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WO2025129130A1 - Antagonisation de gpr25 pour le traitement de maladies auto-immunes - Google Patents

Antagonisation de gpr25 pour le traitement de maladies auto-immunes Download PDF

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WO2025129130A1
WO2025129130A1 PCT/US2024/060223 US2024060223W WO2025129130A1 WO 2025129130 A1 WO2025129130 A1 WO 2025129130A1 US 2024060223 W US2024060223 W US 2024060223W WO 2025129130 A1 WO2025129130 A1 WO 2025129130A1
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cells
gpr25
disease
subject
autoimmune
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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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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • G Protein-Coupled Receptor 25 (GPR25) is expressed in Tissue-resident memory CD8 + T (TRM) 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.
  • TRM Tissue-resident memory CD8 + T
  • 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.
  • a method of diagnosing one or more of asthma, an autoimmune disease, or a fibrotic disease in a subject comprising, consisting of, or consisting essentially of contacting a sample isolated from the subject with an agent that detects the presence of GPR25 in the sample isolated from the subject, wherein the presence of the one or more genes at higher than baseline expression levels is a diagnostic indicator of asthma or the autoimmune or the fibrotic disease or wherein the absence of the one or more genes at higher or lower than baseline expression levels is not diagnostic indicator of the one or more of asthma, the autoimmune disease, or the fibrotic disease.
  • TRMs tissue-resident memory cells
  • a method of determining the responsiveness of a subject having one or more of asthma, an autoimmune disease, or a fibrotic disease to immunotherapy comprising, consisting of, or consisting essentially of contacting tissue-resident memory cells (TRMs) isolated from the subject with an antibody or agent that recognizes and binds a protein encoded by a GPR25 gene to determine the frequency of TRMs in the subject, wherein a high frequency of TRMs indicates lack of responsiveness to immunotherapy.
  • TRMs tissue-resident memory cells
  • a method of determining the responsiveness of a subject having one or more of asthma, an autoimmune disease, or a fibrotic disease to immunotherapy comprising, consisting of, or consisting essentially of contacting tissue-resident memory cells (TRMs) isolated from the subject with an antibody or agent that recognizes and binds a protein encoded by a GPR25 gene and, to determine the frequency of TRMs expressing these proteins, wherein a low frequency of TRMs expressing these proteins indicates responsiveness to immunotherapy.
  • TRMs tissue-resident memory cells
  • a method of determining prognosis of a subject having one or more of asthma, an autoimmune disease, or a fibrotic disease comprising, consisting of, or consisting essentially of measuring the density of CD103 or a protein encoded by a GPR25 gene in a sample isolated from the subject, wherein a low density of TRM indicates a more positive prognosis or wherein a high density or TRM indicates a more negative prognosis.
  • the more negative prognosis comprises, consists of, or consists essentially a decreased probability in the reduction of symptoms of the one or more of asthma, the autoimmune disease, or the fibrotic disease.
  • the lack of low density TRM indicates a more positive prognosis.
  • the more negative prognosis comprises, consists of, or consists essentially of the increased probability reduction of symptoms of the one or more of asthma, the autoimmune disease, or the fibrotic disease. 5 4914-9544-4227.1 Atty Docket No.116639-2730
  • a method of identifying a subject that will or is likely to respond to one or more of an asthma therapy, an autoimmune therapy, or a fibrotic disease therapy comprising, consisting of, or consisting essentially of contacting a sample isolated from the subject with an agent that detects the presence of GPR25 in the sample, wherein the presence of the one or more genes at higher or lower than baseline expression levels indicates that the subject is likely to respond to the one or more of the asthma therapy, the autoimmune therapy, or the fibrotic disease therapy.
  • 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, 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
  • 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.
  • FIG.1D Gpr25 expression levels in CD8 ⁇ + T cells (spleen), CD8 ⁇ + CD69 + and CD8 ⁇ + CD69 ⁇ T cells (liver), CD8 ⁇ + CD8 ⁇ + and CD8 ⁇ + CD8 ⁇ + T cells (siIEL) isolated from unmanipulated wild-type C57BL/6J mice (>90 days old).
  • Graphs in (FIG.1A), and (FIG.1D) depict mean ⁇ S.E.M.; graphs in (FIG.1A), and (FIG.1D) depict mean ⁇ S.D.; all data are representative of two independent experiments, with n 5-11 in each experiment.
  • FIG. 2A 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.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 and frequencies of transferred Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I CD8 + T cells that express CD69 and CXCR6, CD69 and CD103 are shown.
  • 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.).
  • anti-Gr1 were administrated as previous described 25 to eliminate circulating lymphocytes.
  • 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 9 4914-9544-4227.1 Atty Docket No.116639-2730 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.
  • 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.
  • FIG.4A 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.
  • FIG.4B Gene- set enrichment analysis (GSEA) plot shows enrichment of the indicated gene signatures when comparing Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I TRM cells.
  • GSEA Gene- set enrichment analysis
  • 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 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 compared to Gpr25 -/- (KO) OT-I T RM 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.
  • FIG.4K Flow-cytometric analysis of na ⁇ ve murine CD8 + T cells (CD8 + CD44 lo CD62L hi ) that were stimulated with anti-CD3 and anti-CD28 for 24h before transfection with pCMV-Thy1.1 (empty vector; EV) or pCMV-Gpr25-Thy1.1 (Gpr25), and then stimulated with TGF- ⁇ for 24 hours.
  • FIGS.5A – 5H Gpr25 promotes development of lung T RM cells.
  • FIG.5A Illustration of the lung 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 intratracheally at a 1:1 ratio into CD45.1 recipient mice before infection with LCMV-OVA delivered intratracheally (i.t.)
  • FIG. 5B FIG.5C
  • Flow-cytometric analysis of cells isolated from lungs of recipient mice (n 5) at 30 days post infection.
  • 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.
  • FIG.5H Representative histograms and the frequencies of TCF1-expressing cells in Gpr25 +/+ (WT) and Gpr25 -/- (KO) OT-I CD8 + T cells.
  • Atty Docket No.116639-2730 (FIG.5C) are representative of three independent experiments. Data from (FIG.5H) are representative of two independent experiments.
  • 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.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.
  • FIG.6B Bar graph in (FIG.6B) depicts mean, graphs in (FIG.6D), (FIG.6G) depict mean ⁇ S.D., each symbol represents data from an individual 13 4914-9544-4227.1 Atty Docket No.116639-2730 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. Statistical significance for the comparisons in (FIG.6B), (FIG.6D), (FIG.6G) was computed using two-tailed two unequal variance Student t test; *P ⁇ 0.05, **P ⁇ 0.01 and ***P ⁇ 0.001.
  • FIG.7B Flow-cytometric plots show sequential gating strategies for isolating CD8 + CD69 + and CD8 + CD69 ⁇ T cells present in spleen and liver (top panel), CD8 ⁇ + CD8 ⁇ + and CD8 ⁇ + CD8 ⁇ + T cells present in small intestinal intraepithelial lymphocytes (siIEL; bottom panel) of unmanipulated wild-type C57BL/6J mice (> 90 days old), used in FIG.1D.
  • 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.).
  • FIG.8D Congenically distinct Gpr25 +/+ (WT) and Gpr25 -/- (KO) 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.).
  • 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.
  • 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).
  • FIG.14 illustrates a GPR25 Hit ID testing funnel.
  • FIG.15 illustrates a GPR25 lead generation plan.
  • An equivalent of a polynucleotide shares at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) identity to the reference, and encodes the same polypeptide as the one encoded by the reference, or encodes an equivalent of the polypeptide encoded by the reference.
  • a sequence alignment is performed between the test and reference sequences. The positions or segments aligned to each other are determined as equivalents.
  • analogue refers to an equivalent having one or more modified amino acids and one or more amino acids replaced with another amino acid.
  • 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, 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
  • albumin equivalent comprises, or consists essentially of, or yet further consists of, 21 4914-9544-4227.1 Atty Docket No.116639-2730 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 His 6 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 is able to 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.
  • polyhistidine tagged fusion proteins can be recovered by immobilized metal ion chromatography using Ni 2+ or Co 2+ loaded resins, anti- 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 purifying, or “separating” refer 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. However, 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. 22 4914-9544-4227.1 Atty Docket No.116639-2730
  • 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.
