To induce an MCMV memory T cell pool, 4-5 weeks old C57bl6/J (B6) mice were infected and waited a minimum of 2 months to allow the establishment of latency, defined by maintenance of the viral genome without detectable lytic replication. MCMV -infected mice and non-infected control mice were then surgically implanted with KPC1242 tumor cells in their pancreas. When tumors were palpable (~ 9-11 days post tumor cell injection (dpi)), mice were treated bi-weekly with either vehicle (DMSO/PBS) or iRGD plus MCMVp therapy (three 15-mer CD4 T cell peptide epitopes derived from the viral proteins m09, m25, and ml42; and three 8/9- mer CD8 T cell peptide epitopes derived from the viral proteins m38. m45. and IE3) (FIG. 1A). Tumor growth was monitored by ultrasound and mice were weighed twice a week.
Infected mice treated with iRGD + MCMVp therapy showed dramatically reduced tumor growth, while infection alone or treatment of uninfected mice showed no benefit (FIGS. IB and 8A bottom panel). Histological analysis of the tumor tissue showed increased necrosis, cleaved caspase-3, and CD3+ T cell infiltration (FIG. 1C). On the other hand, the same analysis in the liver showed increased T cell infiltration but no cleaved caspase-3 staining (FIG. ID) Despite the beneficial effects of MCMVp therapy, some toxicity resulting in significant weight loss was observed in the infected and treated group, possibly due to the high doses of MCMVp used initially (50pg of each) (FIG. 8A top panel). This was associated with a massive expansion of highly proliferative (Ki67) and activated (CD69) MCMV specific CD8 T cells in the tumor and spleen (>80% of total T cells), while expansion of MCMV CD4 T cells in the spleen was minimal (FIGS. 8B and 8C). Therefore, a dose titration of the MCMVp was performed to test if a therapeutic window with reduced toxicity could be achieved. After testing different doses of CD4 and CD8 peptide epitopes (FIG. 9A) it was determined that Ipg of CD8 peptide epitopes and 50pg of CD4 peptide epitopes (Dose A) was optimal.
Notably, this dose of MCMVp therapy-imposed tumor growth control without inducing measurable weight loss. A survival study conducted at this dose showed a 68% increase in survival with a median of 25 days for infected mice treated with vehicle vs 42 days for infected mice that underwent MCMVp therapy (p=0.0027) (FIGS. 2A and 9A). Next, the effects of iRGD or MCMVp alone on tumor growth and surv ival were tested and compared to the combination. iRGD alone had no beneficial effect, but surprisingly MCMVp alone had the same effect on T cell infiltration, tumor growth, and survival despite the omission of iRGD (FIGS. 9B-9D). Interestingly, 3 immunodominant MCMV-specific T cell populations (m25 CD4 T, and m38/m45 CD8 T cells) were still preferentially enriched in the tumor compared to the spleen and liver despite the absence of iRGD, indicating that an iRGD- independent mechanism was mediating their tumor localization (FIG. 9E).
Since high PD-1 expression was observed in MCMV-specific TILs (FIG. 8C), it was assessed whether checkpoint blockade could further enhance MCMVp therapydependent tumor control. As KPC tumors contain high levels of IL- 10 producing myeloid-derived suppressor cells (MDSC), the treatment was combined with a blocking anti-IL-lOR antibody. However, no additional benefits to MCMVp therapy were observ ed, even when both PD-1 and IL-10R were combined (FIG. 9F).
Other combination therapies were tested such as combination with anti-ILla blocking antibodies, however no benefit was observed (FIGS. 22A and 22C). This was the last treatment study performed in combination with iRGD. The rest of the studies were conducted without the addition of iRGD.
It was next sought to assess whether combining MCMVp therapy with a chemotherapeutic agent could show additional benefit. Gemcitabine (Gem), one of the standard of care drugs in pancreatic cancer therapy, was used which was previously shown to reduce the levels of MDSCs and enhance T cell immunity' when used at low' doses (5mg/kg) (28-30). In this treatment study, it was found that while Gem lacked any anti-tumor effect on its own at 5mg/kg, co-treatment with MCMVp showed significant benefit over either monotherapy, with no apparent toxicity, resulting in a further delay in tumor grow th and significantly decreased tumor weights at end point (FIG. 2B). A repeat experiment of these treatment conditions showed a significant delay in tumor growth and survival in mice treated with MCMV therapy plus low- dose Gem (+/-iRGD) (FIG. 22B). All the tested doses were well tolerated (FIG.
22C).
In order to show7 that the benefits of MCMVp therapy were not restricted to a single pancreatic tumor model, the C57B16/J (B6) x!29SvJ (129) Fl hybrid model was implanted with the syngeneic KPC46 tumor cell line. Similar to KPC 1242 tumors, neither MCMV infection without MCMVp therapy or peptide epitope treatment of uninfected mice impacted tumor growth. Only infected mice treated with MCMVp therapy showed a significant decrease in tumor growth (FIG. 2C). In this model, MCMVp therapy alone showed enhanced efficacy even as compared to KPC1242 in B6 mice, with Gem having no additional benefit. In addition, the effects of C/.-CD40 was evaluated alone or in combination with MCMV therapy. Ultrasound monitoring showed a significantly reduced tumor growth in mice treated with the combination of MCMV therapy and a-CD40, demonstrating their additive effects (FIG. 21A). Similarly, tumor weights at end point showed a reduced tumor weight especially in the a-CD40+ MCMV therapy group (FIG. 21B). Taken together, it was shown that harnessing pre-existing CMV memory T cell responses had major therapeutic benefit in two different aggressive orthotopic pancreatic cancer models.
MCMV T cells expanded following treatment, and preferentially localize to the tumor
To characterize the phenotype and effector function of the MCMV specific T cells in spleen, liver and tumor, various therapeutic regimens were used as described herein (FIGS. 3 and 10). First, the total number of CD8 T cells and CD4 Tconv (CD4T effector cells not including Treg) was determined, and found (i) no differences in the spleen, (ii) increased CD8 and CD4 Tconv in the liver of infected mice treated with the MCMVp therapy (+/-Gem), and (iii) increased CD8 T cells in tumors of the same 2 therapeutic groups. (FIG. 10A). To assure specificity, MCMV-specific T cells were identified using both MHC-I and -II tetramers labeled in both APC and PE, and only double-positive cells were analyzed (FIGS. 3 A and D). MCMV-specific CD4 Tconv cells were detected exclusively in tumors from infected mice treated the MCMVp therapy. The spleen contained a low proportion of MCMV T cells, while the liver and tumor had higher and more comparable levels. The dominant epitope in the liver was ml42, while tumors were more enriched with m25 specific T cells (FIGS. 3A and 3B). Quantification of the absolute number of m09, m25 and ml 42 specific Tconv cells in tumors (FIG. 3C), livers and spleens (FIG. 10B) paralleled their proportions shown in FIG. 3B. In addition, CD4+FoxP3+ MCMV-specific regulatory T cells were analyzed, and showed a much lower proportion than CD4 Tconv (FIG. 10D), suggesting that in vivo expansion of MCMV-specific CD4 T cells resulted in the differentiation of a bonified antiviral effector CD4 T cell rather than an immunoregulatory one.
