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WO2025019872A2 - Identification of novel sequences of the ervmer34-1 human endogenous retroviral sequence family, and modification of those sequences for a more effective anti-cancer vaccine and other potential anti-cancer therapies - Google Patents

Identification of novel sequences of the ervmer34-1 human endogenous retroviral sequence family, and modification of those sequences for a more effective anti-cancer vaccine and other potential anti-cancer therapies Download PDF

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WO2025019872A2
WO2025019872A2 PCT/US2024/039057 US2024039057W WO2025019872A2 WO 2025019872 A2 WO2025019872 A2 WO 2025019872A2 US 2024039057 W US2024039057 W US 2024039057W WO 2025019872 A2 WO2025019872 A2 WO 2025019872A2
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ervmer34
seq
cell
cancer
protein
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WO2025019872A3 (en
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Jeffrey Schlom
Duane H. HAMILTON
Renee N. DONAHUE
Claudia M. Palena
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US Department of Health and Human Services
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US Department of Health and Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • HERVs Human Endogenous Retroviruses
  • HERVs are remnants of retrovirus germ line infections early in primate evolution and are not viruses. Remarkably, HERVs represent approximately 8% of the human genome.
  • HERVs are an extremely diverse group constituted by at least 3 major classes: Class I consisting of HERV-H, HHLA2 and HERV-E, among others; Class II consisting of HERV-K, among others; and a very diverse Class III, which includes ERVMER34-1 (HEMO).
  • HERVs encode env and gag sequences with a similar organization to Retroviridae, and they can be epigenetically regulated.
  • International Patent Publication WO 2021/150694 which is incorporated herein in its entirety describes human immunogenic epitopes of ERVMER34-1 (HEMO) and HHLA2 HERVs, which can be used in vaccines and other compositions for the prevention or therapeutic treatment of cancer.
  • the invention provides a protein or polypeptide derived from ERVMER34-1.
  • the invention further provides a nucleic acid encoding the inventive protein or polypeptide, a vector comprising the nucleic acid, cells comprising the protein or polypeptide, nucleic acid, and/or vector, and composition, such as a pharmaceutical composition, comprising any of the foregoing.
  • Figures 1A through 1E graphically present data concerning the identification of ERVMER34-1 as a potential therapeutic target.
  • Figure 1A RNA expression levels of ERVMER34-1 in multiple human carcinomas (black dots) along with histologically normal tissues adjacent to the tumor when available (grey dots). The shaded area represents the median expression of ERVMER34-1 in normal tissues adjacent to the tumor plus two standard deviations.
  • FIG. 1B Western blot of ERVMER34-1 expression in protein lysates of matched tumor and tumor adjacent tissues collected from patients diagnosed with either lung or colon adenocarcinomas (left panel). ERVMER34-1 expression in lung and colon protein lysates collected from healthy donors (right panel). GAPDH is used as a loading control.
  • Figure 1C Representative images of immunofluorescence-based staining of ERVMER34-1 protein, and the tumor-associated antigens CEA and brachyury in human cerebellum, colon, breast, uterus, and testis. Scale bars indicate 100 ⁇ m.
  • FIG. 1D Representative images of immunohistochemical staining of ERVMER34-1 protein expression in human colon carcinoma, bladder carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, and endometrial carcinoma. Cytokeratin is included as a tumor marker. Scale bars indicate 50 ⁇ m.
  • Figure 1E Representative images of immunohistochemical staining of ERVMER34-1 protein expression in normal lung, histologically normal lung tissues adjacent to the tumor, and lung squamous cell carcinoma tissue. Indicated case numbers correspond to those indicated in Table 6. Scale bars indicate 50 ⁇ m. DAPI staining also is represented.
  • Figures 2A through 2K present data demonstrating that ERVMER34-1 specific immunity can be expanded through in vitro stimulation.
  • Figure 2A Immune reactivity of healthy donor and cancer patient PBMCs against the 15-mer ERVMER34-1 peptide library, as assessed using a 16-hour IFN- ⁇ ELISPOT assay.
  • FIG. 2B Immune reactivity of individual 15-mer ERVMER34-1 peptides comprising the peptide library as assessed in 5 paired healthy donors by a 16-hour IFN- ⁇ ELISPOT assay (left panel) or following 7 days of in vitro stimulation with a ERVMER34-1 peptide library prior to performing a 16-hour IFN- ⁇ ELISPOT assay (right panel).
  • HLA-A alleles of each healthy donor are indicated below.
  • Each column represents a healthy donor, and each row indicates immune reactivity of each single ERVMER34-1 peptide that comprises the ERVMER34-115-mer peptide library.
  • FIG. 2C Immune reactivity of individual 15-mer ERVMER34-1 peptides in carcinoma patients following 7 days of in vitro stimulation with a ERVMER34-1 peptide library prior to performing a 16-hour IFN- ⁇ ELISPOT assay.
  • Figure 2D ERVMER34-1 immunity detected in lung, breast, and colon carcinoma patients as well as male and female healthy donor PBMCs following a 7-day incubation and 24-hour restimulation with a pool of ERVMER34-1 peptides as assessed by an intracellular cytokine assay. Each column indicates cytokine production from a single donor, and each row a single detected analyte.
  • FIG. 2E Representative data from an intracellular cytokine assay comparing relative immunogenicity of ERVMER34-1 to additional tumor- associated antigens.
  • Figure 2F Comparison of immunity obtained following a 7-day stimulation with either ERVMER34-1, CEACAM5, PSA and MUC1 in 12 healthy donors. Mapping the diversity of ERVMER34-1 reactive CD8+ T cells expanded from PBMCs from a healthy donor A ( Figures 2G and 2H) and donor B ( Figures 2I and 2J).
  • Figure 2K Lysis of the parental SW620 and SW620 ERVMER34-1 CRISPR knockout cell lines following overnight incubation with CD8+ T cells expanded from PBMCs from donor A at indicated effector-to- target ratios.
  • Figure 2L Percent specific lysis of the parental SW620 and SW620 ERVMER34- 1 CRISPR knockout cell lines following overnight incubation with CD8+ T cells expanded from PBMCs from donors B-D using an effector-to-target ratio of 5:1. **P ⁇ 0.01; ****P ⁇ 0.0001 by an unpaired T test.
  • Figures 3A through 3G present data demonstrating the generation of a therapeutic cancer vaccine targeting ERVMER34-1.
  • Figure 3A Graphical representations of the full-length and ERVMER34-1 vaccine.
  • Figure 3B Amino acid sequence of ERVMER34-1 with identified protein regions highlighted.
  • FIG. 3C ERVMER34-1 protein expression in human dendritic cells following infection with control, full-length or ERVMER34-1 vaccine adenovirus. Dendritic cells were infected 48 hours prior to protein isolation with viral particles at one of four multiplicities of infection (MOI): 20,000, 6,600, 2,220 or 730, respectively, in lanes from left to right. GAPDH is included as a protein loading control.
  • Figure 3D Graphical representation of experiment (left panel). Detection of soluble ERVMER34-1 protein in the sera of mice 1 day prior to and 3 days following vaccination with control, full-length, or ERVMER34-1 vaccine (right panel).
  • FIG. 3E Enumeration of ERVMER34-1 reactive T cells in splenocytes harvested from animals vaccinated with control, full-length or the ERVMER34-1 vaccine using a 16-hour IFN- ⁇ ELISPOT assay. Each row is an individual animal, and each column is a single peptide comprising the 15-mer peptide library.
  • Figures 3F and 3G Enumeration of ERVMER34-1 specific multi-functional T cells following vaccination with either the full-length ERVMER34-1 adenovirus or the ERVMER34-1 vaccine as assessed by an ex vivo intracellular cytokine production assay. Number indicates the percentage of total T cells responding to ERVMER34-1 peptides.
  • FIG. 3H Relative magnitude of ERVMER34-1 reactive CD4+ and CD8+ T cells.
  • Figure 4 graphically presents data demonstrating that vaccine targeting ERVMER34- 1 mediates tumor control of established EMT6 tumors.
  • Panel A Immunofluorescence staining of ERVMER34-1 protein expression in EMT6 parental and EMT6 pERVMER34-1 tumors. DAPI staining also is represented.
  • Panel B Treatment schema.
  • FIG. 5A through 5I graphically present data demonstrating vaccine targeting ERVMER34-1 mediates regression of established tumors.
  • FIG. 5A Immunofluorescence staining of ERVMER34-1 protein expression in MC38 parental and MC38 pERVMER34-1 tumors. DAPI staining also is represented.
  • Figure 5B Treatment scheme.
  • Figure 5C Survival Leydig 771368 E-056-2023-0-PCT-01 5 curves of mice treated as indicated in in panel
  • Figure 5D Enumeration of tumor infiltrating lymphocytes in mice treated as indicated in panel B.
  • FIG. 5E ERVMER34-1 immune reactivity of splenocytes and expanded tumor infiltrating lymphocytes harvested from animals vaccinated with control adenovirus or ERVMER34-1 vaccine as assessed by a 16-hour IFN- ⁇ ELISPOT assay using individual 15-mer ERVMER34-1 peptides in the assay.
  • Figures 5F and 5G Comparison of immune reactivity of ERVMER34-1, MC38 neoepitopes and p15e of tumor- infiltrating T cells.
  • Figure 5G Assessment of IFN- ⁇ present within the tumor microenvironment in mice treated as shown in panel E.
  • FIG. 5H Expression of PD-L1 on the surface of CD45+CD11b+ cells (left panel) and CD45 ⁇ cells (right panel) within the tumor.
  • Figure 5I Tumor growth curves following tumor rechallenge with parental MC38 cells (left flank) and MC38 pERVMER34-1 tumors (right flank). *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001 by an unpaired T test.
  • Figures 6A through 6F presents information and data concerning experiments demonstrating that a combination of immune checkpoint blockade with ERVMER34-1 vaccine enhances its anti-tumor efficacy.
  • Figure 6A Graphical representation of tumor treatment schedule used for subsequent panels.
  • FIG. 6B Spider plots of growth curves of MC38 pERVMER34-1 tumors treated as indicated in panel A.
  • Figure 6C Immune reactivity of splenocytes harvested from animals treated as indicated using a 16-hour IFN- ⁇ ELISPOT assay with individual 15-mer ERVMER34-1 peptides in the assay.
  • Figure 6D Immune reactivity of splenocytes harvested from animals treated as indicated using a 16-hour IFN- ⁇ ELISPOT assay with individual MC38 neoepitope peptides in the assay.
  • Figure 6E Survival curve of animals treated as indicated in panel A.
  • FIG. 6F Tumor growth curves following tumor rechallenge with parental MC38 cells (left flank) and MC38 pERVMER34-1 tumors (right flank). *P ⁇ 0.05; **P ⁇ 0.01 by an unpaired T test. *P ⁇ 0.05; **P ⁇ 0.01 by an unpaired T test.
  • Figure 7 graphically depicts: (Panel A) RNA expression of indicated endogenous retroviral transcripts in normal tissues in the GTex database.
  • FIG. 6F Tumor growth curves following tumor rechallenge with parental MC38 cells (left flank) and MC38 pERVMER34-1 tumors (right flank). *P ⁇ 0.05; **P ⁇ 0.01 by an unpaired T test.
  • Figure 7 graphically depicts: (Panel A) RNA expression of indicated endogenous retroviral transcripts in normal tissues in the GTex database.
  • Panel B RNA expression of indicated endogenous retroviral transcripts in tumor tissues in the Cancer Genome Atlas (TCGA)
  • Figure C Survival curves of patients diagnosed with uveal melanoma, head and neck squamous and adenoid cystic carcinomas based upon high and low ERVMER34-1 RNA Leydig 771368 E-056-2023-0-PCT-01 6 expression in tumor tissues. Cutoffs for high low ERVMER34-1 expression were set to the top 66% and lower 33%, respectively.
  • Figure 8 graphically depicts representative ELISPOT images.
  • Figure 9 graphically depicts the flow cytometric gating strategy for cytokine- producing T cells (Figure 9A) Human PBMCs, ( Figure 9B) Murine splenocytes.
  • Figure 10 graphically depicts: (Panel A) Western blot of ERVMER34-1 expression in either parental K562 cells or the aAPC cell line. (Panel B) Flow cytometric analysis of CD80 and HLA-A2 on either K562 or aAPC cell lines. (Panel C) Immune-fluorescent analysis of ERVMER34-1 expression in either parental SW620 (left panel) or SW620 ERVMER34-1 CRISPR knock-out cell line (right panel). DAPI staining also is represented.
  • Figure 11 graphically depicts tumor growth curves of MC38 pERVMER34-1 tumors treated with a single dose (dotted line) of either control adenovirus or ERVMER34-1 vaccine.
  • DETAILED DESCRIPTION OF THE INVENTION Proteins and Polypeptides [0017]
  • the invention provides a protein or polypeptide derived from ERVMER34-1.
  • the inventive protein or polypeptide can be either isolated or present in a composition or cell comprising other elements.
  • the term "isolated” as used herein encompasses compounds or compositions that have been removed from a biological environment (e.g., a cell, tissue, culture medium, body fluid, etc.) or otherwise increased in purity to any degree (e.g., isolated from a synthesis medium). Isolated compounds and compositions, thus, can be synthetic or naturally produced.
  • the inventive protein or polypeptide is a protein that comprises, consists essentially of, or consists of a sequence of amino acids substantially as set forth in SEQ ID NO:2. It will be observed that the amino acid sequence of SEQ ID NO:2 is derived from the full length ERVMER34-1 peptide sequence (SEQ ID NO:1).
  • SEQ ID NO:2 lacks the signal domain of the full length ERVMER34-1 amino acid sequence, which can prevent the trafficking of the protein to the cell surface.
  • SEQ ID NO:2 also lacks the cleavage site of the full length ERVMER34-1 amino acid sequence, which can prevent the cleavage of the surface unit Leydig 771368 E-056-2023-0-PCT-01 7 from the transmembrane unit of the 1 protein.
  • SEQ ID NO:2 also lacks predicted immunosuppressive domain of the full length ERVMER34-1 amino acid sequence, which can prevent the potential dampening of the immunity generated against the target antigen.
  • SEQ ID NO:2 lacks the transmembrane domain of the ERVMER34-1 protein, which shares homology with several human proteins.
  • the amino acid sequence of the inventive protein can comprise or consist essentially of SEQ ID NO:2 while not consisting exactly of SEQ ID NO:2.
  • the inventive protein or polypeptide can include one or more additional amino acid mutations (substitutions, insertions or deletions), for example, to inactivate or delete a natural biological function of the full length ERVMER34-1 protein (SEQ ID NO:1) (e.g., to improve expression or enhance safety of the antigen).
  • the inventive protein shares at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 99% sequence identity with, or is 100% identical to (i.e., consisting of) SEQ ID NO:2.
  • the inventive protein or polypeptide is a 15-mer polypeptide that comprises, consists essentially of, or consists of a sequence of amino acids substantially as set forth in SEQ ID NOs:7-99.
  • the amino acid of the inventive polypeptide consists of a sequence of amino acids exactly as set forth in SEQ ID NOs:7-99, although minor deviations from these sequences, when such do not alter the immunogenicity of the polypeptide, are within the scope of the present invention.
  • the inventive protein or polypeptide can be prepared by any method, such as by synthesizing the protein or polypeptide (e.g., solid-state synthesis) or by expressing a nucleic acid encoding an appropriate amino acid sequence for the peptide or polypeptide in a cell and, in some embodiments, harvesting the peptide or polypeptide from the cell.
  • the peptide or polypeptide is not harvested from the cell, such as in embodiments of the invention directed to a yeast-based immunotherapy composition, which is described in detail below.
  • a combination of such methods of production of peptides and polypeptides also can be used.
  • Methods of de novo synthesizing peptides and methods of recombinantly producing peptides or polypeptides are known in the art.
  • Leydig 771368 E-056-2023-0-PCT-01 8 Nucleic Acids [0022]
  • the invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding the inventive protein or polypeptide.
  • the inventive nucleic acid molecule can comprise DNA (genomic or cDNA) or RNA and can be single or double stranded. Furthermore, the inventive nucleic acid molecule can comprise nucleotide analogues or derivatives (e.g., inosine or phosphonothioate nucleotides and the like).
  • the inventive nucleic acid sequence can encode the inventive protein or polypeptide alone or as part of a fusion protein. Persons of ordinary skill are able to deduce the coding sequence of a nucleic acid when given the sequence of the encoded protein or polypeptide.
  • inventive nucleic acid encoding the peptide or polypeptide can be provided as part of a construct comprising the inventive nucleic acid molecule and elements that enable delivery of the nucleic acid molecule to a cell, and/or expression of the nucleic acid molecule in a cell.
  • elements include, for example, expression vectors, promoters, and transcription and/or translation control sequences.
  • constructs can also be referred to as "recombinant nucleic acid molecules.” Suitable vectors, promoters, transcription/translation sequences, and other elements, as well as methods of preparing such nucleic acid molecules and constructs, are known in the art.
  • the inventive nucleic acid can be made by any suitable method, such as solid-state synthesis, known to persons or ordinary skill.
  • the phrases “nucleic acid” and “nucleic acid molecule” primarily refer to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a peptide or polypeptide.
  • nucleic acid molecule primarily refers to a nucleic acid molecule operatively linked to an element such as a transcription control sequence but can be used interchangeably with the phrase "nucleic acid molecule.”
  • nucleic acid molecule primarily refers to a nucleic acid molecule operatively linked to an element such as a transcription control sequence but can be used interchangeably with the phrase “nucleic acid molecule.”
  • Leydig 771368 E-056-2023-0-PCT-01 9 Vectors [0026]
  • the invention further provides a vector comprising the inventive nucleic acid molecule comprising a nucleic acid sequence encoding the inventive protein or polypeptide.
  • plasmids e.g., DNA plasmids
  • bacteria e.g., yeast, listeria
  • viral vectors including poxvirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, polio virus, alphavirus, and baculorvirus, Sindbis virus.
  • the vector is a plasmid
  • the plasmid can be complexed with one or more agents for facilitating transfection of cells or enhancing stability, e.g., chitosan.
  • the inventive protein or polypeptide comprises, consists essentially of, or consists of a sequence of amino acids substantially as set forth in SEQ ID NO:2, the vector is an adenoviral vector.
  • a vector useful in the invention e.g., a plasmid or a viral vector
  • a vector useful in the invention also can comprise a nucleic acid sequence encoding one or more immunostimulatory/regulatory molecules, granulocyte macrophage colony stimulating factor (GM-CSF), cytokines, and/or molecules that can enhance an immune response (e.g., additional tumor-associated antigens).
  • GM-CSF granulocyte macrophage colony stimulating factor
  • cytokines e.g., additional tumor-associated antigens
  • TAAs tumor-associated antigens
  • cancer antigens include, but are not limited to, 5- ⁇ -reductase, ⁇ -fetoprotein (AFP), AM-1, APC, April, B melanoma antigen gene (BAGE), ⁇ -catenin, Bcl12, bcr-abl, Brachyury, CA-125, caspase-8 (CASP-8 also known as FLICE), Cathepsins, CD19, CD20, CD21/complement receptor 2 (CR2), CD22/BL-CAM, CD23/F c ⁇ RII, CD33, CD35/complement receptor 1 (CR1), CD44/PGP-1, CD45/leucocyte common antigen (LCA), CD46/membrane cofactor protein (MCP), CD52/CAMPATH-1, CD55/decay accelerating factor (DAF), CD59/protectin, CDC27, CDK4, carcinoembryonic antigen (CEA), c-my
  • the nucleic acid encoding the inventive protein or polypeptide, as well as any other exogenous gene(s), preferably is/are inserted into a site or region (insertion region) in the vector (e.g., adenovirus) that does not affect virus viability of the resultant recombinant virus.
  • a site or region in the vector (e.g., adenovirus) that does not affect virus viability of the resultant recombinant virus.
  • a site or region e.g., adenovirus
  • the inventive vector can include suitable promoters and regulatory elements, such as a transcriptional regulatory element or an enhancer.
  • suitable promoters include the SV40 early promoter, an RSV promoter, the retrovirus LTR, the adenovirus major late promoter, the human CMV immediate early I promoter, and various poxvirus promoters, such as the Pr7.5K promoter, 30K promoter, 40K promoter, I3 promoter, Prs promoter, PrsSynIIm promoter, PrLE1 promoter, synthetic early/late (sE/L) promoter, HH promoter, 11K promoter, and Pi promoter.
  • a cell e.g., isolated cell
  • inventive protein or polypeptide comprising (1) in inventive protein or polypeptide, (2) an inventive nucleic acid molecule encoding the inventive protein or polypeptide, and/or (3) a vector comprising an inventive nucleic acid molecule
  • Suitable cells include prokaryotic and eukaryotic cells, e.g., mammalian cells, yeast, fungi other than yeast, and bacteria (such as E. coli).
  • the cell can be used in vitro, such as for research or for production of the peptide or polypeptide, or the cell can be used in vivo.
  • the cell is a yeast cell, which may be used to provide a yeast vehicle component of the yeast-based immunotherapy composition as described herein.
  • the cell can be an antigen presenting cell (APC). Suitable APCs include, but are not limited to, dendritic cells, B lymphocytes, monocytes, macrophages, and the like.
  • the cell is dendritic cell. Dendritic cells of different maturation stages can be isolated based on the cell surface expression markers.
  • mature dendritic cells are less able to capture new proteins for presentation but are much better at stimulating resting T cells to grow and differentiate. Thus, mature dendritic cells can be of importance.
  • Mature dendritic cells can be identified by their change in morphology and by the presence of various markers. Such markers include, but are not limited to, cell surface markers such as B7.1, B7.2, CD40, CD11, CD83, and MHC class II. Alternatively, maturation can be identified by observing or measuring the production of pro-inflammatory cytokines.
  • Dendritic cells can be collected and analyzed using typical cytofluorography and cell sorting techniques and devices, such as a fluorescence-activated cell sorter (FACS).
  • FACS fluorescence-activated cell sorter
  • the invention provides a cell, such as an APC, engineered to express an ERVMER34-1 protein and one or both of HLA (such as human HLA-A2) and the CD80 costimulatory molecule.
  • the ERVMER34-1 protein can be or Leydig 771368 E-056-2023-0-PCT-01 12 comprise an amino acid sequence of or the full-length ERVMER34-1 protein (SEQ ID NO:1) or a modified version, such as the inventive protein (SEQ ID NO:2, including variants thereof as discussed above).
  • the cell can be generated, for example, by co-transfecting and/or - infecting an APC cell with vectors for expressing HLA (human HLA-A2 is discussed below in Example 6), CD80, and the desired ERVMER34-1 protein.
  • the cell thereafter can be expanded to generate a cell line, comprising APCs expressing the ERVMER34-1 protein along with HLA- A2 and the CD80 costimulatory molecule.
  • the cell line can be used in immunological research in vitro or, in some applications, therapeutically.
  • a cell comprising (1) the peptide or polypeptide, (2) a nucleic acid molecule encoding the peptide or polypeptide, and/or (3) a vector comprising the nucleic acid molecule also is provided herein.
  • Suitable cells include prokaryotic and eukaryotic cells, e.g., mammalian cells, yeast, fungi other than yeast, and bacteria (such as E. coli). The cell can be used in vitro, such as for research or for production of the peptide or polypeptide, or the cell can be used in vivo.
  • the cell is a yeast cell, which may be used to provide a yeast vehicle component of the yeast-based immunotherapy composition as described herein.
  • the cell can be a peptide-pulsed antigen presenting cell. Suitable antigen presenting cells include, but are not limited to, dendritic cells, B lymphocytes, monocytes, macrophages, and the like.
  • the cell is dendritic cell. Dendritic cells of different maturation stages can be isolated based on the cell surface expression markers. For example, mature dendritic cells are less able to capture new proteins for presentation but are much better at stimulating resting T cells to grow and differentiate. Thus, mature dendritic cells can be of importance.
  • Mature dendritic cells can be identified by their change in morphology and by the presence of various markers. Such markers include, but are not limited to, cell surface markers such as B7.1, B7.2, CD40, CD11, CD83, and MHC class II. Alternatively, maturation can be identified by observing or measuring the production of pro-inflammatory cytokines.
  • Dendritic cells can be collected and analyzed using typical cytofluorography and cell sorting techniques and devices, such as a fluorescence-activated cell sorter (FACS).
  • FACS fluorescence-activated cell sorter
  • compositions [0038]
  • inventive protein or polypeptide, nucleic acid, vector, or cell can be formulated as a composition (e.g., pharmaceutical composition) comprising the protein or polypeptide, nucleic acid, vector, or cell and a carrier (e.g., a pharmaceutically or physiologically acceptable carrier).
  • a carrier e.g., a pharmaceutically or physiologically acceptable carrier
  • the protein or polypeptide, nucleic acid, vector, cell, or composition of the invention can be used in the methods described herein alone or as part of a pharmaceutical formulation.
  • the composition e.g., pharmaceutical composition
  • Vectors and compositions of the invention can further include or can be administered with (concurrently, sequentially, or intermittently with) any other agents or compositions or protocols that are useful for inhibiting, preventing, or treating a disease or clinical condition.
  • the composition can comprise one or more other pharmaceutically active agents or drugs.
  • anticancer agents e.g., chemotherapeutic or radiotherapeutic agents
  • antimetabolites hormones, hormone antagonists, antibiotics, antiviral drugs, antifungal drugs, cyclophosphamide, and combinations thereof.
  • the composition can comprise one or more additional anti-cancer agents.
  • additional anti-cancer agents can be co-administered with the inventive composition, such as in a separate composition.
  • Suitable anticancer agents include, without limitation, alkylating agents, folate antagonists, purine antagonists, pyrimidine antagonists, spindle poisons, topoisomerase inhibitors, apoptosis inducing agents, angiogenesis inhibitors, podophyllotoxins, nitrosoureas, cisplatin, carboplatin, interferon, asparginase, tamoxifen, leuprolide, flutamide, megestrol, Leydig 771368 E-056-2023-0-PCT-01 14 mitomycin, bleomycin, doxorubicin, taxol, geldanamycin (e.g., 17-AAG), and various anti-cancer peptides and antibodies known in the art.
  • alkylating agents include, without limitation, alkylating agents, folate antagonists, purine antagonists, pyrimidine antagonists, spindle poisons, topoisomerase inhibitors, apoptosis inducing agents, an
  • Non-limiting examples of additional anti- cancer peptides that can be used in combination with the protein or polypeptide, nucleic acid, vector, or cell include those disclosed in International Patent Publication WO 2021/150694, which is incorporated herein in its entirety. Some of these peptides are set forth herein as SEQ ID NOs:100-140, and others are identified in the Tables within the specification of WO 2021/150694, which are incorporated herein by reference.
