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WO2025129119A1 - Agonification de gpr25 pour thérapie anticancéreuse - Google Patents

Agonification de gpr25 pour thérapie anticancéreuse Download PDF

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Publication number
WO2025129119A1
WO2025129119A1 PCT/US2024/060202 US2024060202W WO2025129119A1 WO 2025129119 A1 WO2025129119 A1 WO 2025129119A1 US 2024060202 W US2024060202 W US 2024060202W WO 2025129119 A1 WO2025129119 A1 WO 2025129119A1
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Prior art keywords
gpr25
cells
cell
cancer
tumor
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Pandurangan VIJAYANAND
Christian OTTENSMEIER
Han Feng
Sungjun Park
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University of Liverpool
La Jolla Institute for Allergy and Immunology
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University of Liverpool
La Jolla Institute for Allergy and Immunology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • G Protein-Coupled Receptor 25 (GPR25) is expressed in Tissue-resident memory CD8 + T (T RM ) cells, which are a distinct population of memory T cells that primarily resides within tissues and respond immediately against pathogens invading barrier tissues, thus representing the first-line of defense.
  • T RM Tissue-resident memory CD8 + T cells
  • GPRs are the largest superfamily of transmembrane receptors in the genome, they interact with a large number of extracellular ligands and transmit intracellular signals by activating heterotrimeric guanine nucleotide-binding proteins.
  • this disclosure provides methods of treating cancer, eliciting an anti-tumor response in a subject in need thereof, or treating or ameliorating an infection in a subject in need thereof, the methods comprising, or consisting essentially of, or consisting of modulating the expression of G Protein-Coupled Receptor 25 (GPR25) in the patient.
  • the disclosure provides methods of administering an agent capable of modulating the expression or activity of GPR25 in a T cell in the patient.
  • the disclosure provides method of promoting the development Atty. Dkt. No.: 116639-2710 of TRM cells in a subject.
  • the disclosure provides a method of promoting development of stem-like TRM cells in a subject.
  • the methods comprise, or consist essentially of, or yet further consist of administering to the patient an agent that comprises, consists of, or consists essentially of a small molecule or a protein, for example TGF- ⁇ .
  • the T cell is selected from the group of: an activated T cell; a tissue-resident memory (TRM) cell; and a stem T cell.
  • the activated T cell is specific for a tumor- specific antigen or a tumor-associated antigen expressed by the cancer or tumor cell, wherein the tumor-associated antigen is optionally overexpressed by the tumor cell.
  • the modulation comprises, consists of, or consists essentially of activating the T cell by agonizing the expression or activity of GPR25 in the T cell.
  • the agent comprises, consists of, or consists essentially of a bispecific agent that binds to GPR25 and binds to a second receptor expressed by the T cell.
  • the second receptor is selected from the group CXCR5, CXCR6, CD8, CD103, CD49A, CD69, CD3, CD28, CD8 or PD1.
  • the second receptor is CXCR5.
  • the bispecific agent comprises, consists of, or consists essentially of a bispecific antibody that binds to GPR25 and the second receptor expressed by the T cell, e.g., selected from the group CXCR5, CXCR6, CD8, CD103, CD49A, CD69, CD3, CD28, CD8 or PD1.
  • the agent that binds to GPR25 is or, comprises, consists of, or consists essentially of an agonistic antibody.
  • the agent comprises, consists of, or consists essentially of a bispecific antibody that binds to GPR25 and the second molecule expressed by the T cell.
  • the agent binds to GPR25 and binds to a tumor antigen expressed by a tumor cell.
  • the tumor antigen is overexpressed by the tumor cell.
  • the tumor antigen comprises, consists of, or consists essentially of a tumor- associated antigen or a tumor-specific antigen.
  • the tumor antigen comprises, consists of, or consists essentially of a tumor-associated antigen or a tumor-specific antigen.
  • the tumor-associated antigen is overexpressed by the tumor cell.
  • the tumor antigen comprises, consists of, or consists essentially of a lung cancer antigen or a Atty. Dkt. No.: 116639-2710 lymphatic tissue antigen.
  • the tumor antigen is selected from the group of MAGE-D4B, PSMA, HER2, HER3, EGFR, AFP, CEA, CA-125, MUC-1, ETA, MUC-1, BAGE, GAGE-1, MAGE-A1, NY-ESO-1, Gp100, Melan-A/MART-1, Prostate-specific antigen, Mammaglobin-A, Alpha-fetoprotein, HER-2/neu, P53, K-ras, or TRP-2/INT2
  • the cancer or tumor cell and/or the cancer or tumor is or is selected from the group of a tumor cell or tissue from a tumor cell or cancer of: the circulatory system; the respiratory tract; the gastrointestinal system the genitourinary tract; the liver; a bone; the nervous system; the reproductive system; the hematologic system; the oral cavity; skin and other tissues comprising connective and soft tissue, retroperitoneum and peritoneum, eye, intraocular melanoma, and adn
  • the cancer or tumor cell and/or the cancer or tumor comprises, consists of, or consists essentially of a cancer or tumor of a tissue or cell from the respiratory tract or lymph nodes.
  • the cancer comprises, consist of, or consists essentially of a localized or metastatic cancer.
  • the subject being treated is a mammal or animal, e.g., a human patient.
  • the subject or patient has reduced expression of GPR25 compared to that of a healthy, non-diseased subject.
  • the methods further comprise, consist of, or consist essentially of resecting the tumor or cancer prior to modulating the expression or activity of GPR25 in the T cell in the patient.
  • the modulating expression or activity of GPR25 in a T cell is administered as a first-line, a second-line, a third-line, a fourth-line or fifth-line therapy.
  • the methods further comprise, consist of, or consist essentially of administering an effective amount of a different anti-cancer agent to the patient.
  • the treating Atty. Dkt. No.: 116639-2710 cancer or a tumor in a cancer patient comprises, consists of, or consists essentially of one or more of providing to the subject or patient one or more of: a reduction in tumor burden, longer overall survival or prolonged time to tumor progression.
  • a method for screening for a GPR25 anticancer therapy comprising, consisting of, or consisting essentially of contacting a first sample of T cells with an amount of the test agent that binds to GPR25 and a second agent that binds a tumor antigen, and assaying for increased expression of GPR25 in the T cell.
  • increased expression of GPR25 in the T cell is an indication that the agent is a GPR25 anticancer therapy.
  • a method of modulating GPR25 in a subject comprising, consisting of, or consisting essentially of administering a bispecific antibody that targets and binds to GPR25 and a second receptor expressed by a T cell.
  • the receptor is selected from the group of: CXCR5, CXCR6, CD8, CD103, CD49A, CD69, CD3, or PD1.
  • the T cell is a stem T cell.
  • the subject or patient is a human patient.
  • a method of determining prognosis of a subject having cancer comprising, consisting of, or consisting essentially contacting T cells isolated from the subject with an antibody or agent that recognizes and binds to GPR25 to determine the frequency of T cells expressing GPR25, wherein a high density of GPR25 in T cells indicates a more positive prognosis or wherein a low density of GPR25 in T cells indicates a more negative prognosis.
  • the more negative prognosis comprises, consists of, or consists essentially of a decreased probability in survival
  • the more positive prognosis comprises, consists of, or consists essentially of an increased probability in survival.
  • the method can further comprise administering to the subject an effective amount of a therapy that is consistent with the prognosis.
  • a method of determining the responsiveness of a subject to cancer therapy that modulates GPR25 comprising, consisting of, or consisting essentially contacting T cells isolated from the subject with an antibody or agent that recognizes and binds to GPR25 to determine the frequency of GPR25 Atty. Dkt.
  • the cancer therapy comprises, consists of, or consists essentially of an agent that modulates the expression and/or activity of GPR25 in the subject.
  • the density of GPR25 in T cells in the subject is compared to a healthy, non-diseased subject.
  • the methods further comprise, consist of, or consist essentially of administering a cancer therapy that modulates GPR25 to the subject.
  • the cancer therapy comprises, consists of, or consists essentially of an agent that binds to GPR25.
  • the agent comprises, consists of, or consists essentially of an agonistic antibody targeting GPR25.
  • the sample is contacted with an agent.
  • the agent is detectably labeled.
  • the detectable label or tag comprises, consists of, or consists essentially of a radioisotope, a metal, horseradish peroxidase, alkaline phosphatase, avidin or biotin.
  • the agent comprises, consists of, or consists essentially of a polypeptide that binds to an expression product encoded by GPR25, or a polynucleotide that hybridizes to a nucleic acid sequence encoding all or a portion of GPR25.
  • the polypeptide comprises, consists of, or consists essentially an antibody, an antigen binding fragment thereof, or a receptor that binds to the GPR25.
  • the antibody comprises, consists of, or consists essentially of an IgG, IgA, IgM, IgE or IgD, or a subclass thereof.
  • the IgG comprises, consists of, or consists essentially of IgG1, IgG2, IgG3 or IgG4.
  • the antigen binding fragment comprises, consists of, or consists essentially of a Fab, Fab’, F(ab’)2, Fv, Fd, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv) or VL or VH.
  • the agent is contacted with the sample in conditions favoring binding of the agent to GPR25. In some aspects of the methods disclosed herein, the agent is contacted with the sample in conditions favoring binding of the agent to GPR25.
  • the methods comprise, consist of, or consist essentially of detection by immunohistochemistry (IHC), in-situ hybridization (ISH), ELISA, immunoprecipitation, immunofluorescence, chemiluminescence, Atty. Dkt. No.: 116639-2710 radioactivity, X-ray, nucleic acid hybridization, protein-protein interaction, immunoprecipitation, flow cytometry, Western blotting, polymerase chain reaction, DNA transcription, Northern blotting and/or Southern blotting.
  • IHC immunohistochemistry
  • ISH in-situ hybridization
  • ELISA immunoprecipitation
  • immunofluorescence immunofluorescence
  • chemiluminescence Atty. Dkt. No.: 116639-2710 radioactivity
  • X-ray nucleic acid hybridization
  • protein-protein interaction protein-protein interaction
  • immunoprecipitation flow cytometry
  • Western blotting polymerase chain reaction
  • DNA transcription Northern blotting and/or Southern blotting
  • the sample comprises, consists of, or consists essentially of cells, tissue, an organ biopsy, an epithelial tissue, a lung, respiratory or airway tissue or organ, a circulatory tissue or organ, a skin tissue, bone tissue, muscle tissue, head, neck, brain, skin, bone and/or blood sample.
  • a method of treating cancer in a subject comprising, consisting of, or consisting essentially of administering a T cell having increased expression of GPR25 to the subject, optionally as compared to the T cells of a subject having the cancer.
  • the cancer or tumor cell and/or the cancer or tumor comprises, consists of, or consists essentially of a cancer or tumor of a tissue or cell from the respiratory tract or lymph nodes. In some aspects, the cancer or tumor cell and/or the cancer or tumor comprises, consists of, or consists essentially of a cancer or tumor of a tissue or cell from the respiratory tract or lymph nodes a cancer or tumor of a tissue or cell from the respiratory tract. In some aspects, the cancer or tumor cell and/or the cancer or tumor comprises, consists of, or consists essentially of a metastatic cancer. In some aspects, the metastatic cancer comprises, consists of, or consists essentially of metastatic lung cancer. In some aspects, the patient is a human patient.
  • the patient has reduced expression of GPR25 compared to that of a healthy, non-diseased subject.
  • the T cell comprises, consists of, or consists essentially of an activated T cell; a tissue-resident memory (TRM) cell; a stem T cell.
  • the activated T cell is specific for a tumor-specific antigen or a tumor-associated antigen expressed by the tumor cell, wherein the tumor-associated antigen is optionally overexpressed by the tumor cell.
  • the T cell comprises, consists of, or consists essentially of a TRM cell.
  • provided herein are methods of promoting the development of TRM cells and/or stem-like TRM cells in a subject.
  • the methods comprise, consist of, or consist essentially of administering an agent that modulates expression of GPR25 to the subject.
  • the TRM cells are in the lung tissue or liver tissue of the subject.
  • the agent is administered to the lung tissue or liver tissue of the subject.
  • the agent comprises, consists of, or consists essentially of a small molecule or a protein selected from the group of: Transforming Growth Factor- ⁇ (TGF- ⁇ ), Nuclear Factor of Activated T-cells (NFAT), or Suppressor of Mothers against Decapentaplegic family member 1 (SMAD1), or equivalents thereof.
  • FIGS. 1A – 1D TGF- ⁇ induces expression of GPR25 in CD8 + T cells.
  • FIG. 1A Quantitative real-time PCR (qRT-PCR) analysis of GPR25 expression levels in na ⁇ ve human CD8 + T cells stimulated with anti-CD3 and anti-CD28 in the presence or absence of TGF- ⁇ for 24 and 48 hours.
  • qRT-PCR Quantitative real-time PCR
  • FIG. 1B UCSC genome browser tracks for genes in the extended GPR25 locus (50 kb), ATAC-seq tracks in the indicated CD8 + T cell populations (top) and ENCODE chromatin immunoprecipitation sequencing tracks for NFATC1, NFATC3 and SMAD1 (bottom); C1, C2 and C3 cis-regulatory regions are shaded.
  • FIG. 1C Gpr25 expression levels in na ⁇ ve murine CD8 + T (CD8 + CD44 lo CD62L hi ) stimulated with anti-CD3 and anti-CD28 in the presence or absence of TGF- ⁇ at the indicated time points.
  • FIGS. 2A – 2E Gpr25 promotes the development of liver T RM cells.
  • FIG. 2A Illustration of the T RM model in which congenically distinct Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells were co-transferred at a 1:1 ratio into CD45.1 recipient mice before infection with LCMV-OVA (i.p.).
  • FIG. 2B Representative contour plots and the frequencies of transferred Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells are shown.
  • FIG. 2C Representative contour plots Atty. Dkt.
  • FIG. 2D Illustration of parabiosis experimental workflow. Congenically distinct Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells were co-transferred at a 1:1 ratio into CD45.1 (host) mice before infection with LCMV-OVA (i.p.). Thirty days post infection, anti-Gr1 were administrated as previous described 25 to eliminate circulating lymphocytes.