Notably, m38 CD8 T cells (and to a lesser extent IE3) were detectable in tumors from infected mice, even in the absence of MCMVp therapy while m45 remained low, as compared to uninfected mice where virtually no MCMV T cells were detected (FIG. 3E). However, this did not translate to a significantly higher absolute number of these T cells in the tumor (FIG. 3F). This slight tumor infiltration of IE3 and m38 T cells in the absence of MCMVp therapy may be due to the effector memory phenotype and “inflationary ” nature of these memory' T cells that increase in numbers over time, while m45 cells show a central memory phenotype and do not inflate.
After MCMVp therapy7 an expansion of m38 and m45 CD8 T cell proportions in the liver as well as the tumor was observed (FIGS. 3D and 3E), which was commensurate with an increase in their absolute numbers in tumors (FIG. 3F) and livers (FIG. IOC), while IE3 CD8 T cells failed to expand significantly and infiltrate the tumor. Taken together, these data showed that MCMVp therapy of infected mice selectively enhanced the presence of m25, m38 and m45 specific T cells in the tumor and to a lesser extent in the liver. Despite the increase in MCMV-specific T cells in the liver following MCMV therapy, histological analysis by cleaved caspase 3 staining showed no tissue damage, even at the highest doses of MCMVp (FIG. 8D).
MCMV-specific CD 8 T cells showed a marked effector phenotype
The pancreatic cancer TME is highly immunosuppressive, tending to inhibit T cell effector function and promote tumor growth. Consequently, analyzed the phenotype and function of tumor-resident MCMV-specific CD8 T cells were analyzed to assess their potential anti-cancer activity7. The expression levels of activation markers (CD44, CD69), costimulatory receptors (CD226), inhibitory receptors (PD1, LAG3, and TIM3) and cytotoxic molecules (Prf, GzmA, and GzmB) were assessed. All of these markers except GzmA were significantly increased in m45-specific CD8 TILs from infected mice treated with MCMVp therapy (FIG. 4A). Of note, GzmB levels were significantly higher in mice treated with MCMVp+Gem, providing a possible mechanistic explanation for the enhanced tumor control in this group compared to the MCMVp therapy alone. M45-specific CD8 T cells also showed a very similar phenotype in the liver, suggesting a conserved phenoty pe in multiple tissues (FIG. 11A). While PD1, LAG3 and TIM3 are often called “exhaustion markers”, they don’t define functional exhaustion as they are also upregulated early during T cell activation to dampen excessive inflammation (31). Consistently, a strong expression of activation markers and cytotoxic molecules in T cells expressing these immune checkpoint molecules was observed, arguing against an exhausted state. In vitro re-stimulation of the tumor and liver cells with a mix of the 6 MCMV peptide epitopes resulted in robust IFNy and TNF production by CD8 T cells, further indicating these cells were not exhausted (FIGS. 4B and 11D).
Of note, MCMV -specific T cells also established tumor residence in infected mice that did not receive MCMVp therapy, as shown by tetramer binding (FIG. 3E) and cytokine production (FIG. 4B). MCMVp therapy further increased both IFNy and TNF production by these TILs. This enhanced IFNy was most evident in the MCMVp+Gem group, correlating with greater tumor growth control, and the same was true for IFNy production by CD4 Tconv in MCMVp+Gem treated mice (FIG. 4B). Following MCMV infection, m45-specific CD8 T cells showed conventional early expansion/contraction kinetics and displayed a central memory T cell phenotype. Conversely, m38- specific CD8 T cells displayed unique kinetics. They underwent early expansion paralleling m45, but only weakly contracted and continued to “inflate” in numbers over time displaying an effector- memory T cell phenotype.
Despite the intrinsic differences in these two distinct antiviral CD8 T cell populations, the phenoty pe of m38 and m45 populations was highly similar in both liver and tumor (FIGS. 4A and 11A-11C). GzmA and GzmB expressions were significantly higher in liver-resident m38 compared to m45 T cells in infected mice not treated with MCMVp therapy (FIGS. 11A and 11B). However, once treated with MCMVp ± Gem, both CD8 T cell populations showed similar Gzm expression in the liver. In the tumor, both these CD8 T cell populations expressed lower GzmA compared to the liver. Surprisingly, tumor resident m38 T cells downregulated GzmA expression after MCMVp therapy, but upregulated GzmB (FIG. 11C). This downregulation of GzmA may7 result from the TME, but, importantly, did not prevent the beneficial effect of MCMVp therapy.
MCMVp treatment induced profound changes in the tumor microenvironment
To assess the effect of MCMVp therapy on the TME, scRNAseq was performed on whole tumors from MCMV infected mice treated with vehicle or MCMVp (FIG. 5A). The major tumor cell populations were epithelial cells, macrophages, T cells, fibroblasts, and dendritic cells. As shown (FIGS. 5A, B, and 12A), the most significant change in the MCMVp therapy group was a marked increase in T cells (0.27%Veh vs 5.7%MCMVp). Additionally, a trend was seen for decreased pancreatic cancer cells (80%Veh vs 66%MCMVp), and increased proportion of macrophages (17%Veh vs 25%MCMVp) and dendritic cells (0.7%Veh vs 1.87%MCMVp). Fibroblasts and other stromal cell populations are known to support tumor growth and shape the TME (32). GSEA analysis of fibroblasts, macrophages and epithelial cells revealed an increase in type-I and type-II IFN response genes and a decreased expression of genes involved in angiogenesis and epithelial- mesenchymal transition (EMT) (FIGS. 5C and 12B). The KRAS signaling pathway was also decreased in all three cell types, a pathway which is highly active in KPC tumor cells. In epithelial cells, a notable difference was decreased collagen and increased MFIC-I expression (FIG. 12C), two factors that would be consistent with an enhanced anti-tumor immune response.