  • anti-cancer agents that can be included in the inventive composition, or be co-administered with the inventive composition, include, but are not limited to, ⁇ -PD-1, ⁇ -CTLA4, ⁇ -PD-1 + ⁇ -CTLA4
  • a particularly interesting combination for use in the present inventive methods includes SEQ ID NO:2, IL15 superagonist (N803, ANKTIVA (nogapendekin alfa inbakicept-pmln)), and/or anti- PD-L1.
  • Other checkpoint inhibitors can be employed in addition to or in lieu of anti-PD-L1, such as anti-CTLA4 and/or anti-PD-1.
  • alkylating agents include, but are not limited to, nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin, or dacarbazine).
  • nitrogen mustards e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil
  • alkyl sulfonates e.g., busulfan
  • nitrosoureas e.g., carmustine, lomustine, semustine, streptozocin, or dacarbazine.
  • Exemplary antimetabolites include, but are not limited to, folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil (5-FU) or cytarabine), and purine analogs (e.g., mercaptopurine or thioguanine).
  • folic acid analogs e.g., methotrexate
  • pyrimidine analogs e.g., 5-fluorouracil (5-FU) or cytarabine
  • purine analogs e.g., mercaptopurine or thioguanine
  • hormones and hormone antagonists include, but are not limited to, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., tamoxifen), and androgens (e.g., testosterone proprionate and fluoxymesterone).
  • adrenocorticosteroids e.g., prednisone
  • progestins e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate
  • estrogens e.g., diethylstilbestrol and ethinyl estradiol
  • antiestrogens e.g., tamoxi
  • exemplary agents include, but are not limited to, vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, Leydig 771368 E-056-2023-0-PCT-01 15 bleomycin, plicamycin, or mitocycin C), (e.g., L-asparaginase), platinum coordination complexes (e.g., cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (e.g., hydroxyurea), methyl hydrazine derivatives (e.g., procarbazine), and adrenocortical suppressants (e.g., mitotane and aminoglutethimide).
  • vinca alkaloids
  • Chemotherapeutics that can be concurrently, sequentially or intermittently administered with the vectors and compositions disclosed herein include Adriamycin, Alkeran, Ara-C, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin, Enzalutamide (MDV-3100 or XT
  • immunomodulators and/or cytokines include, but are not limited to, AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New La, La.), SK&F 106528, tumor necrosis factor (TNF)- ⁇ , and TNF- ⁇ .
  • compositions or protocols that are useful for the treatment of cancer in conjunction with the inventive protein or polypeptide, nucleic acids, vectors, cells, and compositions of the invention include, but are not limited to, surgical resection of a tumor, radiation therapy, allogeneic or autologous stem cell transplantation, adoptive T cell transfer, chimeric antigen receptor (CAR) T-cell therapy, tumor-infiltrating lymphocytes (TIL), and/or targeted cancer therapies (e.g., small molecule drugs, biologics, or monoclonal antibody therapies that specifically target molecules involved in tumor growth and progression, including, but not limited to, selective estrogen receptor modulators (SERMs), aromatase inhibitors, tyrosine kinase inhibitors, serine/threonine kinase inhibitors, histone deacetylase (HDAC) inhibitors, retinoid receptor activators, apoptosis stimulators, angiogenesis inhibitors, poly (ADP-ADP-asethyl-N-phosphate-
  • the agent can be a cancer vaccine, such as PANVAC, Leydig 771368 E-056-2023-0-PCT-01 16 PROSTVAC, MVA-Brachyury TRICOM, Brachyury, AdCEA Avelumab (Avel) Folfox, CEA-MUC-TRICOM CV301, or Bacillus Calmette-Guerin (BCG) alone or combined with PANVAC.
  • the additional active agent e.g., chemotherapeutic agent
  • one or more (e.g., 2, 3, 4, or 5) chemotherapeutic agents is/are administered in combination with the inventive composition.
  • the additional active agent(s) can be administered alone or in a composition.
  • the additional active agent(s) can be formulated by inclusion in a vector (e.g., plasmid or viral vector), in liposomes (tecemotide, which is also known as STIMUVAX TM , L-BLP25, or BLP25 liposome vaccine), or in nanoparticles (e.g., VERSAMUNE TM nanotechnology).
  • a vector e.g., plasmid or viral vector
  • detectemotide which is also known as STIMUVAX TM , L-BLP25, or BLP25 liposome vaccine
  • nanoparticles e.g., VERSAMUNE TM nanotechnology.
  • the composition additionally or alternatively can comprise one or more immunostimulatory/regulatory molecules.
  • immunostimulatory/regulatory molecule such as interleukin (IL)-2, IL-4, IL-6, IL-12, IL-15, IL-15/IL-15Ra, IL-15/IL-15Ra- Fc, interferon (IFN)- ⁇ , tumor necrosis factor (TNF)- ⁇ , B7.1, B7.2, ICAM-1, ICAM-2, LFA-1, LFA-2, LFA-3, CD70, CD-72, RANTES, G-CSF, GM-CSF, OX-40L, 41 BBL, anti-CTLA-4, IDO inhibitor, anti-PD-L1, anti-PD1, and combinations thereof.
  • IL interleukin
  • IL-4 interleukin
  • IL-6 interferon
  • TNF tumor necrosis factor
  • TNF tumor necrosis factor
  • TNF tumor necrosis factor
  • the IL-12 is NHS-IL12, which is an immunocytokine composed of two IL-12 heterodimers fused to the NHS76 antibody (see Strauss et al., Clinical Cancer Research, 25(1): 99-109 (2019)).
  • the composition comprises a combination of B7.1, ICAM-1, and LFA-3 (also referred to as TRICOM).
  • the one or more immunostimulatory/regulatory molecules can be administered in the form of a vector (e.g., a recombinant viral vector, such as those discussed herein or otherwise known to those of skill in the art) comprising a nucleic acid encoding one or more immunostimulatory/regulatory molecules.
  • the one or more immunostimulatory/regulatory molecules can be administered in the form of a DNA plasmid with or without chitosan.
  • the one or more immunostimulatory/regulatory molecules can be administered as a protein (e.g., recombinant protein), such as a protein (e.g., recombinant IL-12) with or without being admixed with chitosan.
  • a protein e.g., recombinant protein
  • a protein e.g., recombinant IL-12
  • One or more Leydig 771368 E-056-2023-0-PCT-01 17 immunostimulatory/regulatory molecules also be administered in combination with, or concurrently with, a yeast-based immunotherapy composition of the invention.
  • the inventive protein or polypeptide also can be conjugated to helper peptides or to large carrier molecules to enhance the immunogenicity of the peptide or polypeptide.
  • helper peptides or to large carrier molecules include, but are not limited to, influenza peptide, tetanus toxoid, tetanus toxoid CD4 epitope, Pseudomonas exotoxin A, poly-L-lysine, a lipid tail, endoplasmic reticulum (ER) signal sequence, and the like.
  • the inventive peptide or polypeptide (protein) also can be conjugated to an immunoglobulin molecule using art-accepted methods.
  • the immunoglobulin molecule can be specific for a surface receptor present on tumor cells, but absent or in very low amounts on normal cells.
  • the immunoglobulin also can be specific for a specific tissue (e.g., breast, ovarian, colon, or prostate tissue).
  • tissue e.g., breast, ovarian, colon, or prostate tissue.
  • Such a peptide-immunoglobulin conjugate or polypeptide-immunoglobulin conjugate allows for targeting of the peptide to a specific tissue and/or cell.
  • the carrier used in the inventive composition can be any of those conventionally used and is limited only by physio-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration.
  • the pharmaceutically acceptable carriers described herein for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. [0050]
  • the choice of carrier and manner of formulation of the inventive composition will be determined in part by the particular protein or polypeptide, nucleic acid, vector, cell, or composition thereof of the invention and other active agents or drugs used, as well as by the particular method used to administer the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof.
  • compositions can be employed in the inventive compositions, and in carrying out the inventive methods described herein.
  • parenteral, subcutaneous, intravenous, intramuscular, and intraperitoneal administration are exemplary and are in no way limiting.
  • routes of administering the protein or Leydig 771368 E-056-2023-0-PCT-01 18 polypeptide, nucleic acid, vector, cell, or of the invention are known, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective response than another route.
  • injectable formulations are among those formulations that are preferred in accordance with the present invention.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxy
  • Oils which can be used in parenteral formulations, include petroleum, animal, vegetable, and synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-b-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts
  • Preservatives and buffers may be used.
  • such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17.
  • HLB hydrophile-lipophile balance
  • Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • inventive reagents the protein or polypeptide, nucleic acid, vector, cell, or composition
  • the invention provides the therapeutic (including prophylactic) use of such for treatment or prophylaxis of disease, particularly in a human patient.
  • One particularly contemplated type of disease particularly amenable to prophylaxis or therapy using the inventive reagents includes cancers. Accordingly, the invention provides methods of treating a subject suffering from or susceptible to a tumor and/or enhancing an immune response against cancer and/or inhibiting a cancer. Leydig 771368 E-056-2023-0-PCT-01 20 [0058] In a first embodiment, the comprise administering a therapeutically effective amount of one or more of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof to a subject.
  • inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be used to prevent the development of cancer, particularly in an individual at higher risk to develop such cancer than other individuals, or to treat a patient afflicted with cancer.
  • inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof is useful for preventing emergence of cancer, arresting progression of cancer or eliminating cancer. More particularly, the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be used to prevent, inhibit or delay the development of tumors, and/or to prevent, inhibit or delay tumor migration and/or tumor invasion of other tissues (metastases) and/or to generally prevent or inhibit progression of cancer in an individual.
  • inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can also be used to ameliorate at least one symptom of the cancer, such as by reducing tumor burden in the individual; inhibiting tumor growth in the individual; increasing survival of the individual; and/or preventing, inhibiting, reversing or delaying progression of the cancer in the individual.
  • inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be used to treat a subject with any stage of cancer.
  • Administration of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be “prophylactic” or “therapeutic.”
  • inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof is provided in advance of tumor formation, or the detection of the development of tumors, with the goal of preventing, inhibiting or delaying the development of tumors; and/or preventing, inhibiting or delaying metastases of tumors and/or generally preventing or inhibiting progression of cancer in an individual, and generally to allow or improve the ability of the host’s immune system to fight against a tumor that the host is susceptible of developing.
  • the prophylactic administration of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof prevents, ameliorates, or delays the cancer.
  • the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof is provided at or after the diagnosis of the cancer, with the Leydig 771368 E-056-2023-0-PCT-01 21 goal of ameliorating the cancer, such as by tumor burden in the individual; inhibiting tumor growth in the individual; increasing survival of the individual; and/or preventing, inhibiting, reversing or delaying progression of the cancer in the individual.
  • Treatment comprises, but is not limited to, destroying tumor cells, reducing tumor burden, inhibiting tumor growth, reducing the size of the primary tumor, reducing the number of metastatic legions, increasing survival of the individual, delaying, inhibiting, arresting or preventing the onset or development of metastatic cancer (such as by delaying, inhibiting, arresting or preventing the onset of development of tumor migration and/or tumor invasion of tissues outside of primary cancer and/or other processes associated with metastatic progression of cancer), delaying or arresting primary cancer progression, improving immune responses against the tumor, improving long term memory immune responses against the tumor antigens, and/or improving the general health of the individual.
  • metastatic cancer such as by delaying, inhibiting, arresting or preventing the onset of development of tumor migration and/or tumor invasion of tissues outside of primary cancer and/or other processes associated with metastatic progression of cancer
  • the cancer to be treated in accordance with the inventive methods can be any cancer, including, but not limited to, cancer of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, lung, urethra, uterine, bladder, colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine, fallopian tube, heart or adrenals.
  • cancers include solid tumor, sarcoma, carcinomas (including but not limited to cystic carcinomas), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma
  • the administration of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof to a host results in a host cell expressing the inventive peptide and optionally one or more immunostimulatory/regulatory molecules and/or other tumor- associated antigens (such as, but not limited to, AIM-2 (Interferon-inducible protein absent in melanoma 2), ALL (Acute lymphoblastic leukemia), AML (Acute myeloid leukemia), 707-AP (707 alanine proline), anti-PD-1, anti-CTLA4, anti-PD-1 + anti-CTLA4, APL (Acute promyelocytic leukemia), ART-4 (Adenocarcinoma antigen recognized by T cells 4), Adoptive transfer vaccine primed autologous T cells + autologous DC tumor cell lysate vaccine + bevacizumab, Allo-tumor cells (XRT) + GM-CSF + BCG, Autologous DC tumor cell ly
  • AIM-2 Interfer
  • DAM-6 is also called MAGE-B2 and DAM- 10 is also called MAGE-B1), ELF2 (Elongation factor 2), Ep-CAM (Epithelial cell adhesion molecule), EphA2, 3 (Ephrin type-A receptor 2, 3), Ets (E-26 transforming specific (family of Leydig 771368 E-056-2023-0-PCT-01 23 transcription factors)), ETV6-AML1 (Ets gene 6/acute myeloid leukemia 1 gene ETS), FGF-5 (Fibroblast growth factor 5), FN (Fibronectin), G250 (Glycoprotein 250), GAGE (G antigen), GnT-V (N-Acetylglucosaminyltransferase V), Gp100 (Glycoprotein 100 kDa), HAGE (Helicase antigen), HER-2/neu (Human epidermal receptor 2/neurological), HLA-A*0201-R170I (Arginine (R) to iso
  • the inventive peptide can be expressed at the cell surface of the infected host cell.
  • the one or more immunostimulatory/regulatory molecules and/or other tumor-associated antigens, modified versions thereof, and immunogenic epitopes thereof can be expressed at the cell surface or may be actively secreted by the host cell.
  • the expression of both the peptide and the immunostimulatory/regulatory molecule provides the necessary MHC restricted peptide to specific T cells and the appropriate signal to the T cells to aid in antigen recognition and proliferation or clonal expansion of antigen specific T cells.
  • the overall result is an upregulation of the immune system.
  • the upregulation of the immune response is an increase in antigen specific T-helper lymphocytes and/or cytotoxic lymphocytes, which are able to kill or inhibit the growth of a cancer cell.
  • the inventive methods can comprise obtaining (by isolating) dendritic cells from a subject, treating the dendritic cells with one or more of the therapeutically effective amount of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof, and administering the treated dendritic cells to the subject.
  • the inventive methods can comprise (a) obtaining (isolating) peripheral blood mononuclear cells (PBMCs) from a subject, (b) isolating dendritic cells from the PBMCs, (c) treating the dendritic cells with one or more of the therapeutically effective amount of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof ex vivo, (d) activating the PBMCs with the treated dendritic cells ex vivo, and (e) administering the activated PBMCs to the subject.
  • PBMCs peripheral blood mononuclear cells
  • the inventive methods also can comprise a method for inhibiting cancer in a subject comprising (a) obtaining (isolating) PBMCs from a subject, (b) isolating dendritic cells from the PBMCs, (c) treating the dendritic cells with one or more of the therapeutically effective amount of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof ex vivo, (d) activating the PBMCs with the treated dendritic cells ex vivo, (e) isolating T lymphocytes Leydig 771368 E-056-2023-0-PCT-01 25 from the activated PBMCs ex vivo, and (f) the isolated T lymphocytes to the subject.
  • the invention also provides the use of adoptively transferred T cells stimulated in vitro with one or more of the therapeutically effective amount of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof to inhibit cancer in a subject.
  • the invention also provides T-cell receptor (TCR) engineered T cells and TCR engineered NK cells for treating (e.g., inhibiting) cancer in a subject.
  • TCR engineered T cells and TCR engineered NK cells can be prepared by any suitable methods, such as those described in Ping et al., Protein Cell, 9(3) 254-266 (2016) (incorporated herein in its entirety).
  • the TCR engineered T cells and TCR engineered NK cells target cancer cells expressing ERVMER34-1, such as the epitopes of SEQ ID NOs: 2-99 or 100-140.
  • inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be administered to the subject by any method.
  • the inventive protein or polypeptide, or nucleic acid encoding the peptide or polypeptide can be introduced into a cell (e.g., in a host) by any of various techniques, such as by contacting the cell with the peptide, polypeptide, the nucleic acid, or a composition comprising the nucleic acid as part of a construct, as described herein, that enables the delivery and expression of the nucleic acid.
  • a cell e.g., in a host
  • Specific protocols for introducing and expressing nucleic acids in cells are known in the art.
  • Suitable methods of administering peptides, polypeptides (proteins), nucleic acids, vectors, cells, and compositions to hosts (subjects) are known in the art.
  • the host can be any suitable host, such as a mammal (e.g., a rodent, such as a mouse, rat, hamster, or guinea pig, rabbit, cat, dog, pig, goat, cow, horse, primate, or human).
  • a mammal e.g., a rodent, such as a mouse, rat, hamster, or guinea pig, rabbit, cat, dog, pig, goat, cow, horse, primate, or human.
  • inventive protein or polypeptide, nucleic acid, or vector e.g., recombinant poxvirus
  • the peptide, polypeptide, nucleic acid, vector (e.g., recombinant poxvirus) or combination of vectors, cell, and composition can be directly administered (e.g., locally administered) by direct injection into the cancerous lesion or tumor or by topical application (e.g., with a pharmaceutically acceptable carrier).
  • a pharmaceutically acceptable carrier e.g., with a pharmaceutically acceptable carrier.
  • the inventive protein or nucleic acid, vector, cell, or composition thereof can be administered alone or in combination with other anti-cancer agents, adjuvants, incorporated into liposomes (as described in, e.g., U.S.
  • Patent Nos.5,643,599, 5,464,630, 5,059,421, and 4,885,172 which are incorporated herein in their entireties), incorporated into nanoparticles (e.g., VERSAMUNE TM nanotechnology), administered with cytokines, administered with biological response modifiers (e.g., interferon, interleukin-2 (IL-2), administered colony-stimulating factors (CSF, GM-CSF, and G-CSF), and/or administered other reagents in the art that are known to enhance immune response.
  • biological response modifiers e.g., interferon, interleukin-2 (IL-2), administered colony-stimulating factors (CSF, GM-CSF, and G-CSF
  • CSF colony-stimulating factors
  • GM-CSF GM-CSF
  • G-CSF G-CSF
  • a particularly interesting combination for use in the present inventive methods includes SEQ ID NO:2, IL15 superagonist (N803, ANKTIVA (nogapendekin alfa inbakicept-pmln)), and/or anti-PD-L1.
  • Other checkpoint inhibitors can be employed in addition to or in lieu of anti-PD-L1, such as anti-CTLA4 and/or anti-PD-1.
  • Suitable adjuvants include alum, aluminum salts, aluminum phosphate, aluminum hydroxide, aluminum silica, calcium phosphate, incomplete Freund’s adjuvant, saponins, such as QS21 (an immunological adjuvant derived from the bark of the South American tree Quillaja saponaria Molina), monophosphoryl lipid A (MLP-A), and RIBI DETOX TM adjuvant.
  • the adjuvant for use in the invention is the cytokine, GM-CSF.
  • GM- CSF has been shown to be an effective vaccine adjuvant because it enhances antigen processing and presentation by dendritic cells.
  • GM-CSF can be administered using a viral vector or as an isolated protein in a pharmaceutical formulation.
  • GM- CSF can be administered to the host before, during, or after the initial administration of the peptide, polypeptide, nucleic acid, vector, cell, or composition thereof to enhance the antigen- specific immune response in the host.
  • recombinant GM-CSF protein can be administered to the host on each day of vaccination with the peptide, polypeptide, nucleic acid, vector, cell, or composition thereof and for each of the following 3 days (i.e., a total of 4 days). Any suitable dose of GM-CSF can be used.
  • GM-CSF can be administered per day.
  • the GM-CSF can be administered by any method (e.g., subcutaneously) and, preferably, is administered at or near the site of the vaccination of a host with the peptide, polypeptide, nucleic acid, vector, cell, or composition thereof.
  • inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof is administered to a host (e.g., mammal, such as a human) in an amount effective to generate an immune response, preferably a cellular immune response.
  • a host e.g., mammal, such as a human
  • the efficacy of the peptide, polypeptide, nucleic acid, vector, or cell as an immunogen may be determined by in vivo or in vitro parameters as are known in the art. These parameters include but are not limited to antigen-specific cytotoxicity assays, regression of tumors, inhibition of cancer cells, production of cytokines, and the like.
  • any suitable dose of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be administered to a host.
  • the appropriate dose will vary depending upon such factors as the host’s age, weight, height, sex, general medical condition, previous medical history, disease progression, and tumor burden and can be determined by a clinician.
  • the agent to be administered comprises an inventive protein or polypeptide
  • such can be administered in a dose of about 0.05 mg to about 10 mg (e.g., 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, and ranges therebetween) per vaccination of the host (e.g., mammal, such as a human), and preferably about 0.1 mg to about 5 mg per vaccination.
  • Several doses e.g., 1, 2, 3, 4, 5, 6, or more
  • a dose is provided every month for 3 months.
  • a suitable dose can include, by way of non-limiting example, about 1 x 10 5 to about 1 x 10 12 (e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , and ranges therebetween) plaque forming units (pfus), although a lower or higher dose can be administered to a host.
  • about 2 x 10 8 pfus can be administered (e.g., in a volume of about 0.5 mL).
  • the agent to be administered comprises an inventive cell (e.g., cytotoxic T cells), such can be administered, by way of non-limiting example, to a host in a dose of between about 1 x 10 5 and 2 x 10 11 (e.g., 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , and ranges Leydig 771368 E-056-2023-0-PCT-01 28 therebetween) cells per infusion.
  • the cells administered in multiple, for example one to three (e.g., one, two, or three), infusions.
  • the host can be administered a biological response modifier, such as interleukin 2 (IL-2).
  • IL-2 interleukin 2
  • the administration of the cytotoxic T cells can be followed by the administration of the peptide, polypeptide, nucleic acid, vector, or composition thereof in order to prime the cytotoxic T cells to further expand the T cell number in vivo.
  • the cells to be administered are dendritic cells
  • the amount of dendritic cells administered to the subject will vary depending on the condition of the subject and should be determined via consideration of all appropriate factors by the practitioner.
  • dendritic cells are utilized for adult humans. These amounts will vary depending on the age, weight, size, condition, sex of the subject, the type of tumor to be treated, the route of administration, whether the treatment is regional or systemic, and other factors. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the subject.
  • the invention provides a method of generating peptide-specific cytotoxic T lymphocytes in vivo, ex vivo, or in vitro by stimulation of lymphocytes with an effective amount of the inventive protein or polypeptide, nucleic acid, vector, or cell, alone or in a composition with one or more immunostimulatory/regulatory molecules and/or adjuvants or in a liposome formulation.
  • the lymphocytes can be lymphocytes from any suitable source, e.g., peripheral blood, tumor tissues, lymph nodes, and effusions, such as pleural fluid or ascites fluid.
  • the ERVMER34-1 peptide specific cytotoxic T lymphocytes are immunoreactive.
  • the cytotoxic T lymphocytes inhibit the occurrence of tumor cells and cancer and inhibit the growth of, or kill, tumor cells.
  • the cytotoxic T lymphocytes in addition to being antigen specific, can be MHC class (e.g., MHC class I) restricted.
  • the cytotoxic T lymphocytes preferably have a CD8 + phenotype.
  • lymphocytes are removed from the host and stimulated ex vivo with the peptide, polypeptide, nucleic acid, vector, cell, or composition thereof to generate Leydig 771368 E-056-2023-0-PCT-01 29 cytotoxic T lymphocytes.
  • the cytotoxic T can be administered to the host in order to enhance an immune response to cancer, thereby inhibiting the cancer.
  • the invention provides a method of inhibiting cancer in a host comprising (a) obtaining lymphocytes (e.g., from the host), (b) stimulating the lymphocytes with the peptide, polypeptide, nucleic acid, vector, cell, or composition thereof to generate cytotoxic T lymphocytes, and (c) administering the cytotoxic T lymphocytes to the host, wherein the cancer is inhibited.
  • lymphocytes within the host are stimulated by administration to the host of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof to generate cytotoxic T lymphocytes, which cytotoxic T lymphocytes enhance an immune response to cancer, thereby inhibiting the cancer.
  • the inventive therapeutic and prophylactic methods can include a prime-and-boost protocol.
  • the protocol includes an initial “prime” with a composition comprising one or more recombinant vectors encoding the inventive peptide or polypeptide and optionally one or more immunostimulatory/regulatory molecules and/or other tumor-associated antigens , modified versions thereof, and immunogenic epitopes thereof, followed by one or preferably multiple “boosts” with a composition containing the inventive peptide or polypeptide or one or more vectors encoding the inventive protein or polypeptide and optionally one or more immunostimulatory/regulatory molecules and/or other tumor-associated antigens , modified versions thereof, and immunogenic epitopes thereof.
  • the initial priming vaccination can comprise one or more vectors.
  • a single vector is used for delivery of the inventive peptide and one or more immunostimulatory/regulatory molecules and/or other tumor-associated antigens, modified versions thereof, and immunogenic epitopes thereof.
  • two or more vectors comprise the priming vaccination, which are administered simultaneously in a single injection.
  • the boosting vaccinations also can comprise one or more vectors.
  • a single vector is used for delivery of the inventive peptide and the one or more immunostimulatory/regulatory molecules and/or other tumor-associated antigens, modified Leydig 771368 E-056-2023-0-PCT-01 30 versions thereof, and immunogenic epitopes of the boosting vaccination.
  • two or more vectors comprise the boosting vaccination, which are administered simultaneously in a single injection.
  • Different types of vectors can be used to provide a heterologous prime/boost protocol using vectors carrying different sets of therapeutic molecules for inoculations at different time intervals.
  • a first vector e.g., adenovirus, orthopox, vaccinia, and the like
  • a second type of vector composition is used to boost.
  • the schedule for administration of the vectors typically involves repeated administration of the boosting vector.
  • the boosting vector can be administered 1-3 times (e.g., 1, 2, or 3 times) at any suitable time period (e.g., every 2-4 weeks) for any suitable length of time (e.g., 6-12 weeks for a total of at least 5 to 15 boosting vaccinations).
  • the primary vaccination can comprise a recombinant adenovirus, poxvirus, or vaccinia vector followed by multiple booster vaccinations with an adenovirus, poxvirus, or vaccinia vector.
  • the host receives one vaccination with the priming vector, followed every 2 weeks thereafter with the boosting vector for 6 boosts, followed by every 4 weeks thereafter with the boosting vector, and continuing with the boosting vector for a period of time dependent on disease progression.