  • CD8KO mice Seven days after anti-Gr1 administration, CD8KO (recipient) mice were co-joined with CD45.1 mice (host) via parabiosis surgery. Mice were analyzed 30 days after the surgery.
  • FIG. 2E Flow-cytometric analysis of CD8 + T cells isolated from the liver of host mice and spleen of recipient mice at 30 days after parabiosis surgery. Representative contour plots and frequencies of transferred Gpr25 +/+ (WT; CD45.1.2), Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells from the liver of host CD45.1 mice and spleen of recipient mice are shown. Bar graphs in (FIGS. 2B – 2C, FIG.
  • FIGS. 3A – 3E depict mean, each symbol represents data from an individual mouse; all data from (FIGS. 2B – 2C) are representative of three independent experiments; data from (FIG. 2E) are pooled data from two independent experiments. Statistical significance for the comparisons was computed using two- tailed two unequal variance Student t test; *P ⁇ 0.05, **P ⁇ 0.01 and *** P ⁇ 0.001.
  • FIGS. 3A – 3E Gpr25 deficiency impairs early stages of T RM cell development. (FIG.
  • FIG. 3A Illustration of the TRM model in which congenically distinct Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells were co-transferred at a 1:1 ratio into CD45.1 recipient mice before infection with LCMV-OVA (i.p.).
  • FIG.3B For the in vivo T cell proliferation assay, CTV-labeled OT-I cells were adoptively transferred into recipient mice before infection.
  • FIG. 3C Representative contour plots and frequencies of transferred Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I CD8 + T cells that express CD69 and KLRG1 are shown for the indicated time point post infection.
  • FIG. 3D Representative contour plots and the frequencies of transferred Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells are shown.
  • FIGS. 3E Representative contour plots and frequencies of transferred Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I CD8 + T cells that express CD69 and CXCR6 in the spleen and liver are shown; numbers adjacent to outlined areas indicate frequencies of cells co-expressing CD69 and CXCR6.
  • Gpr25 promotes the development of secondary T RM cells.
  • FIG. 4A FIG. 4A
  • FIG. 4B Congenically distinct Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells were co-transferred at a 1:1 ratio into CD45.1 recipient mice before infection with LCMV-OVA (i.p.).
  • Volcano plot shows false discovery rate (FDR) and fold change in expression levels of differentially expressed transcripts (False discovery rate (FDR) ⁇ 0.05, fold change > 0.25) between Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I T RM cells; dashed lines depict the threshold used for fold change and FDR; full list of differentially expressed transcripts is provided in Table 1.
  • FDR false discovery rate
  • FDR False discovery rate
  • FIG. 4F Representative contour plots and the frequencies of transferred Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I T cells in the liver are shown.
  • FIG. 4G Representative contour plots and the frequencies of transferred Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I T cells in the liver are shown.
  • FIG. 4H Representative contour plots and frequencies of transferred Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I TRM cells in the liver that express CD69 and CXCR6 (FIG. 4G), CD69 and KLRG1 (FIG. 4H) are shown; numbers adjacent to outlined areas (FIG. 4G) indicate percentage of cells co-expressing CD69 and CXCR6.
  • FIG. 4I GSEA plot shows positive enrichment of TGF- ⁇ response gene signatures, from the indicated source, in Gpr25 +/+ (WT) OT-I when Atty. Dkt. No.: 116639-2710 compared to Gpr25 -/- (KO) OT-I TRM cells.
  • FIG. 4J Flow-cytometric analysis na ⁇ ve murine CD8 + T cells (CD8 + CD44 lo CD62L hi ) from Gpr25 +/+ (WT) and Gpr25 -/- (KO) mice stimulated with anti-CD3 and anti-CD28 in the presence or absence of TGF- ⁇ for 24 hours. Representative histogram plots showing phosphorylation of Smad2(pS465/pS467)/Smad3(pS423/pS425) in the indicated samples. (FIG.
  • FIGS. 5A – 5H Gpr25 promotes development of lung T RM cells.
  • FIG. 5B Representative contour plots and the frequencies of transferred Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells in lungs are shown.
  • FIG. 5C Representative contour plots and frequencies of transferred Gpr25 +/+ (WT) OT-I CD8 + T cells, Gpr25 -/- (KO) OT-I CD8 + T cells and host CD8 + T cells that express CD69 and CD103 (top), CD69 and CXCR6 (bottom) are shown; numbers adjacent to outlined areas (bottom) indicate frequencies of cells co-expressing CD69 and CXCR6.
  • UMAP uniform manifold approximation and projection
  • Plot shows Z-score average expression (color scale) and percentage of cells (size scale) expressing selected transcripts that Atty. Dkt. No.: 116639-2710 are differentially expressed in the two clusters (FDR ⁇ 0.05, fold change > 0.25; full list of differentially expressed transcripts is provided in.
  • FIG. 5F UMAPs illustrating Seurat- normalized expression levels of Il7r, Xcl1, Gpr183, Zeb2, S1pr5, Gzmb, and Cx3cr1 transcripts in single cells.
  • FIG. 5G Illustration of the lung TRM model in which Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I CD8 + T cells were transferred into independent cohorts of CD8KO recipient mice before infection with LCMV-OVA (i.t.).
  • FIG. 5H Representative histograms and the frequencies of TCF1-expressing cells in Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I CD8 + T cells.
  • FIGS. 6A – 6G Gpr25-deficient T cells fail to control lung metastasis.
  • FIG. 6A Illustration of the lung metastasis model in which Gpr25 +/+ (WT; CD45.1.2) OT-I CD8 + T cells were transferred intratracheally (OT-I transfer group) into CD45.1 recipient mice before infection with LCMV-OVA (i.t.); Thirty days after infection, B16F10-OVA melanoma cells were injected intravenously.
  • FIG. 6B Representative picture of lung metastatic nodules and number of nodules on the surface of 5 lobes assessed 13 days after tumor injection in mice from the OT-I transfer and no transfer group.
  • FIG. 6A Illustration of the lung metastasis model in which congenically distinct Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells were co-transferred intratracheally at a 1:1 ratio into CD45.1 recipient mice before infection with LCMV-OVA (i.t.); Thirty days after infection, B16F10-OVA melanoma cells were injected intravenously into these mice.
  • FIG. 6C Representative contour plots and the frequencies of transferred Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells are shown.
  • FIG. 6E Illustration of the lung metastasis model in which Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I CD8 + T cells were transferred intratracheally into two cohorts of (FIG. 6C) CD45.1 or (FIG. 6E) CD8KO recipient mice before infection with LCMV-OVA (i.t.). Thirty days after infection, B16F10-OVA melanoma cells were injected intravenously into these mice Atty.
  • FIG. 6D Representative picture of lung metastatic nodules and number of nodules on the surface of 5 lobes, assessed 14 days after tumor injection, in the mice receiving Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells.
  • FIG. 6F Survival curve of cohorts of CD8KO recipient mice that received Gpr25 +/+ (WT), Gpr25 -/- (KO) OT-I CD8 + T cells or no adoptive transfer of OT-1 CD8 + T cells.
  • FIG. 6G Representative picture of lung metastatic nodules and number of nodules on the surface of 5 lobes, assessed 14 days after tumor injection, in mice from the OT-I transfer and no OT-1 transfer group.
  • Bar graph in (FIG. 6B) depicts mean, graphs in (FIG. 6D), (FIG. 6G) depict mean ⁇ S.D., each symbol represents data from an individual mouse; all data from (FIG. 6B) are representative of two independent experiments.
  • Data from (FIG. 6F), (FIG. 6G) are pooled data from two independent experiments.
  • FIGS. 7A – 7B Sequence analysis of GPR25 and flow cytometry gating strategies.
  • FIG. 7A Alignment of human and mouse GPR25 (Genebank entries NP_005289.2, NP_001094986.1, respectively). Identical amino acid residues are shown as (*), homology as (:), and different residues shaded.
  • FIGS. 8A – 8E Gpr25 promotes the development of liver T RM cells.
  • OT-I CD8 + T cells were co-transferred at a 1:1 ratio into CD45.1 recipient mice before infection with LCMV-OVA (i.p.).
  • OT-I TRM cells CD69 + CD62L-KLRG1-
  • OT-I TEM CD127 + CD62L-
  • OT-I TCM CD127 + CD62L +
  • FIG. 8B Representative contour plots show expression of CD69 Atty. Dkt. No.: 116639-2710 and CD62L in OT-I cells.
  • c Quantitative real-time PCR (qRT-PCR) analysis of Gpr25 (left) and S1pr1 (right) expression levels in the indicated T cell subsets from Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells.
  • FIG. 8D Quantitative real-time PCR
  • Bar graph in (FIG. 8C) depicts mean ⁇ S.E.M.
  • bar graph in (FIG. 8E) depicts mean, each symbol represents data from an individual mouse.
  • FIGS. 9A – 9D Gpr25 deficiency impairs early stages of T RM cell development.
  • FIGS.9A – 9D Congenically distinct Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells were co-transferred at a 1:1 ratio into CD45.1 recipient mice before infection with LCMV-OVA (i.p.).
  • FIG.9A Frequencies of transferred Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells in liver at 24 and 48 hours post infection are shown.
  • FIG. 9B Representative contour plots and frequencies of transferred Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I CD8 + T cells that express CD66 and CD44 in liver at 24 and 48 hours are shown.
  • FIG.9C Mean fluorescence intensity of CD25 expression in transferred Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I CD8 + T cells at 24 hours post infection is shown.
  • FIG.9D Representative contour plots show frequency of transferred Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I CD8 + T cells in liver that express CD69, CD62L, CD44 and CD127 at 30 days post infection. Bar graphs in a-c depicts mean, each symbol represents data from an individual mouse.
  • FIGS. 10A – 10D Gpr25 promotes the development of secondary T RM cells.
  • FIG. 10B Expression levels of the indicated transcripts in Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I TRM cells are shown.
  • FIG. 10D Congenically distinct Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells were co-transferred at a 1:1 ratio into CD45.1 recipient mice before infection with LCMV- OVA (i.p.).
  • GSEA Gene-set enrichment analysis plot shows enrichment of the indicated gene signatures in Gpr25 +/+ (WT) when compared to Gpr25 -/- (KO) T cells.
  • False discovery rate (FDR) and normalized enrichment score (NES) were determined using fgsea package on R.
  • GSEA plot shows positive enrichment of TGF- ⁇ response gene signatures, from the indicated source, in Gpr25 +/+ (WT) OT-I when compared to Gpr25 -/- (KO) OT-I T cells isolated from the liver 12 days post infection.
  • FIG.11A Illustration of the T RM model in which congenically distinct Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells were co-transferred intravenously at a 1:1 ratio into CD45.1 recipient mice before infection with LCMV-OVA delivered intraperitoneally (i.p.)
  • FIG.11B Representative contour plots and the frequencies of transferred Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells in the lungs are shown.
  • FIG.11C Atty. Dkt. No.: 116639-2710 Representative contour plots and frequencies of transferred Gpr25 +/+ (WT) OT-I CD8 + T cells, Gpr25 -/- (KO) OT-I CD8 + T cells and host CD8 + T cells in the lungs that express CD69 and CD103 are shown.
  • FIGS.5A – 5C Representative contour plots and frequencies of transferred Gpr25 +/+ (WT) OT-I CD8 + T cells, Gpr25 -/- (KO) OT-I CD8 + T cells and host CD8 + T cells that express CD69 and CD103 are shown (independent repeat of experiment shown in FIGS.5A – 5C).
  • FIGS. 12A – 12C Gpr25-deficient T cells fail to control lung metastasis.
  • FIGS. 12A Illustration of the lung metastasis model.
  • OT-I CD8 + T cells were transferred intratracheally (OT-I transfer group) into CD45.1 recipient mice before infection with LCMV-OVA (i.t.); Thirty days after infection, B16F10-OVA melanoma cells were injected intravenously. FTY720 or control PBS was intraperitoneally administrated to mice at indicated time points.
  • FIG. 12B Picture of lung metastatic nodules and number of nodules assessed 13 days after tumor injection.
  • FIG. 12C Representative contour plots and frequencies of total CD8 + T cells in the tumors of mice from the indicated groups. Bar graphs in (FIG. 12B) depicts mean, graph in (FIG.
  • FIG. 12C depict mean ⁇ S.D.; each symbol represents data from an individual mouse; all data from (FIGS. 12B – 12C) are representative of two independent experiments. Statistical significance for the comparisons was computed using two-tailed two unequal variance Student t test; *P ⁇ 0.05, **P ⁇ 0.01. FIG. 13.
  • FIG. 14 illustrates a GPR25 Hit ID testing funnel.
  • FIG. 15 illustrates a GPR25 lead generation plan.
  • FIG. 16. Demonstrates quantitative real-time PCR (qRT-PCR) analysis of GPR25 expression levels in na ⁇ ve human (left) and murine (right) CD8+ T cells stimulated with anti- CD3 and anti-CD28 in the presence or absence of TGF- ⁇ for 24 and 48 hours.
  • No.: 116639-2710 complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
  • all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof.
  • All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of +/- 15 %, or alternatively 10%, or alternatively 5%, or alternatively 2% and such ranges are included.
  • the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others.
  • the transitional phrase consisting essentially of (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the recited embodiment.
  • the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising”. “Consisting of” shall mean Atty. Dkt. No.: 116639-2710 excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein.
  • analogue refers to an equivalent having one or more modified amino acids and one or more amino acids replaced with another amino acid. Such modification may include but is not limited to conjugation with a molecule (for example, a small molecule, a cytotoxic molecule, a linker, a pH-sensitive linker, and/or a thiol linker), sialylation, Atty. Dkt.