CellChat was utilized to investigate how cell-cell interactions may be impacted by MCMVp therapy. This analysis suggested reduced interactions between fibroblasts and both epithelial cells and macrophages, while T cell interactions with macrophages, dendritic cells and epithelial cells were increased (FIG. 5D). This conclusion was based on the increased expression of chemokines, complement, APRIL/7h/s/ 3, and Tnf mediated crosstalk between T cell and macrophages. Decreased TGF-P and SPPI dependent pathways was also observed (FIG. 5E), which are involved in maintaining the immunosuppressive TME phenotype (33, 34). TGF- P dependent signaling netw orks also decreased betw een T cells, epithelial cells and fibroblasts (FIGS. 5E and 5F). In turn, MCMVp therapy increased potential FasLg interactions between T cells and fibroblasts, but not between T cells and epithelial cells. Importantly, fibroblast-epithelial cell interactions involving grow th factors (FGF, VEGF, EGF) were reduced following MCMVp therapy, which would be consistent with a better prognosis (FIG. 5F). Taken together, these results demonstrated that a peptide-based therapy can induce significant changes in the TME that lead to improved survival.
MCMV therapy led to tumor infiltration of activated and cytotoxic MCMV TCRafi cells To further explore the TIL phenotype following the MCMVp therapy, and whether "bystander activation’ of T cells that are not virus specific may occur, a second scRNAseq on FACS sorted CD3+CD4+ and CD3+CD8+ T cells was performed (FIG. 6). Approximately 50,000 T cells were analyzed for both gene expression and V(D)J CDR3 sequences to assess the overall TCRafl repertoire. Bioinformatic analysis yielded 20 distinct T cell clusters (FIG. 6A), with 13 composed by CD8 T cells. 5 representing CD4 Tconv and 2 FoxP3+ CD4 Treg (FIGS. 6A and 6B).
Analysis of T cells in mice that were treated with MCMVp therapy clearly showed the emergence of 5 new clusters (FIGS. 6B and 13A). Clusters 1, 7, 12, 14 and 16 were largely exclusive to the MCMVp therapy group (FIG. 6C), with cluster 7 being CD4 Tconv and the other four composed of CD8 T cells. To assess whether these clusters contained MCMV-specific T cells, their TCRa and TCR0 CDR3 sequences were analyzed (FIG. 6D). In the CD4 Tconv cluster 7, a strong signal for m25 and ml42-associated CDR3s. and an intermediate signal for m09 were observed. In cluster 7, -25% of CD4 T cells in the MCMVp group contained MCMV TCRaP sequences compared to -1% in infected mice not treated with the therapy. Similarly, a large proportion of m45 and m38-associated CDR3s in clusters 1, 16 and 14, and a weaker signal in cluster 12 were detected (FIGS. 6D and 6E). Next, the analysis was repeated by looking exclusively at TCR0 sequences, since more MCMV-specific TCRP sequences are known, compared to the TCRa sequences (FIGS. 6F and 13B). Up to 50% of cluster 1 was composed of MCMV-specific CD8 T cells, half of that population expressed m45-specific CDR3, and the other half expressed CDR3 ascribed to both m45/m38 (FIG. 6F). Since cluster 1 was the second most abundant cluster (FIG. 13C), it was concluded that MCMV-specific CD8 T cells constituted the majority of the tumor infiltrating T cells.
All 20 clusters were analyzed for the expression of key transcription factors, cytotoxic molecules, cytokines and co-signaling receptors that regulate T cell effector function (FIG. 6G). All clusters induced by the MCMVp therapy expressed high levels of Tbx21 (T-bet) and 7/hy. Cluster 1 expressed very high levels oiPrfl, Gzmh. Gzmk and Fasl, indicative of high cytotoxic potential. As seen by flow cytometry (FIGS. 4 and 11), elevated expression of co-inhibitory receptors was observed in cluster 1 (Pdcdl. Haver 2. Lag3), as well as Ctla4 and Tigit. However, enhanced levels of several costimulatory receptors were also observed (Cd226. Klrkl, (2127. Cd28. Tnfsf9 These results were consistent with these m45 and m38 tumor resident T cells being highly activated/differentiated. Notably, cluster 7 which contained the highest numbers of m25 and ml 42 CD4 Tconv, had the most IJhy expression of any cluster, and also showed high expression of costimulatory receptors Cd28, Tnfrsf4 (0X40), Tnfrsfl8 (GITR), and Cd4()lg. which was consistent with a strong effector phenotype. The top 5 differentially expressed genes in cluster 1 were Haver 2 (TIM3), Pdcdl (PD1), Rgsl6^ I.agS, and Cxcr . and in cluster 7 Maf Tnjrsf4, Glrx and Izumolr (FIG. 13D). Interestingly, glutaredoxin (Glrx) regulated redox homeostasis and cellular oxidative stress, which may help sustain T cell function in a stressful environment like the TME.
Vaccine-induced T cells localized to tumors, but had no clinical benefit in the absence of MCMV infection
Because MCMV-specific T cells preferentially localized to tumors in the absence of iRGD, the underlying mechanism was investigated. Mice were infected with MCMV, and latency was established before tumor cell implantation. Therefore, if viral replication occurs directly in the KPC tumor cells, reactivation from latency and subsequent infection of these cells would have to occur. Not unexpectedly, MCMV productively infected the KPC cells in culture, as shown using a MCMV- mCherry reporter virus (FIG. 7A). Next, it was tested whether the MCMV DNA could be detected by qPCR in tumors implanted in the infected mice. qPCR detected MCMV DNA in livers and spleens from day 4 acutely infected mice, but was undetectable in KPC 1242 (FIG. 7B) or KPC46 (FIG. 14A) tumors.
As these analyses showed that MCMV was not directly infecting the tumor and promoting virus-specific T cell recruitment, it was tested whether infection was even required for their tumor-residency. This was done by immunizing mice with MCMV or ovalbumin (OVA)-derived CD8 and CD4 T cell epitopes, implanting tumors, and recalling these vaccine-induced memory T cells with peptide injection once the tumors were palpable (FIG. 7C). Tumor growth was monitored by ultrasound for 2 weeks, and then the mice were sacrificed to analyze the MCMV or OVA-specific T cells from tumors, draining lymph nodes (dLN), and spleens. Contrary to the MCMV infected mice, the MCMVp therapy did not curtail tumor growth to nearly the same extent when memory T cells were induced by vaccination. Recalling the vaccine-induced MCMV or OVA T cells had no impact on tumor volume and showed only a modest reduction of tumor weight in both groups at the experimental end point (FIG. 7D). Somewhat unexpectedly, very high numbers of m45 and m38 CD8 T cells were found in the tumors of the MCMVp immunized group (FIG. 7E), more than that observed in dLN and spleens, with more modest numbers of m25 and m!42 CD4 Tconv (FIG. 14B).