  • EXAMPLE [0088] This Example demonstrates that novel sequences of the ERVMER34-1 human endogenous retroviral sequence family described herein have been identified and shown to be immunogenic in humans via activation of human T cells in vitro.
  • a modified the ERVMER34-1 protein sequence described herein (SEQ ID NO:2) for incorporation into a ERVMER34-1 modified sequence adenovirus has been engineered to (a) remove the signal domain to prevent the trafficking of the protein to the cell surface, (b) remove the cleavage site to prevent the cleavage of the surface unit from the transmembrane unit of the ERVMER34-1 protein, (c) remove the predicted immunosuppressive domain to prevent the potential dampening Leydig 771368 E-056-2023-0-PCT-01 31 of the immunity generated against the target and (d) remove the transmembrane domain because this sequence shares homology with several human proteins.
  • SW620 ERVMER34-1 CRISPR Leydig 771368 E-056-2023-0-PCT-01 32 cell line cells were co-transfected with a Cas9 protein version 2 and a TrueGuide synthetic guide RNA targeting human ERVMER34-1 (ID number CRISPR924929_SGM) using the Lipofectamine CRISPRMAX transfection reagent (ThermoFisher).
  • SW620 ERVMER34-1 knockout CRISPR cells were single-cell sorted and ERVMER34-1 negative cells were identified by western blot.
  • ERVMER34-1 For the generation of the MC38 pERVMER34-1 cell line, the human ERVMER34-1 gene was synthesized (ThermoFisher) and cloned into the pNGFR expression plasmid, which was kindly provided by Warren Pear (Addgene plasmid # 27489). MC38 cells were transfected with pERVMER34-1-NGFR, and single-cell sorted based upon surface expression of NGFR using a MA900 cell sorter (Sony Biotechnology). ERVMER34-1 expression was confirmed by western blot.
  • K562 cells were transfected with plasmids encoding the full-length human HLA-A2 and CD80 (Genecopoeia) along with pERVMER34-1-NGFR. Transfectants were single-cell sorted based upon surface expression of CD80, HLA-A2 and NGFR using a MA900 cell sorter (Sony Biotechnology).
  • aAPC artificial antigen-presenting cell line
  • Hybridomas were generated using splenocytes harvested from BALB/c mice that were immunized with the purified ERVMER34-1 recombinant protein (GenScript).
  • GenScript purified ERVMER34-1 recombinant protein
  • All ERVMER34-1 peptides were chemically synthesized by GenScript at a purity greater than 75% as assessed by HPLC.
  • the peptides comprising the 15-mer ERVMER34- 1 peptide library are identified herein as SEQ ID NOs:7-99.
  • Full-length 15-mer overlapping peptide libraries for brachyury and mucin1 were chemically synthesized by JPT Technologies.
  • PBMCs collected from either healthy donors or cancer patients were separated by Ficoll-Hypaque density gradient separation and cryopreserved in 90% serum and 10% DMSO.
  • De-identified PBMC samples were obtained from healthy volunteers who provided written informed consent at the National Institute of Health (NIH) Clinical Center Blood Bank (protocol NCT00001846).
  • PBMCs were obtained from the blood of patients diagnosed with prostate, lung, breast, and colon cancers enrolled in clinical trials at the National Cancer Institute (NCT00179309, NCT01772004, Leydig 771368 E-056-2023-0-PCT-01 33 NCT03050814, NCT00088413, NCT01519817, NCT00923741, NCT00924092, NCT02840994, NCT00113984).
  • NCT00179309, NCT01772004, Leydig 771368 E-056-2023-0-PCT-01 33 NCT03050814, NCT00088413, NCT01519817, NCT00923741, NCT00924092, NCT02840994, NCT00113984 [0096] In vitro expansion of ERVMER34-1 reactive T cells.
  • PBMCs collected from either healthy donors or cancer patients were plated at 2.5x10 6 cells/well in a 24-well tray in a volume of 1 mL Immunocult X
  • PBMCs were plated at 107 cells/well in 6-well trays for experiments that required larger numbers of T cells to assess immunity generated against individual ERVMER34-1 peptides.
  • the 15-mer overlapping peptide library was added at a concentration of 0.2 ⁇ g/mL/peptide.
  • Cultures were supplemented on days 3 and 5 with cytokines (IL7 and IL15, 10 ng/mL of each; PeproTech). On day 7, cultures were washed and rested in media without the addition of peptide and cytokines. On day 11, cultures were harvested and restimulated for 24 hours to assess ERVMER34-1 specific immunity.
  • ERVMER34-1 specific immune responses were assessed using either an IFN- ⁇ ELISPOT assay or intracellular cytokine staining.
  • ELISPOT assays Antigen-dependent IFN- ⁇ producing cells were enumerated using either human or murine ELISPOT assays purchased from BD Biosciences and used according to the manufacturer’s instructions. For assays using murine splenocytes, 5x10 5 cells/well were incubated overnight with appropriate peptides at concentrations indicated below. For expanded tumor-infiltrating T cell (TIL) cultures, lymphocytes were counted and distributed equally amongst the number of test wells along with 10 6 splenocytes harvested from na ⁇ ve animals.
  • TIL tumor-infiltrating T cell
  • Antigen-specific IFN- ⁇ producing cells from TIL were normalized to antigen-dependent counts per 5x10 5 expanded TIL.
  • Human PBMCs either ex vivo or following a 1-week in vitro stimulation were plated at 5x10 5 cells per well in the ELISPOT assay.
  • peptide concentrations were used: 10 ⁇ g/mL for single peptides and 0.2 ⁇ g/mL/peptide for pooled peptide libraries.
  • Spots were quantified using an ImmunoSpot analyzer (Cellular Technology, Ltd). Antigen-dependent spots were calculated by subtracting any background spots in antigen-negative wells for each sample. [0098] Intracellular cytokine stain.
  • SW620 parental and SW620 ERVMER34-1 knockout CRISPR cells were plated in 8 well chamber slides (Ibidi, 80807). Once attached, cells were fixed with 3.2% paraformaldehyde (Electron Microscopy Sciences, 15714, diluted 1:10 in PBS to make 3.2%), permeabilized in 0.05% Surfact-Amps X (ThermoFisher Scientific, 28314, diluted 1:200 for 0.05%), and blocked in PBS supplemented with 10% goat serum and 1% BSA. Cells were incubated overnight with a mouse primary antibody against ERVMER34-1 (clone 14F-3-1) at 2 ⁇ g/ml.
  • HN802d Head and neck carcinoma
  • CO1004 colon carcinoma
  • BL242 bladder carcinoma
  • lung adenocarcinoma LC1511
  • BC04118a lung squamous cell carcinoma
  • BC09012b endometrium carcinoma
  • BN961b normal adult human tissue microarrays
  • Murine tumor tissue was fixed in Z-fix (Anatech, Battle Creek, MI) overnight, embedded in paraffin, and sectioned onto glass slides (American HistoLabs, Gaithersburg, MD). Slides were stained using the Opal 4-Color Manual IHC Kit (PerkinElmer, Waltham, MA) per the manufacturer’s instructions.
  • slides were deparaffinized and rehydrated with xylene and ethanol gradients, microwaved with Leydig 771368 E-056-2023-0-PCT-01 35 pH6, pH9, or Rodent Decloaker (BioCare Pacheco, CA) antigen retrieval solution, cooled, rinsed with tris-buffered saline, 0.1% tween (TBST), and blocked with BLOXALL Blocking Solution (Vector Laboratories, Burlingame, CA). Staining with primary and secondary antibodies and OPAL fluorophore working solution was conducted following the manufacturer’s instructions.
  • Antibodies including anti-ERVMER34-1 (clone 14F3) and anti-Pan-cytokeratin (clone AE1/AE3; SantaCruz Biotechnology) were used to stain human tissue microarrays.
  • Murine tumors were stained using a rabbit polyclonal ERVMER34-1 antibody (ThermoFisher Scientific). Slide scanning was performed using an Axio Scan.Z1 using a 20x objective (Zeiss).
  • Human tissue microarrays were scored independently by two investigators. Sections with less than 10% specific staining were scored as +, sections where 10-50% of cells had specific staining were scored with ++.
  • Expansion of ERVMER34-1 specific CD8+ cytotoxic T cells PBMCs collected from healthy HLA-A2 donors were enriched for CD8+ T cells using a negative selection isolation kit (Miltenyi Biotec). CD8+ T cells were cultured with irradiated (200 Gy) aAPC cells at a 20:1 ratio. On days 3 and 5 of culture, IL-7 and IL-15 (10 ng/mL of each; PeproTech) were added to the culture. On day 7, culture was washed and restimulated as described above.
  • CD8+ T cells underwent between three and five of these in vitro stimulation cycles prior to being used in subsequent lysis and ELISPOT assays.
  • Lysis assays For flow cytometric-based lysis assays, the SW620 parental cells were labeled with CellTracker CM-DIL (ThermoFisher Scientific) and the SW620 ERVMER34-1 CRISPR cell line was labeled with CellTracker Deep Red (ThermoFisher Scientific) per the manufacturer’s recommended protocol. Labeled SW620 parental and ERVMER34-1 CRISPR cells were mixed at a 1:1 ratio. Effector CD8+ T cells were added to the target cell mixture at a 10:1 ratio.
  • % lysis [1 - (cell count in well of interest/tumor only average cell count)] ⁇ 100.
  • % lysis [1 - (cell count in well of interest/tumor only average cell count)] ⁇ 100.
  • mice Female C57BL/6 mice were obtained from the NCI Frederick Cancer Research Facility. Mice were between 5 and 6 weeks old at the start of experiments and were maintained under pathogen-free conditions in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Tumors were induced by implanting 3 x 10 5 MC38 tumor cells or 1x106 MC38 pERVMER34-1 tumor cells subcutaneously. [00106] Vaccination and treatments.
  • Animals were injected subcutaneously with either 1010 viral particles of control adenovirus (Vector Biolabs), full-length ERVMER34-1 adenovirus, or a modified ERVMER34-1 adenovirus (designated ERVMER34-1 vaccine) diluted in PBS following tumor establishment in mice.
  • Anti-PD-L1 (10F.9G2, BioXCell, 200 ⁇ g) was administered intraperitoneally and diluted in PBS.
  • Spleens were harvested in LPA media (RPMI-1640 supplemented with 10% FBS, 1.5% HEPES Buffer, 1x Sodium Pyruvate, 1x non-essential amino Leydig 771368 E-056-2023-0-PCT-01 37 acids, 1x Glutamine, 1x 1x Gentamicin), dissociated through 70- ⁇ m filters, and subjected to ACK lysis (Quality Biological) to obtain splenocytes for analysis.
  • LPA media RPMI-1640 supplemented with 10% FBS, 1.5% HEPES Buffer, 1x Sodium Pyruvate, 1x non-essential amino Leydig 771368 E-056-2023-0-PCT-01 37 acids, 1x Glutamine, 1x 1x Gentamicin), dissociated through 70- ⁇ m filters, and subjected to ACK lysis (Quality Biological) to obtain splenocytes for analysis.
  • Tumors were harvested in RPMI with 5% FBS media, cut into small pieces, and incubated for 30 minutes at 37 °C and 300 rpm in a digestion cocktail composed of RPMI supplemented with 5% (v/v) FBS, 2 mg/mL Collagenase Type I and IV (Worthington Biochemical Corporation), and 40 U/mL DNase I (Calbiochem). Following digestion, tumors were ground through 70- ⁇ m filters, spun for 5 minutes at 500 g, and resuspended in media for cell counting and subsequent flow cytometry analysis. Mouse sera was collected into serum tubes with separating gel (BD Biosciences), rested for 30 minutes, and centrifuged at 800 g for 2 minutes.
  • separating gel BD Biosciences
  • CD8+T cells Singlets/live/CD45+CD3+CD8+
  • CD4+ T cells singlets/live/CD45+CD3+CD4+FoxP3-
  • Effector memory singlets/live/CD45+/CD8+CD44+CD62L+.
  • Data were collected using an Attune NxT flow cytometer and analyzed using FlowJo (v10, BD Biosciences).
  • FlowJo v10, BD Biosciences.
  • Western blot Following cell lysis, protein samples (50 ⁇ g) were run in 4-12 % Bis- Tris gels (Invitrogen). Transfer was performed using the iBlot2 gel transfer device (Invitrogen).
  • the membrane was then blocked with 5% non-fat dried milk in PBS with 0.1% Tween 20 Leydig 771368 E-056-2023-0-PCT-01 38 (PBST) for 1 hour at room temperature.
  • 1 antibody (2 ⁇ g/mL) (Clone 14F3) and rabbit GAPDH at 1:5000 (Cell signaling) diluted in 5% milk in PBST were added and incubation was performed overnight at 4 °C. Afterwards, membranes were washed three times, 5 minutes each with PBST.
  • Secondary antibodies IRDye 800 Goat anti-mouse (LI-COR) and IRDye 800 Goat anti-rabbit (LI-COR) were diluted 1:5000 in 5% milk PBST and incubated for 1 hour at room temperature.
  • ERVMER34-1 Identification of ERVMER34-1 as a potential therapeutic target. To identify ERVs that could be potentially targeted by a therapeutic cancer vaccine, the expression of ERVs annotated in the GTEx database was analyzed. As shown in Figure 7A, ERVMER34-1 had very low expression in normal adult tissues as compared to other ERVs examined. In addition, ERVMER34-1 was expressed in multiple tumor types as compared to other ERVs with low levels of normal tissue expression (Figure 7B).
  • ERVMER34-1 is expressed in multiple cancer types.
  • the expression of ERVMER34-1 protein in multiple normal and tumor tissues was assessed by immunofluorescence utilizing commercially available tissue arrays.
  • most normal tissues including cerebrum, heart, esophagus, stomach, liver, pancreas, adrenal gland, lung, lymph node, spleen, ovary, prostate, skeletal muscle, diaphragm, omentum, and skin had no detectable ERVMER34-1 expression, except for low levels detected in isolated cells in the cerebellum, small intestine and colon, breast, bronchus, uterus, kidney and testis.
  • Examples of ERVMER34-1 staining in normal adult tissues are presented in Figure 1C.
  • this figure also includes staining of CEA (CEACAM5) and brachyury (TBXT) in normal tissues, both of which are tumor-associated antigens that have been previously targeted clinically using therapeutic cancer vaccines in the absence of toxicities.
  • ERVMER34-1 expression is similar to or less than that seen with CEA and brachyury in these normal adult tissues, suggesting that ERVMER34-1 could be safely targeted by a therapeutic cancer vaccine.
  • ERVMER34-1 protein was also detected in several tumor types, including colon (23/39 cases), head and neck (50/69 cases), bladder (8/17 cases), lung adenocarcinoma (21/49 cases), lung squamous cell carcinoma (33/50), and endometrium cancer (32/59 cases) ( Figure 1D and Tables 2-7). Specifically, ERVMER34-1 expression was shown to be restricted to the tumor, as it co-localized with the tumor marker cytokeratin ( Figure 1D). While ERVMER34-1 protein is not detectable in normal lung tissues, expression is sometimes observed in histologically normal tissues adjacent to the tumor. ( Figure 1E and Tables 5, 6 and 7).
  • ERVMER34-1 specific T cells can be expanded through in vitro stimulation.
  • a 15-mer overlapping peptide library was chemically synthesized, spanning the entire ERVMER34-1 protein sequence minus a 170 amino acid sequence corresponding to a fragment with high homology with other human proteins to enhance the assay's specificity.
  • ERVMER34-1 peptide library immune reactivity in healthy donor and cancer patient PBMCs was assessed using an overnight (16-hour) IFN- ⁇ ELISPOT Leydig 771368 E-056-2023-0-PCT-01 40 assay. As shown in Figure 2A, a greater in the immune response was observed with PBMCs from cancer patients as compared to healthy donor PBMCs, following a 16-hour exposure to ERVMER34-1 peptides.
  • PBMCs were either stimulated with each individual peptide comprising the ERVMER34-1 library for 16 hours in an IFN- ⁇ ELISPOT assay or they were stimulated for 7 days in vitro in the presence of the pooled library, followed by a 16-hour restimulation in an IFN- ⁇ ELISPOT assay.
  • IFN- ⁇ ELISPOT assay While healthy donors did not demonstrate detectable ERVMER34-1 reactive T cells when evaluated in a 16-hour ex vivo assay (Figure 2B, left panel), immune responses could be detected against multiple ERVMER34-115- mer peptides following a 7-day in vitro stimulation (Figure 2B, right panel).
  • ERVMER34-1 reactive T cells To expand ERVMER34-1 reactive T cells, the HLA-deficient human Chronic Myeloid Leukemia cell line K562 was modified to generate an artificial antigen-presenting cell line (termed “aAPC”), which was engineered to express the full-length ERVMER34-1 protein (Figure 10A) along with the HLA-A2 restriction element and the costimulatory molecule CD80 ( Figure 10B).
  • aAPC artificial antigen-presenting cell line
  • ERVMER34-1 vaccine To target ERVMER34-1, two adenoviral vectors were generated, one encoding the full-length ERVMER34-1 protein and another one encoding a rationally modified ERVMER34-1 sequence (hereinafter referred to as ERVMER34-1 vaccine).
  • the signal peptide was removed to ensure that the envelope protein was not expressed on the surface of infected cells ( Figure 3A, B).
  • a region of 170 amino acids characterized by a high degree of homology to other human proteins was removed from the ERVMER34-1 vaccine. The deletion of this 170 amino acid fragment also eliminated the cleavage and immunosuppressive domains of the ERVMER34-1 protein ( Figure 3A and B).
  • mice were primed with control, full-length or ERVMER34-1 vaccine vectors on day 0 and boosted on day 7.
  • ERVMER34-1 immunity was assessed 2 weeks following the second dose of vaccine using a 16- hour IFN- ⁇ ELISPOT assay. Both versions of the vaccine were able to expand T-cell responses with similar diversities and magnitude of response (Figure 3E). Additionally, the full-length adenovirus and the ERVMER34-1 vaccine were able to expand comparable numbers of multifunctional ERVMER34-1 reactive T cells in the spleens of immunized animals (Figure 3F), with the majority of T cells being CD8+ T cells ( Figure 3G).
  • mice bearing EMT6 pERVMER34-1 tumors were treated as indicated in Figure 4B and observed increased tumor control when the ERVMER34-1 vaccine is used as a monotherapy, but we observed the best anti-tumor immunity when the vaccine is combined with PD-L1 blockade ( Figure 4C).
  • Targeting ERVMER34-1 mediates tumor clearance in the MC38 model.
  • the murine MC38 colon carcinoma cell line was engineered to express the full-length human ERVMER34-1 protein (MC38 pERVMER34-1) ( Figure 5A). Mice bearing MC38 pERVMER34-1 tumors were treated with a single dose of either a control adenovirus vector or the ERVMER34-1 vaccine.
  • Tumor clearance associates with an expansion of ERVMER34-1 reactive T cells in both the spleen and within the tumor microenvironment (Figure 5E). Immunity was assessed in animals 6 days following the administration of vaccine using an IFN- ⁇ ELISPOT assay. We observed that vaccination associates with the expansion of ERVMER34-1 reactive T cells both in the spleen and within the tumor microenvironment ( Figure 5E). Tumor regression appears to be mediated primarily by ERVMER34-1 reactive T cells as we do not observe expansion of T cells within the tumor microenvironment following vaccination which are specific for either tumor neoepitopes or the p15e tumor antigen known to be expressed in the MC38 model ( Figure 4F).
  • mice whose MC38 pERVMER34- 1 tumors regressed following a single dose of the ERVMER34-1 vaccine were rechallenged with the parental MC38 tumor cell line on their left flank and the MC38 pERVMER34-1 cell line on their right flank.
  • the cleavage site between surface and transmembrane unit is “CTQG” (SEQ ID NO:4).
  • CQG the putative immune-suppressive domain
  • SRKDHVLDIPTTQRQTA SEQ ID NO:5
  • Transmembrane domain is “WGYVLLIVLFCLFIFVLIYVRVF” (SEQ ID NO:6).

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Abstract

The invention provides a protein or polypeptide derived from ERVMER34-1. The invention further provides a nucleic acid encoding the inventive protein or polypeptide, a vector comprising the nucleic acid, cells comprising the protein or polypeptide, nucleic acid, and/or vector, and composition, such as a pharmaceutical composition, comprising any of the foregoing. These reagents (the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof) are useful for immunological treatment and prophylaxis of diseases, particularly cancers.