  • 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.
  • 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.
  • baseline expression is assessed via immunohistochemistry or flow-cytometry of tissue biopsies (i.e. healthy adjacent tissue) and comprises, consists of, or consists essentially of normalized mean expression.
  • expression of GPR25 will be measured in tumor biopsies and compared to baseline levels, where higher than baseline expression of GPR25 comprises, consists of, or consists essentially of at least about a 2 or more, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15 fold increase in expression relative to baseline expression and/or lower than baseline expression of GPR25 is at least about a 2 or more, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15 fold decrease in expression relative to baseline expression.
  • 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
  • RNA short hairpin RNA
  • small hairpin RNA small hairpin 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.
  • 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.
  • the terms “retain” “similar” and “same” are used interchangeably while describing a function, an activity or an 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 28 4914-9544-4227.1 Atty Docket No.116639-2730 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 using methods disclosed herein.
  • 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 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.
  • oligonucleotide refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide.
  • Oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position. 29 4914-9544-4227.1 Atty Docket No.116639-2730 Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group.
  • One or more bases of the oligonucleotide may also be modified to include a phosphorothioate bond (e.g., one of the two oxygen atoms in the phosphate backbone which is not involved in the internucleotide bridge, is replaced by a sulfur atom) to increase resistance to nuclease degradation.
  • a phosphorothioate bond e.g., one of the two oxygen atoms in the phosphate backbone which is not involved in the internucleotide bridge, is replaced by a sulfur atom
  • the exact size of the oligonucleotide will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide.
  • the oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, restriction endonuclease digestion of plasmids or phage DNA, reverse transcription, PCR, or a combination thereof.
  • the oligonucleotide may be modified e.g., by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • “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. 30 4914-9544-4227.1 Atty Docket No.116639-2730 Examples of stringent hybridization conditions include: incubation temperatures of about 25° C.
  • 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.
  • high stringency conditions include: incubation temperatures of about 55° C.
  • the subunits may be linked by peptide bonds. In another aspect, 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.
  • 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 31 4914-9544-4227.1 Atty Docket No.116639-2730 (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction.
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (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 extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • an extrachromosomal replicon e.g., a plasmid
  • a nuclear or mitochondrial chromosome e.g., a nuclear or mitochondrial chromosome.
  • a number of 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
  • 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 examples include retroviral vectors, adenovirus vectors, adeno- associated virus vectors, herpes simplex virus vectors, alphavirus vectors and the like.
  • animal refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • 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 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.
  • 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.
  • 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, 33 4914-9544-4227.1 Atty Docket No.116639-2730 chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, synovial fluid, mucosal secreti
  • the sample is a diseased tissue biopsy.
  • the terms “disease” “disorder” and “condition” are used interchangeably herein, referring to asthma, an autoimmune disorder, or fibrotic disorder, a status of being diagnosed with asthma, an autoimmune disorder, or fibrotic disorder, or a status of being suspected of having asthma, an autoimmune disorder, or fibrotic disorder.
  • extracellular matrix ECM
  • 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, 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.
  • enzymes which produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish per
  • 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, 34 4914-9544-4227.1 Atty Docket No.116639-2730 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.
  • immunotyping 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.
  • 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.
  • 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.
  • CD4+ cells T-helper cells
  • CD8+ cells cytotoxic T- cells
  • Treg T-regulatory cells
  • TRM cells Tissue-resident memory T 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.
  • 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, 35 4914-9544-4227.1 Atty Docket No.116639-2730 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,
  • Null leukemia cell lines including but not limited to REH, NALL-1, KM-3, L92-221, are a 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, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, U266 myeloma.
  • leukemias and lymphomas such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, 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.
  • Tissue resident memory cell or “TRM” cells refer to a subset of long-lived memory T cells that occupy epithelial and mucosal tissues.
  • a “cytotoxic cell” intends a cell that is capable of killing other cells or microbes. Examples of cytotoxic cells include but are not limited to CD8+ T-cells, certain CD4+ T-cells, double-negative T-cells, gamma delta T-cells, natural-killer (NK) cells, NK T-cells, and neutrophils, which cells are capable of mediating cytotoxicity responses.
  • NK natural-killer
  • CD4+ cytotoxic T-cells refers to a population of T-cells that express CD4 on their surface and, generally, are ThPOK- (downregulated). These CD4+ cytotoxic T-cells can also be characterized by CD8 ⁇ expression, downregulation of Gata3, and upregulation of Runx3 and Tbet.
  • ThPOK Uniprot: O15156 (human); Q64321 (mouse); also known as ZBTB7B
  • Gata3 Uniprot: P23771 (human); P23772 (mouse); P23825 (chicken)
  • Runx3 Uniprot: Q13761 (human); Q64131 (mouse); Q91ZK1 (rat)
  • Tbet Uniprot: Q9UL17 (human); Q5PSB0 (mouse); E1UGZ0 (rainbow trout), also known as TBX21
  • CD4 Uniprot: P01730 (human); P06332 (mouse); P33705 (dog)
  • CD8 ⁇ Uniprot: P01732 (human); P01731 (mouse); P33706 (dog), CD8 ⁇ being a homodimer of this protein).
  • CD4+ T-cells refers to T-cells that express CD4 on their surface and, generally, are ThPOK+ (upregulated); these cells include na ⁇ ve CD4+ T-cells, Th1 T-cells, Th17 T-cells, and T-regulatory cells.
  • surface markers e.g., CD8 ⁇ and CD4, can be identified by antibodies to the listed surface markers, i.e., an anti- CD8 ⁇ antibody or an anti-CD4 antibody.
  • anti- and the descriptor “antibody” refer to an antibody, fragment, derivative, or biological equivalent thereof that recognizes or binds the recited protein, e.g., anti-CD4 antibody recognizes and binds CD4.
  • CD8+ cytotoxic T-cell refers to a cytotoxic T-cell and/or a precursor thereof which is CD8+ and expresses CD8 ⁇ on its surface.
  • CD8 ⁇ refers to a heterodimer of CD8 ⁇ and CD8 ⁇ (also known as CD8b) that is expressed on the surface of 37 4914-9544-4227.1 Atty Docket No.116639-2730 CD8+T-cells.
  • Non-limiting exemplary amino acid sequences for CD8 ⁇ can be found in the Uniprot database under accession numbers P10966 (human CD8 ⁇ ); P10300 (mouse CD8 ⁇ ); P79336 (cat CD8 ⁇ ); other homologs of the same may also be found in the Uniprot database, i.e., at www.uniprot.org.
  • 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).
  • the agent may include a small molecule, an antibody, lentivirus, adeno-associated virus, an antisense oligonucleotide, an siRNA or an miRNA.
  • An antagonistic anti GPR25 antibody intends an antibody, antigen binding fragment, derivative or other modification as described herein that recognizes and binds the GPR25 protein.
  • the antagonizing antibodies or agents described in the application may bind to GPR25 to decrease, eliminate, and/or otherwise modulate the activity of the GPR25 receptor and/or the GPR25 expressing cell. Such activities may include proliferation or inhibition of cell signaling activities of the cell upon which the GPR25 receptor is expressed.
  • the antagonistic antibodies or agents of this disclosure target and specifically bind to GPR25.
  • the antagonistic antibody or agents bind the receptor in a manner that mimics the binding of the physiological ligand resulting in antibody-mediated antagonism.
  • treatment with an antagonistic anti-GPR25 antibodies significantly impedes the progression of asthma, an autoimmune disorder or fibrotic disorder, which may be mediated via inhibition of CD8+ T cells.
  • Immunotherapies utilizing antagonistic 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. Depending on the Immunoglobulin (IgG) antibody subclass, they bind to and can thus get crosslinked by Fcg receptors with different affinities.
  • IgG Immunoglobulin
  • the terms “antibody,” “antibodies” and “immunoglobulin” 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 40 4914-9544-4227.1 Atty Docket No.116639-2730 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.
  • CDR complementarity determining region
  • a heavy or light chain or a ligand binding portion thereof a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, at least one portion of a binding protein, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non-antibody protein.
  • the variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues.
  • 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 fragment refers to at least one portion of an intact antibody, or recombinant variants thereof, and refers to the antigen binding domain, e.g., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen.