These results demonstrated that viral infection is not required for preferential accumulation of MCMV-specific T cells within the tumor, and the same was observed for OVA-specific T cells (FIG. 7F). Phenotyping of tumor resident m45 CD8 T cells showed expressions of CD69, PD1, LAG3, Prf and GzmB (FIG. 7G). Restimulation of CD45+ tumor cells with either OVA or MCMV CD8 T cell peptide epitopes also stimulated IFNy production (FIG. 7H). However, the expression of activation and cytotoxic markers observed in tumor-resident m45 CD8 T cells was lower in the vaccinated mice compared to the infected mice after peptide recall of the memory cells. Specifically, the expressions of CD226, GzmA and GzmB did not increase to nearly the extent in vaccine induced m45 CD8 T cells after MCMVp therapy as observed for the infected mice (e.g. 70% GzmB+ vs 30% GzmB in infection vs immunized groups; FIGS. 71 and 7 J). Taken together, these results suggested that while viral infection was not absolutely required for preferential T cell tumor infiltration after MCMVp therapy (i.e. vaccine-induced T cells also localized to tumors when recalled), the MCMV-specific memory T cells induced by infection were superior at tumor growth control.
Example 2: Testing the effects of CMV peptide epitopes in humanized pancreatic cancer mouse models
To evaluate engraftment kinetics and determine the timeframe for developing Graft Versus Host Disease (GvHD), NOD scid gamma (NSG) mice and NSG-MHC I/II DKO mice were retro-orbitally injected with a patient’s PBMCs for T-cell engraftment. GvHD can kill the animal within 3-5 weeks post PBMC inoculation, particularly in NSG mice. On the other hand, NSG MHCI/II DKO mice show delayed GvHD and may be more suitable for these experiments. These humanized mice were then engrafted with patient’s human pancreatic tumor tissue or cells, and w ere challenged with IV injection of a human CMV peptide pool or specific HCMV peptide epitopes (previously selected based on blood reactivity) to elicit a CMV- specific T-cell killing of the tumor. Tumor models were generated using pancreatic cancer tissues obtained from both CMV-positive and -negative patients with available PBMC samples or PBMCs obtained in serial collections. Tumor characteristics and grow th kinetics w ere evaluated to optimize timing and determine inter-patient tumor variability. CMV+/- tumors were orthotopically implanted into corresponding humanized mice generated using the patient's PBMC. Mice were treated twice a week with CMV-peptide epitopes to elicit CMV-specific T-cell killing of the tumor.
Optimizing PBMC isolation and storage protocols
To isolate PBMCs of the highest quantity and viability’ for humanized mouse model development, PBMCs were isolated using two protocols, SepMate and Easy Sep. The PBMCs were isolated from whole blood obtained from a healthy donor. Table 5 show s the cell counts and viabilities obtained from the tw o protocols. The Easy Sep protocol yielded the highest live cell count, about 9 million more cells in the same total volume of cells, and a viability of about 77 %. In contrast, the SepMate had a lower cell count with a 10 % decrease in cell viability. Therefore, the Easy Sep protocol was used all future PBMC isolations. The isolated PBMCs from the tw o protocols w ere combined and injected retro-orbitally in NSG mice at concentrations of 10 million, 5 million (n = 3 each), and 2.5 million (n = 2).
Table 5. Comparison between two PBMC isolation protocols
To optimize PBMC storage conditions, CryoStor and Cell-Vive storage solutions were used. PBMCs were isolated from whole blood obtained from a PDAC treatment naive patient using the Easy Sep protocol. Initial PBMC counts w ere taken (viability of 98 %). and 7.5, 2.5 million PBMCs were injected into NSG mice (n = 3 each) for engraftment. The remaining PBMCs w ere divided and frozen in the two storage solutions at 20.6 million each in liquid nitrogen. After about six weeks (43 days), the PBMCs were thawed and counted (Table 6). The Cry oStor and the Cell- Vive storage solutions yielded 20 million total cells with 57 % and 69 % viabilities, respectively. The thawed PBMCs were mixed and injected into MHC I/II DKO mice for engraftment. The DKO mice were monitored for T-cell engraftment.
Table 6. Yield and viability of PBMCs after storage in CryoStor and Cell-Vive
CMV Serotyping of patients
ELISA was performed on serum samples obtained from the blood of PDAC patients. The analysis showed a high prevalence of CMV IgG present in the patient population (FIG. 15). PBMC has been isolated from the same patient (Patient ID 121.26) for engraftment in both NSG and NSG-MHC I/II DKO mice.
Developing PDX and organoid models from patient tumors
Tumor sample was obtained from the patient with ID 121.26. Organoids were generated and expanded from the tumor tissues (Figure 16). These organoids can be orthotopically injected into the pancreas of an NSG mouse to develop into tumors.
Additionally, to develop alternative cancer model to test the effects of using preexisting CMV immunity directed against the tumor, two tumor chunks from the patient tumor were orthotopically implanted into NSG mice (n = 2) to develop patient-derived xenograft (PDX) (FIG. 17).
Monitoring the kinetics of PBMC engraftment
Next, to monitor the kinetics of PBMC engraftment in NSG mice, 10 million, 5 million, and 2.5 million PBMCs obtained from a healthy donor were engrafted in NSG mice, and engraftment was monitored weekly using flow cytometry and staining for mouse CD45, human CD45, CD3, CD4, and CD8 (FIG. 18). In this study, engraftment peaked at the week 4 and was coupled with weight loss in mice, a telltale sign of GvHD (FIGS. 19A and 19B). In all cases, more than 90 % of all the hCD45+ cells were CD3+ T-cells (FIGS. 19C-19F). In addition, PBMCs isolated from the CMV+ patient were engrafted in NSG mice and NSG MHCI/II DKO mice (FIGS. 20A and 20B). The patient’s PBMCs were much slower to engraft, with the highest engraftment being 4 % hCD45+ cells. T-cell engraftment kinetics in both mice models were monitored as well as the time it took for the mice to develop GvHD.
References:
1. Schadendorf, D., F. S. Hodi, C. Robert, J. S. Weber, K. Margolin, O. Hamid, D. Patt, T.-T.Chen, D. M. Berman, and J. D. Wolchok. 2015. Pooled Analysis of Long-Term Survival Data From Phase II and Phase III Trials of Ipilimumab in Unresectable or Metastatic Melanoma. JCO 33: 1889-1894.