Description

Leydig 771368 E-056-2023-0-PCT-01 1 IDENTIFICATION OF NOVEL SEQUENCES OF THE ERVMER34-1 HUMAN ENDOGENOUS RETROVIRAL SEQUENCE FAMILY, AND MODIFICATION OF THOSE SEQUENCES FOR A MORE EFFECTIVE ANTI-CANCER VACCINE AND OTHER POTENTIAL ANTI-CANCER THERAPIES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/527,895, filed July 20, 2023, the contents of which are incorporated herein in their entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with Government support under project number ZIABC010944 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention. BACKGROUND OF THE INVENTION [0003] Human Endogenous Retroviruses (HERVs) are remnants of retrovirus germ line infections early in primate evolution and are not viruses. Remarkably, HERVs represent approximately 8% of the human genome. They are an extremely diverse group constituted by at least 3 major classes: Class I consisting of HERV-H, HHLA2 and HERV-E, among others; Class II consisting of HERV-K, among others; and a very diverse Class III, which includes ERVMER34-1 (HEMO). HERVs encode env and gag sequences with a similar organization to Retroviridae, and they can be epigenetically regulated. [0004] International Patent Publication WO 2021/150694, which is incorporated herein in its entirety describes human immunogenic epitopes of ERVMER34-1 (HEMO) and HHLA2 HERVs, which can be used in vaccines and other compositions for the prevention or therapeutic treatment of cancer. Despite this advance, there remains a need for additional reagents derived from ERVMER34-1 (HEMO) and improved immunological reagents. The present invention addresses this clinical need. Leydig 771368 E-056-2023-0-PCT-01 2 BRIEF OF THE INVENTION [0005] The invention provides a protein or polypeptide derived from ERVMER34-1. The invention further provides a nucleic acid encoding the inventive protein or polypeptide, a vector comprising the nucleic acid, cells comprising the protein or polypeptide, nucleic acid, and/or vector, and composition, such as a pharmaceutical composition, comprising any of the foregoing. These reagents (the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof) are useful for immunological treatment and prophylaxis of diseases, particularly cancers. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) [0006] Figures 1A through 1E graphically present data concerning the identification of ERVMER34-1 as a potential therapeutic target. (Figure 1A) RNA expression levels of ERVMER34-1 in multiple human carcinomas (black dots) along with histologically normal tissues adjacent to the tumor when available (grey dots). The shaded area represents the median expression of ERVMER34-1 in normal tissues adjacent to the tumor plus two standard deviations. (Figure 1B) Western blot of ERVMER34-1 expression in protein lysates of matched tumor and tumor adjacent tissues collected from patients diagnosed with either lung or colon adenocarcinomas (left panel). ERVMER34-1 expression in lung and colon protein lysates collected from healthy donors (right panel). GAPDH is used as a loading control. (Figure 1C) Representative images of immunofluorescence-based staining of ERVMER34-1 protein, and the tumor-associated antigens CEA and brachyury in human cerebellum, colon, breast, uterus, and testis. Scale bars indicate 100 μm. (Figure 1D) Representative images of immunohistochemical staining of ERVMER34-1 protein expression in human colon carcinoma, bladder carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, and endometrial carcinoma. Cytokeratin is included as a tumor marker. Scale bars indicate 50 μm. (Figure 1E) Representative images of immunohistochemical staining of ERVMER34-1 protein expression in normal lung, histologically normal lung tissues adjacent to the tumor, and lung squamous cell carcinoma tissue. Indicated case numbers correspond to those indicated in Table 6. Scale bars indicate 50 μm. DAPI staining also is represented. Leydig 771368 E-056-2023-0-PCT-01 3 [0007] Figures 2A through 2K present data demonstrating that ERVMER34-1 specific immunity can be expanded through in vitro stimulation. (Figure 2A) Immune reactivity of healthy donor and cancer patient PBMCs against the 15-mer ERVMER34-1 peptide library, as assessed using a 16-hour IFN-γ ELISPOT assay. (Figure 2B) Immune reactivity of individual 15-mer ERVMER34-1 peptides comprising the peptide library as assessed in 5 paired healthy donors by a 16-hour IFN-γ ELISPOT assay (left panel) or following 7 days of in vitro stimulation with a ERVMER34-1 peptide library prior to performing a 16-hour IFN-γ ELISPOT assay (right panel). HLA-A alleles of each healthy donor are indicated below. Each column represents a healthy donor, and each row indicates immune reactivity of each single ERVMER34-1 peptide that comprises the ERVMER34-115-mer peptide library. (Figure 2C) Immune reactivity of individual 15-mer ERVMER34-1 peptides in carcinoma patients following 7 days of in vitro stimulation with a ERVMER34-1 peptide library prior to performing a 16-hour IFN-γ ELISPOT assay. (Figure 2D) ERVMER34-1 immunity detected in lung, breast, and colon carcinoma patients as well as male and female healthy donor PBMCs following a 7-day incubation and 24-hour restimulation with a pool of ERVMER34-1 peptides as assessed by an intracellular cytokine assay. Each column indicates cytokine production from a single donor, and each row a single detected analyte. (Figure 2E) Representative data from an intracellular cytokine assay comparing relative immunogenicity of ERVMER34-1 to additional tumor- associated antigens. (Figure 2F) Comparison of immunity obtained following a 7-day stimulation with either ERVMER34-1, CEACAM5, PSA and MUC1 in 12 healthy donors. Mapping the diversity of ERVMER34-1 reactive CD8+ T cells expanded from PBMCs from a healthy donor A (Figures 2G and 2H) and donor B (Figures 2I and 2J). (Figure 2K) Lysis of the parental SW620 and SW620 ERVMER34-1 CRISPR knockout cell lines following overnight incubation with CD8+ T cells expanded from PBMCs from donor A at indicated effector-to- target ratios. (Figure 2L) Percent specific lysis of the parental SW620 and SW620 ERVMER34- 1 CRISPR knockout cell lines following overnight incubation with CD8+ T cells expanded from PBMCs from donors B-D using an effector-to-target ratio of 5:1. **P ≤ 0.01; ****P ≤ 0.0001 by an unpaired T test. Leydig 771368 E-056-2023-0-PCT-01 4 [0008] Figures 3A through 3G present data demonstrating the generation of a therapeutic cancer vaccine targeting ERVMER34-1. (Figure 3A) Graphical representations of the full-length and ERVMER34-1 vaccine. (Figure 3B) Amino acid sequence of ERVMER34-1 with identified protein regions highlighted. The sequences removed from the ERVMER34-1 vaccine are underlined. (Figure 3C) ERVMER34-1 protein expression in human dendritic cells following infection with control, full-length or ERVMER34-1 vaccine adenovirus. Dendritic cells were infected 48 hours prior to protein isolation with viral particles at one of four multiplicities of infection (MOI): 20,000, 6,600, 2,220 or 730, respectively, in lanes from left to right. GAPDH is included as a protein loading control. (Figure 3D) Graphical representation of experiment (left panel). Detection of soluble ERVMER34-1 protein in the sera of mice 1 day prior to and 3 days following vaccination with control, full-length, or ERVMER34-1 vaccine (right panel). (Figure 3E) Enumeration of ERVMER34-1 reactive T cells in splenocytes harvested from animals vaccinated with control, full-length or the ERVMER34-1 vaccine using a 16-hour IFN-γ ELISPOT assay. Each row is an individual animal, and each column is a single peptide comprising the 15-mer peptide library. (Figures 3F and 3G) Enumeration of ERVMER34-1 specific multi-functional T cells following vaccination with either the full-length ERVMER34-1 adenovirus or the ERVMER34-1 vaccine as assessed by an ex vivo intracellular cytokine production assay. Number indicates the percentage of total T cells responding to ERVMER34-1 peptides. (Figure 3H) Relative magnitude of ERVMER34-1 reactive CD4+ and CD8+ T cells. [0009] Figure 4 graphically presents data demonstrating that vaccine targeting ERVMER34- 1 mediates tumor control of established EMT6 tumors. (Panel A) Immunofluorescence staining of ERVMER34-1 protein expression in EMT6 parental and EMT6 pERVMER34-1 tumors. DAPI staining also is represented. (Panel B) Treatment schema. (Panel C) Growth of EMT6 pERVMER34-1 tumors in mice treated as indicated. [0010] Figures 5A through 5I graphically present data demonstrating vaccine targeting ERVMER34-1 mediates regression of established tumors. (Figure 5A) Immunofluorescence staining of ERVMER34-1 protein expression in MC38 parental and MC38 pERVMER34-1 tumors. DAPI staining also is represented. (Figure 5B) Treatment scheme. (Figure 5C) Survival Leydig 771368 E-056-2023-0-PCT-01 5 curves of mice treated as indicated in in panel (Figure 5D) Enumeration of tumor infiltrating lymphocytes in mice treated as indicated in panel B. (Figure 5E) ERVMER34-1 immune reactivity of splenocytes and expanded tumor infiltrating lymphocytes harvested from animals vaccinated with control adenovirus or ERVMER34-1 vaccine as assessed by a 16-hour IFN-γ ELISPOT assay using individual 15-mer ERVMER34-1 peptides in the assay. (Figures 5F and 5G) Comparison of immune reactivity of ERVMER34-1, MC38 neoepitopes and p15e of tumor- infiltrating T cells. (Figure 5G) Assessment of IFN-γ present within the tumor microenvironment in mice treated as shown in panel E. (Figure 5H) Expression of PD-L1 on the surface of CD45+CD11b+ cells (left panel) and CD45‒ cells (right panel) within the tumor. (Figure 5I) Tumor growth curves following tumor rechallenge with parental MC38 cells (left flank) and MC38 pERVMER34-1 tumors (right flank). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 by an unpaired T test. [0011] Figures 6A through 6F presents information and data concerning experiments demonstrating that a combination of immune checkpoint blockade with ERVMER34-1 vaccine enhances its anti-tumor efficacy. (Figure 6A) Graphical representation of tumor treatment schedule used for subsequent panels. (Figure 6B) Spider plots of growth curves of MC38 pERVMER34-1 tumors treated as indicated in panel A. (Figure 6C) Immune reactivity of splenocytes harvested from animals treated as indicated using a 16-hour IFN-γ ELISPOT assay with individual 15-mer ERVMER34-1 peptides in the assay. (Figure 6D) Immune reactivity of splenocytes harvested from animals treated as indicated using a 16-hour IFN-γ ELISPOT assay with individual MC38 neoepitope peptides in the assay. (Figure 6E) Survival curve of animals treated as indicated in panel A. (Figure 6F) Tumor growth curves following tumor rechallenge with parental MC38 cells (left flank) and MC38 pERVMER34-1 tumors (right flank). *P ≤ 0.05; **P ≤ 0.01 by an unpaired T test. *P ≤ 0.05; **P ≤ 0.01 by an unpaired T test. [0012] Figure 7 graphically depicts: (Panel A) RNA expression of indicated endogenous retroviral transcripts in normal tissues in the GTex database. (Panel B) RNA expression of indicated endogenous retroviral transcripts in tumor tissues in the Cancer Genome Atlas (TCGA) database. (Panel C) Survival curves of patients diagnosed with uveal melanoma, head and neck squamous and adenoid cystic carcinomas based upon high and low ERVMER34-1 RNA Leydig 771368 E-056-2023-0-PCT-01 6 expression in tumor tissues. Cutoffs for high low ERVMER34-1 expression were set to the top 66% and lower 33%, respectively. [0013] Figure 8 graphically depicts representative ELISPOT images. [0014] Figure 9 graphically depicts the flow cytometric gating strategy for cytokine- producing T cells (Figure 9A) Human PBMCs, (Figure 9B) Murine splenocytes. [0015] Figure 10 graphically depicts: (Panel A) Western blot of ERVMER34-1 expression in either parental K562 cells or the aAPC cell line. (Panel B) Flow cytometric analysis of CD80 and HLA-A2 on either K562 or aAPC cell lines. (Panel C) Immune-fluorescent analysis of ERVMER34-1 expression in either parental SW620 (left panel) or SW620 ERVMER34-1 CRISPR knock-out cell line (right panel). DAPI staining also is represented. [0016] Figure 11 graphically depicts tumor growth curves of MC38 pERVMER34-1 tumors treated with a single dose (dotted line) of either control adenovirus or ERVMER34-1 vaccine. DETAILED DESCRIPTION OF THE INVENTION Proteins and Polypeptides [0017] In one aspect, the invention provides a protein or polypeptide derived from ERVMER34-1. The inventive protein or polypeptide can be either isolated or present in a composition or cell comprising other elements. In the context of the present invention, the term "isolated" as used herein encompasses compounds or compositions that have been removed from a biological environment (e.g., a cell, tissue, culture medium, body fluid, etc.) or otherwise increased in purity to any degree (e.g., isolated from a synthesis medium). Isolated compounds and compositions, thus, can be synthetic or naturally produced. [0018] In one embodiment, the inventive protein or polypeptide is a protein that comprises, consists essentially of, or consists of a sequence of amino acids substantially as set forth in SEQ ID NO:2. It will be observed that the amino acid sequence of SEQ ID NO:2 is derived from the full length ERVMER34-1 peptide sequence (SEQ ID NO:1). In one respect, SEQ ID NO:2 lacks the signal domain of the full length ERVMER34-1 amino acid sequence, which can prevent the trafficking of the protein to the cell surface. SEQ ID NO:2 also lacks the cleavage site of the full length ERVMER34-1 amino acid sequence, which can prevent the cleavage of the surface unit Leydig 771368 E-056-2023-0-PCT-01 7 from the transmembrane unit of the 1 protein. Furthermore, SEQ ID NO:2 also lacks predicted immunosuppressive domain of the full length ERVMER34-1 amino acid sequence, which can prevent the potential dampening of the immunity generated against the target antigen. A fourth modification is that SEQ ID NO:2 lacks the transmembrane domain of the ERVMER34-1 protein, which shares homology with several human proteins. [0019] It will be understood that the amino acid sequence of the inventive protein can comprise or consist essentially of SEQ ID NO:2 while not consisting exactly of SEQ ID NO:2. In this respect, the inventive protein or polypeptide can include one or more additional amino acid mutations (substitutions, insertions or deletions), for example, to inactivate or delete a natural biological function of the full length ERVMER34-1 protein (SEQ ID NO:1) (e.g., to improve expression or enhance safety of the antigen). Preferably, the inventive protein shares at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 99% sequence identity with, or is 100% identical to (i.e., consisting of) SEQ ID NO:2. [0020] In another aspect, the inventive protein or polypeptide is a 15-mer polypeptide that comprises, consists essentially of, or consists of a sequence of amino acids substantially as set forth in SEQ ID NOs:7-99. Preferably, the amino acid of the inventive polypeptide consists of a sequence of amino acids exactly as set forth in SEQ ID NOs:7-99, although minor deviations from these sequences, when such do not alter the immunogenicity of the polypeptide, are within the scope of the present invention. [0021] The inventive protein or polypeptide can be prepared by any method, such as by synthesizing the protein or polypeptide (e.g., solid-state synthesis) or by expressing a nucleic acid encoding an appropriate amino acid sequence for the peptide or polypeptide in a cell and, in some embodiments, harvesting the peptide or polypeptide from the cell. In some embodiments, the peptide or polypeptide is not harvested from the cell, such as in embodiments of the invention directed to a yeast-based immunotherapy composition, which is described in detail below. A combination of such methods of production of peptides and polypeptides also can be used. Methods of de novo synthesizing peptides and methods of recombinantly producing peptides or polypeptides are known in the art. Leydig 771368 E-056-2023-0-PCT-01 8 Nucleic Acids [0022] The invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding the inventive protein or polypeptide. The inventive nucleic acid molecule can comprise DNA (genomic or cDNA) or RNA and can be single or double stranded. Furthermore, the inventive nucleic acid molecule can comprise nucleotide analogues or derivatives (e.g., inosine or phosphonothioate nucleotides and the like). The inventive nucleic acid sequence can encode the inventive protein or polypeptide alone or as part of a fusion protein. Persons of ordinary skill are able to deduce the coding sequence of a nucleic acid when given the sequence of the encoded protein or polypeptide. [0023] The inventive nucleic acid encoding the peptide or polypeptide can be provided as part of a construct comprising the inventive nucleic acid molecule and elements that enable delivery of the nucleic acid molecule to a cell, and/or expression of the nucleic acid molecule in a cell. Such elements include, for example, expression vectors, promoters, and transcription and/or translation control sequences. Such constructs can also be referred to as "recombinant nucleic acid molecules." Suitable vectors, promoters, transcription/translation sequences, and other elements, as well as methods of preparing such nucleic acid molecules and constructs, are known in the art. [0024] The inventive nucleic acid can be made by any suitable method, such as solid-state synthesis, known to persons or ordinary skill. [0025] Although the phrases “nucleic acid” and "nucleic acid molecule" primarily refer to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a peptide or polypeptide. Similarly, the phrase "recombinant nucleic acid molecule" primarily refers to a nucleic acid molecule operatively linked to an element such as a transcription control sequence but can be used interchangeably with the phrase "nucleic acid molecule." Leydig 771368 E-056-2023-0-PCT-01 9 Vectors [0026] The invention further provides a vector comprising the inventive nucleic acid molecule comprising a nucleic acid sequence encoding the inventive protein or polypeptide. Examples of suitable vectors include plasmids (e.g., DNA plasmids), bacteria, yeast, listeria, and viral vectors, including poxvirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, polio virus, alphavirus, and baculorvirus, Sindbis virus. Where the vector is a plasmid, the plasmid can be complexed with one or more agents for facilitating transfection of cells or enhancing stability, e.g., chitosan. [0027] In a preferred embodiment, wherein the inventive protein or polypeptide comprises, consists essentially of, or consists of a sequence of amino acids substantially as set forth in SEQ ID NO:2, the vector is an adenoviral vector. [0028] In addition to the nucleic acid molecule encoding the inventive protein or polypeptide, a vector useful in the invention (e.g., a plasmid or a viral vector) also can comprise a nucleic acid sequence encoding one or more immunostimulatory/regulatory molecules, granulocyte macrophage colony stimulating factor (GM-CSF), cytokines, and/or molecules that can enhance an immune response (e.g., additional tumor-associated antigens). Exemplary additional tumor-associated antigens (TAAs, also referred to as cancer antigens) include, but are not limited to, 5-α-reductase, α-fetoprotein (AFP), AM-1, APC, April, B melanoma antigen gene (BAGE), β-catenin, Bcl12, bcr-abl, Brachyury, CA-125, caspase-8 (CASP-8 also known as FLICE), Cathepsins, CD19, CD20, CD21/complement receptor 2 (CR2), CD22/BL-CAM, CD23/FcεRII, CD33, CD35/complement receptor 1 (CR1), CD44/PGP-1, CD45/leucocyte common antigen (LCA), CD46/membrane cofactor protein (MCP), CD52/CAMPATH-1, CD55/decay accelerating factor (DAF), CD59/protectin, CDC27, CDK4, carcinoembryonic antigen (CEA), c-myc, cyclooxygenase-2 (cox-2), deleted in colorectal cancer gene (DCC), DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, fibroblast growth factor-8a (FGF8a), fibroblast growth factor-8b (FGF8b), FLK-1/KDR, folic acid receptor, G250, G melanoma antigen gene family (GAGE-family), gastrin 17, gastrin-releasing hormone, ganglioside 2 (GD2)/ganglioside 3 (GD3)/ganglioside-monosialic acid-2 (GM2), gonadotropin releasing hormone (GnRH), UDP-GlcNAc:R1Man(α1-6)R2 [GlcNAc to Man(α1-6)] β1,6-N- Leydig 771368 E-056-2023-0-PCT-01 10 acetylglucosaminyltransferase V (GnT V), gp100/Pme117, gp-100-in4, gp15, gp75/tyrosine-related protein-1 (gp75/TRP-1), human chorionic gonadotropin (hCG), heparanase, Her2/neu, human mammary tumor virus (HMTV), 70 KD heat-shock protein (HSP70), human telomerase reverse transcriptase (hTERT), insulin-like growth factor receptor-1 (IGFR-1), interleukin-13 receptor (IL-13R), inducible nitric oxide synthase (iNOS), Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT, melanoma antigen-encoding family (MAGE-family, including at least MAGE-1, MAGE-2, MAGE-3, and MAGE-4), mammaglobin, MAP17, Melan-A/melanoma antigen recognized by T-cells-1 (MART-1), mesothelin, MIC A/B, MT-MMPs, mucin (e.g., MUC1), testes-specific antigen NY-ESO-1, osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1, platelet-derived growth factor (PDGF), µPA, PRAME, probasin, progenipoietin, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), RAGE-1, Rb, RCAS1, mutated Ras, SART-1, SSX-family, STAT3, STn, TAG-72, transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), Thymosin-beta-15, tumor necrosis factor-alpha (TNF-α), TP1, TRP-2, tyrosinase, vascular endothelial growth factor (VEGF), ZAG, p16INK4, and glutathione-S-transferase (GST), as well as modified versions thereof (e.g., CEA-6D). [0029] In the case of a viral vector, the nucleic acid encoding the inventive protein or polypeptide, as well as any other exogenous gene(s), preferably is/are inserted into a site or region (insertion region) in the vector (e.g., adenovirus) that does not affect virus viability of the resultant recombinant virus. Such regions can be readily identified by testing segments of virus DNA for regions that allow recombinant formation without seriously affecting virus viability of the recombinant virus and are generally known to those of skill in the art. For example, a thymidine kinase (TK) gene, such as is present in many viruses, can serve as an insertion region. [0030] The inventive vector can include suitable promoters and regulatory elements, such as a transcriptional regulatory element or an enhancer. Suitable promoters include the SV40 early promoter, an RSV promoter, the retrovirus LTR, the adenovirus major late promoter, the human CMV immediate early I promoter, and various poxvirus promoters, such as the Pr7.5K promoter, 30K promoter, 40K promoter, I3 promoter, Prs promoter, PrsSynIIm promoter, PrLE1 promoter, synthetic early/late (sE/L) promoter, HH promoter, 11K promoter, and Pi promoter. While the Leydig 771368 E-056-2023-0-PCT-01 11 promoters typically will be constitutive inducible promoters also can be used in the inventive vectors. Such inducible systems allow regulation of gene expression. Cells [0031] In one aspect of the invention, a cell (e.g., isolated cell) comprising (1) in inventive protein or polypeptide, (2) an inventive nucleic acid molecule encoding the inventive protein or polypeptide, and/or (3) a vector comprising an inventive nucleic acid molecule also is provided herein. Suitable cells include prokaryotic and eukaryotic cells, e.g., mammalian cells, yeast, fungi other than yeast, and bacteria (such as E. coli). The cell can be used in vitro, such as for research or for production of the peptide or polypeptide, or the cell can be used in vivo. In one embodiment, the cell is a yeast cell, which may be used to provide a yeast vehicle component of the yeast-based immunotherapy composition as described herein. In another embodiment, the cell can be an antigen presenting cell (APC). Suitable APCs include, but are not limited to, dendritic cells, B lymphocytes, monocytes, macrophages, and the like. [0032] In one embodiment, the cell is dendritic cell. Dendritic cells of different maturation stages can be isolated based on the cell surface expression markers. For example, mature dendritic cells are less able to capture new proteins for presentation but are much better at stimulating resting T cells to grow and differentiate. Thus, mature dendritic cells can be of importance. Mature dendritic cells can be identified by their change in morphology and by the presence of various markers. Such markers include, but are not limited to, cell surface markers such as B7.1, B7.2, CD40, CD11, CD83, and MHC class II. Alternatively, maturation can be identified by observing or measuring the production of pro-inflammatory cytokines. [0033] Dendritic cells can be collected and analyzed using typical cytofluorography and cell sorting techniques and devices, such as a fluorescence-activated cell sorter (FACS). Antibodies specific to cell surface antigens of different stages of dendritic cell maturation are commercially available and can be used for identification and purification. [0034] In another embodiment, the invention provides a cell, such as an APC, engineered to express an ERVMER34-1 protein and one or both of HLA (such as human HLA-A2) and the CD80 costimulatory molecule. In this embodiment, the ERVMER34-1 protein can be or Leydig 771368 E-056-2023-0-PCT-01 12 comprise an amino acid sequence of or the full-length ERVMER34-1 protein (SEQ ID NO:1) or a modified version, such as the inventive protein (SEQ ID NO:2, including variants thereof as discussed above). The cell can be generated, for example, by co-transfecting and/or - infecting an APC cell with vectors for expressing HLA (human HLA-A2 is discussed below in Example 6), CD80, and the desired ERVMER34-1 protein. The cell thereafter can be expanded to generate a cell line, comprising APCs expressing the ERVMER34-1 protein along with HLA- A2 and the CD80 costimulatory molecule. The cell line can be used in immunological research in vitro or, in some applications, therapeutically. [0035] In one aspect of the invention, a cell (e.g., isolated cell) comprising (1) the peptide or polypeptide, (2) a nucleic acid molecule encoding the peptide or polypeptide, and/or (3) a vector comprising the nucleic acid molecule also is provided herein. Suitable cells include prokaryotic and eukaryotic cells, e.g., mammalian cells, yeast, fungi other than yeast, and bacteria (such as E. coli). The cell can be used in vitro, such as for research or for production of the peptide or polypeptide, or the cell can be used in vivo. In one embodiment, the cell is a yeast cell, which may be used to provide a yeast vehicle component of the yeast-based immunotherapy composition as described herein. In another embodiment, the cell can be a peptide-pulsed antigen presenting cell. Suitable antigen presenting cells include, but are not limited to, dendritic cells, B lymphocytes, monocytes, macrophages, and the like. [0036] In one embodiment, the cell is dendritic cell. Dendritic cells of different maturation stages can be isolated based on the cell surface expression markers. For example, mature dendritic cells are less able to capture new proteins for presentation but are much better at stimulating resting T cells to grow and differentiate. Thus, mature dendritic cells can be of importance. Mature dendritic cells can be identified by their change in morphology and by the presence of various markers. Such markers include, but are not limited to, cell surface markers such as B7.1, B7.2, CD40, CD11, CD83, and MHC class II. Alternatively, maturation can be identified by observing or measuring the production of pro-inflammatory cytokines. [0037] Dendritic cells can be collected and analyzed using typical cytofluorography and cell sorting techniques and devices, such as a fluorescence-activated cell sorter (FACS). Antibodies Leydig 771368 E-056-2023-0-PCT-01 13 specific to cell surface antigens of different of dendritic cell maturation are commercially available. Compositions [0038] The inventive protein or polypeptide, nucleic acid, vector, or cell can be formulated as a composition (e.g., pharmaceutical composition) comprising the protein or polypeptide, nucleic acid, vector, or cell and a carrier (e.g., a pharmaceutically or physiologically acceptable carrier). Furthermore, the protein or polypeptide, nucleic acid, vector, cell, or composition of the invention can be used in the methods described herein alone or as part of a pharmaceutical formulation. [0039] The composition (e.g., pharmaceutical composition) can comprise more than one protein or polypeptide, nucleic acid, vector, or cell of the invention. Vectors and compositions of the invention can further include or can be administered with (concurrently, sequentially, or intermittently with) any other agents or compositions or protocols that are useful for inhibiting, preventing, or treating a disease or clinical condition. For example, the composition can comprise one or more other pharmaceutically active agents or drugs. Examples of such other pharmaceutically active agents or drugs that may be suitable for use in the pharmaceutical composition include anticancer agents (e.g., chemotherapeutic or radiotherapeutic agents), antimetabolites, hormones, hormone antagonists, antibiotics, antiviral drugs, antifungal drugs, cyclophosphamide, and combinations thereof. [0040] Particularly for applications in which the inventive composition is used for the treatment of cancer, in addition to the protein or polypeptide, nucleic acid, vector, or cell of the invention, the composition can comprise one or more additional anti-cancer agents. Alternatively, such additional anti-cancer agents can be co-administered with the inventive composition, such as in a separate composition. [0041] Suitable anticancer agents include, without limitation, alkylating agents, folate antagonists, purine antagonists, pyrimidine antagonists, spindle poisons, topoisomerase inhibitors, apoptosis inducing agents, angiogenesis inhibitors, podophyllotoxins, nitrosoureas, cisplatin, carboplatin, interferon, asparginase, tamoxifen, leuprolide, flutamide, megestrol, Leydig 771368 E-056-2023-0-PCT-01 14 mitomycin, bleomycin, doxorubicin, taxol, geldanamycin (e.g., 17-AAG), and various anti-cancer peptides and antibodies known in the art. Non-limiting examples of additional anti- cancer peptides that can be used in combination with the protein or polypeptide, nucleic acid, vector, or cell include those disclosed in International Patent Publication WO 2021/150694, which is incorporated herein in its entirety. Some of these peptides are set forth herein as SEQ ID NOs:100-140, and others are identified in the Tables within the specification of WO 2021/150694, which are incorporated herein by reference. Additionally, other anti-cancer agents that can be included in the inventive composition, or be co-administered with the inventive composition, include, but are not limited to, α-PD-1, α-CTLA4, α-PD-1 + α-CTLA4 Adoptive transfer vaccine primed autologous T cells + autologous DC tumor cell lysate vaccine + bevacizumab, Allo-tumor cells (XRT) + GM-CSF + BCG, Autologous DC tumor cell lysate vaccine + bevacizumab +/- low-dose cyclophosphamide, Bintrafusp alfa (α-PD-L1, GFβRII), bevacizumab, MAGE vaccine (Melanoma antigen), and N803 (IL-15 superagonist). A particularly interesting combination for use in the present inventive methods includes SEQ ID NO:2, IL15 superagonist (N803, ANKTIVA (nogapendekin alfa inbakicept-pmln)), and/or anti- PD-L1. Other checkpoint inhibitors can be employed in addition to or in lieu of anti-PD-L1, such as anti-CTLA4 and/or anti-PD-1. [0042] Exemplary alkylating agents include, but are not limited to, nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin, or dacarbazine). Exemplary antimetabolites include, but are not limited to, folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil (5-FU) or cytarabine), and purine analogs (e.g., mercaptopurine or thioguanine). Exemplary hormones and hormone antagonists include, but are not limited to, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., tamoxifen), and androgens (e.g., testosterone proprionate and fluoxymesterone). Other exemplary agents include, but are not limited to, vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, Leydig 771368 E-056-2023-0-PCT-01 15 bleomycin, plicamycin, or mitocycin C), (e.g., L-asparaginase), platinum coordination complexes (e.g., cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (e.g., hydroxyurea), methyl hydrazine derivatives (e.g., procarbazine), and adrenocortical suppressants (e.g., mitotane and aminoglutethimide). [0043] Chemotherapeutics that can be concurrently, sequentially or intermittently administered with the vectors and compositions disclosed herein include Adriamycin, Alkeran, Ara-C, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin, Enzalutamide (MDV-3100 or XTANDITM), and calcitriol. Exemplary immunomodulators and/or cytokines include, but are not limited to, AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, tumor