  • 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 41 4914-9544-4227.1 Atty Docket No.116639-2730 epitope binding fragments of an antibody.
  • an antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Pat. No.6,703,199, which describes fibronectin polypeptide minibodies).
  • Fn3 fibronectin type III
  • Fab means a monovalent antigen- binding fragment of an immunoglobulin that is composed of the light chain and part of the heavy chain.
  • 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 means 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.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise V L -linker-V H or may comprise V H -linker-V L .
  • the term “antibody heavy chain” refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
  • the term “antibody light chain” refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring 42 4914-9544-4227.1 Atty Docket No.116639-2730 conformations.
  • 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.
  • antibody variant includes synthetic and engineered forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain portions but not two complete heavy chains (such as, domain deleted antibodies or minibodies); multi-specific forms of antibodies (e.g., bi-specific, tri-specific, etc.) altered to bind to two or more different antigens or to different epitopes on a single antigen); heavy chain molecules joined to scFv molecules and the like.
  • antibody variant includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three, four or more copies of the same antigen.
  • the term “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.
  • 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 encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Moreover, the skilled artisan will understand 43 4914-9544-4227.1 Atty Docket No.116639-2730 that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample.
  • Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • the term “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.
  • “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population. Monoclonal antibodies are highly specific, as each monoclonal antibody is directed against a single determinant on the antigen.
  • 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.
  • human antibody as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. 44 4914-9544-4227.1 Atty Docket No.116639-2730
  • the 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).
  • the term “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 , CH2, CH3), 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 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. For example, 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.
  • 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 45 4914-9544-4227.1 Atty Docket No.116639-2730 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. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference 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 to making these antibodies are described herein. In one embodiment, an antibody as used herein may be a recombinant antibody.
  • recombinant human antibody includes all human antibodies that are 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.
  • Such 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 to making these antibodies are described herein. 46 4914-9544-4227.1 Atty Docket No.116639-2730 As used 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 antibodies may have conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions.
  • amino acids of the present disclosure may bind to an antigen or molecule.
  • polyclonal antibody or polyclonal antibody composition refer to a preparation of antibodies that are derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope.
  • 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 47 4914-9544-4227.1 Atty Docket No.116639-2730 linking the antibody to a second molecule.
  • CDRs Complementarity determining regions
  • a variable region and a T cell receptor generated by B-cell s and T-cells respectively, wherein these molecules bind to their specific antigen (also called epitope).
  • the terms “variable region” and “variable domain” are used interchangeably, referring to the polypeptide of a light or heavy chain of an antibody that varies greatly in its sequence of amino acid residues from one antibody to another, and that determines the conformation of the combining site which confers the specificity of the antibody for a particular antigen.
  • 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.
  • variable region of an amino acid sequence refers to that the first about 100 amino acids, or alternatively about 110 amino acids, or alternatively about 120 amino acids, or alternatively about 130 amino acids, or alternatively about 140 amino acids, or alternatively about 150 amino acids of the amino acid sequence (including or excluding a signal peptide if applicable) is the variable region.
  • 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.
  • 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 publicly 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 CalssificatiIon (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
  • the antagonizing antibodies described in the application may bind to GPR25to increase, enhance, upregulate, and/or otherwise modulate the activity of the GPR25receptor and/or the GPR25expressing cell. Such activities may include proliferation and cell signaling activities of the cell upon which the GPR25receptor is expressed.
  • the antagonistic antibodies of this disclosure target and specifically bind to GPR25. In some aspects, the antagonistic antibody binds the receptor in a manner that mimics the binding of the physiological ligand resulting in antibody-mediated antagonism.
  • treatment with an antagonistic anti-GPR25antibodies significantly impedes 49 4914-9544-4227.1 Atty Docket No.116639-2730 progression of asthma, an autoimmune disorder, or a fibrotic disorder.
  • Immunotherapies utilizing antagonistic antibodies 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 antagonistic 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, Fc modifications like glycoengineering or point mutations, the antibody subclass and antigen expression.
  • Immunune 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.
  • 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 in vivo or in vitro.
  • the skilled artisan will understand that a variety of macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides and polysaccharides have the potential to be immunogenic.
  • nucleic acids encoding a molecule capable of eliciting an immune response necessarily encode an immunogen.
  • immunogens are not limited to full-length molecules, but may include partial molecules.
  • 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, antagonistic 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.
  • an “immunotherapy agent” means a type of treatment which modulates a patient’s own immune system to treat asthma, an autoimmune disorder, or fibrotic disorder, including but not limited to a physical intervene, a chemical substance, a biological molecule or particle, a cell, a tissue or organ, or any combinations thereof, modulating a patient's immune response against asthma, an autoimmune disorder, or fibrotic disorder.
  • immunotherapy agents include antibodies, immune regulators, checkpoint inhibitors, an antisense oligonucleotide (ASO), a RNA interference (RNAi), a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, a viral vector,, and a CAR therapy.
  • Antisense oligonucleotides are oligonucleotides capable of altering mRNA expression through a variety of mechanisms, including ribonuclease H mediated decay of the pre-mRNA, direct steric blockage, and exon content modulation through splicing site binding on pre-mRNA.
  • Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20–24 (normally 21) base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway.
  • immune checkpoint refers to a regulator and/or modulator of the immune system). Their interaction activates either inhibitory or activating immune signaling 51 4914-9544-4227.1 Atty Docket No.116639-2730 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 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.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes a cell transfected or infected in vivo or in vitro with a recombinant vector, an expression vector, or a nucleic acid encoding an antibody of the present disclosure.
  • a host cell, which comprises a recombinant vector, expression vector, or a nucleic acid encoding an antibody disclosed herein may also be referred to as a “recombinant host cell,” “engineered host cell,” or “engineered cell”.
  • 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- 52 4914-9544-4227.1 Atty Docket No.116639-2730 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.
  • a “composition” is intended to mean a combination of active 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.
  • 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 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
  • 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 53 4914-9544-4227.1 Atty Docket No.116639-2730 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 53 4914-9544-4227.1 Atty Docket No.116639
  • 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 method of determining the most effective route of administration are 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 examples of route 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 54 4914-9544-4227.1 Atty Docket No.116639-2730 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.
  • 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.
  • 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. 55 4914-9544-4227.1 Atty Docket No.116639-2730 As used herein, “treating” or “treatment of” a condition, disease or 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 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 survival of a subject beyond that expected in the absence of treatment.
  • Treating can also mean inhibiting the progression of the condition, disorder or disease, slowing the progression of the condition, disorder or disease temporarily, although in some instances, it involves halting the progression of the condition, disorder or disease permanently.
  • 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. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. Further, as used herein, 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 condition, disease, or disorder capable of being treated by the aspects disclosed herein is an autoimmune or fibrotic disorder, disease, or condition, proinflammatory condition, or an aberrant immune response.
  • the autoimmune or fibrotic disorder, disease, or condition, proinflammatory condition, or an aberrant immune response is selected from the group consisting of polymyositis, vasculitis syndrome, giant cell arteritis, Takayasu arteritis, relapsing, polychondritis, acquired hemophilia A, Still’s disease, adult-onset Still’s disease, amyloid A amyloidosis, polymyalgia rheumatic, 56 4914-9544-4227.1 Atty Docket No.116639-2730 Spondyloarthritides, Pulmonary arterial hypertension, graft-versus-host disease, autoimmune myocarditis, contact hypersensitivity (contact dermatitis), gastro-esophageal reflux disease, erythroderma, Behcet’s disease, amyotrophic lateral sclerosis, transplantation, rheumatoid arthritis, juvenile rheumatoid arthritis, malignant rheumatoid
  • prevention refers to a decrease in the occurrence of a disease or disease symptoms in a patient. As indicated above, the prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment. In one aspect, the term “treatment” excludes “prevention.” In one embodiment, the term “disease” or “disorder” as used herein refers to allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation.
  • the term “disease” or “disorder” as used herein refers to a status of being diagnosed with allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation, a status of being suspect of having allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation, or a status of at high risk of having allergy (optionally, a food allergy or an allergic reaction), hypersensitivity, asthma, inflammatory response or inflammation.
  • a “symptom” of a disease includes any clinical or laboratory manifestation associated with the disease and is not limited to what a subject can feel or observe.