  • No.: 116639-2710 polysialylation, O-glycosylation, N-glycosylation, myristoylation, palmitoylation, isoprenylation or prenylation, glipyatyon, lipoylation, phosphopantetheinylation, ethanolamine phosphoglycerol attachment, diphthamide formation, hypusine formation, acylation, acetylation, formylation, alkylation, methylation, amidation, citrullination, deamidation, eliminylation, ISGylation, SUMOylation, ubiquitination, neddylation, pupylation, biotinylation, carbamylation, oxidation, pegylation, glycation, carbamylation, carbonylation, spontaneous isopeptide bond formation, butyrylation, gamma-carboxylation, malonylation, hydroxylation, iodination, nucleotide addition, phosphate ester (O-linked)
  • albumin equivalent comprises, or consists essentially of, or yet further consists of, polypeptides which can be expressed at a reasonable quantity and which still retains or improves on certain albumin properties, including the binding of the albumin fragment to an FcRn receptor, as is known in the art or described herein.
  • affinity tag refers to a polypeptide that may be included within a fusion protein to allow detection of the fusion protein and/or purification of the fusion protein from the cellular milieu using a ligand that is able to bind to, i.e., has affinity for, the affinity tag.
  • the ligand may be, but is not limited to, an antibody, a resin, or a complementary polypeptide.
  • An affinity tag may comprise a small peptide, commonly a peptide of approximately 4 to 16 amino acids in length, or it may comprise a larger polypeptide.
  • Commonly used affinity tags include polyarginine, FLAG, V5, polyhistidine, c-Myc, Strep II, maltose binding protein (MBP), N-utilization substance protein A (NusA), thioredoxin (Trx), and glutathione S-transferase (GST), among others (for examples, see GST Gene Fusion System Handbook - Sigma-Aldrich).
  • the affinity tag is a polyhistidine tag, for example a His6 tag.
  • an affinity tag in a fusion protein allows the fusion protein to be purified from the cellular milieu by affinity purification, using an affinity medium that can tightly and specifically bind the affinity tag.
  • the affinity medium may comprise, for example, a metal-charged resin or a ligand covalently linked to a stationary phase (matrix) such as agarose or metal beads.
  • matrix such as agarose or metal beads.
  • polyhistidine tagged fusion proteins also referred to as His tagged fusion proteins
  • FLAG affinity gels may be used to capture FLAG tagged fusion proteins, and glutathione cross- linked to a solid support such as agarose may be used to capture GST tagged fusion proteins.
  • purification refers to the process of isolating one or more polypeptides from a complex mixture, such as a cell lysate or a mixture of polypeptides. The purification, separation, or isolation need not be complete, i.e., some components of the complex mixture may remain with the one or more polypeptides after the purification process.
  • the product of purification should be enriched for the one or more polypeptides relative to the complex mixture before purification and a significant portion of the other components initially present within the complex mixture should be removed by the purification process.
  • the term “cell” as used herein may refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source. “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus.
  • the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells.
  • eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human, e.g., HEK293 cells, Chinese Hamster Ovary (CHO) cells and 293T cells.
  • HEK293 cells Chinese Hamster Ovary (CHO) cells and 293T cells.
  • “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called an episome.
  • Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 ⁇ m in diameter and 10 ⁇ m long).
  • Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium. Atty. Dkt.
  • encode refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • isolated or a grammatical variation thereof as used herein refers to molecules or biologicals or cellular materials being substantially free from other materials.
  • the term “functional” may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a particular, specified effect.
  • nucleic acid sequence and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • this term includes, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • the polynucleotide comprises and/or encodes a messenger RNA (mRNA), a short hairpin RNA, and/or small hairpin RNA.
  • mRNA messenger RNA
  • the polynucleotide is or encodes an mRNA.
  • the polynucleotide is a double-strand (ds) DNA, such as an engineered ds DNA or a ds cDNA synthesized from a single-stranded RNA.
  • ds double-strand
  • the terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
  • Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • RNAi transfer RNA
  • RNAi ribosomal RNA
  • RNAi ribozymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non- nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single- stranded molecules.
  • any embodiment disclosed herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule.
  • This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • isolated or recombinant as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule as well as polypeptides.
  • isolated or recombinant nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polynucleotides, polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • isolated or recombinant means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature.
  • an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype.
  • an isolated polynucleotide is separated from the 3′ and 5′ contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require “isolation” to distinguish it from its naturally occurring counterpart.
  • the terms “engineered” “synthetic” “recombinant” and “non- naturally occurring” are interchangeable and indicate intentional human manipulation, for example, a modification from its naturally occurring form, and/or a sequence optimization.
  • equivalent or biological equivalent are used interchangeably when referring to a particular molecule, biological, or cellular material and intend those having minimal homology while still maintaining desired structure or functionality (for example, having a similar functional activity). It should be understood, without being explicitly stated that when referring to an equivalent or biological equivalent to a reference polypeptide, protein, or polynucleotide, that an equivalent or biological equivalent has the recited structural relationship to the reference polypeptide, protein, or polynucleotide and equivalent or substantially equivalent biological activity.
  • non-limiting examples of equivalent polypeptides, proteins, or polynucleotides include a polypeptide, protein, or polynucleotide having at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity thereto or for polypeptide, polynucleotide, or protein sequences across the length of the reference polynucleotide.
  • an equivalent polypeptide is one that is encoded by a polynucleotide or its complement that hybridizes under conditions of high stringency to a polynucleotide encoding such reference polypeptide sequences and that have substantially equivalent or equivalent biological activity. Conditions of high stringency are described herein and incorporated herein by reference.
  • an equivalent thereof is a polypeptide encoded by a polynucleotide or a complement thereto, having at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity, or at least 97% sequence identity across the length of the reference polynucleotide to the reference polynucleotide, e.g., the wild-type polynucleotide.
  • Such equivalent polypeptides have the same biological activity as the reference polynucleotide. Atty. Dkt.
  • Non-limiting examples of equivalent polypeptides include a polynucleotide having at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 97%, identity to a reference polynucleotide.
  • An equivalent also intends a polynucleotide or its complement that hybridizes under conditions of high stringency to a reference polynucleotide. Such equivalent polypeptides have the same biological activity as the reference polynucleotide.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences across the length of the reference polynucleotide.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a non-limiting exemplary alignment program is BLAST, using default parameters.
  • Sequence identity and percent identity can be determined by incorporating them into clustalW (available at the web address:genome.jp/tools/clustalw/, last accessed on Jan. 13, 2017).
  • “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non- Atty. Dkt.
  • No.: 116639-2710 homologous sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.
  • the term “at least 90% identical” refers to an identity of two compared sequences (polynucleotides or polypeptides) of about 90% to about 100%.
  • “Homology” or “identity” or “similarity” can also refer to two nucleic acid molecules that hybridize under stringent conditions.
  • the terms “retain” “similar” and “same” are used interchangeably while describing a function, an activity or a functional activity of a polynucleotide, a protein and/or a peptide, referring to a functional activity of at least about 20% (including but not limited to: at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or about 100%) of the activity of the reference protein, polynucleotide and/or peptide.
  • polypeptide, protein, polynucleotide, or antibody an equivalent or a biologically equivalent of such is intended within the scope of this disclosure.
  • biological equivalent thereof is intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody, fragment, polypeptide, or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof.
  • an equivalent polynucleotide is one that hybridizes under stringent conditions to the polynucleotide or complement of the polynucleotide as described herein for use in the described methods.
  • an equivalent antibody or antigen binding polypeptide intends one that binds with at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least Atty. Dkt. No.: 116639-2710 85%, or alternatively at least 90%, or alternatively at least 95% affinity or higher affinity to a reference antibody or antigen binding fragment.
  • the equivalent thereof competes with the binding of the antibody or antigen binding fragment to its antigen under a competitive ELISA assay.
  • an equivalent intends at least about 80% homology or identity and alternatively, at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid.
  • “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • stringent hybridization conditions include: incubation temperatures of about 25° C. to about 37° C.; hybridization buffer concentrations of about 6 ⁇ SSC to about 10 ⁇ SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4 ⁇ SSC to about 8 ⁇ SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40° C. to about 50° C.; buffer concentrations of about 9 ⁇ SSC to about 2 ⁇ SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5 ⁇ SSC to about 2 ⁇ SSC.
  • Examples of high stringency conditions include: incubation temperatures of about 55° C. to about 68° C.; buffer concentrations of about 1 ⁇ SSC to about 0.1 ⁇ SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1 ⁇ SSC, 0.1 ⁇ SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed. Atty. Dkt. No.: 116639-2710
  • the term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunits of amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds.
  • the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein’s or peptide’s sequence.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • a consecutive amino acid sequence refers to a sequence having at least two amino acids. However, it is noted that a consecutive amino acid sequence of a first part and a second part does not limit the amino acid sequence to have the first part directly conjugated to the second part. It is also possible that the first part is linked to the second part via a third part, such as a link, thus forming one consecutive amino acid sequence.
  • a polynucleotide disclosed herein can be delivered to a cell or tissue using a gene delivery vehicle.
  • Gene delivery “gene transfer” “mRNA-based delivery”, “transducing,” and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction.
  • exogenous polynucleotide sometimes referred to as a “transgene”
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes, including for example protamine complexes, lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, and inorganic nanoparticles, or combinations thereof) as well as techniques facilitating the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • vector-mediated gene transfer by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes, including for example protamine complexes, lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, and inorganic nanoparticles, or combinations thereof
  • the introduced polynucleotide can be unmodified or can comprise one or more modifications; for example, a modified mRNA may comprise ARCA capping; enzymatic polyadenylation to add a tail of 100-250 adenosine residues; and substitution of one or both of cytidine with 5-methylcytidine and/or uridine with pseudouridine.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an Atty. Dkt.
  • extrachromosomal replicon e.g., a plasmid
  • a nuclear or mitochondrial chromosome e.g., a nuclear or mitochondrial chromosome.
  • vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
  • a “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state.
  • Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances.
  • “Plasmids” used in genetic engineering are called “plasmid vectors”. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.
  • MCS multiple cloning site
  • Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest.
  • a “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors include retroviral vectors, adenovirus vectors, adeno- associated virus vectors, herpes simplex virus vectors, alphavirus vectors and the like.
  • the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • the term “mammal” includes both human and non-human mammals.
  • a “subject” or “patient” to whom the therapies such as for example a combination of anti-GPR25 therapy and immune checkpoint inhibitor is administered is Atty. Dkt. No.: 116639-2710 preferably a mammal such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human).
  • the subject or patient can be a human, such as an adult patient or a pediatric patient.
  • the term “subject” refers includes but is not limited to a subject at risk of cancer or an infection as well as a subject that has already developed cancer or infection.
  • Such subjects include mammalian animals (mammals), such as a non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans.
  • Subjects include animal disease models, for example, mouse and other animal models of cancer or an age-associated genome dysfunction, disorder, or disease known in the art. Accordingly, subjects appropriate for treatment include those having or at risk of cancer or an infection, also referred to as subjects in need of treatment.
  • Subjects in need of treatment therefore include subjects that have been previously had cancer or an infection, or that have an ongoing cancer or an infection or have developed one or more adverse symptoms caused by or associated with cancer or an infection regardless of the type, timing or degree of onset, progression, severity, frequency, duration of the symptoms.
  • Prophylactic uses and methods are therefore included.
  • Target subjects for prophylaxis may be at increased risk (probability or susceptibility) of developing cancer or an infection. Such subjects are considered in need of treatment due to being at risk.
  • Subjects for prophylaxis need not be at increased risk but may be from the general population in which it is desired to protect a subject against cancer or an infection for example. Such a subject that is desired to be protected against cancer or an infection can be administered treatment or agent described herein.
  • a subject that is not specifically at risk for cancer or an infection, but nevertheless desires protection against cancer or an infection can be administered a composition or agent as described herein. Such subjects are also considered in need of treatment.
  • Atty. Dkt. No.: 116639-2710 “Prophylaxis” and grammatical variations thereof mean a method in which contact, administration or in vivo delivery to a subject is prior to development of cancer or an infection. In certain situations, it may not be known that a subject has developed cancer or an infection, but administration or in vivo delivery to a subject can be performed prior to manifestation of disease pathology or an associated adverse symptom, condition, complication, etc. caused by or associated with cancer or an infection.
  • a composition or method of the present disclosure can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility to cancer or an infection, or an adverse symptom, condition or complication associated with or caused by cancer or an infection.
  • “Prophylaxis” can also refer to a method in which contact, administration or in vivo delivery to a subject is prior to a secondary or subsequent exposure or infection. In such a situation, a subject may have had a prior cancer or an infection or prior adverse symptom, condition or complication associated with or caused by cancer or an infection. Treatment by administration or in vivo delivery to such a subject, can be performed prior to a secondary or subsequent cancer or an infection.
  • Such a method can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards a secondary or subsequent cancer or an infection, or an adverse symptom, condition or complication associated with or caused by or associated with a secondary or subsequent cancer or an infection.
  • Treatment of cancer or an infection can be at any time during the cancer or an infection.
  • Certain embodiments of the present disclosure can be administered as a combination (e.g., with a second active), or separately concurrently or in sequence (sequentially) in accordance with the methods described herein as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by cancer or an infection, or an adverse symptom, condition or complication associated with or caused by cancer or an infection.
  • a method can be practiced one or more times (e.g., 1-10, 1-5 or 1-3 times) an hour, day, week, month, or year.
  • a non-limiting dosage schedule is 1-7 times per week, for 1, 2, 3, Atty. Dkt. No.: 116639-2710 4, 5, 6, 7, 8, 9, 10, 15, 20 or more weeks, and any numerical value or range or value within such ranges.
  • An “effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two or more agents, that, when administered for the treatment of a mammal or other subject, is sufficient to effect such treatment for the disease. The “effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.
  • Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, whether a subject has previously had cancer or an infection, the onset, progression, severity, frequency, duration probability of or susceptibility of the symptom, condition, pathology or complication, the treatment protocol and compositions, the clinical endpoint desired, the occurrence of previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit. The dose amount, number, frequency, or duration may be proportionally increased or reduced, as indicated by the status of the subject.
  • the subject has previously had cancer or an infection, whether the subject is merely at risk of cancer or an infection, exposure or infection, whether the subject has been previously treated for cancer or an infection.
  • the dose amount, number, frequency, or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy.