necrosis factor (TNF)-α, and TNF-β. [0044] Other agents, compositions or protocols (e.g., therapeutic protocols) that are useful for the treatment of cancer in conjunction with the inventive protein or polypeptide, nucleic acids, vectors, cells, and compositions of the invention include, but are not limited to, surgical resection of a tumor, radiation therapy, allogeneic or autologous stem cell transplantation, adoptive T cell transfer, chimeric antigen receptor (CAR) T-cell therapy, tumor-infiltrating lymphocytes (TIL), and/or targeted cancer therapies (e.g., small molecule drugs, biologics, or monoclonal antibody therapies that specifically target molecules involved in tumor growth and progression, including, but not limited to, selective estrogen receptor modulators (SERMs), aromatase inhibitors, tyrosine kinase inhibitors, serine/threonine kinase inhibitors, histone deacetylase (HDAC) inhibitors, retinoid receptor activators, apoptosis stimulators, angiogenesis inhibitors, poly (ADP-ribose) polymerase (PARP) inhibitors, or immunostimulators). Additionally, or alternatively, the agent can be a cancer vaccine, such as PANVAC, Leydig 771368 E-056-2023-0-PCT-01 16 PROSTVAC, MVA-Brachyury TRICOM, Brachyury, AdCEA Avelumab (Avel) Folfox, CEA-MUC-TRICOM CV301, or Bacillus Calmette-Guerin (BCG) alone or combined with PANVAC. [0045] The additional active agent (e.g., chemotherapeutic agent) can be administered before, concurrently with (including simultaneously), alternating with, sequentially, or after administration with the inventive composition. In certain embodiments, one or more (e.g., 2, 3, 4, or 5) chemotherapeutic agents is/are administered in combination with the inventive composition. [0046] The additional active agent(s) can be administered alone or in a composition. The additional active agent(s) can be formulated by inclusion in a vector (e.g., plasmid or viral vector), in liposomes (tecemotide, which is also known as STIMUVAXTM, L-BLP25, or BLP25 liposome vaccine), or in nanoparticles (e.g., VERSAMUNETM nanotechnology). [0047] The composition additionally or alternatively can comprise one or more immunostimulatory/regulatory molecules. Any suitable immunostimulatory/regulatory molecule can be used, such as interleukin (IL)-2, IL-4, IL-6, IL-12, IL-15, IL-15/IL-15Ra, IL-15/IL-15Ra- Fc, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, B7.1, B7.2, ICAM-1, ICAM-2, LFA-1, LFA-2, LFA-3, CD70, CD-72, RANTES, G-CSF, GM-CSF, OX-40L, 41 BBL, anti-CTLA-4, IDO inhibitor, anti-PD-L1, anti-PD1, and combinations thereof. In one embodiment, the IL-12 is NHS-IL12, which is an immunocytokine composed of two IL-12 heterodimers fused to the NHS76 antibody (see Strauss et al., Clinical Cancer Research, 25(1): 99-109 (2019)). Preferably, the composition comprises a combination of B7.1, ICAM-1, and LFA-3 (also referred to as TRICOM). The one or more immunostimulatory/regulatory molecules can be administered in the form of a vector (e.g., a recombinant viral vector, such as those discussed herein or otherwise known to those of skill in the art) comprising a nucleic acid encoding one or more immunostimulatory/regulatory molecules. For example, the one or more immunostimulatory/regulatory molecules (e.g., IL-12) can be administered in the form of a DNA plasmid with or without chitosan. Alternatively, the one or more immunostimulatory/regulatory molecules can be administered as a protein (e.g., recombinant protein), such as a protein (e.g., recombinant IL-12) with or without being admixed with chitosan. One or more Leydig 771368 E-056-2023-0-PCT-01 17 immunostimulatory/regulatory molecules also be administered in combination with, or concurrently with, a yeast-based immunotherapy composition of the invention. [0048] In formulation and for use in the inventive methods discussed herein, the inventive protein or polypeptide also can be conjugated to helper peptides or to large carrier molecules to enhance the immunogenicity of the peptide or polypeptide. These molecules include, but are not limited to, influenza peptide, tetanus toxoid, tetanus toxoid CD4 epitope, Pseudomonas exotoxin A, poly-L-lysine, a lipid tail, endoplasmic reticulum (ER) signal sequence, and the like. The inventive peptide or polypeptide (protein) also can be conjugated to an immunoglobulin molecule using art-accepted methods. The immunoglobulin molecule can be specific for a surface receptor present on tumor cells, but absent or in very low amounts on normal cells. The immunoglobulin also can be specific for a specific tissue (e.g., breast, ovarian, colon, or prostate tissue). Such a peptide-immunoglobulin conjugate or polypeptide-immunoglobulin conjugate allows for targeting of the peptide to a specific tissue and/or cell. [0049] The carrier used in the inventive composition can be any of those conventionally used and is limited only by physio-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. [0050] The choice of carrier and manner of formulation of the inventive composition will be determined in part by the particular protein or polypeptide, nucleic acid, vector, cell, or composition thereof of the invention and other active agents or drugs used, as well as by the particular method used to administer the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof. A variety of suitable formulations of the pharmaceutical composition, thus, can be employed in the inventive compositions, and in carrying out the inventive methods described herein. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, and intraperitoneal administration are exemplary and are in no way limiting. One skilled in the art will appreciate that these routes of administering the protein or Leydig 771368 E-056-2023-0-PCT-01 18 polypeptide, nucleic acid, vector, cell, or of the invention are known, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective response than another route. [0051] Injectable formulations are among those formulations that are preferred in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622- 630 (1986)). [0052] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. [0053] Oils, which can be used in parenteral formulations, include petroleum, animal, vegetable, and synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Leydig 771368 E-056-2023-0-PCT-01 19 [0054] Suitable soaps for use in include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-b-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof. [0055] Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. [0056] The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Uses [0057] Using the inventive reagents (the protein or polypeptide, nucleic acid, vector, cell, or composition), the invention provides the therapeutic (including prophylactic) use of such for treatment or prophylaxis of disease, particularly in a human patient. One particularly contemplated type of disease particularly amenable to prophylaxis or therapy using the inventive reagents includes cancers. Accordingly, the invention provides methods of treating a subject suffering from or susceptible to a tumor and/or enhancing an immune response against cancer and/or inhibiting a cancer. Leydig 771368 E-056-2023-0-PCT-01 20 [0058] In a first embodiment, the comprise administering a therapeutically effective amount of one or more of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof to a subject. The inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be used to prevent the development of cancer, particularly in an individual at higher risk to develop such cancer than other individuals, or to treat a patient afflicted with cancer. The inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof is useful for preventing emergence of cancer, arresting progression of cancer or eliminating cancer. More particularly, the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be used to prevent, inhibit or delay the development of tumors, and/or to prevent, inhibit or delay tumor migration and/or tumor invasion of other tissues (metastases) and/or to generally prevent or inhibit progression of cancer in an individual. The inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can also be used to ameliorate at least one symptom of the cancer, such as by reducing tumor burden in the individual; inhibiting tumor growth in the individual; increasing survival of the individual; and/or preventing, inhibiting, reversing or delaying progression of the cancer in the individual. The inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be used to treat a subject with any stage of cancer. [0059] Administration of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be “prophylactic” or “therapeutic.” When provided prophylactically, the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof is provided in advance of tumor formation, or the detection of the development of tumors, with the goal of preventing, inhibiting or delaying the development of tumors; and/or preventing, inhibiting or delaying metastases of tumors and/or generally preventing or inhibiting progression of cancer in an individual, and generally to allow or improve the ability of the host’s immune system to fight against a tumor that the host is susceptible of developing. The prophylactic administration of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof prevents, ameliorates, or delays the cancer. [0060] When provided therapeutically, the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof is provided at or after the diagnosis of the cancer, with the Leydig 771368 E-056-2023-0-PCT-01 21 goal of ameliorating the cancer, such as by tumor burden in the individual; inhibiting tumor growth in the individual; increasing survival of the individual; and/or preventing, inhibiting, reversing or delaying progression of the cancer in the individual. [0061] Treatment (e.g., inhibiting cancer and/or enhancing an immune response against cancer) comprises, but is not limited to, destroying tumor cells, reducing tumor burden, inhibiting tumor growth, reducing the size of the primary tumor, reducing the number of metastatic legions, increasing survival of the individual, delaying, inhibiting, arresting or preventing the onset or development of metastatic cancer (such as by delaying, inhibiting, arresting or preventing the onset of development of tumor migration and/or tumor invasion of tissues outside of primary cancer and/or other processes associated with metastatic progression of cancer), delaying or arresting primary cancer progression, improving immune responses against the tumor, improving long term memory immune responses against the tumor antigens, and/or improving the general health of the individual. It will be appreciated that tumor cell death can occur without a substantial decrease in tumor size due to, for instance, the presence of supporting cells, vascularization, fibrous matrices, etc. Accordingly, while reduction in tumor size is preferred, it is not required in the treatment of cancer. [0062] The cancer to be treated in accordance with the inventive methods can be any cancer, including, but not limited to, cancer of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, lung, urethra, uterine, bladder, colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine, fallopian tube, heart or adrenals. More particularly, cancers include solid tumor, sarcoma, carcinomas (including but not limited to cystic carcinomas), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ Leydig 771368 E-056-2023-0-PCT-01 22 tumor, cervical cancer, testicular tumor, lung small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, medulloblastoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi’s sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma (including but not limited to uveal melanoma), neuroblastoma, retinoblastoma, a blood-born tumor, acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, or multiple myeloma. [0063] In one aspect, the administration of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof to a host results in a host cell expressing the inventive peptide and optionally one or more immunostimulatory/regulatory molecules and/or other tumor- associated antigens (such as, but not limited to, AIM-2 (Interferon-inducible protein absent in melanoma 2), ALL (Acute lymphoblastic leukemia), AML (Acute myeloid leukemia), 707-AP (707 alanine proline), anti-PD-1, anti-CTLA4, anti-PD-1 + anti-CTLA4, APL (Acute promyelocytic leukemia), ART-4 (Adenocarcinoma antigen recognized by T cells 4), Adoptive transfer vaccine primed autologous T cells + autologous DC tumor cell lysate vaccine + bevacizumab, Allo-tumor cells (XRT) + GM-CSF + BCG, Autologous DC tumor cell lysate vaccine + bevacizumab +/- low-dose cyclophosphamide, BAGE (B antigen), bcr-abl (Breakpoint cluster region-Abelson), Bintrafusp alfa (α-PD-L1, TGFβRII), bevacizumab, Brachyury, CAMEL (CTL-recognized antigen on melanoma), CAP-1 (Carcinoembryonic antigen peptide-1), CASP-8 (Caspase 8), CDC27 (Cell division cycle 27), CDK4 (Cyclin-dependent kinase 4), CEA (Carcinoembryonic antigen), CLCA2 (Calcium-activated chloride channel 2), CML (Chronic myelogenous leukemia), CT (Cancer-testis (antigen)), CTL (Cytotoxic T lymphocytes), Cyp-B (Cyclophilin B), DAM (Differentiation antigen melanoma (the epitopes of DAM-6 and DAM-10 are equivalent, but the gene sequences are different. DAM-6 is also called MAGE-B2 and DAM- 10 is also called MAGE-B1), ELF2 (Elongation factor 2), Ep-CAM (Epithelial cell adhesion molecule), EphA2, 3 (Ephrin type-A receptor 2, 3), Ets (E-26 transforming specific (family of Leydig 771368 E-056-2023-0-PCT-01 23 transcription factors)), ETV6-AML1 (Ets gene 6/acute myeloid leukemia 1 gene ETS), FGF-5 (Fibroblast growth factor 5), FN (Fibronectin), G250 (Glycoprotein 250), GAGE (G antigen), GnT-V (N-Acetylglucosaminyltransferase V), Gp100 (Glycoprotein 100 kDa), HAGE (Helicase antigen), HER-2/neu (Human epidermal receptor 2/neurological), HLA-A*0201-R170I (Arginine (R) to isoleucine (I) exchange at residue 170 of the α-helix of the α2-domain in the HLA-A2 gene), H/N (Head and neck), HSP70-2 M (Heat shock protein 70-2 mutated), HST-2 (Human signet-ring tumor 2), hTERT (Human telomerase reverse transcriptase), iCE (Intestinal carboxyl esterase), IL-13Rα2 (Interleukin 13 receptor α2 chain), KIAA0205 (Name of the gene as it appears in databases), LAGE (L antigen), LDLR/FUT (Low density lipid receptor/GDP-L- fucose:β-D-galactosidase 2-α-L-fucosyltransferase), MAGE vaccine (Melanoma antigen), MART-1/Melan-A (Melanoma antigen recognized by T cells-1 / melanoma antigen A), MART-2 (Melanoma Ag recognized by T cells-2), MC1R (Melanocortin 1 receptor), M-CSF (Macrophage colony-stimulating factor gene), MHC (Major histocompatibility complex), MSI (Microsatellite instability), MUC1, 2 (Mucin 1, 2), MUM-1, -2, -3 (Melanoma ubiquitous mutated 1, 2, 3), NA88-A (NA cDNA clone of patient M88), Neo-PAP (Neo-poly(A) polymerase, NPM/ALK (Nucleophosmin/anaplastic lymphoma kinase fusion protein), NSCLC (Non–small cell lung carcinoma), NY-ESO-1 (New York esophageous 1), OA1 (Ocular albinism type 1 protein), OGT (O-Linked N-acetylglucosamine transferase gene), ORF (Open reading frame), OS-9, Name of the gene as it appears in databases, P15 (Protein 15), p190 minor bcr-abl (Protein of 190-kDa bcr-abl), Pml/RARα (Promyelocytic leukemia / retinoic acid receptor α), PRAME (Preferentially expressed antigen of melanoma), PSA (Prostate-specific antigen), PSMA (Prostate-specific membrane antigen), PTPRK (Receptor-type protein-tyrosine phosphatase kappa), RAGE (Renal antigen), RCC (Renal cell carcinoma), RU1, 2 (Renal ubiquitous 1, 2), SAGE (Sarcoma antigen), SART-1, -2, -3 (Squamous antigen rejecting tumor 1, 2, 3), SCC (Squamous cell carcinoma), SSX-2 (Synovial sarcoma), X breakpoint 2, Survivin-2B (Intron 2-retaining surviving), SYT/SSX (Synaptotagmin I / synovial sarcoma, X fusion protein), TAA (Tumor-associated antigen), TEL/AML1 (Translocation Ets-family leukemia/acute myeloid leukemia 1), TGFβRII (Transforming growth factor β receptor 2), TPI (Triosephosphate isomerase), TRAG-3 (Taxol resistant associated protein 3), TRG (Testin-related gene), TRP-1 (Tyrosinase-related protein 1, Leydig 771368 E-056-2023-0-PCT-01 24 or gp75), TRP-2 (Tyrosinase-related protein , TRP-2/INT2 (TRP-2/intron 2), TRP-2/6b (TRP- 2/novel exon 6b), TSTA (Tumor-specific transplantation antigens), WT1 (Wilms’ tumor gene)), and the like, and including modified versions thereof, and immunogenic epitopes thereof that are co-administered. [0064] The inventive peptide can be expressed at the cell surface of the infected host cell. The one or more immunostimulatory/regulatory molecules and/or other tumor-associated antigens, modified versions thereof, and immunogenic epitopes thereof can be expressed at the cell surface or may be actively secreted by the host cell. The expression of both the peptide and the immunostimulatory/regulatory molecule provides the necessary MHC restricted peptide to specific T cells and the appropriate signal to the T cells to aid in antigen recognition and proliferation or clonal expansion of antigen specific T cells. The overall result is an upregulation of the immune system. Preferably, the upregulation of the immune response is an increase in antigen specific T-helper lymphocytes and/or cytotoxic lymphocytes, which are able to kill or inhibit the growth of a cancer cell. [0065] In one aspect, the inventive methods can comprise obtaining (by isolating) dendritic cells from a subject, treating the dendritic cells with one or more of the therapeutically effective amount of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof, and administering the treated dendritic cells to the subject. [0066] Similarly, the inventive methods can comprise (a) obtaining (isolating) peripheral blood mononuclear cells (PBMCs) from a subject, (b) isolating dendritic cells from the PBMCs, (c) treating the dendritic cells with one or more of the therapeutically effective amount of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof ex vivo, (d) activating the PBMCs with the treated dendritic cells ex vivo, and (e) administering the activated PBMCs to the subject. [0067] The inventive methods also can comprise a method for inhibiting cancer in a subject comprising (a) obtaining (isolating) PBMCs from a subject, (b) isolating dendritic cells from the PBMCs, (c) treating the dendritic cells with one or more of the therapeutically effective amount of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof ex vivo, (d) activating the PBMCs with the treated dendritic cells ex vivo, (e) isolating T lymphocytes Leydig 771368 E-056-2023-0-PCT-01 25 from the activated PBMCs ex vivo, and (f) the isolated T lymphocytes to the subject. The invention also provides the use of adoptively transferred T cells stimulated in vitro with one or more of the therapeutically effective amount of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof to inhibit cancer in a subject. [0068] The invention also provides T-cell receptor (TCR) engineered T cells and TCR engineered NK cells for treating (e.g., inhibiting) cancer in a subject. TCR engineered T cells and TCR engineered NK cells can be prepared by any suitable methods, such as those described in Ping et al., Protein Cell, 9(3) 254-266 (2018) (incorporated herein in its entirety). In one embodiment, the TCR engineered T cells and TCR engineered NK cells target cancer cells expressing ERVMER34-1, such as the epitopes of SEQ ID NOs: 2-99 or 100-140. [0069] The inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be administered to the subject by any method. For example, the inventive protein or polypeptide, or nucleic acid encoding the peptide or polypeptide (e.g., as a vector) can be introduced into a cell (e.g., in a host) by any of various techniques, such as by contacting the cell with the peptide, polypeptide, the nucleic acid, or a composition comprising the nucleic acid as part of a construct, as described herein, that enables the delivery and expression of the nucleic acid. Specific protocols for introducing and expressing nucleic acids in cells are known in the art. [0070] Suitable methods of administering peptides, polypeptides (proteins), nucleic acids, vectors, cells, and compositions to hosts (subjects) are known in the art. The host (subject or individual) can be any suitable host, such as a mammal (e.g., a rodent, such as a mouse, rat, hamster, or guinea pig, rabbit, cat, dog, pig, goat, cow, horse, primate, or human). For example, the inventive protein or polypeptide, nucleic acid, or vector (e.g., recombinant poxvirus) can be administered to a host by exposure of tumor cells to the peptide, polypeptide, nucleic acid, or vector ex vivo or by injection of the peptide, polypeptide, nucleic acid, or vector into the host. The peptide, polypeptide, nucleic acid, vector (e.g., recombinant poxvirus) or combination of vectors, cell, and composition can be directly administered (e.g., locally administered) by direct injection into the cancerous lesion or tumor or by topical application (e.g., with a pharmaceutically acceptable carrier). Leydig 771368 E-056-2023-0-PCT-01 26 [0071] The inventive protein or nucleic acid, vector, cell, or composition thereof can be administered alone or in combination with other anti-cancer agents, adjuvants, incorporated into liposomes (as described in, e.g., U.S. Patent Nos.5,643,599, 5,464,630, 5,059,421, and 4,885,172, which are incorporated herein in their entireties), incorporated into nanoparticles (e.g., VERSAMUNETM nanotechnology), administered with cytokines, administered with biological response modifiers (e.g., interferon, interleukin-2 (IL-2), administered colony-stimulating factors (CSF, GM-CSF, and G-CSF), and/or administered other reagents in the art that are known to enhance immune response. A particularly interesting combination for use in the present inventive methods includes SEQ ID NO:2, IL15 superagonist (N803, ANKTIVA (nogapendekin alfa inbakicept-pmln)), and/or anti-PD-L1. Other checkpoint inhibitors can be employed in addition to or in lieu of anti-PD-L1, such as anti-CTLA4 and/or anti-PD-1. [0072] Examples of suitable adjuvants include alum, aluminum salts, aluminum phosphate, aluminum hydroxide, aluminum silica, calcium phosphate, incomplete Freund’s adjuvant, saponins, such as QS21 (an immunological adjuvant derived from the bark of the South American tree Quillaja saponaria Molina), monophosphoryl lipid A (MLP-A), and RIBI DETOXTM adjuvant. [0073] In one aspect, the adjuvant for use in the invention is the cytokine, GM-CSF. GM- CSF has been shown to be an effective vaccine adjuvant because it enhances antigen processing and presentation by dendritic cells. Experimental and clinical studies suggest that recombinant GM-CSF can boost host immunity directed at a variety of immunogens. GM-CSF can be administered using a viral vector or as an isolated protein in a pharmaceutical formulation. GM- CSF can be administered to the host before, during, or after the initial administration of the peptide, polypeptide, nucleic acid, vector, cell, or composition thereof to enhance the antigen- specific immune response in the host. For example, recombinant GM-CSF protein can be administered to the host on each day of vaccination with the peptide, polypeptide, nucleic acid, vector, cell, or composition thereof and for each of the following 3 days (i.e., a total of 4 days). Any suitable dose of GM-CSF can be used. For instance, 50-500 µg (e.g., 100 µg, 200 µg, 300 µg, 400 µg, and ranges therebetween) of recombinant GM-CSF can be administered per day. Leydig 771368 E-056-2023-0-PCT-01 27 The GM-CSF can be administered by any method (e.g., subcutaneously) and, preferably, is administered at or near the site of the vaccination of a host with the peptide, polypeptide, nucleic acid, vector, cell, or composition thereof. [0074] The inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof is administered to a host (e.g., mammal, such as a human) in an amount effective to generate an immune response, preferably a cellular immune response. The efficacy of the peptide, polypeptide, nucleic acid, vector, or cell as an immunogen may be determined by in vivo or in vitro parameters as are known in the art. These parameters include but are not limited to antigen-specific cytotoxicity assays, regression of tumors, inhibition of cancer cells, production of cytokines, and the like. [0075] For use in the inventive methods, any suitable dose of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof can be administered to a host. The appropriate dose will vary depending upon such factors as the host’s age, weight, height, sex, general medical condition, previous medical history, disease progression, and tumor burden and can be determined by a clinician. For example, when the agent to be administered comprises an inventive protein or polypeptide, such can be administered in a dose of about 0.05 mg to about 10 mg (e.g., 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, and ranges therebetween) per vaccination of the host (e.g., mammal, such as a human), and preferably about 0.1 mg to about 5 mg per vaccination. Several doses (e.g., 1, 2, 3, 4, 5, 6, or more) can be provided (e.g., over a period of weeks or months). In one embodiment a dose is provided every month for 3 months. [0076] When the agent to be administered comprises an inventive viral vector, a suitable dose can include, by way of non-limiting example, about 1 x 105 to about 1 x 1012 (e.g., 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010, 1 x 1011, and ranges therebetween) plaque forming units (pfus), although a lower or higher dose can be administered to a host. For example, about 2 x 108 pfus can be administered (e.g., in a volume of about 0.5 mL). [0077] When the agent to be administered comprises an inventive cell (e.g., cytotoxic T cells), such can be administered, by way of non-limiting example, to a host in a dose of between about 1 x 105 and 2 x 1011 (e.g., 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010, and ranges Leydig 771368 E-056-2023-0-PCT-01 28 therebetween) cells per infusion. The cells administered in multiple, for example one to three (e.g., one, two, or three), infusions. In addition to the administration of the cells, the host can be administered a biological response modifier, such as interleukin 2 (IL-2). When the cells to be administered are cytotoxic T cells, the administration of the cytotoxic T cells can be followed by the administration of the peptide, polypeptide, nucleic acid, vector, or composition thereof in order to prime the cytotoxic T cells to further expand the T cell number in vivo. [0078] When the cells to be administered are dendritic cells, the amount of dendritic cells administered to the subject will vary depending on the condition of the subject and should be determined via consideration of all appropriate factors by the practitioner. Preferably, about 1x106 to about 1x1012 (e.g., about 1x107, about 1x108, about 1x109, about 1x1010, or about 1x1011 including ranges between of any of the cell numbers described herein) dendritic cells are utilized for adult humans. These amounts will vary depending on the age, weight, size, condition, sex of the subject, the type of tumor to be treated, the route of administration, whether the treatment is regional or systemic, and other factors. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the subject. [0079] In another aspect, the invention provides a method of generating peptide-specific cytotoxic T lymphocytes in vivo, ex vivo, or in vitro by stimulation of lymphocytes with an effective amount of the inventive protein or polypeptide, nucleic acid, vector, or cell, alone or in a composition with one or more immunostimulatory/regulatory molecules and/or adjuvants or in a liposome formulation. The lymphocytes can be lymphocytes from any suitable source, e.g., peripheral blood, tumor tissues, lymph nodes, and effusions, such as pleural fluid or ascites fluid. [0080] The ERVMER34-1 peptide specific cytotoxic T lymphocytes are immunoreactive. Preferably, the cytotoxic T lymphocytes inhibit the occurrence of tumor cells and cancer and inhibit the growth of, or kill, tumor cells. The cytotoxic T lymphocytes, in addition to being antigen specific, can be MHC class (e.g., MHC class I) restricted. The cytotoxic T lymphocytes preferably have a CD8+ phenotype. [0081] In one embodiment, lymphocytes are removed from the host and stimulated ex vivo with the peptide, polypeptide, nucleic acid, vector, cell, or composition thereof to generate Leydig 771368 E-056-2023-0-PCT-01 29 cytotoxic T lymphocytes. The cytotoxic T can be administered to the host in order to enhance an immune response to cancer, thereby inhibiting the cancer. Accordingly, the invention provides a method of inhibiting cancer in a host comprising (a) obtaining lymphocytes (e.g., from the host), (b) stimulating the lymphocytes with the peptide, polypeptide, nucleic acid, vector, cell, or composition thereof to generate cytotoxic T lymphocytes, and (c) administering the cytotoxic T lymphocytes to the host, wherein the cancer is inhibited. [0082] In another embodiment, lymphocytes within the host are stimulated by administration to the host of the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof to generate cytotoxic T lymphocytes, which cytotoxic T lymphocytes enhance an immune response to cancer, thereby inhibiting the cancer. [0083] The inventive therapeutic and prophylactic methods can include a prime-and-boost protocol. In particular, in one embodiment related to the inventive protein or polypeptide, nucleic acid, vector, cell, or composition thereof of the invention, the protocol includes an initial “prime” with a composition comprising one or more recombinant vectors encoding the inventive peptide or polypeptide and optionally one or more immunostimulatory/regulatory molecules and/or other tumor-associated antigens , modified versions thereof, and immunogenic epitopes thereof, followed by one or preferably multiple “boosts” with a composition containing the inventive peptide or polypeptide or one or more vectors encoding the inventive protein or polypeptide and optionally one or more immunostimulatory/regulatory molecules and/or other tumor-associated antigens , modified versions thereof, and immunogenic epitopes thereof. [0084] In this embodiment, the initial priming vaccination can comprise one or more vectors. In one embodiment, a single vector is used for delivery of the inventive peptide and one or more immunostimulatory/regulatory molecules and/or other tumor-associated antigens, modified versions thereof, and immunogenic epitopes thereof. In another embodiment, two or more vectors comprise the priming vaccination, which are administered simultaneously in a single injection. [0085] The boosting vaccinations also can comprise one or more vectors. In one embodiment, a single vector is used for delivery of the inventive peptide and the one or more immunostimulatory/regulatory molecules and/or other tumor-associated antigens, modified Leydig 771368 E-056-2023-0-PCT-01 30 versions thereof, and immunogenic epitopes of the boosting vaccination. In another embodiment, two or more vectors comprise the boosting vaccination, which are administered simultaneously in a single injection. [0086] Different types of vectors (such as those described above and otherwise known to those of skill in the art) can be used to provide a heterologous prime/boost protocol using vectors carrying different sets of therapeutic molecules for inoculations at different time intervals. For example, in one heterologous prime/boost combination, a first vector (e.g., adenovirus, orthopox, vaccinia, and the like) composition is used to prime, and a second type of vector composition is used to boost. [0087] The schedule for administration of the vectors typically involves repeated administration of the boosting vector. The boosting vector can be administered 1-3 times (e.g., 1, 2, or 3 times) at any suitable time period (e.g., every 2-4 weeks) for any suitable length of time (e.g., 6-12 weeks for a total of at least 5 to 15 boosting vaccinations). For example, the primary vaccination can comprise a recombinant adenovirus, poxvirus, or vaccinia vector followed by multiple booster vaccinations with an adenovirus, poxvirus, or vaccinia vector. In a particular embodiment, the host receives one vaccination with the priming vector, followed every 2 weeks thereafter with the boosting vector for 6 boosts, followed by every 4 weeks thereafter with the boosting vector, and continuing with the boosting vector for a period of time dependent on disease progression. EXAMPLE [0088] This Example demonstrates that novel sequences of the ERVMER34-1 human endogenous retroviral sequence family described herein have been identified and shown to be immunogenic in humans via activation of human T cells in vitro. In addition, a modified the ERVMER34-1 protein sequence described herein (SEQ ID NO:2) for incorporation into a ERVMER34-1 modified sequence adenovirus has been engineered to (a) remove the signal domain