  • 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 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 58 4914-9544-4227.1 Atty Docket No.116639-2730 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.
  • the term “specifically binds,” with respect to an antibody means an antibody or binding fragment thereof (e.g., Fv fragment or scFv) which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, a chimeric antigen receptor, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, a chimeric antigen receptor recognizes and binds to a specific protein structure rather than to proteins generally.
  • the Fv fragment further comprises a linker domain to generate a single chain variable fragment (scFv).
  • the linker domain is operably linked to the heavy chain variable domain and the light chain variable domain.
  • the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived.
  • the flexible polypeptide linker includes, but are not limited to, (Gly4 Ser)4 or (Gly4 Ser)3.
  • the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser).
  • compositions Disclosed herein are methods of treating one or more of asthma, an autoimmune disease, or a fibrotic disease in a subject, determining prognosis of a subject having one or more of asthma, an autoimmune disease, or a fibrotic disease, determining the responsiveness of a subject to one or more of asthma, an autoimmune disease, or a fibrotic disease therapies in a subject that modulates GPR25. Also disclosed herein are methods for screening for a GPR25 asthma, an autoimmune disease, or a fibrotic treatment in a subject.
  • provided herein is a method of treating autoimmune disease, inflammatory disease, and/or aberrant immune responses, including asthma, or eliciting an anti- inflammatory response in a subject in need thereof.
  • methods of inhibiting the proliferation of TRM cells in a subject are provided herein.
  • methods of inhibiting the development of stem-like TRM cells in a subject comprise, consist of, or consist essentially of modulating the expression of G Protein-Coupled Receptor 25 (GPR25) in the patient.
  • GPR25 G Protein-Coupled Receptor 25
  • the methods comprise, consist of, or consist essentially of administering to the subject an effective amount of an agent to inhibit the activity of a population of T-cells expressing GPR25, thereby treating the one or more of asthma, the autoimmune disease, or fibrotic disease in the subject.
  • the methods comprise, consist of, or consist essentially of administering to the subject an effective amount of an agent that reduces expression of GPR25 in T-cells, thereby treating the one or more of asthma, the autoimmune disease, or the fibrotic disease in the subject.
  • the T cells comprise, consist of, or consist essentially of CD4+ or CD8+ T-cells.
  • the T cells comprise, consist of, or consist essentially of CD8+ T-cells.
  • the T cells comprise, consist of, or consist essentially of tissue resident memory (TRM) cells. 60 4914-9544-4227.1 Atty Docket No.116639-2730
  • the agent comprises, consists of, or consists essentially of a small molecule, an antibody, lentivirus, adeno-associated virus, an antisense oligonucleotide, an siRNA or an miRNA.
  • the siRNA or miRNA is complementary to at least a fragment of a polynucleotide encoding GPR25.
  • administration of the agent induces lower than baseline expression of GPR25 in the T cells.
  • baseline expression comprises, consists of, or consists essentially of normalized mean gene expression.
  • the methods further comprise, consist of, or consist essentially of, administering to the subject an additional therapy for the one or more of asthma, the autoimmune disease, or the fibrotic disease.
  • the additional therapy comprises, consists of, or consists essentially of one or more of bronchodilators, corticosteroids, and/or monoclonal antibodies for the treatment of one or more of asthma, the autoimmune, or the fibrotic disease.
  • a method of diagnosing one or more of asthma, an autoimmune disease, or a fibrotic disease in a subject comprising, consisting of, or consisting essentially of contacting a sample isolated from the subject with an agent that detects the presence of GPR25 in the sample isolated from the subject, wherein the presence of the one or more genes at higher than baseline expression levels is a diagnostic indicator of asthma or the autoimmune or the fibrotic disease or wherein the absence of the one or more genes at higher or lower than baseline expression levels is not diagnostic indicator of the one or more of asthma, the autoimmune disease, or the fibrotic disease.
  • the sample comprises, consists of, or consists essentially of a TRM cell.
  • a method of diagnosing one or more of asthma, an autoimmune disease, or a fibrotic disease in a subject comprising, consisting of, or consisting essentially of contacting tissue-resident memory cells (TRMs) isolated from the subject or a sample isolated from the subject, with an antibody or agent that recognizes and binds 61 4914-9544-4227.1 Atty Docket No.116639-2730 to GPR25 to determine the frequency of TRMs expressing these proteins, wherein a high frequency of TRMs expressing these proteins is diagnostic of the one or more of asthma, the autoimmune disease, or the fibrotic disease.
  • TRMs tissue-resident memory cells
  • TRMs tissue-resident memory cells
  • a method of determining prognosis of a subject having a one or more of asthma, an autoimmune disease, or a fibrotic disease comprising, consisting of, or consisting essentially of measuring the density of tissue-resident memory cells (TRM) in a sample isolated from the subject, wherein a low density of TRM indicates a more positive prognosis or wherein a high density of TRM indicates a more negative prognosis, optionally wherein the more negative prognosis comprises a decreased probability in the reduction of symptoms of asthma, the autoimmune, or the fibrotic disease and a lack of low density TRM indicates a more positive prognosis.
  • TRM tissue-resident memory cells
  • a method of determining prognosis of a subject suffering from one or more of asthma, an autoimmune disease, or a fibrotic disease comprising, consisting of, or consisting essentially of contacting tissue-resident memory cells (TRMs) isolated from the subject with one or more of: an antibody or agent that recognizes and a protein encoded by a GPR25 gene, wherein a low density of TRMs indicates a more positive prognosis or wherein a high density of TRMs indicates a more negative prognosis.
  • TRMs tissue-resident memory cells
  • the more negative prognosis comprises a decreased probability in the reduction of symptoms of asthma, the autoimmune or the fibrotic disease
  • the more positive prognosis comprises an increased probability in the reduction of symptoms of the one or more of asthma, the autoimmune disease, or the fibrotic disease.
  • a method of determining prognosis of a subject having one or more of asthma, an autoimmune disease, or a fibrotic disease comprising, 62 4914-9544-4227.1 Atty Docket No.116639-2730 consisting of, or consisting essentially of contacting tissue-resident memory cells (TRMs) isolated from the subject with an antibody or agent that recognizes and binds a protein encoded by a GPR25 gene to determine the frequency of TRMs expressing these proteins, wherein a low density of TRMs indicates a more positive prognosis or wherein a high density of TRMs indicates a more negative prognosis.
  • TRMs tissue-resident memory cells
  • the more negative prognosis comprises a decreased probability in the reduction of symptoms of the autoimmune or fibrotic disease
  • the more positive prognosis comprises an increased probability in the reduction of symptoms of the one or more of asthma, the autoimmune disease, or the fibrotic disease.
  • a method of determining prognosis of a subject having one or more of asthma, an autoimmune disease, or a fibrotic disease comprising contacting tissue-resident memory cells (TRMs) isolated from the subject with an antibody or agent that recognizes and binds CD103 to determine the frequency of CD103+ TRMs or an antibody that recognizes and binds a protein encoded by a GPR25 gene to determine the frequency of TRMs expressing the protein, wherein a low density of TRMs indicates a more positive prognosis or wherein a high density of TRMs indicates a more negative prognosis, optionally wherein the more negative prognosis comprises a decreased probability in the reduction of symptoms of asthma, the autoimmune disease, or the fibrotic disease, and wherein the more positive prognosis comprises an increased probability in the reduction of symptoms of the one or more of asthma, the autoimmune disease, or the fibrotic disease.
  • TRMs tissue-resident memory cells
  • a method of determining the responsiveness of a subject having one or more of asthma, an autoimmune disease, or a fibrotic disease to immunotherapy comprising, consisting of, or consisting essentially of contacting tissue-resident memory cells (TRMs) isolated from the subject with an antibody or agent that recognizes and binds a protein encoded by a GPR25 gene to determine the frequency of TRMs in the subject, wherein a high frequency of TRMs indicates lack of responsiveness to immunotherapy.