  • the route, dose, number and frequency of administrations, treatments, and timing/intervals between treatment and disease development can be modified.
  • a desirable treatment of the present disclosure will elicit robust, long-lasting immunity against cancer or an infection.
  • disclosure methods, uses and compositions provide long-lasting immunity to cancer or an infection. Atty. Dkt.
  • a biological sample, or a sample can be obtained from a subject, cell line or cultured cell or tissue.
  • exemplary samples include, but are not limited to, cell sample, tissue sample, tumor biopsy, liquid samples such as blood and other liquid samples of biological origin (including, but not limited to, ocular fluids (aqueous and vitreous humor), peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal
  • the sample is a tumor/cancer biopsy.
  • a “solid tumor” is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. The solid tumor can be localized or metastatic.
  • the terms “disease” “disorder” and “condition” are used interchangeably herein, referring to a cancer, a status of being diagnosed with a cancer, or a status of being suspected of having a cancer.
  • extracellular matrix is a three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins, that provide structural and biochemical support to surrounding cells. It is an essential component of the tumor microenvironment. Cancer development and progression are associated with increased ECM deposition and crosslink, while the chemical and physical signals elicited from ECM are necessary for cancer cell proliferation and invasion.
  • the ECM of a cancer comprises a peri-cancerous cell or tissue.
  • the term “detectable marker” refers to at least one marker capable of directly or indirectly, producing a detectable signal.
  • a non-exhaustive list of this marker includes enzymes which produce a detectable signal, for example by colorimetry, fluorescence, Atty. Dkt. No.: 116639-2710 luminescence, such as horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose-6 phosphate, dehydrogenase, chromophores such as fluorescent, luminescent dyes, groups with electron density detected by electron microscopy or by their electrical property such as conductivity, amperometry, voltammetry, impedance, detectable groups, for example whose molecules are of sufficient size to induce detectable modifications in their physical and/or chemical properties, such detection may be accomplished by optical methods such as diffraction, surface plasmon resonance, surface variation, the contact angle change or physical methods such as atomic force spectroscopy, tunnel effect, or radioactive molecules such as 32 P, 35 S , 89 Zr or 125 I.
  • luminescence such as horseradish peroxidase
  • purification marker refers to at least one marker useful for purification or identification.
  • a non-exhaustive list of this marker includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S-protein.
  • Suitable direct or indirect fluorescence marker comprise FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin, Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten.
  • immunophenotyping refers to the analysis of heterogeneous populations of cells for the purpose of identifying the presence and proportions of the various populations in the sample. Antibodies are used to identify cells by detecting specific antigens (termed markers) expressed by these cells. In an aspect, the cell samples are characterized by immunophenotyping using techniques such as flow cytometry.
  • characterizations of the various cell types, (such as T cells, B cells and their subsets) present in a cell sample may be carried out using any suitable methodology such as reverse transcriptase polymerase chain reaction (RT-PCR) or immunocytochemistry (IHC).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • IHC immunocytochemistry
  • first line or second line or third line or “fourth line” or “fifth line” refers to the order of treatment received by a patient.
  • First-line therapy regimens are treatments given first, whereas second or third-line therapy are given after the first-line therapy or after the second-line therapy, respectively.
  • the National Cancer Institute defines first-line therapy as “the first treatment for a disease or condition. In patients with cancer, primary Atty. Dkt.
  • T cell refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of a T-cell receptor on the cell surface. T- cells may either be isolated or obtained from a commercially available source.
  • T cell includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T- cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg), Tissue-resident memory T cells (TRM cells), stem T cells and gamma-delta T cells.
  • a “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses.
  • Non-limiting examples of commercially available T-cell lines include lines BCL2 (AAA) Jurkat (ATCC® CRL-2902TM), BCL2 (S70A) Jurkat (ATCC® CRL-2900TM), BCL2 (S87A) Jurkat (ATCC® CRL-2901TM), BCL2 Jurkat (ATCC® CRL-2899TM), Neo Jurkat (ATCC® CRL-2898TM), TALL-104 cytotoxic human T cell line (ATCC # CRL-11386).
  • T-cell lines e.g., such as Deglis, EBT-8, HPB- MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1 and T34; and immature T- cell lines, e.g., ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L- KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13, MOLT-16, MT-1, MT-ALL, P12/Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1 to
  • Null leukemia cell lines including but not limited to REH, NALL-1, KM-3, L92-221, are another commercially available source of immune cells, as are cell lines derived from other leukemias and lymphomas, such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, Atty. Dkt. No.: 116639-2710 HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, U266 myeloma.
  • TRM cells refer to a subset of long-lived memory T cells that occupy various tissues without recirculating. TRM cells reside in tissues that create barriers against the outside environment and thus provide defense against repeatedly incoming pathogens. TRMs also play a role in the protection against malignancies. Three cell surface markers that has been associated with TRM are CD69, CD49a, and CD103.
  • Transforming Growth Factor Beta is a growth and differentiation factor encoded by 33 genes in mammals and comprises homo- and heterodimers. “Frequency” of cells expressing any one particular molecule, biomarker, or antigen refers to the likelihood of or ratio of cells expressing the molecule, biomarker, or antigen compared to a population of T cells at large.
  • G Protein-Coupled Receptor 25 (“GPR25) is a protein encoding gene that encodes a member of the G-protein coupled receptor 1 family. G-protein coupled receptors are membrane proteins which activate signaling cascades as a response to extracellular stress. Among its related pathways are GPCR downstream signaling and Class A/1 (Rhodopsin-like receptors).
  • FIG. 7A Mouse MQSTEPWSPSWGTLSWDYSGSGSLDQVELCPAWNLPYGHAIIPALYLAA FAVGLPGNAFVVWLLSRQRGPRRLVDTFVLHLAAADLGFVLTLPLWAAAEARGGLWP FGDGLCKVSSFALAVTRCAGALLLAGMSVDRYLAVGRPLSARPLRSARCVRAVCGAA WAAAFLAGLPALLYRGLQPSLDGVGSQCAEEPWEALQGVGLLLLLLTFALPLAVTLICY WRVSRRLPRVGRARSNSLRIIFTVESVFVGCWLPFGVLRSLFHLARLQALPLPCSLLLAL RWGLTVTTCLAFVNSSANPVIYLLLDRSFRARARFGLCARAGRQVRRISSASSLSRDDSS VFRGRSPKVNSASATW Human Atty.
  • agonistic antibodies and/or agents have the ability to bind and activate the target receptor in a way that mimics the activity of the ligand.
  • the agent may include a small molecule, an antibody, lentivirus, adeno-associated virus, an antisense oligonucleotide, an siRNA or an miRNA.
  • An agonistic anti GPR25 antibody intends an antibody, antigen binding fragment, derivative or other modification as described herein that recognizes and binds the GPR25 protein.
  • the agonizing antibodies or agents described in the application may bind to GPR25 to increase, enhance, upregulate, and/or otherwise modulate the activity of the GPR25 receptor and/or the GPR25 expressing cell. Such activities may include proliferation and cell signaling activities of the cell upon which the GPR25 receptor is expressed.
  • the agonistic antibodies or agents of this disclosure target and specifically bind to GPR25.
  • the agonistic antibody or agents bind the receptor in a manner that mimics the binding of the physiological ligand resulting in antibody-mediated agonism.
  • treatment with an agonistic anti- GPR25 antibodies significantly impedes tumor growth, which may be mediated via stimulation of tumor infiltrating CD8+ T cells.
  • Immunotherapies utilizing agonistic antibodies or agents especially those targeting trimeric receptors like 4-1BB or CD40, require antibody crosslinking via Fcg receptors expressed on APCs for efficient T cell activation.
  • IgG Immunoglobulin
  • the antibody subclass and the availability, type, and degree of expression of Fcg receptors on APCs are critical determinants of immunotherapy treatment efficacy.
  • the agonistic activity of antibodies targeting co-stimulatory receptors depends on a variety of factors and does not always require Fc cross-linking. Such factors include but are not limited to antibody affinity, Atty. Dkt. No.: 116639-2710 Fc modifications like glycoengineering or point mutations, the antibody subclass and antigen expression.
  • antibody includes whole antibodies and any antigen binding fragment or a single chain thereof.
  • antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule.
  • antibody also include immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fab′, F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies and kappa bodies; multispecific antibody fragments formed from antibody fragments and one or more isolated.
  • anti-GPR25 when used before a protein name, anti-GPR25 for example, refers to a monoclonal or polyclonal antibody that binds and/or has an affinity to a particular protein.
  • the antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.
  • Antibodies can be isolated from any suitable biological source, e.g., murine, rat, sheep and canine.
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multi- specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • F(ab′)2 means a bivalent antigen-binding fragment of an immunoglobulin that contains both light chains and part of both heavy chains.
  • Fv fragment or “variable domain fragment” refers to a VH domain and a VL domain of an antibody specifically binding to an antigen, both domains forming together a Fv fragment.
  • Fv fragments mean an antibody fragment comprising the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv fragment polypeptide further comprises a polypeptide linker between the VH and VL domains polypeptide that enables the scFv to form.
  • Kappa ( ⁇ ) and lambda ( ⁇ ) light chains refer to the two major antibody light chain isotypes.
  • synthetic antibody means an antibody, which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage.
  • the term should also be construed to mean an antibody, which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • antigen or “Ag” is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • any macromolecule including virtually all proteins or peptides, can serve as an antigen.
  • antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encoding an Atty. Dkt. No.: 116639-2710 “antigen” as that term is used herein.
  • bispecific antibody refers to an antibody that can simultaneously bind to two different receptors, epitopes or antigens.
  • the bispecific antibodies of the instant disclosure may target and bind antigens on the same cells or different cells.
  • the bispecific antibodies bind to GPR25 and a second molecule on the T cell.
  • GPR25 may be expressed on a T cell.
  • the second molecule is expressed on the same T cell.
  • the bispecific antibodies of the claimed disclosure increase target specificity for GPR25 expressing T cells, while limiting undesirable off-target activity.
  • the bispecific bind and modulate the expression or activity of GPR25 in or on the T cell or the GPR25 expressing T cells.
  • the bispecific antibodies bind to GPR25 and a tumor or cancer antigen expressed by a tumor or cancer cell, including but not limited to tumor-associated antigens or tumor-specific antigens.
  • the bispecific antibody may simultaneously bind and activate the GPR25 expressing T cell, while also binding to a tumor or cancer antigen.
  • the activated T cell is able to target the tumor or cancer cells expressing the antigen.
  • the antigen is overexpressed or specifically expressed by the tumor or cancer cell.
  • the antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
  • the antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like.
  • the antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like.
  • Monoclonal antibodies may be generated using hybridoma techniques or recombinant DNA methods known in the art.
  • a hybridoma is a cell that is produced in the laboratory from the fusion of an antibody-producing lymphocyte and a non-antibody producing cancer cell, usually a myeloma or lymphoma.
  • a hybridoma proliferates and produces a continuous sample of a specific monoclonal antibody.
  • Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to antigens of interest, and screening of antibody display libraries in cells, phage, or similar systems.
  • the term “human antibody” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • human antibody refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, C L , C H domains (e.g., C H1 , C H2 , C H3 ), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations.
  • antibodies designated primate monkey, baboon, chimpanzee, etc.
  • rodent mouse, rat, rabbit, guinea pig, hamster, and the like
  • other mammals designate such species, sub-genus, genus, sub-family, family-specific antibodies.
  • chimeric antibodies include any combination of the above.
  • a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non-human animal or prokaryotic Atty. Dkt. No.: 116639-2710 or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single-chain antibody, it can comprise a linker peptide that is not found in native human antibodies.
  • an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
  • linker peptides are considered to be of human origin.
  • a human antibody is “derived from” a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, e.g., by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library.
  • a human antibody that is “derived from” a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of human germline immunoglobulins.
  • a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences).
  • a human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.
  • a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene.
  • the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.
  • a “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The term also intends recombinant human antibodies. Methods for making these antibodies are described herein.
  • an antibody as used herein may be a recombinant antibody.
  • the term “recombinant human antibody”, as used herein, includes all human antibodies that are Atty. Dkt.
  • No.: 116639-2710 prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. Methods for making these antibodies are described herein.
  • chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.
  • humanized antibody or “humanized immunoglobulin” refers to a human/non-human chimeric antibody that contains a minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a variable region of the recipient are replaced by residues from a variable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity and capacity.
  • Humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody.
  • the humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, a non-human antibody containing one or more amino acids in a framework region, a constant region or a CDR, that have been substituted with a correspondingly positioned amino acid from a human antibody.
  • Fc immunoglobulin constant region
  • humanized antibodies are expected to produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody.
  • the humanized Atty. Dkt. No.: 116639-2710 antibodies may have conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions.
  • Conservative substitutions groupings include: glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, serine-threonine and asparagine-glutamine.
  • the antibodies of the present disclosure may bind to an antigen or molecule.
  • the antigens or molecules are expressed in a T cell, tumor cell, or tissue of a subject.
  • polyclonal antibody or “polyclonal antibody composition” as used herein refer to a preparation of antibodies that are derived from different B-cell lines.
  • antibody derivative comprises a full-length antibody or a fragment of an antibody, wherein one or more of the amino acids are chemically modified by alkylation, pegylation, acylation, ester formation or amide formation or the like, e.g., for linking the antibody to a second molecule.
  • CDRs Complementarity-determining regions
  • variable region is about 90 amino acids long to about 200 amino acids long, including but not limited to about 100 amino acids long, or alternatively about 110 amino acids long, or alternatively about 120 amino acids long, or alternatively about 130 amino acids long, or alternatively about 140 amino acids long, or alternatively about 150 amino acids long, or alternatively about 160 amino acids long, or alternatively about 170 amino acids long, or alternatively about 180 amino acids long, or alternatively about 190 amino acids long.
  • a variable region of an amino acid sequence refers to the first Atty. Dkt.
  • a set of CDRs constitutes a paratope also called an antigen-binding site, which is a part of an antibody that recognizes and binds to an antigen.