to prevent the trafficking of the protein to the cell surface, (b) remove the cleavage site to prevent the cleavage of the surface unit from the transmembrane unit of the ERVMER34-1 protein, (c) remove the predicted immunosuppressive domain to prevent the potential dampening Leydig 771368 E-056-2023-0-PCT-01 31 of the immunity generated against the target and (d) remove the transmembrane domain because this sequence shares homology with several human proteins. [0089] In addition, the Example demonstrates: (a) lack of ERVMER34-1 expression in normal human immune cell subsets, (b) efficacy of ERVMER34-1 vaccination in the MC38 murine colon carcinoma model, (c) immunity generated following vaccination with either the full-length or the modified sequence ERVMER34-1 therapeutic cancer vaccines, (d) ERVMER34-1 reactive T cells can be expanded from the blood of human cancer patients, (e) ERVMER34-1 reactive T cells can be expanded from the blood of healthy human donors, (f) a diverse and robust T-cell response can be elicited against ERVMER34-1 in vitro using healthy donor-derived PBMCs, (g) characterization of novel monoclonal ERVMER34-1 reactive antibodies, (h) the generation of an artificial antigen-presenting cell expressing the full-length ERVMER34-1 protein along with HLA-A2 and the CD80 costimulatory molecule, (i) specific lysis of a human carcinoma cell line by ERVMER34-1 reactive T cells expanded from a healthy human donor using an artificial APC-ERVMER34-1, and (j) the combination of immune checkpoint blockade with ERVMER34-1 vaccination enhances anti-tumor immunity, promotes the expansion of neoepitope-reactive T cells, and improves survival. [0090] This Example is presented to highlight some of this experimental work, and to further illustrate the invention but, of course, this should not be construed as in any way limiting its scope. METHODS [0091] The following experimental methods were employed. [0092] Cell lines. The C57BL/6-derived colon MC38 cell line was cultured as previously described. The human colon SW620 cell line was purchased from the American Type Culture Collection and cultured in RPM1-1640 media supplemented with 10% fetal bovine serum. The human Chronic Myeloid Leukemia K562 cell line was purchased from ATCC and cultured in DMEM media supplemented with 10% fetal bovine serum. Cell lines were determined to be mycoplasma free by using a MycoAlert Mycoplasma Detection Kit (Lonza) and used at low passage number from the date of acquisition. To generate the SW620 ERVMER34-1 CRISPR Leydig 771368 E-056-2023-0-PCT-01 32 cell line, cells were co-transfected with a Cas9 protein version 2 and a TrueGuide synthetic guide RNA targeting human ERVMER34-1 (ID number CRISPR924929_SGM) using the Lipofectamine CRISPRMAX transfection reagent (ThermoFisher). SW620 ERVMER34-1 knockout CRISPR cells were single-cell sorted and ERVMER34-1 negative cells were identified by western blot. For the generation of the MC38 pERVMER34-1 cell line, the human ERVMER34-1 gene was synthesized (ThermoFisher) and cloned into the pNGFR expression plasmid, which was kindly provided by Warren Pear (Addgene plasmid # 27489). MC38 cells were transfected with pERVMER34-1-NGFR, and single-cell sorted based upon surface expression of NGFR using a MA900 cell sorter (Sony Biotechnology). ERVMER34-1 expression was confirmed by western blot. To generate the artificial antigen-presenting cell line (aAPC), K562 cells were transfected with plasmids encoding the full-length human HLA-A2 and CD80 (Genecopoeia) along with pERVMER34-1-NGFR. Transfectants were single-cell sorted based upon surface expression of CD80, HLA-A2 and NGFR using a MA900 cell sorter (Sony Biotechnology). [0093] Generation of ERVMER34-1 antibody. A recombinant His8-tagged, ERVMER34- 1 protein spanning amino acids 27-432 was produced via an expression system in SF21 insect cells. Hybridomas were generated using splenocytes harvested from BALB/c mice that were immunized with the purified ERVMER34-1 recombinant protein (GenScript). [0094] Peptides. All ERVMER34-1 peptides were chemically synthesized by GenScript at a purity greater than 75% as assessed by HPLC. The peptides comprising the 15-mer ERVMER34- 1 peptide library are identified herein as SEQ ID NOs:7-99. Full-length 15-mer overlapping peptide libraries for brachyury and mucin1 were chemically synthesized by JPT Technologies. [0095] Peripheral blood from healthy donors and cancer patients. PBMCs collected from either healthy donors or cancer patients were separated by Ficoll-Hypaque density gradient separation and cryopreserved in 90% serum and 10% DMSO. De-identified PBMC samples were obtained from healthy volunteers who provided written informed consent at the National Institute of Health (NIH) Clinical Center Blood Bank (protocol NCT00001846). PBMCs were obtained from the blood of patients diagnosed with prostate, lung, breast, and colon cancers enrolled in clinical trials at the National Cancer Institute (NCT00179309, NCT01772004, Leydig 771368 E-056-2023-0-PCT-01 33 NCT03050814, NCT00088413, NCT01519817, NCT00923741, NCT00924092, NCT02840994, NCT00113984). [0096] In vitro expansion of ERVMER34-1 reactive T cells. PBMCs collected from either healthy donors or cancer patients were plated at 2.5x106 cells/well in a 24-well tray in a volume of 1 mL Immunocult XF T Cell Expansion Medium (Stemcell Technologies). Alternatively, PBMCs were plated at 107 cells/well in 6-well trays for experiments that required larger numbers of T cells to assess immunity generated against individual ERVMER34-1 peptides. For all cultures, the 15-mer overlapping peptide library was added at a concentration of 0.2 μg/mL/peptide. Cultures were supplemented on days 3 and 5 with cytokines (IL7 and IL15, 10 ng/mL of each; PeproTech). On day 7, cultures were washed and rested in media without the addition of peptide and cytokines. On day 11, cultures were harvested and restimulated for 24 hours to assess ERVMER34-1 specific immunity. ERVMER34-1 specific immune responses were assessed using either an IFN-γ ELISPOT assay or intracellular cytokine staining. [0097] ELISPOT assays. Antigen-dependent IFN-γ producing cells were enumerated using either human or murine ELISPOT assays purchased from BD Biosciences and used according to the manufacturer’s instructions. For assays using murine splenocytes, 5x105 cells/well were incubated overnight with appropriate peptides at concentrations indicated below. For expanded tumor-infiltrating T cell (TIL) cultures, lymphocytes were counted and distributed equally amongst the number of test wells along with 106 splenocytes harvested from naïve animals. Antigen-specific IFN-γ producing cells from TIL were normalized to antigen-dependent counts per 5x105 expanded TIL. Human PBMCs either ex vivo or following a 1-week in vitro stimulation were plated at 5x105 cells per well in the ELISPOT assay. For all assays the following peptide concentrations were used: 10 μg/mL for single peptides and 0.2 μg/mL/peptide for pooled peptide libraries. Spots were quantified using an ImmunoSpot analyzer (Cellular Technology, Ltd). Antigen-dependent spots were calculated by subtracting any background spots in antigen-negative wells for each sample. [0098] Intracellular cytokine stain. Between 1 to 2 million cells were plated in each well of a 96-well round-bottom tray along with the ERVMER34-1 peptide library at a final concentration of 0.2 μg/mL/peptide. Following 2 hours of incubation, 1 μL/mL brefeldin A and Leydig 771368 E-056-2023-0-PCT-01 34 0.7 μL/mL monensin (BD Biosciences) were and cultures were incubated for an additional 22 hours. Following the restimulation, cells were stained with Fixable Viability Dye eFluor™ 506 (ThermoFisher Scientific). Human and mouse cultures were stained using appropriate antibodies listed in Table 8. Samples were run on an Attune NxT flow cytometer (ThermoFisher Scientific), and data were analyzed using FlowJo software package (BD Biosciences). Antigen- dependent cytokine producing cells were calculated by subtracting the number of cytokine- producing cells observed in paired antigen-negative wells for each sample. [0099] Survival analysis. Survival analysis of cancer patients was performed using Gene Expression Profiling Interactive Analysis (GEPIA, gepia .cancer - pku.cn/). Cutoffs for high and low ERVMER34-1 expression were set to 66% and 33%, respectively. [00100] ERVMER34-1 immunofluorescence. SW620 parental and SW620 ERVMER34-1 knockout CRISPR cells were plated in 8 well chamber slides (Ibidi, 80807). Once attached, cells were fixed with 3.2% paraformaldehyde (Electron Microscopy Sciences, 15714, diluted 1:10 in PBS to make 3.2%), permeabilized in 0.05% Surfact-Amps X (ThermoFisher Scientific, 28314, diluted 1:200 for 0.05%), and blocked in PBS supplemented with 10% goat serum and 1% BSA. Cells were incubated overnight with a mouse primary antibody against ERVMER34-1 (clone 14F-3-1) at 2 µg/ml. Slides were then washed with PBS and incubated with goat anti-mouse IgG Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (ThermoFisher Scientific, A32723). Nuclei were stained using Spectral DAPI (Akoya Biosciences, FP1490). Images were acquired immediately after staining using a Zeiss Axio Observer microscope using a 20x objective. [00101] OPAL immunofluorescence. Head and neck carcinoma (HN802d), colon carcinoma (CO1004), bladder carcinoma (BL242), lung adenocarcinoma (LC1531), lung squamous cell carcinoma (BC04118a), endometrium carcinoma (BC09012b), and normal adult (BN961b) human tissue microarrays were purchased from Tissuearray.com. Murine tumor tissue was fixed in Z-fix (Anatech, Battle Creek, MI) overnight, embedded in paraffin, and sectioned onto glass slides (American HistoLabs, Gaithersburg, MD). Slides were stained using the Opal 4-Color Manual IHC Kit (PerkinElmer, Waltham, MA) per the manufacturer’s instructions. Briefly, slides were deparaffinized and rehydrated with xylene and ethanol gradients, microwaved with Leydig 771368 E-056-2023-0-PCT-01 35 pH6, pH9, or Rodent Decloaker (BioCare Pacheco, CA) antigen retrieval solution, cooled, rinsed with tris-buffered saline, 0.1% tween (TBST), and blocked with BLOXALL Blocking Solution (Vector Laboratories, Burlingame, CA). Staining with primary and secondary antibodies and OPAL fluorophore working solution was conducted following the manufacturer’s instructions. Antibodies including anti-ERVMER34-1 (clone 14F3) and anti-Pan-cytokeratin (clone AE1/AE3; SantaCruz Biotechnology) were used to stain human tissue microarrays. Murine tumors were stained using a rabbit polyclonal ERVMER34-1 antibody (ThermoFisher Scientific). Slide scanning was performed using an Axio Scan.Z1 using a 20x objective (Zeiss). Human tissue microarrays were scored independently by two investigators. Sections with less than 10% specific staining were scored as +, sections where 10-50% of cells had specific staining were scored with ++. Sections where greater than 50% of cells had specific staining using the ERVMER34-1 antibody as compared to staining using a control mouse IgG antibody (Abcam) were scored as +++. [00102] Expansion of ERVMER34-1 specific CD8+ cytotoxic T cells. PBMCs collected from healthy HLA-A2 donors were enriched for CD8+ T cells using a negative selection isolation kit (Miltenyi Biotec). CD8+ T cells were cultured with irradiated (200 Gy) aAPC cells at a 20:1 ratio. On days 3 and 5 of culture, IL-7 and IL-15 (10 ng/mL of each; PeproTech) were added to the culture. On day 7, culture was washed and restimulated as described above. CD8+ T cells underwent between three and five of these in vitro stimulation cycles prior to being used in subsequent lysis and ELISPOT assays. [00103] Lysis assays. For flow cytometric-based lysis assays, the SW620 parental cells were labeled with CellTracker CM-DIL (ThermoFisher Scientific) and the SW620 ERVMER34-1 CRISPR cell line was labeled with CellTracker Deep Red (ThermoFisher Scientific) per the manufacturer’s recommended protocol. Labeled SW620 parental and ERVMER34-1 CRISPR cells were mixed at a 1:1 ratio. Effector CD8+ T cells were added to the target cell mixture at a 10:1 ratio. Following an overnight incubation at 37 °C, samples were stained with Fixable Viability Dye eFluor™ 506 (ThermoFisher Scientific), run on an Attune NxT flow cytometer (ThermoFisher Scientific) and analyzed using FlowJo (BD Biosciences). For image-based lysis assays, target cells were harvested, washed, labeled with 10 μM calcein-AM (Invitrogen) for 20 Leydig 771368 E-056-2023-0-PCT-01 36 minutes at 37 °C, washed, and plated at 3000 per well in 384-well flat-bottom culture plates. Effector cells from various donors were added at an effector-to-target ratio of 20:1. Following an 18-hour incubation, viable calcein-AM positive cells were counted using a Celigo Image Cytometer (PerkinElmer). Percent lysis was calculated as follows: % lysis = [1 - (cell count in well of interest/tumor only average cell count)] × 100. [00104] Detection of soluble ERVMER34-1 in serum. Blood samples were collected from animals 1 day prior to vaccination with control adenovirus, full-length ERVMER34-1 adenovirus, or ERVMER34-1 vaccine. Subsequently, blood samples were taken 3 days after vaccination, 1 day prior to boost, and 2 days after boost. Clear flat-bottom Immulon plates (ThermoFisher Scientific) were coated with 100 μg/well of anti-ERVMER34-1 (clone 4H5). Washes were performed with 0.05% Tween 20 in phosphate buffered saline and wells were blocked with 10% fetal bovine serum. Sera from individual animals were diluted 1:50 in duplicates. HRP-conjugated anti-ERVMER34-1 (clone 14F3) secondary antibody was prepared using HRP Conjugation Kit – Lightning-Link (Abcam) according to the manufacturer’s instructions and used at a concentration of 0.5 μg/mL. TMB substrate (ThermoFisher Scientific) was used to develop the reaction and sulfuric acid was added to stop the reaction. Absorbance was measured at 450nm utilizing an EnVision plate reader (PerkinElmer). [00105] Mice. Female C57BL/6 mice were obtained from the NCI Frederick Cancer Research Facility. Mice were between 5 and 6 weeks old at the start of experiments and were maintained under pathogen-free conditions in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Tumors were induced by implanting 3 x 105 MC38 tumor cells or 1x106 MC38 pERVMER34-1 tumor cells subcutaneously. [00106] Vaccination and treatments. Animals were injected subcutaneously with either 1010 viral particles of control adenovirus (Vector Biolabs), full-length ERVMER34-1 adenovirus, or a modified ERVMER34-1 adenovirus (designated ERVMER34-1 vaccine) diluted in PBS following tumor establishment in mice. Anti-PD-L1 (10F.9G2, BioXCell, 200 μg) was administered intraperitoneally and diluted in PBS. [00107] Cell isolation and preparation. Spleens were harvested in LPA media (RPMI-1640 supplemented with 10% FBS, 1.5% HEPES Buffer, 1x Sodium Pyruvate, 1x non-essential amino Leydig 771368 E-056-2023-0-PCT-01 37 acids, 1x Glutamine, 1x 1x Gentamicin), dissociated through 70-μm filters, and subjected to ACK lysis (Quality Biological) to obtain splenocytes for analysis. Tumors were harvested in RPMI with 5% FBS media, cut into small pieces, and incubated for 30 minutes at 37 °C and 300 rpm in a digestion cocktail composed of RPMI supplemented with 5% (v/v) FBS, 2 mg/mL Collagenase Type I and IV (Worthington Biochemical Corporation), and 40 U/mL DNase I (Calbiochem). Following digestion, tumors were ground through 70-μm filters, spun for 5 minutes at 500 g, and resuspended in media for cell counting and subsequent flow cytometry analysis. Mouse sera was collected into serum tubes with separating gel (BD Biosciences), rested for 30 minutes, and centrifuged at 800 g for 2 minutes. Supernatant was stored at -20 °C until analysis. For the expansion of tumor-infiltrating lymphocytes, excised tumors were diced into 2 mm3 pieces and plated in a 24-well plate containing RPMI complete medium supplemented with recombinant human IL-2 (6000IU/mL). Tissue culture was incubated at 37 °C for 5 days. Subsequently, media supplemented with IL-2 was changed twice per week by removing half of the spent media and TIL were split when nearly confluent. Tumor pieces were removed 3 weeks into culture. [00108] Cytokine assessment in tumors. Tumors were collected into 500 μL of PBS and minced with scissors into several small pieces. Afterwards, samples were spun for 5 minutes at 500 g. Supernatants were then transferred into new tubes and centrifuged at 13,000 g for 10 minutes. Tumor supernatants were then stored at -80 °C until further analysis. Cytokines were then assessed using the LegendPlex Mouse Inflammation Panel (Biolegend) following the manufacturer’s instructions. [00109] Flow cytometric assays. All antibodies used for flow cytometric analysis are listed in Table 8. Cell populations were identified as follows: CD8+T cells: Singlets/live/CD45+CD3+CD8+; CD4+ T cells: singlets/live/CD45+CD3+CD4+FoxP3-; Effector memory: singlets/live/CD45+/CD8+CD44+CD62L+. Data were collected using an Attune NxT flow cytometer and analyzed using FlowJo (v10, BD Biosciences). [00110] Western blot. Following cell lysis, protein samples (50 μg) were run in 4-12 % Bis- Tris gels (Invitrogen). Transfer was performed using the iBlot2 gel transfer device (Invitrogen). The membrane was then blocked with 5% non-fat dried milk in PBS with 0.1% Tween 20 Leydig 771368 E-056-2023-0-PCT-01 38 (PBST) for 1 hour at room temperature. 1 antibody (2 μg/mL) (Clone 14F3) and rabbit GAPDH at 1:5000 (Cell signaling) diluted in 5% milk in PBST were added and incubation was performed overnight at 4 °C. Afterwards, membranes were washed three times, 5 minutes each with PBST. Secondary antibodies IRDye 800 Goat anti-mouse (LI-COR) and IRDye 800 Goat anti-rabbit (LI-COR) were diluted 1:5000 in 5% milk PBST and incubated for 1 hour at room temperature. Membranes were then washed three times, 5 minutes each with PBST, and imaging was performed using an Odyssey Imager (LI-COR). RESULTS [00111] Identification of ERVMER34-1 as a potential therapeutic target. To identify ERVs that could be potentially targeted by a therapeutic cancer vaccine, the expression of ERVs annotated in the GTEx database was analyzed. As shown in Figure 7A, ERVMER34-1 had very low expression in normal adult tissues as compared to other ERVs examined. In addition, ERVMER34-1 was expressed in multiple tumor types as compared to other ERVs with low levels of normal tissue expression (Figure 7B). In examining the TCGA database for ERV expression in tumors and tumor-adjacent histologically normal tissues, relatively low ERVMER34-1 expression was observed in tumor-adjacent tissues, compared to higher levels of expression in various tumor types (Figure 1A). To assess ERVMER34-1 expression at the protein level, protein lysates were analyzed by western blot using a novel mouse monoclonal anti-ERVMER34-1 antibody. Increased protein expression in lung and colon adenocarcinoma samples as compared to matched tumor-adjacent normal tissues was observed (Figure 1B left panel). As tumor-adjacent tissues may not be representative of true normal tissue expression, a western blot analysis was also performed on protein lysates generated from lung and colon normal tissues obtained from healthy donors and no ERVMER34-1 protein expression was observed (Figure 1B right panel). Analysis of data in the TCGA database also demonstrated an association between high levels of ERVMER34-1 expression and decreased survival in uveal melanoma, head and neck squamous cell carcinoma, and adenoid cystic carcinoma, suggesting ERVMER34-1 expression may play a role in tumor progression in some cancer types (Figure Leydig 771368 E-056-2023-0-PCT-01 39 7C). Based on these results, the hypothesis that ERVMER34-1 might be a promising immunotherapeutic target. [00112] ERVMER34-1 is expressed in multiple cancer types. The expression of ERVMER34-1 protein in multiple normal and tumor tissues was assessed by immunofluorescence utilizing commercially available tissue arrays. As shown in Table 1, most normal tissues, including cerebrum, heart, esophagus, stomach, liver, pancreas, adrenal gland, lung, lymph node, spleen, ovary, prostate, skeletal muscle, diaphragm, omentum, and skin had no detectable ERVMER34-1 expression, except for low levels detected in isolated cells in the cerebellum, small intestine and colon, breast, bronchus, uterus, kidney and testis. Examples of ERVMER34-1 staining in normal adult tissues are presented in Figure 1C. For comparison, this figure also includes staining of CEA (CEACAM5) and brachyury (TBXT) in normal tissues, both of which are tumor-associated antigens that have been previously targeted clinically using therapeutic cancer vaccines in the absence of toxicities. ERVMER34-1 expression is similar to or less than that seen with CEA and brachyury in these normal adult tissues, suggesting that ERVMER34-1 could be safely targeted by a therapeutic cancer vaccine. ERVMER34-1 protein was also detected in several tumor types, including colon (23/39 cases), head and neck (50/69 cases), bladder (8/17 cases), lung adenocarcinoma (21/49 cases), lung squamous cell carcinoma (33/50), and endometrium cancer (32/59 cases) (Figure 1D and Tables 2-7). Specifically, ERVMER34-1 expression was shown to be restricted to the tumor, as it co-localized with the tumor marker cytokeratin (Figure 1D). While ERVMER34-1 protein is not detectable in normal lung tissues, expression is sometimes observed in histologically normal tissues adjacent to the tumor. (Figure 1E and Tables 5, 6 and 7). This expression of ERVMER34-1 in tumor adjacent tissues is only present in cases where the matched tumor expresses ERVMER34-1 (Table 7). [00113] ERVMER34-1 specific T cells can be expanded through in vitro stimulation. To assess ERVMER34-1 immunity, a 15-mer overlapping peptide library was chemically synthesized, spanning the entire ERVMER34-1 protein sequence minus a 170 amino acid sequence corresponding to a fragment with high homology with other human proteins to enhance the assay's specificity. Using this ERVMER34-1 peptide library, immune reactivity in healthy donor and cancer patient PBMCs was assessed using an overnight (16-hour) IFN-γ ELISPOT Leydig 771368 E-056-2023-0-PCT-01 40 assay. As shown in Figure 2A, a greater in the immune response was observed with PBMCs from cancer patients as compared to healthy donor PBMCs, following a 16-hour exposure to ERVMER34-1 peptides. To further assess the immunogenicity of ERVMER34-1 in healthy donors, two different assay conditions were evaluated; PBMCs were either stimulated with each individual peptide comprising the ERVMER34-1 library for 16 hours in an IFN-γ ELISPOT assay or they were stimulated for 7 days in vitro in the presence of the pooled library, followed by a 16-hour restimulation in an IFN-γ ELISPOT assay. While healthy donors did not demonstrate detectable ERVMER34-1 reactive T cells when evaluated in a 16-hour ex vivo assay (Figure 2B, left panel), immune responses could be detected against multiple ERVMER34-115- mer peptides following a 7-day in vitro stimulation (Figure 2B, right panel). We also performed a similar 7-day stimulation assay using PBMCs from cancer patients and observed a similar diverse ERVMER34-1 immune response, demonstrating immunogenicity of ERVMER34-1 in carcinoma patients. Representative ELISPOT images are presented in Figure 8. Using an intracellular cytokine assay as a readout, the expansion of ERVMER34-1 reactive CD4+ and CD8+ T cells following a 7-day in vitro stimulation was demonstrated and both cancer patients and healthy donors exhibited a similar response to ERVMER34-1 peptides (Figure 2D). For comparison, we directly compared the immunogenicity of 15-mer overlapping peptide libraries of ERVMER34-1, CEACAM5, MUC1 and PSA following a 7-day in-vitro stimulation. As expected, with a peptide library, we do observe the predominant expansion of antigen-specific CD4+ cells as compared to CD8+ cells, however we observe the ERVMER34-1 peptide is more efficient at expanding reactive T cells than other tumor associated antigens assessed (Figures 2E and 2F; gating strategy presented in Figure 9A). [00114] Expansion of ERVMER34-1 specific CD8+ cytotoxic T cells. To expand ERVMER34-1 reactive T cells, the HLA-deficient human Chronic Myeloid Leukemia cell line K562 was modified to generate an artificial antigen-presenting cell line (termed “aAPC”), which was engineered to express the full-length ERVMER34-1 protein (Figure 10A) along with the HLA-A2 restriction element and the costimulatory molecule CD80 (Figure 10B). ERVMER34-1 reactive T cells were expanded from healthy HLA-A2 donors by incubating enriched CD8+ T cells with irradiated aAPCs. Following three weekly stimulation cycles, the specificity of CD8+ Leydig 771368 E-056-2023-0-PCT-01 41 T cell cultures was assessed by an ELISPOT using individual peptides comprising the 15- mer peptide library. Interestingly, analysis of T cells expanded from a healthy donor (donor A) demonstrated primary reactivity against a single 15-mer peptide, MGSLSNYALLQLTLT (SEQ ID NO:7) (Figure 2G). In subsequent studies using 9-mer peptides contained within this 15-mer peptide sequence, YALLQLTLT (SEQ ID NO:112) was identified as the nominal peptide recognized by the ERVMER34-1 reactive T cells (Figure 2H). We similarly identified the nominal peptides being recognized by an additional donor (Figure 2I and 2J). To assess the ability of these expanded ERVMER34-1 reactive CD8+ T cells to specifically lyse human carcinoma cells, the HLA-A2 positive human SW620 colon carcinoma cell line, which naturally expresses the ERVMER34-1 protein, was used. To define the specificity of the T cell lysis, a SW620 cell line was generated in which the expression of ERVMER34-1 was silenced by using a CRISPR-based strategy (Figure 10C). With this SW620 isogenic cell pair, it was demonstrated that ERVMER34-1 specific CD8+ cytotoxic T cells lysed the parental SW620 cells at both effector-to-target ratios evaluated but failed to lyse SW620 targets silenced for ERVMER34-1 (donor A, Figure 2K). The specific killing of SW620 cells was demonstrated with ERVMER34- 1-reactive CD8+ T cells expanded from three additional healthy donors (donors B-D, Figure 2L). [00115] Generation of a therapeutic cancer vaccine targeting ERVMER34-1. To target ERVMER34-1, two adenoviral vectors were generated, one encoding the full-length ERVMER34-1 protein and another one encoding a rationally modified ERVMER34-1 sequence (hereinafter referred to as ERVMER34-1 vaccine). In the ERVMER34-1 vaccine, the signal peptide was removed to ensure that the envelope protein was not expressed on the surface of infected cells (Figure 3A, B). In addition, a region of 170 amino acids characterized by a high degree of homology to other human proteins was removed from the ERVMER34-1 vaccine. The deletion of this 170 amino acid fragment also eliminated the cleavage and immunosuppressive domains of the ERVMER34-1 protein (Figure 3A and B). Both versions of these adenoviral vectors were able to induce protein expression of their respective targets following infection of human dendritic cells differentiated from PBMCs collected from a healthy donor (Figure 3C). As the surface unit of the ERVMER34-1 protein can be shed from the cell surface, an ELISA assay was performed to detect ERVMER34-1 expression in the sera of mice prior to and 3 days Leydig 771368 E-056-2023-0-PCT-01 42 following the administration of either the or the ERVMER34-1 vaccine. ERVMER34-1 protein was detected in the sera of 3/5 animals injected with the full-length version, but not in animals given either the control or the ERVMER34-1 vaccine (Figure 3D). To assess the ability of the vectors to expand ERVMER34-1 reactive T cells, mice were primed with control, full-length or ERVMER34-1 vaccine vectors on day 0 and boosted on day 7. ERVMER34-1 immunity was assessed 2 weeks following the second dose of vaccine using a 16- hour IFN-γ ELISPOT assay. Both versions of the vaccine were able to expand T-cell responses with similar diversities and magnitude of response (Figure 3E). Additionally, the full-length adenovirus and the ERVMER34-1 vaccine were able to expand comparable numbers of multifunctional ERVMER34-1 reactive T cells in the spleens of immunized animals (Figure 3F), with the majority of T cells being CD8+ T cells (Figure 3G). Based on these results, subsequent experiments were performed using the ERVMER34-1 vaccine. [00116] Targeting ERVMER34-1 mediates tumor control in the EMT6 model. To assess the anti-tumor efficacy of the therapeutic cancer vaccine targeting ERVMER34-1, the murine EMT6 breast carcinoma cell line which is known to be refractory to checkpoint therapy. We engineered the EMT6 to express the full-length ERVMER34-1 protein (EMT6 pERVMER34-1) (Figure 4A). Mice bearing EMT6 pERVMER34-1 tumors were treated as indicated in Figure 4B and observed increased tumor control when the ERVMER34-1 vaccine is used as a monotherapy, but we observed the best anti-tumor immunity when the vaccine is combined with PD-L1 blockade (Figure 4C). [00117] Targeting ERVMER34-1 mediates tumor clearance in the MC38 model. The murine MC38 colon carcinoma cell line was engineered to express the full-length human ERVMER34-1 protein (MC38 pERVMER34-1) (Figure 5A). Mice bearing MC38 pERVMER34-1 tumors were treated with a single dose of either a control adenovirus vector or the ERVMER34-1 vaccine. Tumor clearance was detected in 4/9 animals treated with the ERVMER34-1 vaccine as compared to 1/10 in animals treated with the control adenovirus (Figure 11), resulting in a significant increase in survival for animals treated with the ERVMER34-1 vaccine (Figure 5C). Vaccination of mice with the ERVMER34-1 vaccine increased the infiltration of CD8+ T cells in the tumor microenvironment (TME), and these cells Leydig 771368 E-056-2023-0-PCT-01 43 were more proliferative and tended to be of memory phenotype than in animals receiving control adenovirus (Figure 5D). No increase in tumor infiltrating CD4+ T cells following vaccination was observed. Tumor clearance associates with an expansion of ERVMER34-1 reactive T cells in both the spleen and within the tumor microenvironment (Figure 5E). Immunity was assessed in animals 6 days following the administration of vaccine using an IFN-γ ELISPOT assay. We observed that vaccination associates with the expansion of ERVMER34-1 reactive T cells both in the spleen and within the tumor microenvironment (Figure 5E). Tumor regression appears to be mediated primarily by ERVMER34-1 reactive T cells as we do not observe expansion of T cells within the tumor microenvironment following vaccination which are specific for either tumor neoepitopes or the p15e tumor antigen known to be expressed in the MC38 model (Figure 4F). [00118] Interestingly, we observed an increase in IFN-γ present within the TME following vaccination with the ERVMER34-1 vaccine (Figure 5G). The increased presence of IFN-γ associated with an increased expression of PD-L1 by both monocytes and tumor cells in animals treated with the ERVMER34-1 vaccine (Figure 5H). Will be including O-link data from when we receive the results from Frederick. In a subsequent study, mice whose MC38 pERVMER34- 1 tumors regressed following a single dose of the ERVMER34-1 vaccine were rechallenged with the parental MC38 tumor cell line on their left flank and the MC38 pERVMER34-1 cell line on their right flank. All rechallenged animals were able to control the growth of MC38 pERVMER34-1 tumors (16/16). In addition, only 9/16 animals were able to control the growth of the parental MC38 cell line (Figure 5I), an effect likely caused by MC38 antigen spreading in some treated animals. [00119] Combination of immune checkpoint blockade with ERVMER34-1 vaccination enhances anti-tumor immunity, promotes the expansion of neoepitope-reactive T cells, and improves survival. Considering the enhanced PD-L1 expression within the TME, the question arose whether the addition of PD-L1 blockade would further improve anti-tumor efficacy when combined with the ERVMER34-1 vaccine. To test this hypothesis, a single dose of ERVMER34-1 vaccine was administered in mice bearing large MC38 pERVMER34-1 tumors (300 mm3), followed by three doses of anti-PD-L1 (Figure 6A). While neither control Leydig 771368 E-056-2023-0-PCT-01 44 adenovirus nor control adenovirus in with PD-L1 blockade was able to induce tumor clearance, the combination of ERVMER34-1 vaccine with anti-PD-L1 was able to induce tumor clearance in 9/10 animals as compared to 0/9 animals treated with the ERVMER34-1 vaccine alone (Figure 6B). The combination of PD-L1 blockade with the ERVMER34-1 vaccine did not enhance the expansion of ERVMER34-1 reactive T cells as compared vaccine alone (Figure 6C), but rather promoted the expansion of neoepitope reactive T cells (Figure 6D). Interestingly, this expansion of neoepitope-reactive T cells associated with the ability of mice to control a rechallenge with both the MC38 pERVMER34-1 cell line (7/7 mice) along with the parental MC38 cell line (6/7 mice) (Figure 6E).