  • TRMs tissue-resident memory cells
  • a method of determining the responsiveness of a subject having one or more of asthma, an autoimmune disease, or a fibrotic disease to immunotherapy comprising, consisting of, or consisting essentially of contacting 63 4914-9544-4227.1 Atty Docket No.116639-2730 tissue-resident memory cells (TRMs) isolated from the subject with an antibody or agent that recognizes and binds a protein encoded by a GPR25 gene and, to determine the frequency of TRMs expressing these proteins, wherein a low frequency of TRMs expressing these proteins indicates responsiveness to immunotherapy.
  • TRMs tissue-resident memory cells
  • a method of determining prognosis of a subject having one or more of asthma, an autoimmune disease, or a fibrotic disease comprising, consisting of, or consisting essentially of measuring the density of CD103 or a protein encoded by a GPR25 gene in a sample isolated from the subject, wherein a low density of TRM indicates a more positive prognosis or wherein a high density or TRM indicates a more negative prognosis.
  • the more negative prognosis comprises, consists of, or consists essentially a decreased probability in the reduction of symptoms of the one or more of asthma, the autoimmune disease, or the fibrotic disease.
  • the lack of low density TRM indicates a more positive prognosis.
  • the more negative prognosis comprises, consists of, or consists essentially of the increased probability reduction of symptoms of the one or more of asthma, the autoimmune disease, or the fibrotic disease.
  • a method of identifying a subject that will or is likely to respond to one or more of an asthma therapy, an autoimmune therapy, or a fibrotic disease therapy comprising, consisting of, or consisting essentially of contacting a sample isolated from the subject with an agent that detects the presence of GPR25 in the sample, wherein the presence of the one or more genes at higher or lower than baseline expression levels indicates that the subject is likely to respond to the one or more of the asthma therapy, the autoimmune therapy, or the fibrotic disease therapy.
  • baseline expression comprises, consists of, or consists essentially of normalized mean gene expression.
  • higher than baseline expression of the one or more genes is at least about a 2-fold increase in expression relative to baseline expression and/or lower than baseline expression of the one or more genes is at least about a 2-fold decrease in expression relative to baseline expression.
  • the methods further comprise, consist of, or consist essentially of administering to a patient in need thereof an asthma therapy, an autoimmune therapy, or a 64 4914-9544-4227.1 Atty Docket No.116639-2730 fibrotic disease therapy to the subject.
  • the asthma therapy, the autoimmune therapy, or the fibrotic disease therapy comprises, consists of, or consists essentially of one or more of hormonal therapy, immunotherapy, bronchodilators, corticosteroids, or monoclonal antibodies.
  • the sample is contacted with an agent.
  • the agent comprises, consists of, or consists essentially of a detectable lab 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 comprises a polypeptide that binds to an expression product encoded by the gene, or a polynucleotide that hybridizes to a nucleic acid sequence encoding all or a portion of the gene.
  • 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 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.
  • the one or more of the asthma or autoimmune disease, or fibrotic disease comprises, consists of, or consists essentially of polymyositis, vasculitis syndrome, giant cell arteritis, Takayasu arteritis, relapsing, polychondritis, acquired hemophilia A, Still's disease, adult-onset Still's disease, amyloid A amyloidosis, polymyalgia rheumatic, Spondyloarthritides, Pulmonary arterial hypertension, graft-versus-host disease, autoimmune myocarditis, contact hypersensitivity (contact dermatitis), gastro-esophageal reflux disease, erythroderma, Behcet's disease, amyotrophic lateral sclerosis, transplantation, rheumatoid arthritis, juvenile rheumatoid arthritis, malignant rheumatoid arthritis, Drug-
  • 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 asthma, or an autoimmune disease, or a fibrotic disease 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 buffering agent, a preservative or a protein-stabilizing agent.
  • TRM Tissue-resident memory CD8 + T
  • 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 (TCM) and effector memory T (TEM) cells.
  • TRM 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.
  • TRM cells Although the molecules that play an important role in the development and function of T RM cells are not fully understood, several studies have defined the key transcriptional regulators such as Hobit, Blimp-1, Runx3, Etv5, Id3, fatty acid binding proteins 68 4914-9544-4227.1 Atty Docket No.116639-2730 (FABPs) 10,20-23 , and cytokines such as interleukin (IL)-15 and transforming growth factor beta (TGF- ⁇ ) that contribute to TRM cell development and maintenance 4,24-26 . Besides their protective role in infections, it is now well-appreciated that TRM cells also play a critical role in cancer immunosurveillance 27,28 .
  • FABPs Atty Docket No.116639-2730
  • cytokines such as interleukin (IL)-15 and transforming growth factor beta (TGF- ⁇ ) that contribute to TRM cell development and maintenance 4,24-26 .
  • IL interleukin
  • TGF- ⁇ transforming growth factor
  • 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 .
  • GPR G protein-coupled receptor
  • GPR25 is expressed at significantly higher levels in TRM cells compared with non-TRM 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
  • Gpr25 also has been reported to have a ligand-independent constitutive activity 47 .
  • TRM Tissue-resident memory
  • TGF- ⁇ a key cytokine involved in the development of T RM 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 TRM 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 T RM cells displayed defects in their ability to differentiate into secondary TRM cells and maintain the TRM 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.
  • Example 2 GPR25 Antagonism in Tissue resident memory T Cells TGF- ⁇ induces expression of GPR25 in CD8 + T cells Applicant previously found that GPR25 expression was significantly increased in both lung and tumor-infiltrating CD8 + TRM cells when compared to non-TRM cells 36 . Notably, the expression of GPR25 was strongly correlated with the expression of several key T RM -associated genes, such as ZNF683, RBPJ, ITGAE, ITGA1, S1PR1 and S1PR5 36 .
  • TGF- ⁇ is known to tightly regulate the expression of many T RM -associated genes 4 .
  • Applicant tested its role in regulating the expression of GPR25.
  • TGF- ⁇ treatment resulted in rapid induction of GPR25 in 70 4914-9544-4227.1
  • Atty Docket No.116639-2730 primary human CD8 + T cells that were polyclonally activated in vitro by using anti-CD3 and ant- CD28 antibodies (FIG.1A).
  • FOG.1A anti-CD3 and ant- CD28 antibodies
  • C1, C2, C3 upstream of GPR25 transcription start site displayed prominent transposase accessible peaks (ATAC-seq peaks) in tumor-infiltrating CD8 + TRM 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. Applicant’s 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.
  • GPR25 is a TGF- ⁇ inducible gene that is selectively expressed in TRM cells.
  • LCMV lymphocytic choriomeningitis virus
  • TRM central memory
  • TRM cells CD45.1.2 + CD62L ⁇ CD69 + T cells in the liver
  • OT-I CD8 + T cells expressed higher levels of Gpr25 transcripts when compared to OT-I T CM and T EM cells (CD45.1.2 + CD62L + CD69- and CD45.1.2 + CD62L ⁇ CD69 ⁇ T cells in the spleen, respectively)
  • FIGGS.8A – 8C 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).
  • S1PR1PR1 sphigosine-1-phosphate receptor
  • Applicant found relatively equal proportions of Gpr25 +/+ and Gpr25 -/- OT-I T cells in the spleen, liver and siIEL (FIG.2B), which suggested that the Gpr25 deficiency did not influence the accumulation of T cells in these tissue compartments. Further, Gpr25 deficiency also did not impact the memory phenotypes of transferred T cells in the spleen (FIG. 8E), a tissue compartment that is largely devoid of T RM cells.
  • the liver TRM cells (CD69 + ), as expected, co-expressed CXCR6 15,20 , but lacked expression of CD103 (FIG.2C), a T RM marker for cells localized in the epithelial compartment 10,15,20 , and KLRG1 (FIG.8E), a marker of effector memory T cells 54 , as reported previously 15,20 .
  • FIG. 2C The liver TRM cells
  • FIG.8E KLRG1
  • T RM cells were generated in vivo by transferring equal numbers of congenically distinct Gpr25 +/+ OT-I (WT) and Gpr25 -/- OT-I (KO) T cells into CD45.1 recipient mice (host) before infection with LCMV-OVA.
  • WT congenically distinct Gpr25 +/+ OT-I
  • KO Gpr25 -/- OT-I
  • circulatory OT-I cells were first depleted by administrating anti-Gr-1 antibody 25 30 days post infection and then host mice were conjoined with na ⁇ ve CD8KO mice (parabiont recipient) that lack CD8 + T cells (FIG.2D).