  • CDRn refers to a CDRn in an immunoglobulin chain or derived from an immunoglobulin chain, wherein the number n is selected from 1-3.
  • CDRLn refers to a CDRn in a light chain or derived from a light chain, wherein the number n is selected from 1-3; while CDRHn refers to a CDRn in a heavy chain or derived from a heavy chain, wherein the number n is selected from 1-3.
  • framework region (FR) refers to the part of a variable region which is not a CDR.
  • FRn refers to a FR in a heavy chain or a light chain or derived from a heavy chain or a light chain, and wherein the number n is selected from 1-4.
  • a variable region comprises or consists essentially of, or yet further consists of the following (optionally following the order as provided, and further optionally from the amino terminus to the carboxyl terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • Variable regions and/or CDRs of an antibody or a fragment thereof can be determined by one of skill in the art, for example, using publically or commercially available tools.
  • Non-limiting examples of such tools include, IgBlast (accessible at www.ncbi.nlm.nih.gov/igblast/), Scaligner (available from drugdesigntech at www.scaligner.com/), IMGT rules and/or tools (see, for example, www.imgt.org/IMGTScientificChart/Nomenclature/IMGT-FRCDRdefinition.html, also accessible at www.imgt.org/), Chothia Canonical Assignment (accessible at www.bioinf.org.uk/abs/chothia.html), Antigen receptor Numbering And Receptor Classification (ANARCI, accessible at opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/anarci/), the Kabat numbering method/scheme (e.g., Kabat, E.A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.
  • agonistic antibodies have the ability to bind and activate the target receptor in a way that mimics the activity of the ligand.
  • An agonistic anti-GPR25 antibody intends an antibody, antigen-binding fragment, derivative or other modification as described herein that recognizes and binds the GPR25protein.
  • the agonizing antibodies described in the application may bind to GPR25 to increase, enhance, upregulate, and/or otherwise modulate the activity of the GPR25 receptor and/or the GPR25 expressing cell. Such activities may include proliferation and cell signaling activities of the cell upon which the GPR25 receptor is expressed.
  • the agonistic antibodies of this disclosure target and specifically bind to GPR25. In some aspects, the agonistic antibody binds the receptor in a manner that mimics the binding of the physiological ligand resulting in antibody-mediated agonism. In some aspects, treatment with an agonistic anti-GPR25 antibodies significantly impedes tumor growth, which may be mediated via stimulation of tumor-infiltrating CD8+ T cells.
  • Immunotherapies utilizing agonistic antibodies require antibody crosslinking via Fcg receptors expressed on APCs for efficient T cell activation.
  • Fcg receptors expressed on APCs for efficient T cell activation.
  • IgG Immunoglobulin
  • they bind to and can thus get crosslinked by Fcg receptors with different affinities.
  • the antibody subclass and the availability, type and degree of expression of Fcg receptors on APCs are critical determinants of immunotherapy treatment efficacy.
  • Immuno response broadly refers to the antigen-specific responses of lymphocytes to foreign substances.
  • immunogen and “immunogenic” refer to molecules with the capacity to elicit an immune response. All immunogens are antigens, however, not all antigens are immunogenic.
  • An immune response disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response may occur Atty. Dkt.
  • the term “inducing an immune response in a subject” or “modulating an immune response” are terms well understood in the art and intends that an increase or decrease of at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold or more in an immune response (i.e. T cell or antibody response) to an antigen (or epitope) and can be detected or measured by various methods known in the art.
  • the frequency or activity of antigen-specific T cells can be measured by multiple methods, including, but not limited to, flow cytometry, RNA-sequencing or in vitro assays.
  • modulating activity refers to increasing or decreasing the activity of specific T cell populations associated with an immune response. Modulating of activity may be accomplished by the administration of agents, including antibodies, that target and bind to specific T cell receptors in order to activate the T cell population expressing that molecule. Modulation may occur when the T cells are engaged by costimulatory ligands, agonistic antibodies or cytokines. In some aspects, modulating activity may include the administration of an agent that targets a molecule on a T cell. In some aspects, the molecule is GPR25and the agent is an antibody that targets GPR25, thus activating the GPR25expressing T cell.
  • immunotherapy agent means a type of cancer treatment which uses a patient’s own immune system to fight cancer, including but not limited to a physical intervention, a chemical substance, a biological molecule or particle, a cell, a tissue or an organ, or any combinations thereof, enhancing or activating or initiating a patient's immune response against cancer.
  • immunotherapy agents include antibodies, immune regulators, checkpoint inhibitors, an antisense oligonucleotide (ASO), an RNA interference Atty. Dkt.
  • RNAi RNAi
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • a viral vector e.g., a viral vector, an anti-cancer cell therapy (e.g., transplanting an anti-cancer immune cell optionally amplified and/or activated in vivo, or administering an immune cell expressing a chimeric antigen receptor (CAR)), a CAR therapy, and cancer vaccines.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • immune checkpoint refers to a regulator and/or modulator of the immune system (such as an immune response, an anti-tumor immune response, a nascent anti- tumor immune response, an anti-tumor immune cell response, an anti-tumor T cell response, and/or an antigen recognition of T cell receptor in the process of immune response). Their interaction activates either inhibitory or activating immune signaling pathways.
  • a checkpoint may contain one of the two signals: an stimulatory immune checkpoint that stimulates an immune response, and an inhibitory immune checkpoint inhibiting an immune response.
  • the immune checkpoint is crucial for self-tolerance, which prevents the immune system from attacking cells indiscriminately.
  • the immune checkpoints are present on T cells, antigen-presenting cells (APCs) and/or tumor cells.
  • APCs antigen-presenting cells
  • the term “recombinant host cell,” “recombinant cell,” “engineered host cell,” or “engineered cell,” means a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” or “cell” as used herein.
  • the term “host cell” refers to a cell, which may be used in a process for purifying an immunogenic protein or recombinant antibody in accordance with the present disclosure. Such host cell expresses the protein of interest (the antibody disclosed herein). A host cell may also be referred to as a protein-expressing cell. “Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell may be, but is not limited to, prokaryotic cells, eukaryotic cells, archeobacteria, bacterial cells, insect cells, yeast, mammal cells, and/or plant cells.
  • Bacteria envisioned as host cells can be either gram-negative or gram- positive, e.g. Escherichia coli, Erwinia sp., Klebsellia sp., Lactobacillus sp. or Bacillus subtilis.
  • the host cell is a yeast cell.
  • the yeast host cell is selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, and Pichia pastoris.
  • composition is intended to mean a combination of active an agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant , diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
  • inert for example, a detectable agent or label
  • active such as an adjuvant , diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
  • Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/antibody components which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to Atty. Dkt.
  • 116639-2710 monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
  • monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
  • disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like
  • a “pharmaceutical composition” is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein.
  • Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. “Administration” can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art.
  • Route of administration can also be determined and the method of determining the most effective route of administration is known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue.
  • Non-limiting Atty. Dkt. No.: 116639-2710 examples of routes of administration include oral administration, nasal administration, injection, and topical application.
  • An agent of the present disclosure can be administered for therapy by any suitable route of administration. It will also be appreciated that the optimal route will vary with the condition and age of the recipient, and the disease being treated.
  • the term “effective amount” refers to a quantity sufficient to achieve a desired effect.
  • the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions.
  • the effective amount will depend on the intended use, the degree of immunogenicity of a particular antigenic compound, and the health/responsiveness of the subject's immune system, in addition to the factors described above. The skilled artisan will be able to determine appropriate amounts depending on these and other factors.
  • the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use.
  • the effective amount may comprise one or more administrations of a composition depending on the embodiment.
  • “Simultaneous use” as used herein refers to the administration of the two compounds of the composition according to the disclosure in a single and identical pharmaceutical form or at the same time in two distinct pharmaceutical forms.
  • “Separate use” as used herein refers to the administration, at the same time, of the two compounds of the composition according to the disclosure in distinct pharmaceutical forms.
  • “Sequential use” as used herein refers to the successive administration of the two compounds of the composition according to the disclosure, each in a distinct pharmaceutical form. Atty. Dkt.
  • adjuvant therapy refers to administration of a therapy or chemotherapeutic regimen to a patient in addition to the primary or initial treatment, such as after removal of a tumor by surgery.
  • Adjuvant therapy is typically given to minimize or prevent a possible cancer reoccurrence.
  • nonadjuvant therapy refers to administration of therapy or chemotherapeutic regimen before surgery, typically in an attempt to shrink the tumor prior to a surgical procedure to minimize the extent of tissue removed during the procedure.
  • adjuvant therapy potentials i.e., sensitizes the subject to the original therapy
  • the subject may help reach one or more of clinical end points of the cancer treatment.
  • tissue is used herein to refer to tissue of a living or deceased organism or any tissue derived from or designed to mimic a living or deceased organism.
  • the tissue may be healthy, diseased, and/or have genetic mutations.
  • the biological tissue may include any single tissue (e.g., a collection of cells that may be interconnected) or a group of tissues making up an organ, or part or region of the body of an organism.
  • the tissue may comprise a homogeneous cellular material or it may be a composite structure such as that found in regions of the body including the thorax which for instance can include lung tissue, skeletal tissue, and/or muscle tissue.
  • Exemplary tissues include, but are not limited to those derived from liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidneys, brain, biliary tree, duodenum, abdominal aorta, iliac vein, heart and intestines, including any combination thereof.
  • “treating” or “treatment of” a condition, disease, disorder or symptoms associated with a condition, disease or disorder refers to an approach for obtaining beneficial or desired results, including clinical results.
  • Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of condition, disorder or disease, stabilization of the state of condition, disorder or disease, prevention of development of condition, disorder or disease, prevention of the spread of condition, disorder or disease, delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset, amelioration or palliation of the condition, disorder or disease state, and remission, whether partial or total. “Treating” can also mean prolonging the survival of a subject beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of the condition, Atty. Dkt.
  • treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the disease or condition.
  • a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control.
  • the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels.
  • treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
  • references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination.
  • the compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage.
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of the extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
  • Treatments containing the disclosed compositions and methods can be first-line, second-line, third-line, fourth-line, fifth-line therapy and are intended to be used as a sole therapy or in combination with other appropriate therapies.
  • the disclosure provides methods of administering an agent capable of modulating the expression or activity of GPR25 in a T cell in the patient.
  • this method comprises, or consists essentially of, or yet further consists of administering to the patient an agent that comprises, consists of, or consists essentially of a small molecule or a protein selected from the group of TGF- ⁇ .
  • the T cell comprises, consists of, or consists essentially of: an activated T cell; a tissue-resident memory (TRM) cell; and a stem T cell.
  • the activated T cell is specific for a tumor-specific antigen or a tumor- associated antigen expressed by the cancer or tumor cell, wherein the tumor-associated antigen is optionally overexpressed by the tumor cell.
  • the modulation comprises, consists of, or consists essentially of activating the T cell by agonizing the expression or activity of GPR25 in the T cell.
  • the agent comprises, consists of, or consists essentially of a bispecific agent that binds to GPR25 and binds to a second receptor expressed by the T cell.
  • the second receptor comprises, consists of, or consists essentially of CXCR5, CXCR6, CD8, CD103, CD49A, CD69, CD3, CD28, CD8 or PD1.
  • the tumor antigen comprises, consists of, or consists essentially of MAGE-D4B, PSMA, HER2, HER3, EGFR, AFP, CEA, CA-125, MUC-1, ETA, MUC-1, BAGE, GAGE-1, MAGE-A1, NY-ESO-1, Gp100, Melan-A/MART-1, Prostate-specific antigen, Mammaglobin-A, Alpha-fetoprotein, HER-2/neu, P53, K-ras, or TRP-2/INT2
  • the cancer or tumor cell and/or the cancer or tumor comprises, consists of, or consists essentially of cancer or tumor of a tissue or cell from the group of: circulatory system; respiratory tract; gastrointestinal system genitourinary tract; live; bone; nervous system; reproductive system; hematologic system; oral cavity; skin and other tissues comprising connective and soft tissue, retroperitoneum and peritoneum, eye, intraocular melanoma, and adnexa
  • the cancer or tumor cell and/or the cancer or tumor comprises, consists of, or consists essentially of a cancer or tumor of a tissue or cell from the respiratory tract or lymph nodes.
  • the cancer comprises, consists of, or consists essentially of a localized or metastatic cancer. Atty. Dkt. No.: 116639-2710
  • the patient is a human patient.
  • the patient has reduced expression of GPR25 compared to that of a healthy, non-diseased subject.
  • the methods further comprise, consist of, or consist essentially of resecting the tumor or cancer prior to modulating the expression or activity of GPR25 in the T cell in the patient.
  • the modulating expression or activity of GPR25 in a T cell is administered as a first-line, a second-line, a third-line, a fourth-line or fifth-line therapy.
  • the methods further comprise, consist of, or consist essentially of administering an effective amount of a different anti-cancer agent to the patient.
  • the treating cancer or a tumor in a cancer patient comprises, consists of, or consists essentially of one or more of a reduction in tumor burden, longer overall survival or prolonged time to tumor progression.
  • a method for screening for a GPR25 anticancer therapy comprising, consisting of, or consisting essentially of contacting a first sample of T cells with an amount of the test agent that binds to GPR25 and a second agent that binds a tumor antigen, and assaying for increased expression of GPR25 in the T cell.
  • increased expression of GPR25 in the T cell is an indication that the agent is a GPR25 anticancer therapy.
  • a method of modulating GPR25 in a subject comprising, consisting of, or consisting essentially of administering a bispecific antibody that targets and binds to GPR25 and a second receptor expressed by a T cell.
  • the receptor comprises, consists of, or consists essentially of CXCR5, CXCR6, CD8, CD103, CD49A, CD69, CD3, or PD1.
  • the T cell comprises, consists of, or consists essentially of a stem T cell.
  • the patient is a human patient.