Leydig 771368 E-056-2023-0-PCT-01 45 Table 1 Expression of ERVMER34-1 in healthy adult tissues Case Age Sex Organ/Anatomic site ERVMER34-1 Notes 1 38 F Cerebrum -
Figure imgf000046_0001
to are with less than 10% specific staining were scored as +, sections where 10-50% of cells had specific staining were scored with ++. Sections where greater than 50% of cells had specific staining using the ERVMER34-1 antibody as compared to staining using a control mouse IgG antibody (Abcam) were scored as +++. Leydig 771368 E-056-2023-0-PCT-01 46 2 Expression of ERVMER34-1 in head and neck carcinomas and normal tissue Case Age Sex Anatomic site Diagnosis TNM Grade Stage ERVMER34 -1 expression Squamous cell carcinoma 1 45 F Nose of nasal cavity T1N0 M0 1-2 I +++
Figure imgf000047_0001
Leydig 771368 E-056-2023-0-PCT-01 47 Case Age Sex Anatomic site TNM Grade Stage ERVMER34 -1 expression 21 51 M Larynx Squamous cell T2N0 carcinoma M0 2 II - Larynx Squamous carcinoma
Figure imgf000048_0001
Glottis
Figure imgf000048_0002
Squamous
Figure imgf000048_0003
carcinoma 1M0 24 Larynx Squamous carcinoma 25
Figure imgf000048_0004
Figure imgf000048_0005
Squamous
Figure imgf000048_0006
carcinoma M0 ++ 26 Squamous carcinoma
Figure imgf000048_0007
Sq
Figure imgf000048_0008
27 M Larynx uamous carcinoma M0 I +++ Squamous carcinoma Squamous
Figure imgf000048_0009
carcinoma M0 2 III ++ Squamous
Figure imgf000048_0010
carcinoma Sq
Figure imgf000048_0011
Larynx uamous carcinoma 1M0 2 IVA - Squamous carcinoma Squamous
Figure imgf000048_0012
carcinoma M0 2 III +++ Squamous
Figure imgf000048_0014
carcinoma
Figure imgf000048_0015
Larynx Squamous carcinoma M0 3 II +++ Squamous
Figure imgf000048_0013
Figure imgf000048_0016
carcinoma
Figure imgf000048_0017
37 68 M Larynx Squamous cell T4aN carcinoma 3 IVA ++ 38 Squamous
Figure imgf000048_0018
Figure imgf000048_0019
carcinoma
Figure imgf000048_0020
39 47 M Larynx Squamous cell T4aN carcinoma 3 IVA +++ 40 Squamous
Figure imgf000048_0021
Figure imgf000048_0022
carcinoma
Figure imgf000048_0023
41 46 M Larynx Squamous cell T3N0 carcinoma 3 III - Verrucous
Figure imgf000048_0024
Figure imgf000048_0025
cell
Figure imgf000048_0026
43 57 M Verrucous squamous T3N0 cell carcinoma 2 III - 44 Basaloid
Figure imgf000048_0027
Figure imgf000048_0028
cell carcinoma
Figure imgf000048_0029
Figure imgf000048_0030
Leydig 771368 E-056-2023-0-PCT-01 48 Case Age Sex Anatomic site TNM Grade Stage ERVMER34 -1 expression 45 64 M Larynx Basaloid squamous T4aN cell carcinoma 0M0 2 IVA +++ 46 Larynx Basaloid cell carcinoma 47
Figure imgf000049_0001
Larynx
Figure imgf000049_0002
Basaloid
Figure imgf000049_0003
cell carcinoma M0
Figure imgf000049_0011
Figure imgf000049_0013
53 37 M Cheek carcinoma M0 1 II - 54 49 M Cheek Squamous cell T1N0 carcinoma M0 1 I ++ 55 70 M Cheek Squamous cell T2N0 carcinoma M0 1 II ++ 56 50 M Tongue Squamous cell T2N0 carcinoma M0 1 II - 57 60 F Lower lip Squamous cell T2N0 carcinoma M0 1 II - Squamous cell 58 28 M Tongue carcinoma (squamous T2N1 M0 * III - epithelial hyperplasia) 59 83 M Face Squamous cell T3N0 carcinoma M0 1 III +
Figure imgf000049_0012
65 33 F Parotid gland Adenoid cystic T3N0 carcinoma - III +++ 66 Lowe Adenoid
Figure imgf000049_0004
r jaw
Figure imgf000049_0005
carcinoma
Figure imgf000049_0006
67 35 F Maxillary sinus Adenoid cystic T3N0 - III +
Figure imgf000049_0007
Figure imgf000049_0008
Figure imgf000049_0009
Figure imgf000049_0010
Leydig 771368 E-056-2023-0-PCT-01 49 Case Age Sex Anatomic site TNM Grade Stage ERVMER34 -1 expression 68 40 M Tongue Adenoid cystic T3N0 carcinoma M0 - III + 69 33 M Parotid Adenoid cystic T2N0 - II +++
Figure imgf000050_0012
77 Tongue Not evaluable
Figure imgf000050_0001
Figure imgf000050_0002
Figure imgf000050_0003
78 22 M gland gland tissue - - - ++
Figure imgf000050_0004
Figure imgf000050_0005
Figure imgf000050_0006
79 43 M gland gland tissue - - -
Figure imgf000050_0007
+ 80 Submandibular
Figure imgf000050_0008
gland
Figure imgf000050_0009
tissue
Figure imgf000050_0010
[00121] Sections with less than 10% specific staining were scored as +, sections where 10- 50% of cells had specific staining were scored with ++. Sections where greater than 50% of cells had specific staining using the ERVMER34-1 antibody as compared to staining using a control mouse IgG antibody (Abcam) were scored as +++. Table 3 Expression of ERVMER34-1 in colon carcinoma and normal tissue Case Age Sex Anatomic Diagnosis TNM Grade Stage ERVMER34-1 site expression 1 80 M Colon Adenocarcinoma T4N2M0 2 III +
Figure imgf000050_0011
Leydig 771368 E-056-2023-0-PCT-01 50 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 13 82 F Colon Adenocarcinoma T3N0M0 2 II Missing 14 78 F Colon Adenocarcinoma T3N1M0 2 II - 15 Colon 16
Figure imgf000051_0001
Colon
Figure imgf000051_0002
Figure imgf000051_0003
Figure imgf000051_0004
Figure imgf000051_0005
27 59 M Colon (mucous tissue and T3N1M0 - II +++ necrotic Mucinous
Figure imgf000051_0006
29 Mucin
Figure imgf000051_0007
73 F Colon ous adenocarcinoma T3N0M0 3 II - 30 66 31 65
Figure imgf000051_0008
32 42 33
Figure imgf000051_0009
51
Figure imgf000051_0010
Colon
Figure imgf000051_0012
Figure imgf000051_0011
(sparse) 34 Colon 35
Figure imgf000051_0013
Colon
Figure imgf000051_0014
Figure imgf000051_0015
Figure imgf000051_0016
38 Colon 39
Figure imgf000051_0018
Colon
Figure imgf000051_0019
Signet-ring cell
Figure imgf000051_0017
carcinoma - - 40 Colon 41
Figure imgf000051_0020
Colon
Figure imgf000051_0021
Normal colonic
Figure imgf000051_0022
42 Colon Normal colonic 43
Figure imgf000051_0023
Colon
Figure imgf000051_0024
Normal colonic
Figure imgf000051_0025
44 Colon Normal colonic 45
Figure imgf000051_0026
Colon
Figure imgf000051_0027
Normal colonic
Figure imgf000051_0028
46 Colon Normal colonic 47
Figure imgf000051_0029
Colon
Figure imgf000051_0030
Normal colonic
Figure imgf000051_0031
48 Colon Normal colonic
Figure imgf000051_0032
Figure imgf000051_0033
Norm
Figure imgf000051_0034
49 M Colon al colonic (smooth muscle) - - -
Figure imgf000051_0035
Figure imgf000051_0036
Figure imgf000051_0037
Figure imgf000051_0038
Leydig 771368 E-056-2023-0-PCT-01 51 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression Normal colonic tissue 50 35 M Colon (chronic inflammation - - - - of colon mucosa) [00122] Sections with less than 10% specific staining were scored as +, sections where 10- 50% of cells had specific staining were scored with ++. Sections where greater than 50% of cells had specific staining using the ERVMER34-1 antibody as compared to staining using a control mouse IgG antibody (Abcam) were scored as +++. Table 4 Expression of ERVMER34-1 in bladder carcinoma and normal tissue Case Age Sex Anatomic Diagnosis TNM Grade Stage ERVMER34-1 site expression 1 56 F Bladder Transitional cell T2N0M carcinoma 0 1 II - Transitional
Figure imgf000052_0001
carcinoma Tr
Figure imgf000052_0002
Bladder ansitional carcinoma 0 1 II ++ Bladder Transitional
Figure imgf000052_0003
carcinoma
Figure imgf000052_0004
Figure imgf000052_0005
Bladder Transitional carcinoma 0 1 II - Bladder Transitional
Figure imgf000052_0007
carcinoma
Figure imgf000052_0008
Bladder Transitional carcinoma 0 1 II -
Figure imgf000052_0006
Bladder
Figure imgf000052_0009
Figure imgf000052_0011
0
Figure imgf000052_0010
9 66 M Bladder Transitional cell T2N0M carcinoma 2 II ++ Transitional
Figure imgf000052_0012
carcinoma
Figure imgf000052_0013
11 63 M Bladder Transitional cell T2N0M carcinoma 2 II - 12 Transitional
Figure imgf000052_0014
Figure imgf000052_0015
carcinoma
Figure imgf000052_0016
13 45 F Bladder Transitional cell T2N0M carcinoma 2 II ++ 14 Transitional
Figure imgf000052_0017
Figure imgf000052_0018
carcinoma
Figure imgf000052_0019
15 64 M Bladder Transitional cell T3N0M 3 II +++
Figure imgf000052_0020
Figure imgf000052_0021
Figure imgf000052_0022
Figure imgf000052_0023
Leydig 771368 E-056-2023-0-PCT-01 52 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 16 74 M Bladder Transitional cell T3N0M carcinoma 0 3 II + 17 38 M Bladder Transitional cell T3bN0 carcinoma M0 3 III ++ 18 (sparse)
Figure imgf000053_0001
Figure imgf000053_0002
necrosis
Figure imgf000053_0003
Figure imgf000053_0004
19 35 M Bladder Normal bladder tissue - - - - 20 50 M Bladder Normal bladder tissue - - - - 21 Bladder Normal bladder 22
Figure imgf000053_0005
Bladder
Figure imgf000053_0006
Normal bladder
Figure imgf000053_0007
23 Bladder Normal bladder
Figure imgf000053_0008
(smooth muscle)
Figure imgf000053_0009
24 Bladder Normal bladder (sparse)
Figure imgf000053_0010
Figure imgf000053_0011
Figure imgf000053_0012
[00123] Sections with less than 10% specific staining were scored as +, sections where 10- 50% of cells had specific staining were scored with ++. Sections where greater than 50% of cells had specific staining using the ERVMER34-1 antibody as compared to staining using a control mouse IgG antibody (Abcam) were scored as +++. Table 5 Expression of ERVMER34-1 in lung adenocarcinoma with matched cancer-adjacent tissues Case Age Sex Anatomic Diagnosis TNM Grade Stage ERVMER34-1 site expression 1 69 F Lung Adenocarcinoma T2aN1M0 2 IIB ++ Lung Cancer
Figure imgf000053_0013
lung tissue
Figure imgf000053_0014
Lung Papillary T1aN0M0 1 IA - Lung Cancer
Figure imgf000053_0015
lung
Figure imgf000053_0016
-
Figure imgf000053_0017
Lung Adenocarcinoma T3N2M0 1 IIIB - Lung Cancer
Figure imgf000053_0019
lung tissue
Figure imgf000053_0020
Lung Papillary T1cN0M0 1 IA3 + Lung
Figure imgf000053_0021
Lung Adenocarcinoma T2bN0M0 2 IIA - Cancer adjacent
Figure imgf000053_0018
Lung
Figure imgf000053_0022
lung tissue
Figure imgf000053_0023
Leydig 771368 E-056-2023-0-PCT-01 53 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 11 53 M Lung Adenocarcinoma T3N0M0 2 IIB - 12 53 M Lung Cancer adjacent lung tissue - - - - 13
Figure imgf000054_0001
Figure imgf000054_0002
14 51 M Lung Cancer adjacent lung tissue - - - ++ 15
Figure imgf000054_0003
Lung
Figure imgf000054_0004
Figure imgf000054_0005
16 59 M Cancer adjacent lung tissue - - - -
Figure imgf000054_0006
Papilla
Figure imgf000054_0007
17 ry adenocarcinoma - 18 Lung 19
Figure imgf000054_0008
Lung
Figure imgf000054_0009
Figure imgf000054_0011
Figure imgf000054_0010
20 M Lung Cancer adjacent lung tissue - - - -
Figure imgf000054_0012
Figure imgf000054_0013
Figure imgf000054_0014
21 75 M Lung (cartilage tissue) T2N0M0 * IB Missing 22 75 Lung Cancer lung tissue + 23 56
Figure imgf000054_0015
Lung
Figure imgf000054_0016
Figure imgf000054_0017
-
Figure imgf000054_0018
24 Lung Cancer adjacent
Figure imgf000054_0019
Figure imgf000054_0020
lung tissue
Figure imgf000054_0021
25 Lung - 26 Lung Cancer adjacent
Figure imgf000054_0022
Figure imgf000054_0023
lung tissue
Figure imgf000054_0024
27 Lung - 28 Lung Cancer
Figure imgf000054_0025
Figure imgf000054_0026
lung tissue
Figure imgf000054_0027
29 F Lung IIB ++ 30 Lung Cancer
Figure imgf000054_0028
Figure imgf000054_0029
lung tissue
Figure imgf000054_0030
31 37 F Lung Adenocarcinoma T2bN0M0 2 IIA + 32 Cancer
Figure imgf000054_0031
Lung
Figure imgf000054_0032
lung tissue
Figure imgf000054_0033
33 40 M Lung Adenocarcinoma T2aN0M0 3 IB + 34 Canc
Figure imgf000054_0034
Lung er
Figure imgf000054_0035
lung tissue
Figure imgf000054_0036
35 49 F Lung Adenocarcinoma T2aN0M0 3 IB +++ 36 Cancer
Figure imgf000054_0037
Lung
Figure imgf000054_0038
lung tissue
Figure imgf000054_0039
37 39 M Lung Adenocarcinoma T3N0M0 3 IIB - 38 Lun Cancer
Figure imgf000054_0040
g
Figure imgf000054_0041
lung tissue
Figure imgf000054_0042
39 61 M Lung Adenocarcinoma T2N1M0 2 IIB +++ 40 Cancer
Figure imgf000054_0043
Lung
Figure imgf000054_0044
lung tissue
Figure imgf000054_0045
41 48 F Lung Adenocarcinoma T2bN0M0 3 IIA +++ 42 Lung Cancer
Figure imgf000054_0046
Figure imgf000054_0047
Figure imgf000054_0048
Leydig 771368 E-056-2023-0-PCT-01 54 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression lung tissue 43 59 F Lung Adenocarcinoma T2aN1M0 2 IIB - 44 59 F Lung Cancer adjacent lung tissue - - - - Lung
Figure imgf000055_0001
Figure imgf000055_0002
Lung Cancer adjacent lung tissue - - - - Lung
Figure imgf000055_0003
Figure imgf000055_0004
Lung Cancer adjacent lung tissue - - - -
Figure imgf000055_0005
Figure imgf000055_0006
Lung (degeneration) - Lung Cancer lung tissue Lung
Figure imgf000055_0007
Figure imgf000055_0008
Lung Cancer adjacent lung tiss - - -
Figure imgf000055_0009
ue Papillar
Figure imgf000055_0010
Lung y adenocarcinoma T2bN2M0 2 IIIA +++ Lung Cancer lung - Lung
Figure imgf000055_0011
Figure imgf000055_0012
Figure imgf000055_0013
- Lung Cancer adjacent
Figure imgf000055_0014
lung tissue
Figure imgf000055_0015
Lung Lung Cancer
Figure imgf000055_0016
lung tissue
Figure imgf000055_0017
Lung - Lung Cancer
Figure imgf000055_0019
lung tissue
Figure imgf000055_0020
Lung IB - Lung Cancer
Figure imgf000055_0021
lung tissue
Figure imgf000055_0022
Lung Adenocarcinoma T2bN0M0 2 IIA +++ Lung Cancer
Figure imgf000055_0023
lung tissue
Figure imgf000055_0024
Lung Adenocarcinoma T2aN0M0 2 IB - Lung Cancer
Figure imgf000055_0025
lung tissue
Figure imgf000055_0026
Lung Adenocarcinoma T3N0M0 3 IIB ++ Lung Cancer
Figure imgf000055_0027
lung tissue
Figure imgf000055_0028
Lung Adenocarcinoma T3N0M0 3 IIB - Lung Cancer
Figure imgf000055_0029
lung tissue
Figure imgf000055_0030
Lung Adenocarcinoma T3N1M0 3 IIIA - Lung Cancer
Figure imgf000055_0031
lung tissue
Figure imgf000055_0032
Lung Adenocarcinoma T2aN0M0 3 IB +
Figure imgf000055_0018
Figure imgf000055_0033
Figure imgf000055_0034
Leydig 771368 E-056-2023-0-PCT-01 55 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 74 51 F Lung Cancer adjacent lung tissue - - - - 75 65 M Lung Adenocarcinoma T4N0M0 3 IIIA - 76 65 M Lung Cancer adjacent lung tissue - - - ++ 77
Figure imgf000056_0001
Lung
Figure imgf000056_0002
Figure imgf000056_0003
78 53 M Lung Cancer adjacent lung tissue - - - + 79
Figure imgf000056_0004
Lung
Figure imgf000056_0005
Figure imgf000056_0006
80 51 M Lung Cancer adjacent lung tissue - - - - 81
Figure imgf000056_0007
Lung
Figure imgf000056_0008
Figure imgf000056_0009
82 55 M Lung Cancer adjacent lung tissue - - - - 83
Figure imgf000056_0010
Lung
Figure imgf000056_0011
Figure imgf000056_0012
84 F Lung Cancer adjacent l - - - - 85
Figure imgf000056_0013
g
Figure imgf000056_0014
ung tissue Lun
Figure imgf000056_0015
86 Lung Cancer adjacent lung - - - -
Figure imgf000056_0016
Figure imgf000056_0017
tissue 87 Lung
Figure imgf000056_0018
- 88 Lung Cancer adjacent
Figure imgf000056_0019
Figure imgf000056_0020
lung tissue
Figure imgf000056_0021
89 Lung - 90 Lung Cancer adjacent
Figure imgf000056_0022
Figure imgf000056_0023
lung tissue
Figure imgf000056_0024
91 Lung - 92 Lung Cancer
Figure imgf000056_0025
Figure imgf000056_0026
lung tissue
Figure imgf000056_0027
93 M Lung - 94 Lung Cancer
Figure imgf000056_0028
Figure imgf000056_0029
lung tissue
Figure imgf000056_0030
95 60 M Lung Adenocarcinoma T2aN0M0 3 IB +++ 96 Lun Cancer
Figure imgf000056_0031
g
Figure imgf000056_0032
lung tissue
Figure imgf000056_0033
97 66 M Lung Adenocarcinoma T3N0M0 3 IIB +++ 98 Lung Cancer
Figure imgf000056_0034
Figure imgf000056_0035
lung tissue
Figure imgf000056_0036
99 66 F Lung Adenocarcinoma T2bN1M0 3 IIB Not evaluable 100 Can
Figure imgf000056_0037
Lung cer
Figure imgf000056_0038
lung tissue
Figure imgf000056_0039
Missing 101 59 M Lung Adenocarcinoma T3N1M0 3 IIIA - 102 Lu Cancer
Figure imgf000056_0040
ng
Figure imgf000056_0041
lung tissue
Figure imgf000056_0042
[00124] Sections with less than 10% specific staining were scored as +, sections where 10- 50% of cells had specific staining were scored with ++. Sections where greater than 50% of cells Leydig 771368 E-056-2023-0-PCT-01 56 had specific staining using the ERVMER34-1 as compared to staining using a control mouse IgG antibody (Abcam) were scored as +++. Table 6 Expression of ERVMER34-1 in lung squamous cell carcinoma with matched cancer- adjacent tissues Case Age Sex Anatomic Diagnosis TNM Grade Stage ERVMER34-1 site expression 1 43 F Lung Keratinizing squamous cell T2N1 carcinoma M0 1 IIB -
Figure imgf000057_0001
Leydig 771368 E-056-2023-0-PCT-01 57 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 20 77 M Lung Keratinizing squamous cell T2N1 carcinoma M0 2 IIB - cell T2N1
Figure imgf000058_0001
Leydig 771368 E-056-2023-0-PCT-01 58 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 44 47 M Lung Non-seratinizing T3N0 squamous cell carcinoma M0 3 IIB +++
Figure imgf000059_0001
Leydig 771368 E-056-2023-0-PCT-01 59 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 68 62 M Lung Cancer adjacent lung tissue of case 18 - - - - 69 30 M Lung Cancer adjacent lung tissue of case 19 - - - - 70 Lung Cancer adjacent tissue of case 20 71
Figure imgf000060_0001
Lung
Figure imgf000060_0002
Cancer adjacent
Figure imgf000060_0003
tissue of case 21 - - - - 72 Lung Cancer adjacent tissue of case 22
Figure imgf000060_0004
Figure imgf000060_0005
Cancer adjacent
Figure imgf000060_0006
73 Lung tissue of case 23 - - - - 74 Lung Cancer adjacent tissue of case 24
Figure imgf000060_0007
Figure imgf000060_0008
75 Can
Figure imgf000060_0009
M Lung cer adjacent tissue of case 25 - - - - 76 Lung Cancer adjacent
Figure imgf000060_0010
Figure imgf000060_0011
tissue of case 26 Cancer a
Figure imgf000060_0012
77 57 M Lung djacent tissue of case 27 - - - - 78 Lung Cancer adjacent
Figure imgf000060_0013
Figure imgf000060_0014
tissue of case 28 Cancer adj