  • Gpr25 +/+ and Gpr25 -/- OT-I T cells accumulated in equal proportions in the host liver (FIG.2E).
  • Applicant observed a significantly higher proportion of Gpr25 -/- OT-I (CD45.2) cells to Gpr25 +/+ OT-I (CD45.1.2) cells (FIG.2E).
  • This finding suggested that Gpr25-deficient TRM cells had lesser tissue-resident potential i.e., were able to enter the circulation of parabiont recipient mice, when compared to Gpr25-sufficient T RM cells.
  • Gpr25 deficiency resulted in a reduction of the frequency of these cells and a corresponding increase in KLRG1 + effectors cells (FIG.3C).
  • Applicant found relatively equal proportions of Gpr25 +/+ and Gpr25 -/- OT-I T cells in the liver on days 5, 12, and 30 post infection (FIG.3D), suggesting that Gpr25 has no impact on the accumulation and development of memory OT-I cells.
  • day 30 when the infection is cleared and memory responses are well-established 55,56 , the effects of Gpr25 on the formation of T RM cells (CD69 + CXCR6 + cells) and effector memory T cells (CD69 ⁇ KLRG1 + cells) were maintained (FIG.3E).
  • Gpr25 +/+ and Gpr25 -/- TRM cells isolated from the liver of recipient mice 30 days post LCMV infection revealed marked differences in transcriptional profiles between Gpr25 +/+ and Gpr25 -/- TRM cells isolated from the liver of recipient mice 30 days post LCMV infection (FIG.3A, FIG.4A, FIG.10A and Table 1).
  • Applicant found that Gpr25 +/+ TRM cells displayed significant positive enrichment of gene signatures linked to stem- like T cells compared to Gpr25 -/- T RM cells (FIG.4B and Table 3), while also displaying negative enrichment for gene signatures linked to effector cells.
  • Gpr25 +/+ T RM cells expressed lower levels of transcripts encoding for cytotoxicity-related effector cell molecules 74 4914-9544-4227.1 Atty Docket No.116639-2730 like granzyme B, granzyme K and perforin, and higher levels of transcripts encoding for transcription factors like T cell factor 1 (Tcf1, encoded by Tcf7) and lymphoid enhancer-binding factor 1 (Lef1) (FIG.10A), which have been shown to play important roles in the initiation and maintenance of stem-like and self-renewal potential of memory T cells as well as restraining their effector differentiation 57-59 .
  • Tcf1 T cell factor 1
  • Lef1 lymphoid enhancer-binding factor 1
  • Applicant confirmed by real-time quantitative polymerase chain reaction (qPCR) and intracellular staining that TCF1 encoding transcripts and TCF1 protein levels are higher in Gpr25 +/+ compared to Gpr25 -/- liver T RM cells (FIG.4C, FIG.4D).
  • qPCR quantitative polymerase chain reaction
  • FIG.4C FIG.4D
  • transferred Gpr25 +/+ OT-1 T cells in the liver of recipient mice were significantly enriched for stem-like T cell gene signatures (FIG.10B and Table 3), indicating that Gpr25 is likely to promote a stem-like program in the early stages of memory T cell development.
  • Gpr25 -/- T RM cells display defects in the generation of stem-like TCF1- expressing cells
  • adoptively transferred TRM cells including liver T RM cells, to migrate and form secondary T RM cells at the same tissue of origin after re-infection 15,23,60,61 .
  • Applicant utilized this secondary transfer approach to test whether Gpr25 deficiency impacted the generation of secondary T RM cells in the liver.
  • Gpr25 is likely to play an 75 4914-9544-4227.1 Atty Docket No.116639-2730 important role not only in the development of primary TRM cells but also in the generation of secondary TRM cell population in the liver. Gpr25 enhances T cell responses to TGF- ⁇ To assess how Gpr25 may influence the generation of stem-like TRM cells, Applicant focused on TGF- ⁇ signaling, which it is known to play an important role both in the generation of T RM cells 62-67 and the maintenance of TCF1-dependent stem-like program in CD8 + memory T cells 68 .
  • Gpr25 likely amplifies TGF- ⁇ signaling to influence the development of stem-like TRM cells.
  • Gpr25 promotes development of lung T RM cells
  • Applicant utilized the LCMV-OVA infection model to ask if Gpr25 played an important role in the generation of lung TRM cells (FIG.11A).
  • Applicant found equal proportions of adoptively transferred Gpr25 +/+ and Gpr25 -/- OT-I T cells in the lungs at day 30 and day 120 following infection with LMCV-OVA delivered intra-peritoneally (FIG.11B).
  • Applicant utilized the intratracheal (i.t.) route for both co- 76 4914-9544-4227.1 Atty Docket No.116639-2730 transferring OT-I T cells (Gpr25 +/+ and Gpr25 -/- ) and infecting the na ⁇ ve CD45.1 recipient mice with LCMV-OVA 22,72-74 (FIG.5A). Relatively equal proportions of Gpr25 +/+ and Gpr25 -/- OT-I T cells were seen in the lungs (FIG.5B).
  • 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.5D).
  • cluster 0 cells which 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 properties such as TCF1 and IL7R (FIG.5E, FIG.5F, and FIG.11E),
  • This finding suggested that, similar to Applicant’s findings in the liver tissue, Gpr25 is likely to promoted the development stem-like TCF-1-expressing T cells as opposed to effector T cells.
  • T cells in cluster 0 which were enriched for Gpr25 +/+ T cells when 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 tissues 75 (FIG.5E and FIG.5F).
  • a recent study reported that the robust generation of TRM cells required TGF- ⁇ induced down-regulation of the 77 4914-9544-4227.1 Atty Docket No.116639-2730 transcription factor ZEB2 and its target gene S1PR5 13 , which in turn promoted the sequestration of cells in the tissues.
  • Gpr25 may also play a role in the downregulation of ZEB2-S1PR5 axis, potentially by modulating TGF- ⁇ signaling and thus favoring the generation of T RM cells in the lungs.
  • Gpr25-deficient T cells fail to control lung metastasis.
  • Applicant examined their capacity to control the growth of lung metastases in a model of secondary challenge with OVA-expressing B16F10 melanoma (B16F10-OVA) cells.
  • OT-I memory T cells OT-1 transfer
  • 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).
  • FIG.6C two independent cohorts of CD45.1 wildtype (FIG.6C) recipient 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 TRM-associated cis-regulatory regions in the GPR25 locus can bind to SMAD1, the key transcription factor downstream of TGF- ⁇ signaling, Applicant propose that GPR25 is likely to be a direct target gene of TGF- ⁇ .
  • Gpr25 plays a key role in supporting the development of TCF1-expressing stem-like T RM cells in liver and lungs but not those that 79 4914-9544-4227.1 Atty Docket No.116639-2730 develop in the siIEL compartment.
  • Gpr25-sufficient TRM 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 TRM cell population with some T RM cells exhibiting more stem-like features, including expression of TCF1, that may contribute to the maintenance of the T RM cell pool and enabling rapid re-expansion upon antigen re-challenge.
  • Applicant’s results suggest that Gpr25 supports the generation of TCF1-expressing TRM cells, i.e., a stem-like differentiation program while limiting the effector differentiation of T RM 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- 80 4914-9544-4227.1 Atty Docket No.116639-2730 intestinal mucosa and CNS 46 .
  • Gpr25 has important non- chemotactic functions and supports the generation and maintenance of TRM cells in the liver and lung. Further functional studies under physiological conditions and employing genetic knock-out models (Gpr25-deficient mice) are likely to clarify the relative contribution of the chemotactic versus non-chemotactic function of Gpr25 in mediating protective immunity in infections and cancer. Discussion Applicant’s single-cell transcriptomic analysis of Gpr25-sufficient and Gpr25- deficient memory T cells provided several mechanistic insights.
  • Gpr25-sufficient TRM cells were enriched for molecules that promote a stem-like memory program
  • Gpr25- deficient TRM cells were enriched for molecules linked to effector program, which suggests that Gpr25 is likely to support a stem-like differentiation program whilst restraining the effector differentiation of T RM cells.
  • 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
  • Gpr25 may mediate its effects on TRM 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.