2. Reck, M., D. Rodriguez-Abreu, A. G. Robinson, R. Hui, T. Csoszi, A. Fill dp, M. Gottfried, N. Peled, A. Tafreshi, S. Cuffe, M. O’Brien. S. Rao, K. Hotta, M. A. Leiby, G. M. Lubiniecki, Y. Shentu, R. Rangwala, and J. R. Brahmer. 2016. Pembrolizumab versus Chemotherapy for PD- Ll-Positive Non-Small- Cell Lung Cancer. N Engl J Med 375: 1823-1833.
3. Garon, E. B., N. A. Rizvi, R. Hui, N. Leighl. A. S. Balmanoukian, J. P. Eder, A. Patnaik, C. AggarwaL M. Gubens, L. Hom, E. Carcereny, M.-J. Ahn, E. Felip, J.-S. Lee, M. D. Hellmann, O. Hamid, J. W. Goldman, J.-C. Soria, M. Dolled-Filhart, R. Z. Rutledge, J. Zhang, J. K. Lunceford, R. Rangwala, G. M. Lubiniecki, C. Roach, K. Emancipator, and L. Gandhi. 2015. Pembrolizumab for the Treatment of Non-Small-Cell Lung Cancer. N Engl J Med 372: 2018- 2028.
4. Wei, S. C., C. R. Duffy, and J. P. Allison. 2018. Fundamental Mechanisms of Immune Checkpoint Blockade Therapy. Cancer Discovery 8: 1069-1086.
5. Australian Pancreatic Cancer Genome Initiative. ICGC Breast Cancer Consortium, ICGC MMML-Seq Consortium, ICGC PedBrain, L. B. Alexandrov, S. Nik-Zainal, D. C. Wedge, S. A.J. R. Aparicio. S. Behjati, A. V. Biankin, G. R. Bignell, N. Bolli, A. Borg, A.-L. Borresen-Dale,S. Boyault, B. Burkhardt, A. P. Butler, C. Caldas. H. R. Davies, C. Desmedt, R. Eils, J. E. Eyfjdrd, J. A. Foekens, M. Greaves, F. Hosoda, B. Hutter, T. Ilicic, S. Imbeaud, M. Imielinski, N. Jager, D. T. W. Jones, D. Jones. S. Knappskog, M. Kool, S. R. Lakhani, C. Lopez-Otin, S. Martin, N. C. Munshi, H. Nakamura, P. A. Northcott, M. Pajic, E. Papaemmanuil. A. Paradiso, J.V. Pearson, X. S. Puente, K. Raine, M. Ramakrishna, A. L. Richardson, J. Richter, P. Rosenstiel, M. Schlesner, T. N. Schumacher, P. N. Span, J. W. Teague, Y. Totoki, A. N. J. Tutt, R. Valdes- Mas, M. M. van Buuren, L. van ’t Veer, A. Vincent-Salomon, N. Waddell. L. R. Yates, J.Zucman-Rossi, P. Andrew Futreal, U. McDermott, P. Lichter, M. Meyerson, S. M. Grimmond, R. Siebert. E. Campo, T. Shibata, S. M. Pfister, P. J. Campbell, and M. R. Stratton. 2013. Signatures of mutational processes in human cancer. Nature 500: 415-421.
6. Schumacher, T. N., and R. D. Schreiber. 2015. Neoantigens in cancer immunotherapy. Science 348: 69-74.
7. Evans, R. A., M. S. Diamond, A. J. Rech, T. Chao, M. W. Richardson, J. H. Lin, D. L. Bajor, K. T. Byrne, B. Z. Stanger, J. L. Riley, N. Markosyan, R. Winograd, and R. H. Vonderheide. 2016. Lack of immunoediting in murine pancreatic cancer reversed with neoantigen. JCI Insight 1. Hiraoka, N., K. Onozato, T. Kosuge, and S. Hirohashi. 2006. Prevalence of FOXP3+ Regulatory T Cells Increases During the Progression of Pancreatic Ductal Adenocarcinoma and Its Premalignant Lesions. Clinical Cancel- Research 12: 5423-5434. Ino, Y., R. Yamazaki-Itoh, K. Shimada, M. Iwasaki, T. Kosuge, Y. Kanai, and
N. Hiraoka. 2013. Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer. Br J Cancer 108: 914-923.
10. Brahmer. J. R.. S. S. Tykodi. L. Q. M. Chow, W.-J. Hwu, S. L. Topalian. P. Hwu, C. G. Drake, L. H. Camacho, J. Kauh, K. Odunsi, H. C. Pitot, O. Hamid, S. Bhatia. R. Martins, K. Eaton, S. Chen, T. M. Salay. S. Alaparthy, J. F. Grosso, A. J. Korman, S. M. Parker, S. Agrawal, S. M. Goldberg, D. M. Pardoll. A. Gupta, and J. M. Wigginton. 2012. Safety and Activity of Anti- PD-L1 Antibody in Patients with Advanced Cancer. N Engl J Med 366: 2455- 2465. Foley, K., V. Kim, E. Jaffee, and L. Zheng. 2016. Current progress in immunotherapy for pancreatic cancer. Cancer Letters 381: 244-251. Royal, R. E., C. Levy, K. Turner, A. Mathur, M. Hughes, U. S. Kammula, R. M. Sherry’, S. L. Topalian, J. C. Yang, I. Lowy, and S. A. Rosenberg. 2010. Phase 2 Trial of Single Agent Ipilimumab (Anti-CTLA-4) for Locally Advanced or Metastatic Pancreatic Adenocarcinoma. Journal of Immunotherapy 33: 828-833. Balachandran, V. P., M. Luksza, J. N. Zhao, V. Makarov, J. A. Moral, R. Remark, B. Herbst, G. Askan, U. Bhanot, Y. Senbabaoglu, D. K. Wells, C. I.