Figure imgf000060_0015
79 64 M Lung acent tissue of case 29 - - - - 80 Lung Cancer adjacent
Figure imgf000060_0016
Figure imgf000060_0017
tissue of case 30
Figure imgf000060_0018
81 65 M Lung Cancer adjacent tissue of case 31 - - - - 82 Lung Cancer adjacent
Figure imgf000060_0019
Figure imgf000060_0020
tissue of case 32
Figure imgf000060_0021
83 50 M Lung Cancer adjacent tissue of case 33 - - - - Cancer adjacent tissue of case 34
Figure imgf000060_0022
Figure imgf000060_0023
Cancer adjacent lung tissue of case 35 - - - +++ 86 Cancer adjacent
Figure imgf000060_0024
Figure imgf000060_0025
tissue of case 36
Figure imgf000060_0026
87 67 M Lung Cancer adjacent lung tissue of case 37 - - - - 88 Ca
Figure imgf000060_0027
Lung ncer adjacent
Figure imgf000060_0028
tissue of case 38
Figure imgf000060_0029
89 56 F Lung Cancer adjacent lung tissue of case 39 - - - - 90 Cancer adjacent
Figure imgf000060_0030
Lung
Figure imgf000060_0031
tissue of case 40
Figure imgf000060_0032
91 52 F Cancer adjacent lung tissue of case 41 - - - -
Figure imgf000060_0033
Figure imgf000060_0034
Leydig 771368 E-056-2023-0-PCT-01 60 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 92 63 M Lung Cancer adjacent lung tissue of case 42 - - - - - - - -
Figure imgf000061_0001
- - - -
Figure imgf000061_0002
- - - -
Figure imgf000061_0003
- - - -
Figure imgf000061_0004
Figure imgf000061_0010
were scored as +, sections where 10- 50% of cells had specific staining were scored with ++. Sections where greater than 50% of cells had specific staining using the ERVMER34-1 antibody as compared to staining using a control mouse IgG antibody (Abcam) were scored as +++. Table 7 Expression of ERVMER34-1 in lung squamous cell carcinoma with matched cancer- adjacent tissues Case Age Sex Anatomic Diagnosis TNM Grade Stage ERVMER34-1 site expression Endometrioid T1bN0
Figure imgf000061_0011
5 51 F Uterus Endometrioid T1bN0 1 IB +++ Endometrioid
Figure imgf000061_0005
Figure imgf000061_0006
Uterus Endometrioid T1bN0 1 IB ++
Figure imgf000061_0007
Figure imgf000061_0008
Figure imgf000061_0009
Leydig 771368 E-056-2023-0-PCT-01 61 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 8 38 F Uterus Endometrioid T1bN0 adenocarcinoma M0 1 IB +++ 9 53 F Uterus Endometrioid T1bN0 1 IB -
Figure imgf000062_0001
Leydig 771368 E-056-2023-0-PCT-01 62 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 32 62 F Uterus Endometrioid T1bN0 adenocarcinoma M0 1 IB - 33 50 F Uterus Endometrioid T1bN0 1 IB -
Figure imgf000063_0001
Leydig 771368 E-056-2023-0-PCT-01 63 Case Age Sex Anatomic TNM Grade Stage ERVMER34-1 site expression 56 54 F Uterus Endometrioid T1bN0 adenocarcinoma M0 3 IB ++ 57 58 F Uterus Endometrioid T1bN0 adenocarcinoma M0 3 IB +++ Endometrioid 58 adenocarcinoma
Figure imgf000064_0001
Figure imgf000064_0002
squamous
Figure imgf000064_0003
59 67 F Uterus Adenosquamous T2N0M carcinoma 0 - II - 60 Invasive mole with
Figure imgf000064_0004
Figure imgf000064_0005
61 s Cance
Figure imgf000064_0006
F Uteru r endometrium tissue - - -
Figure imgf000064_0007
- Cancer adjacent endometrium
Figure imgf000064_0008
63 8 F Uterus C
Figure imgf000064_0009
5 ancer adjacent endometrium tissue - - - -
Figure imgf000064_0010
Figure imgf000064_0011
10% specific
Figure imgf000064_0012
50% of cells had specific staining were scored with ++. Sections where greater than 50% of cells had specific staining using the ERVMER34-1 antibody as compared to staining using a control mouse IgG antibody (Abcam) were scored as +++. Table 8 Antibodies and reagents used in flow cytometric assays Target Species Fluorochrome Clone Vendor Catalogue number CD80 Human PE L307.4 BD Biosciences 557227
Figure imgf000064_0013
IL-17A Human Alexa-Fluor eBio64DE ThermoFisher 53-7179-42
Figure imgf000064_0019
TNFa Human PE BD IFNγ Human PE-Cyanine7
Figure imgf000064_0014
BD
Figure imgf000064_0015
Figure imgf000064_0016
Figure imgf000064_0017
Figure imgf000064_0018
Leydig 771368 E-056-2023-0-PCT-01 64 Target Species Fluorochrome Vendor Catalogue number CD4 Human APC-eFluor OKT4 ThermoFisher 47-0048-42 780 CD8 Human Super Bright RPA-T8 ThermoFisher 78-0088-42 780 CD4 Human PerCP-Cy5.5 CD8 Human
Figure imgf000065_0001
AF700
Figure imgf000065_0002
BD
Figure imgf000065_0003
Figure imgf000065_0004
CD8 Mouse Super Bright 67-
Figure imgf000065_0005
Figure imgf000065_0006
Figure imgf000065_0007
CD4 Mouse ThermoFisher 78- 780 TNFa Mouse PE-eFluor 610 MP6-XT22 ThermoFisher 61-7321-82 IFNγ Mouse PE-Cyanine7 XMG1.2 ThermoFisher 25-7311-82 CD44 Mouse PerCP- IM7 ThermoFisher 45-0441-82 Mouse
Figure imgf000065_0008
SolA15
Figure imgf000065_0009
Figure imgf000065_0010
700 Brilliant BD
Figure imgf000065_0011
421
Figure imgf000065_0012
Figure imgf000065_0013
Mouse Super Bright 30-F11 ThermoFisher 63-0451-82 600 Mouse Super Bright
Figure imgf000065_0014
780
Figure imgf000065_0015
Figure imgf000065_0016
Mouse PE SolA15 ThermoFisher 12-0081-85 Mouse Mouse
Figure imgf000065_0018
Figure imgf000065_0019
Mouse
Figure imgf000065_0020
Figure imgf000065_0021
Mouse Super Bright M1/70 ThermoFisher 78-0112-82 780 N/A eFluor506
Figure imgf000065_0017
Figure imgf000065_0022
Figure imgf000065_0023
Figure imgf000065_0024
SEQUENCES [00127] The following sequences are pertinent to the present disclosure and the disclosure herein. SEQ ID NO:1 – Full length ERVMER34-1 peptide sequence: MGSLSNYALLQLTLTAFLTILVQPQHLLAPVFRTLSILTNQSNCWLCEHLDNAEQPELVFVPAS ASTWWTYSGQWMYERVWYPQAEVQNHSTSSYRKVTWHWEASMEAQGLSFAQVRLLEGNFSLCVE NKNGSGPFLGNIPKQYCNQILWFDSTDGTFMPSIDVTNESRNDDDDTSVCLGTRQCSWFAGCTN RTWNSSAVPLIGLPNTQDYKWVDRNSGLTWSGNDTCLYSCQNQTKGLLYQLFRNLFCSYGLTEA Leydig 771368 E-056-2023-0-PCT-01 65 YLVPSLTRYLTLNASQITNLRSFIHKVTPHRCTQGDTDNPPLYCNPKDNSTIRALFPSLGTYDL EKAILNISKAMEQEFSATKQTLEAHQSKVSSLASASRKDHVLDIPTTQRQTACGTVGKQCCLYI NYSEEIKSNIQRLHEASENLKNVPLLDWQGIFAKVGDWFRSWGYVLLIVLFCLFIFVLIYVRVF RKSRRSLNSQPLNLALSPQQSAQLLVSETSCQVSNRAMKGLTTHQYDTSLL [00128] Within this sequence, the signal peptide is “MGSLSNYALLQLTLTAFLTILVQPQH” (SEQ ID NO:3). Within this sequence, the cleavage site between surface and transmembrane unit is “CTQG” (SEQ ID NO:4). Within this sequence, the putative immune-suppressive domain is “SRKDHVLDIPTTQRQTA” (SEQ ID NO:5). Within this sequence, the Transmembrane domain is “WGYVLLIVLFCLFIFVLIYVRVF” (SEQ ID NO:6). SEQ ID NO:2 – Modified ERVMER34-1 protein sequence incorporated into the ERVMER34-1 modified sequence adenovirus: MLLAPVFRTLSILTNQSNCWLCEHLDNAEQPELVFVPASASTWWTYSGQWMYERVWYPQAEVQN HSTSSYRKVTWHWEASMEAQGLSFAQVRLLEGNFSLCVENKNGSGPFLGNIPKQYCNQILWFDS TDGTFMPSIDVTNESRNDDDDTSVCLGTRQCSWFAGCTNRTWNSSAVPLIGLPNTQDYKWVDRN SGLTWSGNDTCLYSCQNQTKGLLYQLFRNLFCSYGLTEAHGKWRCADASITNDKGHDGHRTPTW WLTGSNLTLSVNNSGLFFLVPLLDWQGIFAKVGDWFRSWGYVLLIVLFCLFIFVLIYVRVFRKS RRSLNSQPLNLALSPQQSAQLLVSETSCQVSNRAMKGLTTHQYDTSLL
Leydig 771368 E-056-2023-0-PCT-01 66 SEQ ID NOs:7-99 – ERVMER34-115-mer sequences shown to be immunogenic in normal human donors SEQ ID NO:7: MGSLSNYALLQLTLT SEQ ID NO:54: GCTNRTWNSSAVPLI SEQ ID NO:8: SNYALLQLTLTAFLT SEQ ID NO:55: RTWNSSAVPLIGLPN SEQ ID NO:9: LLQLTLTAFLTILVQ SEQ ID NO:56: SSAVPLIGLPNTQDY SEQ ID NO:10: TLTAFLTILVQPQHL SEQ ID NO:57: PLIGLPNTQDYKWVD SEQ ID NO:11: FLTILVQPQHLLAPV SEQ ID NO:58: LPNTQDYKWVDRNSG SEQ ID NO:12: LVQPQHLLAPVFRTL SEQ ID NO:59: QDYKWVDRNSGLTWS SEQ ID NO:13: QHLLAPVFRTLSILT SEQ ID NO:60: WVDRNSGLTWSGNDT SEQ ID NO:14: APVFRTLSILTNQSN SEQ ID NO:61: NSGLTWSGNDTCLYS SEQ ID NO:15: RTLSILTNQSNCWLC SEQ ID NO:62: TWSGNDTCLYSCQNQ SEQ ID NO:16: ILTNQSNCWLCEHLD SEQ ID NO:63: NDTCLYSCQNQTKGL SEQ ID NO:17: QSNCWLCEHLDNAEQ SEQ ID NO:64: LYSCQNQTKGLLYQL SEQ ID NO:18: WLCEHLDNAEQPELV SEQ ID NO:65: QNQTKGLLYQLFRNL SEQ ID NO:19: HLDNAEQPELVFVPA SEQ ID NO:66: KGLLYQLFRNLFCSY SEQ ID NO:20: AEQPELVFVPASAST SEQ ID NO:67: YQLFRNLFCSYGLTE SEQ ID NO:21: ELVFVPASASTWWTY SEQ ID NO:68: RNLFCSYGLTEAHGK SEQ ID NO:22: VPASASTWWTYSGQW SEQ ID NO:69: CSYGLTEAHGKWRCA SEQ ID NO:23: ASTWWTYSGQWMYER SEQ ID NO:70: LTEAHGKWRCADASI SEQ ID NO:24: WTYSGQWMYERVWYP SEQ ID NO:71: HGKWRCADASITNDK SEQ ID NO:25: GQWMYERVWYPQAEV SEQ ID NO:72: RCADASITNDKGHDG SEQ ID NO:26: YERVWYPQAEVQNHS SEQ ID NO:73: ASITNDKGHDGHRTP SEQ ID NO:27: WYPQAEVQNHSTSSY SEQ ID NO:74: NDKGHDGHRTPTWWL SEQ ID NO:28: AEVQNHSTSSYRKVT SEQ ID NO:75: HDGHRTPTWWLTGSN SEQ ID NO:29: NHSTSSYRKVTWHWE SEQ ID NO:76: RTPTWWLTGSNLTLS SEQ ID NO:30: SSYRKVTWHWEASME SEQ ID NO:77: WWLTGSNLTLSVNNS SEQ ID NO:31: KVTWHWEASMEAQGL SEQ ID NO:78: GSNLTLSVNNSGLFF SEQ ID NO:32: HWEASMEAQGLSFAQ SEQ ID NO:79: VPLLDWQGIFAKVGD SEQ ID NO:33: SMEAQGLSFAQVRLL SEQ ID NO:80: DWQGIFAKVGDWFRS SEQ ID NO:34: QGLSFAQVRLLEGNF SEQ ID NO:81: IFAKVGDWFRSWGYV SEQ ID NO:35: FAQVRLLEGNFSLCV SEQ ID NO:82: VGDWFRSWGYVLLIV SEQ ID NO:36: RLLEGNFSLCVENKN SEQ ID NO:83: FRSWGYVLLIVLFCL SEQ ID NO:37: GNFSLCVENKNGSGP SEQ ID NO:84: GYVLLIVLFCLFIFV SEQ ID NO:38: LCVENKNGSGPFLGN SEQ ID NO:85: LIVLFCLFIFVLIYV SEQ ID NO:39: NKNGSGPFLGNIPKQ SEQ ID NO:86: FCLFIFVLIYVRVFR SEQ ID NO:40: SGPFLGNIPKQYCNQ SEQ ID NO:87: IFVLIYVRVFRKSRR SEQ ID NO:41: LGNIPKQYCNQILWF SEQ ID NO:88: IYVRVFRKSRRSLNS SEQ ID NO:42: PKQYCNQILWFDSTD SEQ ID NO:89: VFRKSRRSLNSQPLN SEQ ID NO:43: CNQILWFDSTDGTFM SEQ ID NO:90: SRRSLNSQPLNLALS SEQ ID NO:44: LWFDSTDGTFMPSID SEQ ID NO:91: LNSQPLNLALSPQQS SEQ ID NO:45: STDGTFMPSIDVTNE SEQ ID NO:92: PLNLALSPQQSAQLL SEQ ID NO:46: TFMPSIDVTNESRND SEQ ID NO:93: ALSPQQSAQLLVSET SEQ ID NO:47: SIDVTNESRNDDDDT SEQ ID NO:94: QQSAQLLVSETSCQV SEQ ID NO:48: TNESRNDDDDTSVCL SEQ ID NO:95: QLLVSETSCQVSNRA SEQ ID NO:49: RNDDDDTSVCLGTRQ SEQ ID NO:96: SETSCQVSNRAMKGL SEQ ID NO:50: DDTSVCLGTRQCSWF SEQ ID NO:97: CQVSNRAMKGLTTHQ SEQ ID NO:51: VCLGTRQCSWFAGCT SEQ ID NO:98: NRAMKGLTTHQYDTS SEQ ID NO:52: TRQCSWFAGCTNRTW SEQ ID NO:99: AMKGLTTHQYDTSLL SEQ ID NO:53: SWFAGCTNRTWNSSA Leydig 771368 E-056-2023-0-PCT-01 67 SEQ ID NOs:100-140 – Other Polypeptide Sequences (check these) SEQ ID NO:100: RLLEGNFSL SEQ ID NO:127: KWSGRCGLGYLVPSV SEQ ID NO:101: GLGYLVPSL SEQ ID NO:128: RCGLGYLVPSVTRYL SEQ ID NO:102: ALLQLTLTA SEQ ID NO:129: LSNYALLQLTLTAFV SEQ ID NO:103: LQLTLTAFL SEQ ID NO:130: GSLSNYALLQLTLTAFL SEQ ID NO:104: WMYERVWYP TILV SEQ ID NO:105: WLTGSNLTL SEQ ID NO:131: WTYSGQWMYERVWYPQA SEQ ID NO:106: RLLEGNFSV EVQ SEQ ID NO:107: GLGYLVPSV SEQ ID NO:132: LSFAQVRLLEGNFSLCV SEQ ID NO:108: ALLQLTLTV ENK SEQ ID NO:109: LQLTLTAFV SEQ ID NO:133: HRTPTWWLTGSNLTLSV SEQ ID NO:110: WMYERVWYV NNS SEQ ID NO:111: WLTGSNLTV SEQ ID NO:134: KWSGRCGLGYLVPSLTR SEQ ID NO:112: YALLQLTLT YLT SEQ ID NO:113: LSNYALLQLTLTAFL SEQ ID NO:135: GSLSNYALLQLTLTAFV SEQ ID NO:114: WTYSGQWMYERVWYP TILV SEQ ID NO:115: WMYERVWYPQAEVQN SEQ ID NO:136: WTYSGQWMYERVWYVQA SEQ ID NO:116: SFAQVRLLEGNFSLC EVQ SEQ ID NO:117: RTPTWWLTGSNLTLS SEQ ID NO:137: LSFAQVRLLEGNFSVCV SEQ ID NO:118: PTWWLTGSNLTLSVN ENK SEQ ID NO:119: KWSGRCGLGYLVPSL SEQ ID NO:138: HRTPTWWLTGSNLTVSV SEQ ID NO:120: RCGLGYLVPSLTRYL NNS SEQ ID NO:121: LSNYALLQLTLTVFL SEQ ID NO:139: KWSGRCGLGYLVPSVTR SEQ ID NO:122: WTYSGQWMYERVWYV YLT SEQ ID NO:123: WMYERVWYVQAEVQN SEQ ID NO:140: GSLSNYALLQLTLTVFL SEQ ID NO:124: SFAQVRLLEGNFSVC TILV SEQ ID NO:125: RTPTWWLTGSNLTVS SEQ ID NO:126: PTWWLTGSNLTVSVN
Leydig 771368 E-056-2023-0-PCT-01 68 [00129] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [00130] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [00131] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Leydig 771368 E-056-2023-0-PCT-01 69 CLAIM(S): 1. A protein or polypeptide consisting essentially of a sequence of amino acids selected from the group selected from SEQ ID NO:2 or SEQ ID NOs:7-99, wherein SEQ ID NOs:7-99 consists of 15 amino acids. 2. The protein or polypeptide of claim 1, which is a 15-mer polypeptide consisting of a sequence of amino acids selected from the group selected from SEQ ID NOs: 7-99. 3. A protein comprising a sequence of amino acids as set forth in SEQ ID NO:2 or having at least 95% identity to SEQ ID NO:2. 4. A nucleic acid encoding the protein or polypeptide of any of claims 1-3. 5. A recombinant genetic vector comprising the nucleic acid of claim 4. 6. The recombinant genetic vector of claim 5, which is a viral vector. 7. The recombinant genetic vector of claim 5 or 6, which is an adenoviral vector. 8. A recombinant cell comprising (i) a protein or polypeptide of any one of claims 1-3, (ii) a nucleic acid of claim 4, or (iii) a vector of any one of claims 5-7. 9. The cell of claim 8, wherein the cell is human. 10. The cell of claim 8 or 9, wherein the cell is an antigen presenting cell or tumor cell. 11. The recombinant cell of any one of claims 8-10, which expresses the protein or polypeptide. 12. A recombinant cell, which produces a peptide comprising, consisting essentially of, or consisting of a sequence of amino acids as set forth in SEQ ID NOs: 1 or 2 or having at least 95% identity to SEQ ID NOs:1 or 2, the CD80 costimulatory molecule, and an HLA molecule. 13. The cell of claim 12, wherein the HLA molecule is human HLA-2. 14. An isolated or substantially pure T cell, which is directed against a polypeptide consisting essentially of a sequence of amino acids selected from the group selected from Sequences comprising at least 95% identity to SEQ ID NO:2 or SEQ ID NOs:7-99. 15. A pharmaceutical composition comprising (a) the protein or polypeptide of any of claims 1-3, (b) the polynucleotide of claim 4, (c) the recombinant genetic vector of any one of claims 5-7, and/or (d) recombinant cell of any one of claims 8-14, or combination thereof, and a pharmaceutically acceptable carrier. Leydig 771368 E-056-2023-0-PCT-01 70 16. The pharmaceutical composition of claim 15, which comprises (a) a polypeptide selected from SEQ ID NOs:100-140 (b) a acid encoding (a), (c) a cell comprising (a) or (b), or a combination thereof. 17. The pharmaceutical composition of claim 15 or 16, further comprising an immunostimulatory/regulatory molecule. 18. The pharmaceutical composition of claim 17, wherein the immunostimulatory/regulatory molecule is selected from the group consisting of interleukin (IL)-2, IL-4, IL-6, IL-12, IL-15, IL-15/IL15Ra, IL-15/IL-15Ra-Fc, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, B7.1, B7.2, ICAM-1, LFA-3, CD70, RANTES, G-CSF, OX-40L, 41 BBL, anti- CTLA-4, IDO inhibitor, anti-PD-L1, anti-PD1, and combinations thereof. 19. The pharmaceutical composition of claim 18, wherein the IL-12 is in the form of an immunocytokine composed of two IL-12 heterodimers fused to the NHS76 antibody. 20. The pharmaceutical composition of any one of claims 15-19, further comprising a chemotherapeutic drug, radioactive agent, antimetabolite, hormone, hormone antagonist, antibiotic, antiviral drug, antifungal drug, cyclophosphamide, or a combination thereof. 21. The pharmaceutical composition of any one of claims 15-20, further comprising an alkylating agent, folate antagonist, purine antagonist, pyrimidine antagonist, spindle poison, topoisomerase inhibitor, apoptosis inducing agent, angiogenesis inhibitor, podophyllotoxin, nitrosourea, cisplatin, carboplatin, interferon, asparginase, tamoxifen, leuprolide, flutamide, megestrol, mitomycin, bleomycin, doxorubicin, irinotecan, taxol, geldanamycin, or a combination thereof. 22. The pharmaceutical composition of any one of claims 15-21, further comprising a vector comprising a nucleic acid encoding tumor associated antigen selected from the group consisting of CEA, MUC1, PSA, and/or Brachyury. 23. The pharmaceutical composition of any one of claims 15-22, further comprising one or more adjuvants. 24. The pharmaceutical composition of claim 23, wherein one or more adjuvants is selected from the group consisting of alum, aluminum salts, aluminum phosphate, aluminum hydroxide, aluminum silica, calcium phosphate, incomplete Freund’s adjuvant, QS21, MPL-A, RIBI DETOXTM, and combinations thereof. 25. The pharmaceutical composition of any one of claims 15-24, further comprising granulocyte monocyte colony stimulating factor (GM-CSF). 26. The pharmaceutical composition of any one of claims 15-25, further comprising liposomes. 27. The pharmaceutical composition of any one of claims 15-26, which is formulated for injection. Leydig 771368 E-056-2023-0-PCT-01 71 28. A method of vaccinating a patient against a cancer, comprising administering the pharmaceutical composition of any claims 15-27 to a patient having or at risk of having a cancer under conditions suitable for the patient to mount an immune response to the polypeptide. 29. A method of treating cancer in a patient, comprising administering the pharmaceutical composition of any one of claims 15-27 to a patient having a cancer under conditions suitable for cancer to be treated within the patient. 30. Use of the pharmaceutical composition of any one of claims 15-27 for vaccinating a patient against a cancer. 31. Use of the pharmaceutical composition of any one of claims 15-27 for treating cancer in a patient. 32. The method of claim 28 or claim 29, or the use of claim 30 or 31, wherein the composition comprises a protein comprising a sequence of amino acids as set forth in SEQ ID NO:2 or having at least 95% identity to SEQ ID NO:2. 33. The method or use of any one of claims 28-32, wherein the patient is administered anti- PD-L1. 34. The method or use of any one of claims 28-33, wherein the patient is administered N803 (ANKTIVA (nogapendekin alfa inbakicept-pmln))). 35. The method or use of any one of claims 28-34, wherein the patient is a human patient.
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