  • Apelin and Apela 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 T RM responses in health and disease. 81 4914-9544-4227.1 Atty Docket No.116639-2730 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 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 OVA257-264, 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
  • CD8 + T cells were isolated from spleen and liver single-cell suspensions to > 95% purity using a CD8 + T cell negative selection kit (Stem Cell Technologies).
  • HBSS Hank’s balanced salt solution
  • 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
  • the na ⁇ ve CD8 + T cells (0.3 x 10 6 per well) were stimulated in a 48-well plate pre-coated with anti-CD3 (1 ⁇ g/mL, Cat. No.16-0037-85, eBioscience) and anti-CD28 (1 ⁇ g/mL, Cat. No.16-0289-85, eBioscience) in the presence or absence of recombinant human TGF- ⁇ (20 ng/mL, Cat. No.240-B, R&D Systems) for 24 hours and 48 hours.
  • Na ⁇ ve mouse CD8 + T cells (CD8 + CD44 lo CD62L hi ) were isolated from 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.50-898-0, Invitrogen) for 24 hours and 48 hours or the indicated time points.
  • 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.
  • 1 x 10 6 B16F10-OVA cells were injected (i.v.) into either na ⁇ ve mice or mice previously infected with LCMV-OVA.
  • 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 85 4914-9544-4227.1 Atty Docket No.116639-2730 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 Single-cell transcriptome analysis was performed on cells from two experiments: (i) Liver dataset: Applicant sorted and pooled Gpr25 +/+ (WT; CD45.1.2) and Gpr25 -/- (KO; CD45.2) OT-I TRM cells (CD69 + CD62L ⁇ KLRG1 ⁇ ) from CD8 + T cells in the liver of recipient 8 mice at 30 days post LCMV infection.
  • UMI Unique Molecular Identifier
  • Cells were clustered using Seurat’s functions FindNeighbors and FindClusters with a resolution of 0.2 and 0.4 for the liver and lung datasets, respectively.
  • Pairwise single-cell differential gene expression analysis was performed using the R package MAST (v1.14.0) 83 with cellular detection rate (CDR) as a covariate, after normalizing the data to log2 counts per million (log2(CPM+1)).
  • CDR cellular detection rate
  • a gene was considered as differentially expressed if its Benjamini-Hochberg adjusted P-value (called false discovery rate) was ⁇ 0.05 and log2 fold change was >0.25.
  • Cluster-specific markers were determined by MAST using the Seurat function FindAllMarkers with default parameters.
  • 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).
  • Applicant then shifted all the reads aligned to the positive (+) strand by +4 bp, and the reads aligned to the negative (-) strand by -5 bp, using deepTools’ “alignmentSieve” routine 89 . Applicant also discarded reads overlapping with the blacklisted regions (provided in https://github.com/Boyle- Lab/Blacklist/tree/master/lists).
  • 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 87 4914-9544-4227.1 Atty Docket No.116639-2730 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 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. Details regarding the number of samples or mice per group, replicates in independent experiments and statistical tests can be found in the figure legends.
  • GPR25(W164A)-PK are transfected into the PathHunter® U2OS Endosomal-EA (Endo-EA) cells (DiscoverX). These 88 4914-9544-4227.1 Atty Docket No.116639-2730 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).
  • 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.
  • Stable U2OS LIFT cells expressing the mutant target GPR25 are first dispensed at a range of cell seeding densities (500–1500 cells/well). Bortezomib (10 ⁇ M) is used to induce trafficking of the mutant oGPCR and produce EFC-derived luminescence. Next, the effect of serum (0.0-10.0% FBS) and DMSO (0.0–2.0% final v/v) on assay performance is determined. The variability of the assay is determined by running triplicate max/mid/min plates on three days and then calculating the Z’ factor 112 . The minimal performance criteria (Z’ ⁇ 0.5, S/N ⁇ 2.0, %CV ⁇ 10) is required for a HTS.
  • 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). 90 4914-9544-4227.1 Atty Docket No.116639-2730
  • Example 4 GPR25 antagonist 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 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 leverages 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 conducts assays on human T cells treated with TGF- ⁇ , which induces GPR25 expression (FIG.16), to monitor their effects on activation, proliferation, cytokine release, and their ability to enhance responses to TGF-b.
  • (iii) Determine physicochemical and pharmacological properties for in vivo development. Leads identified in this effort are intended for probing receptor function in cellular systems and with further refinement use in vivo.
  • Applicant uses a series of standard in vitro ADME/T and physicochemical profiling 91 4914-9544-4227.1 Atty Docket No.116639-2730 assays in place.
  • aqueous solubility data are determined at pH 7.4 with UV detection. Lipophilicity is determined using a high-throughput shake-flask method with octanol and water. The cLogD and polar surface area are calculated through use of ChemAxon software. Metabolic stability in human- and mouse-liver microsomes is determined by incubating compounds in 1 mg/mL microsomes; the metabolites are quantitated using liquid chromatography/mass spectroscopy methods. Cellular permeability are determined using a monolayer of MDCK1 cells.
  • 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.
  • humanized mouse models can be used to determine activity of GPR25 in vivo.
  • Applicant completes lead generation efforts culminating in the identification of 1- 3 hGPR25 antagonist for functional testing in vitro (human T cells) and in vivo (mouse models).
  • Applicant performs comprehensive efficacy and toxicity studies in mouse models for further advancing the drug-development pathway towards clinical testing. Feasibility in an academic setting. Applicant has extensive experience in HTS and lead optimization of screening hits identified from oGPCR LIFT screens.
  • GPR25 was strongly correlated with the expression of several key TRM- associated genes, such as ZNF683 (Hobit), RBPJ, ITGAE (CD103), S1PR1 and S1PR533. Because TGF- ⁇ is known to tightly regulate the expression of many TRM-associated genes69, Applicant investigated its role in regulating the expression of GPR25. Applicant found that 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).
  • TGF- ⁇ signaling regulates expression of GPR25 Applicant analyzed assay for transposase-accessible chromatin using sequencing (ATAC-seq) profile of the GPR25 locus in human CD8+ T cell subsets33,117.
  • the C2 region displayed prominent transposase accessibility in activated CD8+ T cells.
  • ChIP-seq assays are performed in primary human and murine CD8+ T cells, utilizing 10 million cells/per assay.
  • Primary CD8+ T cells are polyclonally activated in vitro by using anti-CD3 and ant-CD28 antibodies, and treated with TGF- ⁇ (20ng/ml) for 6 hour and 24 hours.
  • TGF- ⁇ (20ng/ml) for 6 hour and 24 hours.
  • Applicant varies fixation conditions and post-immunoprecipitation washing steps to improve the signal to noise ratio, which are assessed by ChIP-PCR for enrichment in binding sites known to be occupied by these TFs. With good ChIP-grade antibodies.
  • ChIPmentation121,122 efficiently prepares libraries from ChIP DNA for sequencing, even from low DNA amounts. Applicant sequences these libraries using the Illumina NovaSeq 6000 platform, generating >40 million 100bp paired-end reads per assay. Analysis. Applicant has extensive experience in analyzing ChIP-seq datasets123- 125. Applicant will follow ENCODE guidelines126 for mapping and peak calling and include independent biological replicates for all ChIP-seq assays to assess the reproducibility. As controls, Applicant includes T cells not treated with TGF- ⁇ .
  • 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.
  • Applicant utilizes non-targeting sgRNAs. Applicant has optimized this assay to work in primary human T cells, and has tested ⁇ 10 cis-regulatory elements in a few months, as described in Chandra et al Nature Genetics 2021122..
  • dCas9 catalytically dead Cas9 protein
  • tiling guide RNAs to target multiple regions
  • dCas9 lacks any silencing domain, binds to targeted chromatin with high affinity and effectively prevent the binding of SMAD proteins.
  • GPR25 Mediates its effect on TRM cells Applicant’s studies have shown that Gpr25 plays a key role in supporting the development and maintenance of TRM cells in the liver and lungs. However, the signaling pathways and mechanisms though 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.
  • GPR25 is an orphan GPCR with no known endogenous ligand, effects of ligand-dependent signaling cannot be tested.
  • GPR25 exhibited significant ligand- independent i.e., constitutive inhibition of cAMP signaling under both baseline and stimulated conditions (using forskolin, an exogenous agent that activates adenylate cyclase and cAMP signaling)72.