O. Cary, O. Grbovic-Huezo, M. Attiyeh. B. Medina. J. Zhang, J. Loo. J. Saglimbeni, M. Abu-Akeel, R. Zappasodi, N. Riaz, M. Smoragiewicz, Z. L. Kelley, O. Basturk. Australian Pancreatic Cancer Genome Initiative, Garvan Institute of Medical Research, Prince of Wales Hospital, Royal North Shore Hospital, University of Glasgow, St Vincent’s Hospital, QIMR Berghofer Medical Research Institute, University of Melbourne, Centre for Cancer Research. University of Queensland, Institute for Molecular Bioscience, Banks town Hospital, Liverpool Hospital, Royal Prince Alfred Hospital, Chris O’Brien Lifehouse. Westmead Hospital, Fremantle Hospital, St John of God Healthcare, Royal Adelaide Hospital, Flinders Medical Centre, Envoi Pathology', Princess Alexandria Hospital, Austin Hospital, Johns Hopkins Medical Institutes, ARC -Net Centre for Applied Research on Cancer, M. Gonen, A. J. Levine, P. J. Allen, D. T. Fearon, M. Merad, S. Gnjatic, C. A. lacobuzio- Donahue, J. D. Wolchok, R. P. DeMatteo, T. A. Chan, B. D. Greenbaum, T. Merghoub, and S. D. Leach. 2017. Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer. Nature 551: 512-516. Rosato. P. C.. S. Wijeyesinghe, J. M. Stolley, C. E. Nelson, R. L. Davis, L. S. Manlove, C. A. Pennell, B. R. Blazar, C. C. Chen, M. A. Geller, V. Vezys, and D. Masopust. 2019. Virus-specific memory T cells populate tumors and can be repurposed for tumor immunotherapy. Nat Commun 10: 567. Quburu, N., L. Bialkowski. S. M. Pontejo. S. K. Sethi. A. T. F. Bell, R. Kim, C. D. Thompson, D. R. Lowy, and J. T. Schiller. 2022. Harnessing anticy tomegalovirus immunity for local immunotherapy against solid tumors. Proc. Natl. Acad. Sci. U.S.A. 119: e2116738119. 16. Teesalu, T., K. N. Sugahara, V. R. Kotamraju, and E. Ruoslahti. 2009. C-end rule peptides mediate neuropilin- 1 -dependent cell, vascular, and tissue penetration. Proc. Natl. Acad. Sci. U.S.A. 106: 16157-16162.
17. Sugahara, K. N., T. Teesalu, P. P. Karmali, V. R. Kotamraju, L. Agemy, D. R. Greenwald, and E. Ruoslahti. 2010. Coadministration of a Tumor-Penetrating Peptide Enhances the Efficacy of Cancer Drugs. Science 328: 1031-1035.
18. Hurtado de Mendoza, T., E. S. Mose, G. P. Botta, G. B. Braun, V. R. Kotamraju. R. P. French. K. Suzuki. N. Miyamura, T. Teesalu, E. Ruoslahti, A. M. Lowy. and K. N. Sugahara. 2021. Tumor-penetrating therapy for P5 integrin-rich pancreas cancer. Nature Communications 12: 1541.
19. Sugahara, K. N., G. B. Braun, T. H. de Mendoza, V. R. Kotamraju, R. P. French. A. M. Lowy. T. Teesalu. and E. Ruoslahti. 2015. Tumor-Penetrating iRGD Peptide Inhibits Metastasis. Molecular Cancer Therapeutics 14: 120- 128.
20. Liu, X., P. Lin, I. Perrett, J. Lin, Y.-P. Liao, C. H. Chang, J. Jiang, N. Wu, T. Donahue. Z. Wainberg, A. E. Nel, and H. Meng. 2017. Tumor-penetrating peptide enhances transcytosis of silicasome-based chemotherapy for pancreatic cancer. Journal of Clinical Investigation 127: 2007-2018.
21. Song, W., M. Li, Z. Tang, Q. Li, Y. Yang, H. Liu, T. Duan, H. Hong, and X. Chen. 2012. Methoxy poly (ethylene glycol) -block- Poly( L -glutamic acid)- Loaded Cisplatin and a Combination With iRGD for the Treatment of Non- Small-Cell Lung Cancers. Macromol. Biosci. 12: 1514-1523.
22. Deng, C„ M. Jia, G. Wei, T. Tan, Y. Fu, H. Gao, X. Sun, Q. Zhang, T. Gong, and Z. Zhang. 2017. Inducing Optimal Antitumor Immune Response through Coadministering iRGD with Pirarubicin Loaded Nanostructured Lipid Carriers for Breast Cancer Therapy. Mol. Pharmaceutics 14: 296-309.
23. Sha, H., R. Li, X. Bian, Q. Liu, C. Xie, X. Xin, W. Kong, X. Qian, X. Jiang, W. Hu, and B. Liu. 2015. A tumor-penetrating recombinant protein anti- EGFR-iRGD enhance efficacy of paclitaxel in 3D multicellular spheroids and gastric cancer in vivo. European Journal of Pharmaceutical Sciences 77: 60- 72.
24. Ding, N., Z. Zou, H. Sha, S. Su, H. Qian, F. Meng, F. Chen, S. Du, S. Zhou, H. Chen, L. Zhang, J. Yang, J. Wei, and B. Liu. 2019. iRGD synergizes with PD-1 knockout immunotherapy by enhancing lymphocyte infiltration in gastric cancer. Nat Commun 10: 1336.
25. Agemy, L., D. Friedmann-Morvinski, V. R. Kotamraju, L. Roth, K. N. Sugahara, O. M. Girard, R. F. Mattrey, I. M. Verma, and E. Ruoslahti. 2011. Targeted nanoparticle enhanced proapoptotic peptide as potential therapy for glioblastoma. Proc. Natl. Acad. Sci. U.S.A. 108: 17450-17455.
26. Dhanwani, R., S. K. Dhanda, J. Pham, G. P. Williams, J. Sidney, A. Grifoni, G. Picarda. C. S. Lindestam Arlehamn, A. Sette. and C. A. Benedict. 2021. Profiling Human Cytomegalovirus- Specific T Cell Responses Reveals Novel Immunogenic Open Reading Frames. J Virol 95: e0094021.
27. Picarda, G., and C. A. Benedict. 2018. Cytomegalovirus: Shape- Shifting the Immune System. J Immunol 200: 3881-3889.
28. Suzuki, E., V. Kapoor, A. S. Jassar, L. R. Kaiser, and S. M. Albelda. 2005. Gemcitabine selectively eliminates splenic Gr-H7CD1 lb+ myeloid suppressor cells in tumor-bearing animals and enhances antitumor immune activity. Clin Cancer Res 11: 6713-6721. 29. Sasso, M. S., G. Lollo, M. Pitorre, S. Solito, L. Pinton, S. Valpione, G. Bastiat, S. Mandruzzato, V. Bronte, I. Marigo, and J.-P. Benoit. 2016. Low dose gemcitabine-loaded lipid nanocapsules target monocytic myeloid-derived suppressor cells and potentiate cancer immunotherapy. Biomaterials 96: 47- 62.
30. Eriksson, E., J. Wenthe, S. Irenaeus, A. Loskog, and G. Ullenhag. 2016. Gemcitabine reduces MDSCs, tregs and TGFp-1 while restoring the teff/treg ratio in patients with pancreatic cancer. J Transl Med 14: 282.