  • a method of determining prognosis of a subject having cancer comprising, consisting of, or consisting essentially contacting T cells isolated from the subject with an antibody or agent that recognizes and binds to GPR25 to determine the frequency of T cells expressing GPR25 in tumor cells, wherein a high density of GPR25 in T cells indicates a more positive prognosis or wherein a low density of GPR25 in T cells indicates a more negative prognosis.
  • the more negative prognosis Atty. Dkt.
  • No.: 116639-2710 comprises, consists of, or consists essentially of a decreased probability of survival, and wherein the more positive prognosis comprises, consists of, or consists essentially of an increased probability of survival.
  • a method of determining the responsiveness of a subject to cancer therapy that modulates GPR25 comprising, consisting of, or consisting essentially contacting T cells isolated from the subject with an antibody or agent that recognizes and binds to GPR25 to determine the frequency of GPR25 expressing T cells in the subject, wherein a high frequency of GPR25 T cells indicates an increased likelihood of responsiveness to a cancer therapy.
  • the cancer therapy comprises, consists of, or consists essentially of an agent that modulates the expression and/or activity of GPR25 in the subject.
  • the density of GPR25 in T cells in the subject is compared to a healthy, non-diseased subject.
  • the methods further comprise, consist of, or consist essentially of administering a cancer therapy that modulates GPR25 to the subject.
  • the cancer therapy comprises, consists of, or consists essentially of an agent that binds to GPR25.
  • the agent comprises, consists of, or consists essentially of an agonistic antibody targeting GPR25.
  • the sample is contacted with an agent.
  • the agent comprises, consists of, or consists essentially of a detectable label or tag.
  • the detectable label or tag comprises, consists of, or consists essentially of a radioisotope, a metal, horseradish peroxidase, alkaline phosphatase, avidin or biotin.
  • the agent comprises, consists of, or consists essentially of a polypeptide that binds to an expression product encoded by GPR25, or a polynucleotide that hybridizes to a nucleic acid sequence encoding all or a portion of GPR25.
  • the polypeptide comprises, consists of, or consists essentially an antibody, an antigen-binding fragment thereof, or a receptor that binds to the GPR25.
  • the antibody comprises, consists of, or consists essentially of an IgG, IgA, IgM, IgE or IgD, or a subclass thereof.
  • the IgG comprises, consists of, or consists essentially of IgG1, IgG2, IgG3 or IgG4.
  • the antigen binding fragment Atty. Dkt.
  • No.: 116639-2710 comprises, consists of, or consists essentially of a Fab, Fab’, F(ab’)2, Fv, Fd, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv) or VL or VH.
  • the agent is contacted with the sample in conditions favoring the binding of the agent to GPR25. In some aspects of the methods disclosed herein, the agent is contacted with the sample in conditions favoring the binding of the agent to GPR25.
  • the methods comprise, consist of, or consist essentially of detection by immunohistochemistry (IHC), in-situ hybridization (ISH), ELISA, immunoprecipitation, immunofluorescence, chemiluminescence, radioactivity, X-ray, nucleic acid hybridization, protein-protein interaction, immunoprecipitation, flow cytometry, Western blotting, polymerase chain reaction, DNA transcription, Northern blotting and/or Southern blotting.
  • IHC immunohistochemistry
  • ISH in-situ hybridization
  • ELISA immunoprecipitation
  • immunofluorescence immunofluorescence
  • chemiluminescence chemiluminescence
  • radioactivity X-ray
  • the sample comprises, consists of, or consists essentially of cells, tissue, an organ biopsy, an epithelial tissue, a lung, respiratory or airway tissue or organ, a circulatory tissue or organ, a skin tissue, bone tissue, muscle tissue, head, neck, brain, skin, bone and/or blood sample.
  • a method of treating cancer in a subject comprising, consisting of, or consisting essentially of administering a T cell having increased expression of GPR25 to the subject.
  • the cancer or tumor cell and/or the cancer or tumor comprises, consists of, or consists essentially of a cancer or tumor of a tissue or cell from the respiratory tract or lymph nodes.
  • the cancer or tumor cell and/or the cancer or tumor comprises, consists of, or consists essentially of a cancer or tumor of a tissue or cell from the respiratory tract or lymph nodes a cancer or tumor of a tissue or cell from the respiratory tract.
  • the cancer or tumor cell and/or the cancer or tumor comprises, consists of, or consists essentially of a metastatic cancer.
  • the metastatic cancer comprises, consists of, or consists essentially of metastatic lung cancer.
  • the patient is a human patient. In some aspects, the patient has reduced expression of GPR25 compared to that of a healthy, non-diseased subject.
  • the T cell comprises, consists of, or consists essentially of an activated T cell; a tissue-resident memory (TRM) cell; a stem T cell.
  • the activated T cell is specific for a tumor-specific antigen or a Atty. Dkt. No.: 116639-2710 tumor-associated antigen expressed by the tumor cell, wherein the tumor-associated antigen is optionally overexpressed by the tumor cell.
  • the T cell comprises, consists of, or consists essentially of a TRM cell.
  • provided herein are methods of promoting the development of TRM cells and/or stem-like TRM cells in a subject.
  • the methods comprise, consist of, or consist essentially of administering an agent that modulates expression of GPR25 to the subject.
  • the TRM cells are in the lung tissue or liver tissue of the subject.
  • the agent is administered to the lung tissue or liver tissue of the subject.
  • the agent comprises, consists of, or consists essentially of a small molecule or a protein selected from the group of: Transforming Growth Factor- ⁇ (TGF- ⁇ ), Nuclear Factor of Activated T-cells (NFAT), or Suppressor of Mothers against Decapentaplegic family member 1 (SMAD1), or equivalents thereof.
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • NFAT Nuclear Factor of Activated T-cells
  • Suppressor of Mothers against Decapentaplegic family member 1 Suppressor of Mothers against Decapentaplegic family member 1
  • kits comprising, or consisting essentially of, or yet further consisting of one or more of the isolated T-cells and/or the composition of this disclosure and instructions for use.
  • the present disclosure provides kits for performing the methods of this disclosure as well as instructions for carrying out the methods of the present disclosure.
  • the kits are useful for diagnosing cancer in a subject from a biological sample taken from the subject e.g., any bodily fluid including, but not limited to, e.g., sputum, serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, acitic fluid or blood and including biopsy samples of body tissue.
  • test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.
  • Methods for preparing protein extracts or membrane extracts of cells are known in the art and can be readily adapted in order to obtain a sample which is compatible with the system utilized.
  • the kit components e.g., reagents
  • the kit can also comprise, or alternatively consist essentially of, or yet further consist of, e.g., a Atty. Dkt. No.: 116639-2710 buffering agent, a preservative or a protein-stabilizing agent.
  • the kit can further comprise, or alternatively consist essentially of, or yet further consist of components necessary for detecting the detectable label, e.g., an enzyme or a substrate.
  • the kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample.
  • Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
  • the kits of the present disclosure may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit. As amenable, these suggested kit components may be packaged in a manner customary for use by those of skill in the art.
  • T RM Tissue-resident memory CD8 + T cells
  • TRM cells are a distinct population of memory T cells that primarily reside within tissues and respond immediately against pathogens invading barrier tissues, thus representing the first line of defense 1,2 .
  • TRM cells are phenotypically, functionally, transcriptionally, and metabolically distinct from circulating memory T cell subsets 8-12 like central memory T (T CM ) and effector memory T (T EM ) cells.
  • T RM cells lack expression of molecules required for tissue egress and trafficking to lymph nodes like S1PR1, S1PR5, C-C motif chemokine receptor (CCR)7 and CD62L 2,13 , and mostly express high levels of CD69, CD49a, and C-X-C motif chemokine receptor (CXCR)6, which play important roles in the tissue retention and localization of TRM cells 14-16 .
  • CXCR C-X-C motif chemokine receptor
  • TRM cells that reside within the epithelium also express the ⁇ E integrin, CD103 8,17 , whereas, TRM cells that reside in non-epithelial tissues, such as liver 10,15 and kidney 18,19 do not express high levels of CD103 10,15,20,21 , presumably reflecting the lack of tight junctions in the sinusoidal endothelium.
  • ⁇ E integrin CD103 817
  • non-epithelial tissues such as liver 10,15 and kidney 18,19
  • T RM cells have shown a positive association between tumor T RM cells and favorable clinical outcome in patients with breast cancer 29,30 , cervical cancer 31 , esophageal cancer 32 , gastric cancer 33 , liver cancer 34 , head and neck cancer 35 , lung cancer 36 , melanoma 37 , and pancreatic cancer 38 .
  • These results suggest that the abundance of T RM cells in the tumor tissue is a prognostic indicator for survival outcomes.
  • Functional studies in murine tumor models have further confirmed the pivotal role of T RM cells in driving anti-tumor immune responses 22,23 .
  • targets that modulate the magnitude and quality of TRM responses are likely to have a potential therapeutic role in certain infections and cancer types as well as in autoimmune diseases, where T RM cells play a pathogenic role 39 .
  • GPR25 41 G protein-coupled receptor
  • GPR25 is expressed at significantly higher levels in T RM cells compared with non-T RM cells in the human lung and tumor tissues 36 .
  • GPR25 has long been considered an orphan receptor with no known endogenous ligand.
  • CXCL17 C-X-C motif chemokine 17
  • CXCL17 C-X-C motif chemokine 17
  • Gpr25 also has been reported to have a ligand-independent constitutive activity 47 .
  • Applicant demonstrate that Gpr25 plays an important role in the development and function of T RM cells in the liver and lung.
  • Atty. Dkt. No.: 116639-2710 Tissue-resident memory (TRM) CD8 + T cells are key players that orchestrate protective anti-viral and anti-tumor immune responses 1-3 .
  • the molecules that support their development and function are not fully defined.
  • Applicant report on the regulation and function of a G-protein coupled receptor, GPR25 that is expressed at high levels in TRM cells compared to non-TRM cells.
  • TGF- ⁇ a key cytokine involved in the development of TRM cells 4 , induces the expression of GPR25 in CD8 + T cells, and SMAD1, a key transcription factor downstream of TGF- ⁇ signaling 5 , binds to T RM -associated cis-regulatory elements in the GPR25 locus.
  • Adoptively transferred T cells from Gpr25-deficient mice showed no significant differences in their accumulation in the liver and lung of recipient mice after viral infection, but they exhibited impaired development into T RM cells. In a tumor challenge model, Gpr25- deficient memory T cells showed significantly reduced expansion and a diminished capacity to control lung metastasis compared to Gpr25-sufficient T cells.
  • TCF1 a key transcription factor promoting stemness in T cells 6 .
  • Gpr25-deficient TRM cells displayed defects in their ability to differentiate into secondary T RM cells and maintain the T RM cell population.
  • Gpr25-deficient T cells showed negative enrichment for TGF- ⁇ signature genes and impaired responses to TGF- ⁇ , indicating that Gpr25 enhances TGF- ⁇ signaling to promote the development of T RM cells.
  • TGF- ⁇ is known to tightly regulate the expression of many TRM-associated genes 4 .
  • TGF- ⁇ treatment resulted in rapid induction of GPR25 in primary human Atty. Dkt. No.: 116639-2710 CD8 + T cells that were polyclonally activated in vitro by using anti-CD3 and ant-CD28 antibodies (FIG. 1A).
  • TGF- ⁇ signaling regulates expression of GPR25 we analyzed assay for transposase-accessible chromatin using sequencing (ATAC-seq) profile of the GPR25 locus in human CD8 + T cell subsets 36,48 .
  • C1, C2, C3 upstream of GPR25 transcription start site displayed prominent transposase accessible peaks (ATAC-seq peaks) in tumor-infiltrating CD8 + T RM cells compared to cell types not expressing GPR25 like non-T RM cells or circulating na ⁇ ve CD8 + T cells (FIG. 1B, top panel).
  • the C2 region displayed prominent transposase accessibility in activated CD8 + T cells.
  • Our analysis suggested that these three cis-regulatory regions are likely to be involved in the regulation of GPR25 with C1,2,3 regions associated with T RM cells and C2 region with T cell receptor (TCR) activation.
  • mice By breeding CD45.1.2 Gpr25 +/+ (Gpr25 wild-type, WT) mice and CD45.2 Gpr25 -/- (Gpr25 knock out, KO) mice with OT-I TCR transgenic mice, which express the transgenic T cell receptor (TCR) specific to SIINFEKL peptide of ovalbumin (OVA) 51 , we generated mice with congenically marked Gpr25-sufficient (Gpr25 +/+ ) and Gpr25-deficient (Gpr25 -/- ) OT-I CD8 + T cells (see Methods).
  • TCR transgenic T cell receptor
  • TRM cells CD45.1.2 + CD62L ⁇ CD69 + T cells in the liver
  • TEM cells CD45.1.2 + CD62L + CD69- and CD45.1.2 + CD62L ⁇ CD69 ⁇ T cells in the spleen, respectively
  • T RM cells expressed significantly lower levels of transcripts encoding for sphigosine- 1-phosphate receptor (S1PR1), which promotes egress of T cells from tissue compartments 53 (FIG. 8C, right panel).
  • S1PR1 sphigosine- 1-phosphate receptor
  • Gpr25 supports the differentiation of T RM cells over effector memory T cells even during the early stages of memory T cell development.
  • Gpr25 supports the generation of stem-like TRM cells
  • T RM cells that develop from Gpr25-deficient OT-I T cells exhibit qualitative differences when compared to those that develop from Gpr25-sufficient OT-I T cells.
  • Differential gene expression analysis revealed marked differences in transcriptional profiles between Gpr25 +/+ and Gpr25 -/- T RM cells isolated from the liver of recipient mice 30 days post LCMV infection (FIG. 3A, FIG. 4A, FIG. 10A and Table 1).
  • FIG. 12A provides better control of lung metastasis when compared to endogenous anti-tumor responses in mice that did not receive OT-I cells (no OT-1 transfer group) (FIG. 12A, FIG. 12B).
  • FTY720 a sphingosine-1-phosphate receptor antagonist that blocks egress of cells from lymph nodes 76 , thus allowing us to primarily assess the functional role of T cells resident in the lungs (FIG. 12A).