  • Another report showed that non-mammalian vertebrate GPR25 can inhibit cAMP signaling in a ligand-dependent manner131. Based on these data, Applicant reasons that GPR25 signaling is likely to inhibit cAMP pathway and its downstream targets in T cells. cAMP pathway inhibits TGF- ⁇ signaling.
  • 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.
  • TGF ⁇ directly regulates expression of the following key genes55,67,127-130,136-139: (i) CD103, encodes for ⁇ E integrin, that is expressed by TRM cells at barrier sites; (ii) KLF2, encodes a transcription factor that regulates expression of S1PR1, a sphingosine-1-phosphate receptor that promotes egress of cells from tissues140; (iii) ZEB2, a transcription factor that regulates expression of S1PR5, and (iv) IL-15 receptor (IL-15RA) expression that supports responses to IL-15.
  • CD103 encodes for ⁇ E integrin, that is expressed by TRM cells at barrier sites
  • KLF2 encodes a transcription factor that regulates expression of S1PR1, a sphingosine-1-phosphate receptor that promotes egress of cells from tissues140
  • ZEB2 a transcription factor that regulates expression of S1PR5
  • IL-15RA IL-15 receptor
  • 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.5F and 5E), which suggested that Gpr25 is likely to promote the formation of long-lived memory cells with stem-like properties as opposed to the differentiation into short-lived effectors.
  • 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.
  • 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
  • loss-of-function Gpr25-/- T cells i.e., genetic knockout
  • rescue studies In vitro studies. While TRM cells express Gpr25, their relatively low abundance in vivo makes it challenging to isolate a sufficient number of cells required for 97 4914-9544-4227.1 Atty Docket No.116639-2730 extensive in vitro studies. Instead, Applicant uses na ⁇ ve CD8+ T cells for these assays.
  • Gpr25 is not expressed by na ⁇ ve CD8+ T cells
  • Applicant forces expression of Gpr25 using viral transduction methods. Briefly, mouse Gpr25 cDNA construct was cloned into pMSCV-Thy1.1 retroviral vector. Empty vector or Gpr25 plasmid was transfected into Plat-E cells to generate retroviral supernatants, which was used for transducing activated T cells. Transduced T cells were expanded for 2-3 days in IL-2, and Thy1.1-expressing cells sorted for further functional studies. Applicant achieve 30% transduction efficiency in primary CD8+ T cells (not shown). Assays and Readouts.
  • 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 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 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); (b) Single-cell RNA-seq, as described in FIG.5F, at early (day 14) and late (day 30) timepoints to determine if forced Gpr25 expression skews the development of memory T cells towards TRM phenotype by restraining the expression of TGF- ⁇ target genes that promote tissue egress (Klf2-S1pr1 and Zeb2-S1pr5 axis). Applicant examines if signature genes in TGF- ⁇ pathway are enriched in T cells with forced Gpr25 expression.
  • TGF- ⁇ RICA constitutively active TGF- ⁇ receptor
  • Gpr25-deficient and Gpr25-sufficient (controls) OT-1 T cells assess its impact on lung TRM cell development in the LCMV-infection model, as described (FIG.5).
  • Constitutively active 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.
  • IL-15 signaling pathway is modulated by Gpr25 to promote TRM development’.
  • the cAMP signaling pathway which is inhibited by Gpr25, has been shown to inhibit IL-2 and its downstream signaling pathway (JAK3/STAT5)143. Considering that 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
  • IL15 receptor- deficient T cells display profound defects in the development and maintenance of TRM cells in vivo127-129. Based on this rationale, here, Applicant will test if IL-15 signaling pathway is also modulated by Gpr25 (alternative hypothesis).
  • Applicant determines if forced expression of Gpr25 impacts target genes and enhancers that are down-stream of IL-15 signaling pathway.
  • 99 4914-9544-4227.1 Atty Docket No.116639-2730 Applicant treats T cells with forced expression of Gpr25 and control T cells (empty vector transduced cells) with IL-15 and assess 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 down-stream 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. Subsequently, Applicant utilizes CRISPR-based assays to assess if SMAD-bound regions enhance the expression of GPR25 in T cells.
  • Migratory DCs activate TGF-beta to precondition naive CD8(+) T cells for tissue-resident memory fate. Science 366, doi:10.1126/science.aav5728 (2019). 5. Massague, J. TGF-beta signal transduction. Annu Rev Biochem 67, 753-791, doi:10.1146/annurev.biochem.67.1.753 (1998). 6. Zhao, X., Shan, Q. & Xue, H. H. TCF1 in T cell immunity: a broadened frontier. Nat Rev Immunol 22, 147-157, doi:10.1038/s41577-021-00563-6 (2022). 7. Sallusto, F., Lanzavecchia, A., Araki, K.
  • TGF-beta Controls the Formation of Kidney-Resident T Cells via Promoting Effector T Cell Extravasation.
  • Capicua/ETS Translocation Variant 5 Axis Regulates Liver-Resident Memory CD8(+) T-Cell Development and the Pathogenesis of Liver Injury. Hepatology 70, 358-371, doi:10.1002/hep.30594 (2019). 21. Frizzell, H. et al. Organ-specific isoform selection of fatty acid-binding proteins in tissue- resident lymphocytes. Sci Immunol 5, doi:10.1126/sciimmunol.aay9283 (2020). 22. Milner, J. J. et al. Runx3 programs CD8(+) T cell residency in non-lymphoid tissues and tumours. Nature 552, 253-257, doi:10.1038/nature24993 (2017).
  • CD103+ tumor-infiltrating lymphocytes are tumor-reactive intraepithelial CD8+ T cells associated with prognostic benefit and therapy response in cervical cancer. Oncoimmunology 6, e1338230, doi:10.1080/2162402X.2017.1338230 (2017).
  • Han, L. et al. Characterization of CD103(+) CD8(+) tissue-resident T cells in esophageal squamous cell carcinoma may be tumor reactive and resurrected by anti-PD-1 blockade.
  • Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427, 355-360, doi:10.1038/nature02284 (2004). 54. Herndler-Brandstetter, D. et al. KLRG1(+) Effector CD8(+) T Cells Lose KLRG1, Differentiate into All Memory T Cell Lineages, and Convey Enhanced Protective Immunity. Immunity 48, 716-729 e718, doi:10.1016/j.immuni.2018.03.015 (2018). 55. Murali-Krishna, K. et al. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection.
  • 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). 59. 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). 60. 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).
  • DPC4 a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271, 350-353, doi:10.1126/science.271.5247.350 (1996). 72. Anderson, K. G. et al. Cutting edge: intravascular staining redefines lung CD8 T cell responses. J Immunol 189, 2702-2706, doi:10.4049/jimmunol.1201682 (2012). 73. Anderson, K. G. et al. Intravascular staining for discrimination of vascular and tissue leukocytes. Nat Protoc 9, 209-222, doi:10.1038/nprot.2014.005 (2014). 109 4914-9544-4227.1 Atty Docket No.116639-2730 74.
  • 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). 85. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-359, doi:10.1038/nmeth.1923 (2012). 110 4914-9544-4227.1 Atty Docket No.116639-2730 86. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.

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Abstract

L'invention concerne des méthodes de traitement de l'asthme, et/ou d'une maladie auto-immune et/ou d'une maladie fibrotique chez un sujet. Les méthodes peuvent consister notamment, consister, ou consister essentiellement à administrer au sujet une quantité efficace d'un agent de façon à inhiber l'activité d'une population de lymphocytes T exprimant GPR25, ce qui permet de traiter l'asthme, et/ou la maladie auto-immune et/ou la maladie fibrotique chez le sujet.
PCT/US2024/060223 2023-12-15 2024-12-13 Antagonisation de gpr25 pour le traitement de maladies auto-immunes Pending WO2025129130A1 (fr)

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US202363611064P 2023-12-15 2023-12-15
US63/611,064 2023-12-15

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WO2025129130A1 true WO2025129130A1 (fr) 2025-06-19

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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)
US20190117689A1 (en) * 2016-04-04 2019-04-25 The Regents Of The University Of California Compositions and methods related to polycytotoxic t cells

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)
US20190117689A1 (en) * 2016-04-04 2019-04-25 The Regents Of The University Of California Compositions and methods related to polycytotoxic t cells

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