31. Kissick, H., and R. Ahmed. 2022. New epigenetic regulators of T cell exhaustion. Cancer Cell 40: 708-710.
32. Kalluri, R. 201 . The biology' and function of fibroblasts in cancer. Nat Rev Cancer 16: 582- 598.
33. Du, Y„ Y. Lin, L. Gan, S. Wang, S. Chen, C. Li, S. Hou, B. Hu, B. Wang, Y. Ye. and Z. Shen. 2024. Potential crosstalk between SPP1 + TAMs and
CD8 + exhausted T cells promotes an immunosuppressive environment in gastric metastatic cancer. J Transl Med 22: 158.
34. Wu, J., Y. Shen, G. Zeng, Y. Liang, and G. Liao. 2024. SPP1+ TAM subpopulations in tumor microenvironment promote intravasation and metastasis of head and neck squamous cell carcinoma. Cancer Gene Ther 31: 311-321.
35. Smith, C. J., V. Venturi, M. F. Quigley, H. Turula, E. Gostick, K. Ladell, B. J. Hill, D. Himelfarb, K. M. Quinn, H. Y. Greenaway, T. H. Y. Dang, R. A. Seder, D. C. Douek, A. B. Hill, M. P. Davenport, D. A. Price, and C. M. Snyder. 2020. Stochastic Expansions Maintain the Clonal Stability of CD8+ T Cell Populations Undergoing Memory Inflation Driven by Murine Cytomegalovirus. J Immunol 204: 112-121.
36. Dash, P., A. J. Fiore-Gartland, T. Hertz, G. C. Wang, S. Sharma, A. Souquette,
J. C. Crawford, E. B. Clemens. T. H. O. Nguyen, K. Kedzierska, N. L. La Gruta, P. Bradley, and P. G. Thomas. 2017. Quantifiable predictive features define epitope-specific T cell receptor repertoires. Nature 547: 89-93.
37. Krenzlin, H., P. Behera, V. Lorenz, C. Passaro, M. Zdioruk, M. O. Nowicki,
K. Grauwet, H. Zhang, M. Skubal, H. Ito, R. Zane, M. Gutknecht, M. B. Griessl, F. Ricklefs, L. Ding, S. Peled, A. Rooj, C. D. James, C. S. Cobbs, C. H. Cook, E. A. Chiocca, and S. E. Lawler. 2019. Cytomegalovirus promotes murine glioblastoma growth via pericyte recruitment and angiogenesis. Journal of Clinical Investigation 129: 1671-1683.
38. Munks, M. W., K. S. Cho, A. K. Pinto, S. Sierro, P. Klenerman, and A. B. Hill. 2006. Four Distinct Patterns of Memory' CD8 T Cell Responses to Chronic Murine Cytomegalovirus Infection. The Journal of Immunology 177: 450-458.
39. Panagioti, E.. A. Redeker, S. Van Duikeren. K. L. Franken, J. W. Drijfhout, S. H. Van Der Burg, and R. Arens. 2016. The Breadth of Synthetic Long Peptide Vaccine-Induced CD8+ T Cell Responses Determines the Efficacy against Mouse Cytomegalovirus Infection. PLoS Pathog 12: el005895.
40. Commisso. C., S. M. Davidson, R. G. Soydaner-Azeloglu, S. J. Parker, J. J. Kamphorst, S. Hackett, E. Grabocka, M. Nofal, J. A. Drebin, C. B. Thompson, J. D. Rabinowitz. C. M. Metallo. M. G. Vander Heiden. and D. Bar-Sagi. 2013. Macropinocytosis of protein is an amino acid supply route in Ras- transformed cells. Nature 497: 633-637. 41. Qiu, Z., W. Liu, Q. Zhu, K. Ke, Q. Zhu, W. Jin, S. Yu, Z. Yang, L. Li, X. Sun, S. Ren, Y. Liu, Z. Zhu, J. Zeng, X. Huang, Y. Huang, L. Wei, M. Ma, J. Lu, X. Chen, Y. Mou, T. Xie, and X. Sui. 2022. The Role and Therapeutic Potential of Macropinocytosis in Cancer. Front. Pharmacol. 13: 919819.
42. Andreasson, C., D. Ansari, F. Ekbom, and R. Andersson. 2021.
Macropinocytosis: the Achilles’ heel of pancreatic cancer? Scandinavian Journal of Gastroenterology 56: 177-179.
43. Brunel. S., G. Picarda, A. Gupta. R. Ghosh, B. McDonald, R. El Morabiti, W. Jiang, J. A. Greenbaum, B. Adler, G. Seumois, M. Croft, P. Vijayanand, and C. A. Benedict. 2024. Late- rising CD4 T cells resolve mouse cytomegalovirus persistent replication in the salivary7 gland. PLoS Pathog 20: el011852.
44. Picarda, G., R. Ghosh, B. McDonald, S. Verma. N. Thiault, R. El Morabiti. T. S. Griffith, and C. A. Benedict. 2019. Cytomegalovirus Evades TRAIL- Mediated Innate Lymphoid Cell 1 Defenses. J Virol 93: e00617-19.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method for treating a cancer in a subject having pre-existing cytomegalovirus (CMV) immunity, the method comprising administering a therapeutically effective amount of a composition comprising one or more of human cytomegalovirus (HCMV) peptide epitopes to the subject having preexisting cytomegalovirus immunity.
2. A method for inducing an immunogenic response to a cancer in a subject having pre-existing cytomegalovirus (CMV) immunity, the method comprising administering a therapeutically effective amount of a composition comprising one or more human cytomegalovirus (HCMV) peptide epitopes to the subject having pre-existing cytomegalovirus immunity.
3. A method for treating a cancer in a subject with no pre-existing cytomegalovirus (CMV) immunity, the method comprising:
(a) inducing cytomegalovirus immunity in the subject with no pre-existing cytomegalovirus immunity;
(b) administering a therapeutically effective amount of a composition comprising one or more human cytomegalovirus (HCMV) peptide epitopes to the subject, thereby treating the cancer in the subject.
4. A method for inducing an immunogenic response to a cancer in a subject with no pre-existing cytomegalovirus (CMV) immunity’, the method comprising:
(a) inducing cytomegalovirus immunity in the subject with no pre-existing cytomegalovirus immunity; and
(b) administering a therapeutically effective amount of a composition comprising one or more human cytomegalovirus (HCMV) peptide epitopes to the subject, thereby inducing an immunogenic response to the cancer in the subject.
5. A method for treating a cancer in a subject in need thereof, the method comprising: (a) exposing isolated PBMCs from a subject to one or more human cytomegalovirus (HCMV) peptide epitopes;
(b) determining HCMV T cell reactivity within the isolated PBMCs to one or more HCMV peptide epitopes; and
(c) administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells of step (b), thereby inducing an immunogenic response against the tumor.