  • mice that received FTY720 treatment developed more metastatic nodulus compared to mice that that did not receive FTY720 treatments, highlighting the relative contribution of circulating T cells to control of lung metastasis (FIG. 12B). More importantly, mice that received FTY720 treatment developed lesser number of metastatic nodules compared to cohorts of mice that did not receive OT-1 T cells, which suggested that lung memory T cells per se can provide control of metastasis independent of replenishment from circulating T cells (FIG. 12B, FIG. 12C).
  • mice received either Gpr25 +/+ OT-I or Gpr25 -/- OT-I CD8 + T cells prior to i.t. infection with LCMV-OVA, then 30 days after infection, mice were challenged i.v. with B16F10-OVA cells and assessed for degree of lung metastasis following FTY720 treatment. Applicant found that the number of metastatic nodules was significantly lower in mice that received Gpr25- sufficient OT-I T cells when compared to those that received Gpr25-deficient OT-I cells (FIG. 6D).
  • CD8KO mice utilized CD8KO mice as recipients and tested the effects of Gpr25 +/+ OT-I or Gpr25 -/- OT-I CD8 + T cells in controlling lung metastasis (FIG. 6E).
  • cohorts of CD8KO mice that did not receive T cells were also included to specifically assess the contribution of transferred OT-I T cells on outcomes.
  • Applicant found that CD8KO mice receiving Gpr25-deficient OT-I cells had poor survival outcomes, with nearly 50% death rate, while most CD8KO mice receiving Gpr25- sufficient OT-I cells survived at the time of analysis (FIG. 6F).
  • GPR25 expression is induced in CD8 + T cells by TGF- ⁇ signaling. Because three T RM -associated cis-regulatory regions in the GPR25 locus can bind to SMAD1, the key transcription factor downstream of TGF- ⁇ signaling, Applicant proposes that GPR25 is likely to be a direct target gene of TGF- ⁇ . In an LCMV infection model, Applicant demonstrates that Gpr25 plays a key role in supporting the development of TCF1-expressing stem-like TRM cells in liver and lungs but not those that develop in the siIEL compartment.
  • Gpr25-sufficient T RM cells were enriched for molecules that promote a stem-like memory program, whereas Gpr25- deficient TRM cells were enriched for molecules linked to an effector program.
  • These results provide an interesting insight, suggesting potential heterogeneity within T RM cell population with some T RM cells exhibiting more stem-like features, including expression of TCF1, that may contribute to the maintenance of the TRM cell pool and enabling rapid re-expansion upon antigen re-challenge.
  • Applicant’s results suggest that Gpr25 supports the generation of TCF1-expressing T RM cells, i.e., a stem-like differentiation program while limiting the effector differentiation of TRM cells by augmenting TGF- ⁇ signaling 68 .
  • GPR25 has been shown to have constitutive activity without ligand engagement 41,47 , identification of the endogenous ligand is likely to shed light on endogenous signals that modulate GPR25 activity in T cells.
  • a recent study identified a chemoattractant chemokine CXCL17 as the ligand for GPR25, and reported that CXCL17- GPR25 functions as a chemoaffinity axis that mediates lymphocytes homing to lung, non- intestinal mucosa and CNS 46 .
  • Applicant’s study highlights that Gpr25 has important non- chemotactic functions and supports the generation and maintenance of T RM cells in the liver and lung. Further functional studies under physiological conditions and employing genetic knock-out Atty. Dkt.
  • Gpr25-deficient memory T cells also displayed defects in downregulating the ZEB2-S1PR5 axis that is required for preventing egress of T cells from the tissues, suggesting that Gpr25 signaling is likely to enhance tissue retention of TRM cells.
  • TGF- ⁇ signaling is essential for the maintenance of both Tcf-1- dependent stem-like program in CD8 + memory T cells as well as for the down-regulation of ZEB2-S1PR5 axis to promote tissue retention 9 . Applicant hypothesizes that Gpr25 may mediate its effects on T RM development by either directly or indirectly modulating TGF- ⁇ signaling, which should be tested in future studies.
  • GPR25 is an orphan receptor and has been shown to have a constitutive activity without ligand engagement 38,43 , identification of the endogenous ligand is likely to shed light on endogenous signals that modulate GPR25 activity in T cells.
  • Two potential ligands, Apelin and Apela are known to activate GPR25 and decrease intracellular cAMP levels in non-mammalian vertebrates 66 , however, their function in human T cells is not known. Future studies to identify physiological ligand(s) that selectively modulate GPR25 signaling in humans and mice are likely to facilitate the development of therapies that target the GPR25 signaling pathway for controlling TRM responses in health and disease. Methods Mice.
  • Gpr25 germline deletion mice (Gpr25 -/- ; RRID:MMRRC_047952-UCD) were generated by the Knock out Mouse Project (KOMP) and obtained from Mutant Mouse Resource and Resource Centers (MMRRC). Briefly, CRISPR guide(s) targeting of Gpr25 exon and Cas9 protein were microinjected or electroporated into C57BL/6N zygotes to generate the Atty. Dkt. No.: 116639-2710 Gpr25 em2(IMPC)Bay allele (Gpr25 -/- ) and progeny were screened for the desired mutation.
  • KOMP Knock out Mouse Project
  • MMRRC Mutant Mouse Resource and Resource Centers
  • Gpr25 -/- mice were viable, and were crossed with OT-I mice 51 , which express the transgenic TCR recognizing the OVA-specific class 1 epitope OVA 257-264 , to generate mice with Gpr25-deficient OT-I cells.
  • OT-I mice stock no.003831
  • CD45.2 C57BL/6J mice stock no.000664
  • CD45.1 mice B6.SJL-Prprc a Pepc b /BoyJ, stock no. 002014
  • CD8aKO mice B6.129S2- Cd8atm1Mak/J, stock no. 002665
  • mice were maintained in specific-pathogen-free conditions in accordance with the Institutional Animal Care and Use Committees (IACUC) of the La Jolla Institute for Immunology (LJI). All mice were maintained on a C57BL/6 background and under a standard 12-hour light / 12-hour dark cycle. Mice were fed standard rodent chow and water ad libitum. Age- (8-12 weeks old) and sex- matched mice (female and male) were used in all experiments. All procedures were approved by the La Jolla Institute for Immunology Animal Ethics Committee. Infection model and adoptive T cell transfer.
  • IACUC Institutional Animal Care and Use Committees
  • Lymphocytic choriomeningitis virus Armstrong strain (LCMV) expressing OVA was a gift from Ananda Goldrath (University of California, San Diego, CA) and generated by Juan-Carlos de la Torre (The Scripps Research Institute, La Jolla, CA) 77 .
  • congenically distinct Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I CD8 + T cells were mixed in a 1:1 ratio and 1 x 10 6 total cells were adoptively co-transferred intravenously into CD45.1 recipient mice.
  • mice were then infected with 2 x 10 5 plaque-forming units (PFU) of LCMV- OVA by intraperitoneal (i.p.) and memory T cell responses analyzed at indicated time points.
  • PFU plaque-forming units
  • RT room temperature
  • RBC red blood cell
  • FACS fluorescence-activated cell sorting
  • Isotype controls for each of antibodies were used for separating negative and positive populations and all gates to define cell subsets were based on this method.
  • all samples were sorted on a BD FACS Fusion system or acquired on a BD FACS Fortessa system (BD Biosciences).
  • In vitro T cell activation assays Using the “Na ⁇ ve CD8 + T cell Isolation Kit” (Cat. No. 130-093-244, Miltenyi Biotec) na ⁇ ve human CD8 + T cells were isolated from peripheral blood mononuclear cells, obtained from blood samples of de-identified donors from the San Diego Blood Bank (SDBB).
  • SDBB San Diego Blood Bank
  • Na ⁇ ve mouse CD8 + T cells (CD8 + CD44 lo CD62L hi ) were isolated from the spleen, and 0.5 x 10 6 cells were stimulated in a 48-well plate pre-coated with anti-CD3 (1 ⁇ g/mL, Cat. No. BE0001-1, BioXCell) and anti-CD28 (1 ⁇ g/mL, Cat. No. 16-0281-85, eBioscience) in RPMI-1640 medium supplemented with 10% FBS and TGF- ⁇ (10 ng/mL, Cat. No. 763102, BioLegend) or mouse IL-15/IL-15R complex (0.4 ng/mL, Cat. No.
  • mice primer sequences used in the qRT-PCR were as follows: Hprt forward, 5’- CTGGTGAAAAGGACCTCTCGAAG-3’, Hprt reverse, 5’- CCAGTTTCACTAATGACACAAACG-3’, S1pr1 forward, 5'- GTGTAGACCCAGAGTCCTGCG-3', S1pr1 reverse, 5'- AGCTTTTCCTTGGCTGGAGAG-3', Gpr25 forward, 5'- TCACCCTGATCTGCTACTGG-3', Gpr25 reverse, 5'- CGCACTGCTATTGACGAAAGC-3'.
  • the expression levels of human GPR25 were normalized to HPRT1 transcript levels.
  • the human primer sequences used in the qRT-PCR were as follows: HPRT1 forward, 5’- ACCAGTCAACAGGGGACATAA-3’, HPRT1 reverse, 5’- CTTCGTGGGGTCCTTTTCACC-3’, GPR25 forward, 5’- CGCTCATCTACCTCCTGCTG-3’, GPR25 reverse, 5’- ACACGGAACTGTCGTCCCT-3’.
  • Tumor model OVA-expressing B16F10 (B16F10-OVA) cells were a gift from the J. Linden laboratory (LJI) and tested negatively for mycoplasma infection. Plasmocin (InvivoGen) was used as a routine addition to culture media to prevent mycoplasma contamination.
  • mice were intraperitoneally injected with FTY720 diluted in sterile PBS at 1mg/kg at indicated time points. Mice were sacrificed on day 14 after injection of tumor cells, and lung tissues were harvested and subjected to FACS analysis. Lung metastatic nodules present on the surface of the 5 lobes of the lungs per mouse were counted. Single-cell transcriptome assay and analysis.
  • UMI Unique Molecular Identifier
  • Shape of violin plots represents the distribution of expression (based on a Gaussian Kernel density estimation model) of cells, including cells with no expression. Violin plots are colored according to the percentage of cells expressing the transcript of interest. UMAPs are colored according to the normalized transcript counts of the genes of interest.
  • ATAC-seq data analysis ATAC-seq data of resting and activated na ⁇ ve CD8 + T cells, TRM and non-TRM CD8 + T cells were analyzed as described previously 36,84 using custom ATAC-seq data processing pipeline ATACproc (https://github.com/ay-lab/ATACProc).
  • Coverage tracks were normalized by scaling factor, bins per million mapped reads (BPM), using the “BamCoverage” routine from deepTools 89 using the arguments “--binSize 50 --normalizeUsing BPM --extendReads”.
  • MACS2 (version 2.1.0) 90 was used for peak calling, with parameters: “-g hs –q 0.05 -- nomodel --nolambda --keep-dup all -- call-summits --shift -100 --extsize 200”.
  • UCSC Genome Browser was used to display the tracks. Transcription factor (TF) ChIP-seq data analysis.
  • TF ChIP-seq data for NFATC1, NFATC3 and SMAD1 for GM12878 cells were retrieved from the ENCODE database 91 . Briefly, reads were aligned using Bowtie2 (version 2.3.3.1) 85,86 to hg19 reference Atty. Dkt. No.: 116639-2710 genome, and coverage tracks were normalized by scaling factor, bins per million mapped reads (BPM), using the “BamCoverage” routine from deepTools 89 using the arguments “--binSize 10 -- normalizeUsing BPM --extendReads 200”. UCSC Genome Browser was used to display the tracks. Quantification and statistical analysis.
  • GPR25(W164A)-PK are transfected into the PathHunter® U2OS Endosomal-EA (Endo-EA) cells (DiscoverX). These cells express the remaining portion of the ⁇ -gal enzyme fused with the early endosomal protein EEA1. With ⁇ -gal split into two fragments: PK and EA, and separated within the cell, no functional ⁇ -gal is produced. When brought together, they form a functional enzyme capable of converting substrate to light (FIG. 13). Atty. Dkt.
  • GPR25(W164A)-PK are expected to show high basal activity (no exogenous EA added). Activity increases with the addition of exogenous EA. This determines (i) the fraction of the receptor that migrates to the membrane despite the presence of the ER-retaining mutations, and, more importantly, (ii) the level of increased signal resulting from the addition of exogenous EA, which will demonstrate robust expression of GPR25. Applicant also assesses retention of GPR25(W164A)-PK in the ER using a monoclonal antibody targeting PK, and confocal microscopy.
  • Applicant establishes the primary HTS assay based on the LIFT platform and develops downstream secondary assays to enable a hit ID testing funnel (FIG. 14).
  • Example 4 GPR25 agonist lead generation and characterization for functional studies in vitro and in vivo.
  • SAR structure-activity relationship
  • Applicant starts by purchasing compounds that differ from hits in select chemical groups, which are acquired from trusted commercial vendors. In cases where Applicant is unable to expand the hit set by purchase, Applicant directly begins the process of hit expansion Atty. Dkt. No.: 116639-2710 through initiating a classic Hit to Lead medicinal chemistry campaign.
  • Applicant uses the panel of cell-based GPR25 assays to assess potency and selectivity with the goal of optimizing SAR for whole-cell activity.
  • Hit prioritization and expansion efforts leverage the chemistry expertise of Dr. Patrick Kenny (Co-I), the lead medicinal chemist at SBP for the NIH-supported initiative to find chemical probes directed to 10 brain-expressed oGPCRs.
  • Applicant focuses on analogs that show enhanced potency from the original hit, while retaining the same mechanism of action.
  • Applicant performs multiple rounds of analysis of analogs for each hit, and based on this data, additional rounds of analogs are synthesized and tested to define the most critical functional groups responsible for evoking the biological effect at GPR25.
  • Applicant profiles ⁇ 25-35 compounds in these assays, and lead compounds with appropriate potency and in vitro ADME properties are dosed (IV/PO/IP) in mice to determine pharmacokinetics.