6. The method of claim 5, further comprising,
(a) expanding the HCMV reactive T cells ex vivo-, and
(b) administering the HCMV reactive T cells to the subject.
7. A method for treating a cancer in a subject in need thereof, the method comprising:
(a) exposing the isolated PBMCs from a subject to one or more human cytomegalovirus (HCMV) peptide epitopes;
(b) determining which HCMV T cells of the isolated PBMCs are reactive to one or more HCMV peptide epitopes;
(c) genetically modifying T cells to express a chimeric antigen receptor (CAR) that targets one or more HCMV peptide epitopes of step (b);
(d) administering the genetically modified T cells of step (c) to the subject; and
(e) administering to the subject one or more HCMV peptide epitopes recognized by the HCMV reactive T cells of step (b), thereby inducing an immunogenic response against the tumor.
8. The method of any of the above claims, wherein inducing cytomegalovirus immunity to the subject with no pre-existing cytomegalovirus immunity includes administering to the subject one or more human cytomegalovirus, one or more human cytomegalovirus (HCMV) peptide epitopes, or one or more mRNA molecules encoding one or more HCMV polypeptides.
9. The method of any of the above claims, wherein the one or more human cytomegalovirus includes an attenuated virus.
10. The method of any of the above claims, wherein the one or more human cytomegalovirus (HCMV) peptide epitopes include a 9-15mer peptide.
11. The method of any one of claims 1-4, wherein the composition comprising one or more human cytomegalovirus (HCMV) peptide epitopes include a 9- 15mer peptide.
12. The method of any one of claims 10 or 11. wherein the 9-15mer peptide includes a CD8 T cell epitope and/or a CD4 T cell epitope.
13. The method of any of the above claims, wherein the one or more human cytomegalovirus (HCMV) peptide epitopes include one or more amino acid sequences set forth in SEQ ID NOS: 1-422.
14. The method of any of the above claims, wherein the composition comprising one or more HCMV peptide epitopes include one or more amino acid sequences set forth in SEQ ID NOS: 1-422.
15. The method of any of the above claims, wherein the one or more human cytomegalovirus (HCMV) peptide epitopes is administered intraperitoneally, intravenously, or subcutaneously.
1 . The method of any of the above claims, wherein the composition is administered intraperitoneally, intravenously, or subcutaneously.
17. The method of any of the above claims, wherein the composition further includes an adjuvant.
18. The method of any of the above claims, wherein the adjuvant includes unmethylated cytosine-guanine dinucleotide (CpG), Polyinosinic- Poly cytidylic Acid Stabilized with Polylysine and Carboxymethylcellulose (Poly ICLC), montanide. or combinations thereof.
19. The method of the above claims, further comprising administering a tumor targeting agent.
20. The method of claim 19, wherein the tumor targeting agent includes certepetide.
21. The method of claim 20, wherein the tumor targeting agent includes a polypeptide, a nanoparticle, or a viral particle.
22. The method of any of the above claims, wherein the tumor targeting agent is co-administered with the composition.
23. The method of any of the above claims, wherein tumor targeting agent is administered before the administration of the composition.
24. The method of any of the above claims, wherein the composition is conjugated to a tumor targeting agent or a cancer therapy.
25. The method of any of the above claims, wherein the immunogenic response includes T cell expansion, T cell activation, T cell tumor infiltration, NK cell activation, expansion of y5 T cells, and/or tumor apoptosis.
26. The method of any of the above claims, further comprising determining the expression level of Neutropilin-1 and/or Integrin P5 in the subject in need thereof.
27. The method of any of the above claims, further comprising administering to the subject a cancer therapy, wherein the cancer therapy is a chemotherapeutic agent or an immunotherapeutic agent.
28. The method of any of the above claims, wherein the chemotherapeutic agent includes a DNA crosslinking agent, an alkylating agent, an anti-metabolite, an anti-microtubule agent, a topoisomerase inhibitor, or a cytotoxic antibiotic.
29. The method of claim any of the above claim, wherein the immunotherapeutic agent is a checkpoint inhibitor, an immunomodulator, a cytokine, a cancer vaccine, a monoclonal antibody, an oncolytic virus, an adoptive cell therapy, a CAR T cell therapy, or combinations thereof.
30. The method of any of the above claims, wherein the cancer therapy is coadministered with the composition.
31. The method of any of the above claims, wherein the cancer therapy is administered before the administration of the composition.
32. The method of any of the above claims, wherein the cancer therapy is administered after the administration of the composition.
33. The method of any of the above claims, wherein the subject is a mammal.
34. The method of any of the above claims, wherein the cancer is a solid tumor.
35. The method of claim 34, wherein the solid tumor is a gastric cancer, a breast cancer, a lung cancer, a prostate cancer, a glioma, a melanoma, an ovarian cancer, a liver cancer, a pancreatic cancer, a kidney cancer, a bladder cancer, a thyroid cancer, a sarcoma, a stomach cancer, a head and neck cancer, a cervical cancer, an endometrial cancer, an esophageal cancer, a thymoma, a soft tissue sarcoma, a bone cancer, a testicular cancer, a penile cancer, a gallbladder cancer, a uterine sarcoma, an adrenal gland cancer, an ampullary cancer, a hepatic angiosarcoma, a nasal, or a paranasal sinus cancer.
36. The method of the above claims, wherein the cancer is a blood cancer.
37. The method of claim 36, wherein the blood cancer is a leukemia, a lymphoma, a myeloma, a myelodysplastic syndrome, or a myeloproliferative neoplasm.
PCT/US2025/020840 2024-03-22 2025-03-21 Use of cytomegalovirus immunity against tumors Pending WO2025199395A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230407332A1 (en) * 2019-05-30 2023-12-21 Gritstone Bio, Inc. Modified adenoviruses

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230407332A1 (en) * 2019-05-30 2023-12-21 Gritstone Bio, Inc. Modified adenoviruses

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BRITSCH ISABEL, VAN WIJNGAARDEN ANNE P., KE XIURONG, HENDRIKS MARK. A.J.M., SAMPLONIUS DOUWE F., PLOEG EMILY M., HELFRICH WIJNAND: "Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selectively eliminate carcinoma cells", ONCOIMMUNOLOGY, TAYLOR & FRANCIS, UNITED STATES, vol. 12, no. 1, 31 December 2023 (2023-12-31), United States, pages 2207868, XP093360791, ISSN: 2162-402X, DOI: 10.1080/2162402X.2023.2207868 *

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