  • Lead selection and characterization in vitro and in vivo The overall testing funnel for identifying lead compound(s) is depicted in FIG. 15. Applicant uses all the data generated in the various assays in an ongoing fashion to guide the design of analogs and enhance/evolve the SAR. With each iteration of analog refinement the potency threshold becomes increasingly rigorous.
  • TGF- ⁇ treatment resulted in rapid induction of GPR25 in primary human CD8+ T cells that were polyclonally activated in vitro by using anti-CD3 and ant-CD28 antibodies (FIG. 1C).
  • Gpr25 expression was also readily induced by TGF- ⁇ in murine CD8+ T cells (FIG. 1A).
  • Applicant analyzed assay for transposase-accessible chromatin using sequencing (ATAC-seq) profile of the GPR25 locus in human CD8+ T cell subsets33,117.
  • SMAD proteins or NFAT to cis-regulatory regions (C1,C2,C3) in the GPR25 locus does not imply that SMAD is regulating GPR25 expression. It is important to show that these SMAD-bound regions are indeed functioning as enhancers for GPR25.
  • Applicant utilizes CRISPRi (dCas9-KRAB) assays, as described122, to silence the cis-regulatory elements (C1, C2 or C3) that bind SMAD or NFAT in activated CD8+ T cells treated with TGF- ⁇ and then determines the effects on GPR25 gene expression by qPCR. As controls, Applicant utilizes non-targeting sgRNAs.
  • Applicant has optimized this assay to work in primary human T cells, and have tested ⁇ 10 cis-regulatory elements in a few months, as described in Chandra et al Nature Genetics 2021122.. To ascertain whether SMAD binding is indispensable for the activity of these enhancers, Applicant will utilize only the catalytically dead Cas9 protein (dCas9) along with tiling guide RNAs to target multiple regions) in and around the predicted SMAD binding sites in C1, C2 or C3 enhancer. Because dCas9 lacks any silencing domain, binds to targeted chromatin with high affinity and effectively prevents the binding of SMAD proteins.
  • dCas9 catalytically dead Cas9 protein
  • Example 5 GPR25 Mediates its effect on TRM cells Applicant’s studies have shown that Gpr25 plays a plays a key role in supporting the development and maintenance of TRM cells in the liver and lungs. However, the signaling pathways and mechanisms through which Gpr25 mediates its effects are not known. Applicant demonstrates Gpr25 modulates TGF- ⁇ signaling to influence the development of TRM cells. GPR25 inhibits cAMP signaling. GPR25 is coupled to the G ⁇ i transducer protein that inhibits adenylate cyclase activity and thus reduces cAMP signaling34. Because GPR25 is an orphan GPCR with no known endogenous ligand, effects of ligand-dependent signaling Atty. Dkt.
  • cAMP signaling can inhibit the expression of TGF- ⁇ target genes by modulating SMAD signaling132-135.
  • heightened cAMP signaling activates cAMP-response element binding protein (CREB), which in turn recruits the transcriptional co-activator p300 away from SMADs, thereby restricting the transcriptional activity of SMAD i.e., TGF- ⁇ signaling133.
  • TGF- ⁇ signaling regulates TRM cell development.
  • the two cytokines, TGF ⁇ and IL-15 play central roles in shaping the TRM gene expression programs.
  • OT-I T cells clustered into two major subsets; notably, a greater fraction of Gpr25+/+ versus Gpr25-/- OT-I memory T cells (62% versus 38%) were observed in cluster 0 (FIG. 5F, left).
  • cluster 0 cells that were enriched for Gpr25+/+ T cells expressed lower levels of transcripts encoding for molecules linked to effector properties like perforin, granzyme B, granzyme A, and higher levels of transcripts encoding for molecules linked to stem-like memory properties such as Tcf1 and IL7R (FIG.
  • T cells in cluster 0 compared to cluster 1 displayed lower expression of transcripts encoding for the transcription factors ZEB2 and S1PR5, a sphingone-1-phosphate receptor that promotes egress of cells from tissues140 (FIG. 5E, 5F).
  • a recent study showed that the robust generation of TRM cells requires down- regulation of transcription factor ZEB2 and its target gene S1PR5 by TGF- ⁇ signaling in the tissues55, which in turn promotes sequestration of cells in the tissues.
  • Gpr25 may play a role in the downregulation of ZEB2-S1PR5 axis, potentially by modulating TGF- ⁇ signaling and thus favoring the generation of TRM cells in the lungs.
  • Strategy and Methods To determine if Gpr25 enhances TGF- ⁇ signaling in T cells, Applicant performs gain-of-function (forced expression of Gpr25), loss-of-function (Gpr25-/- T cells i.e., genetic knockout) and rescue studies.
  • gain-of-function forced expression of Gpr25
  • Gpr25-/- T cells i.e., genetic knockout
  • Applicant treats CD8+ T cells with forced expression of Gpr25 and control T cells (empty vector transduced cells) with TGF- ⁇ (20 ng/ml) and compares their responses at different time points (4, 24 and 48 hours) using multiple assays/readouts that include: (a) Flowcytometry and qPCR to assess expression of key TGF- ⁇ target genes such as Atty. Dkt. No.: 116639-2710 CD103, Klf2, S1pr1, S1pr5, and Zeb2.
  • Applicant assess if forced expression of Gpr25 in T cells leads to increased expression of TGF- ⁇ receptor II (TGF- ⁇ R2)141, potentially augmenting TGF- ⁇ sensing.
  • Applicant transfers congenically distinct OT-1 T cells with forced Gpr25 expression (generated as above), as well as control groups with an empty vector expressing cells and Gpr25-/- OT-I T cells, into separate cohorts of mice prior to LCMV infection (as shown in FIG. 5).
  • Applicant evaluates the phenotype and transcriptional features of congenically-marked transferred cells in the lungs at day 14 and day 30 following infection. Readouts.
  • (a) Phenotype. Applicant examines expression of marker genes linked to TRM cells (CD69, CD103, CXCR6) and TCM cells (CD62L, CD127);
  • TGF- ⁇ signaling restores the capacity of Gpr25-deficient T cells to develop into lung TRM cells by restricting their capacity to egress from the lungs. Forced expression of Gpr25 results in enhancement of TGF- ⁇ signaling in T cells, which is evident through changes in the expression levels, SMAD binding and chromatin accessibility of TGF- ⁇ target genes.
  • enhanced TGF- ⁇ signaling is Applicant’s primary hypothesis, Applicant’s alternate hypothesis is ‘IL-15 signaling pathway is modulated by Gpr25 to promote TRM development’.
  • IL-15 utilizes similar receptors and engages downstream STAT3/5 pathway144, it is highly plausible that cAMP may also inhibit IL-15 signaling.
  • IL-15 signaling has been shown to support the expansion and survival of developing TRM cells127-129, while also inducing the expression of Hobit53, a critical TRM transcription factor that down-regulates expression of genes involved in tissue egress/TCM such as S1PR1 and CCR753.
  • Hobit53 a critical TRM transcription factor that down-regulates expression of genes involved in tissue egress/TCM such as S1PR1 and CCR753.
  • Applicant will test if IL-15 signaling pathway is also modulated by Gpr25 (alternative hypothesis). Because of Applicant’s unbiased approaches (RNA-seq, ATAC-seq), Applicant determines if forced expression of Gpr25 impacts target genes and enhancers that are downstream of IL-15 signaling pathway. In the in vitro models, Applicant treats T cells with forced expression of Gpr25 and controls T cells (empty vector transduced cells) with IL-15 and assesses its effects (as for TGF- ⁇ ), including phosphorylation of STAT5, the key downstream signaling mediator of IL-15.
  • Applicant Utilizing Applicant’s unbiased datasets, Applicant is able to determine if Gpr25 modulates the activities of other transcriptional regulators involved in the development of TRM cells such as Blimp-1, Runx3 and Id3.
  • Organ-specific effects Due to the critical role of lung TRM cells in infection control and mucosal immunity, Applicant’s focuses on examining the downstream effects of Gpr25 in lung TRM cells.
  • TGF- ⁇ Regulates Expression of GRP25 Applicant performs ChIP-seq studies to determine if SMAD proteins, downstream of TGF- ⁇ signaling, bind to cis-regulatory elements in the GPR25 locus.
  • Applicant utilizes CRISPR-based assays to assess if SMAD-bound regions enhance the expression of GPR25 in T cells.
  • Applicant ‘s preliminary studies (FIG. 5) support the hypothesis that Gpr25 may modulate TGF- ⁇ signaling to influence the development of TRM cells. Applicant performs gain-of-function (forced expression of Gpr25), loss-of-function (Gpr25-/- T cells i.e., genetic knockout) and rescue studies (forced expression of constitutively active TGF- ⁇ RICA) both in vitro and in vivo, using mouse models of viral infection. Table 1 24 h 48 h 8 1 9 7 2 3 4 8 4 2 Atty. Dkt.
  • CD49a Expression Defines Tissue-Resident CD8(+) T Cells Poised for Cytotoxic Function in Human Skin. Immunity 46, 287-300, doi:10.1016/j.immuni.2017.01.009 (2017). Fernandez-Ruiz, D. et al. Liver-Resident Memory CD8(+) T Cells Form a Front-Line Defense against Malaria Liver-Stage Infection. Immunity 45, 889-902, doi:10.1016/j.immuni.2016.08.011 (2016). Mackay, L. K. et al. Cutting edge: CD69 interference with sphingosine-1-phosphate receptor function regulates peripheral T cell retention.
  • T-box Transcription Factors Combine with the Cytokines TGF-beta and IL-15 to Control Tissue-Resident Memory T Cell Fate. Immunity 43, 1101-1111, doi:10.1016/j.immuni.2015.11.008 (2015). Christo, S. N. et al. Discrete tissue microenvironments instruct diversity in resident memory T cell function and plasticity. Nat Immunol 22, 1140-1151, doi:10.1038/s41590-021-01004-1 (2021). Atty. Dkt. No.: 116639-2710 Pallett, L. J. et al. IL-2(high) tissue-resident T cells in the human liver: Sentinels for hepatotropic infection.
  • CD103(+) CD8(+) tissue-resident T cells in esophageal squamous cell carcinoma may be tumor reactive and resurrected by anti-PD-1 blockade.
  • Tissue-resident memory CD8(+) T cells amplify anti-tumor immunity by triggering antigen spreading through dendritic cells.
  • Atty. Dkt. No.: 116639-2710 Lohneis, P. et al. Cytotoxic tumour-infiltrating T lymphocytes influence outcome in resected pancreatic ductal adenocarcinoma.
  • G-protein-coupled receptors from structural insights to functional mechanisms. Biochem Soc Trans 41, 135-136, doi:10.1042/BST20120344 (2013). Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schioth, H. B. & Gloriam, D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov 16, 829- 842, doi:10.1038/nrd.2017.178 (2017). Ocon, B. et al. A lymphocyte chemoaffinity axis for lung, non-intestinal mucosae and CNS. Nature, doi:10.1038/s41586-024-08043-2 (2024). Martin, A.
  • Tcf1 and Lef1 provide constant supervision to mature CD8(+) T cell identity and function by organizing genomic architecture. Nature communications 12, 5863, doi:10.1038/s41467-021-26159-1 (2021). Shan, Q. et al. Ectopic Tcf1 expression instills a stem-like program in exhausted CD8(+) T cells to enhance viral and tumor immunity. Cell Mol Immunol 18, 1262-1277, doi:10.1038/s41423-020-0436-5 (2021). Behr, F. M. et al. Tissue-resident memory CD8(+) T cells shape local and systemic secondary T cell responses. Nat Immunol 21, 1070-1081, doi:10.1038/s41590-020-0723-4 (2020). Fonseca, R.
  • Intratumoral follicular regulatory T cells curtail anti-PD-1 treatment efficacy. Nat Immunol 22, 1052-1063, doi:10.1038/s41590-021-00958-6 (2021). Stuart, T. et al. Comprehensive Integration of Single-Cell Data. Cell 177, 1888-1902 e1821, doi:10.1016/j.cell.2019.05.031 (2019). Wolock, S. L., Lopez, R. & Klein, A. M. Scrublet: Computational Identification of Cell Doublets in Single-Cell Transcriptomic Data. Cell Syst 8, 281-291 e289, doi:10.1016/j.cels.2018.11.005 (2019). Finak, G. et al.
  • MAST a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol 16, 278, doi:10.1186/s13059-015-0844-5 (2015). Schmiedel, B. J. et al. COVID-19 genetic risk variants are associated with expression of multiple genes in diverse immune cell types. Nature Communications 12, 6760, doi:10.1038/s41467-021-26888-3 (2021). Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-359, doi:10.1038/nmeth.1923 (2012). Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L.

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Abstract

L'invention concerne des méthodes de modulation d'une réponse immunitaire à une tumeur ou à une cellule cancéreuse chez un patient, de traitement du cancer ou d'une tumeur chez un patient cancéreux, de déclenchement d'une réponse anticancéreuse ou tumorale chez un patient, ou de traitement ou d'amélioration d'une infection chez un sujet en ayant besoin. Les méthodes comprennent la modulation de l'expression ou de l'activité du récepteur 25 couplé à la protéine G (GPR25) dans un lymphocyte T chez le patient.
PCT/US2024/060202 2023-12-15 2024-12-13 Agonification de gpr25 pour thérapie anticancéreuse Pending WO2025129119A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005040822A2 (fr) * 2003-10-24 2005-05-06 Bayer Healthcare Ag Diagnostics et therapeutiques pour des maladies associees au recepteur 25 couple a la proteine g (gpr25)
US20210355443A1 (en) * 2018-10-18 2021-11-18 Board Of Regents, The University Of Texas System Methods for production of tissue resident memory-like t cells and use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005040822A2 (fr) * 2003-10-24 2005-05-06 Bayer Healthcare Ag Diagnostics et therapeutiques pour des maladies associees au recepteur 25 couple a la proteine g (gpr25)
US20210355443A1 (en) * 2018-10-18 2021-11-18 Board Of Regents, The University Of Texas System Methods for production of tissue resident memory-like t cells and use thereof

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