[go: up one dir, main page]

WO2025104233A1 - Inhibition of prostaglandin d2 receptor 1 as means for treating cancer - Google Patents

Inhibition of prostaglandin d2 receptor 1 as means for treating cancer Download PDF

Info

Publication number
WO2025104233A1
WO2025104233A1 PCT/EP2024/082474 EP2024082474W WO2025104233A1 WO 2025104233 A1 WO2025104233 A1 WO 2025104233A1 EP 2024082474 W EP2024082474 W EP 2024082474W WO 2025104233 A1 WO2025104233 A1 WO 2025104233A1
Authority
WO
WIPO (PCT)
Prior art keywords
tumor
hpgds
cells
cancer
dpi
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/082474
Other languages
French (fr)
Inventor
Massimiliano Mazzone
Rosa TROTTA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Katholieke Universiteit Leuven
Vlaams Instituut voor Biotechnologie VIB
Original Assignee
Katholieke Universiteit Leuven
Vlaams Instituut voor Biotechnologie VIB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Katholieke Universiteit Leuven, Vlaams Instituut voor Biotechnologie VIB filed Critical Katholieke Universiteit Leuven
Publication of WO2025104233A1 publication Critical patent/WO2025104233A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/9116—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • G01N2333/91165—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5) general (2.5.1)
    • G01N2333/91171—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5) general (2.5.1) with definite EC number (2.5.1.-)
    • G01N2333/91177—Glutathione transferases (2.5.1.18)
    • 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

  • the disclosure relates to the medical use of inhibitors of expression or function of the prostaglandin D2 receptor 1 in the treatment of cancer in general or more in particular in the treatment of cancer in which HPGDS is predominantly expressed by tumor-associated macrophages in the tumor micro-environment.
  • the disclosure further relates to isolated immune cells in which prostaglandin D2 receptor 1 expression or function is inhibited, such as for medical use.
  • Prostaglandin D2 (PGD2; CAS No 41598-07-6), an arachidonic acid metabolite produced by hematopoietic PGD synthase (H-PGDS or HPGDS) and by lipocalin-type PGD Synthase (L-PGDS or LPGDS), is mainly interacting with two receptors: DPI (DP, PTGDR1) and DP2 (CRTH2, PTGDR2, CD294).
  • DPI DP, PTGDR1
  • DP2 CRTH2, PTGDR2, CD294
  • HPGDS Hematopoietic Prostaglandin D2 signaling has been explored as a therapeutic target for treating allergic diseases including allergic asthma, rhinitis, atopic dermatitis, food allergy, gastrointestinal allergic disorder, and anaphylaxis; and several small molecule HPGDS inhibitors have been reported (reviewed in e.g. Beura and Chetti 2022, J Mol Structure 1259:132704).
  • PGD2 itself was reported to inhibit gastric cancer whereas DP2 knock-down promoted growth (CN106619651; Zhang et al. 2018, Stem Cells 36:990-1003; subcutaneous cancer model). Knock-down of HPGDS was further reported to promote intestinal adenomas (Park et al. 2007, Cancer Res 67:881), whereas treatment of leukemia with an agonist of a prostaglandin D receptor (DPI or DP2) was claimed in US9623031. Mary et al. 2022 (Cancer Immunol Res 10:900-916) reported on the possible involvement of T follicular helper (Tfh) cells in HPGDS/CRTH2-mediated tumor metastasis, and on the combination of anti-IL4 antibodies with inhibition of HPGDS.
  • Tfh T follicular helper
  • the current disclosure relates to inhibitors of prostaglandin D2 receptor 1 (DPI) for use in treating a tumor or cancer, inhibiting a tumor or cancer, or inhibiting progression of a tumor or cancer. More in particular, the tumor or cancer can be a primary tumor or cancer.
  • DPI prostaglandin D2 receptor 1
  • the tumor or cancer micro-environment of the tumor or cancer is characterized in that the total amount of expression of HPGDS in the tumor micro-environment compartment is predominantly contributed by tumor- or cancer-associated macrophages.
  • the inhibitor of DPI is selectively inhibiting function or expression of DPI. In a further embodiment, the inhibitor of DPI is for use in treating a tumor or cancer, inhibiting a tumor or cancer, or inhibiting progression of a tumor or cancer in combination with a further anti-tumor or anti-cancer agent, and/or in combination with surgery or radiation.
  • the current disclosure further relates to isolated macrophages characterized by substantially lacking functional DPI, as well as their use as a medicament, as well as to pharmaceutical compositions comprising such macrophages.
  • the current disclosure further relates to isolated CD8+ T-cells characterized by substantially lacking functional DPI, as well as their use as a medicament, as well as to pharmaceutical compositions comprising such CD8+ T-cells.
  • the current disclosure further relates to methods for selecting a subject having cancer for therapy including an inhibitor of DPI, an isolated macrophage substantially lacking functional DPI or an isolated CD8+ T-cell substantially lacking functional DPI, comprising: assessing the expression of HPGDS in a tumor biopsy sample obtained from the subject, and selecting a subject having cancer for therapy, when the total amount of expression of HPGDS in the tumor micro-environment compartment of the tumor biopsy is predominantly contributed by or occurring in tumor- or cancer- associated macrophages.
  • FIGURE 1 Identification of HPGDS expression on immunosuppressive TAMs
  • FIGURE 2 Hpgds inhibition re-educates macrophages towards an anti-tumoral Ml-like phenotype
  • H FACS analysis of the expression of Ml (CD80) or M2 (CD206) polarization markers in bone marrow derived macrophages (BMDMs) treated for 48hr with 1 pM PGDj.
  • I-K Quantification of the total sprout length (I), number of sprouts (J), and representative images (K) of HUVEC spheroids embedded in collagen I (“HUVEC”) and of HUVEC spheroids co-incubated with BMDMs in which siHpgds was introduced (Hpgds expression silenced; "siHpgds”), or in which a scramble siRNA was introduced (Hpgds expression not silenced; "scramble”).
  • N FACS analysis of the expression of Ml (CDllc) or M2 (CD206) polarization markers in MO-like macrophages in which siHpgds was introduced (Hpgds expression silenced), or in which a scramble siRNA was introduced (Hpgds expression not silenced).
  • FIGURE 3 Hpgds deletion in TAMs potently inhibits tumor growth and reshapes the TME
  • F-G Flow cytometric quantification of CD8 + T cells (F) and activated CD8 + T cells (CD8 + IFNy + ; CD8 + GZMB + ) (G) in WT->WT and Hpgds KO-> WT chimeras (see Example 3).
  • H Quantification of CD8 + T cells infiltration in the tumor core in WT->WT and Hpgds KO-> WT chimeras (see Example 3).
  • K-M FACS analysis of the percentage of TAMs (CD45 + , CDllb + , F480 + ) (K) and of the expression of different Ml (MHC-ll high , CDllc, CD86) (L) or M2 (MHC-ll low , CD206, CD204) (M) polarization markers from CD64-Cre; Hpgds +/+ or CD64-Cre; Hpgds L/L YU M M 1.7 melanoma bearing mice.
  • T quantification of the number of YUMM 1.7 melanoma cancer cells migrated towards macrophages (Ctrl or Hpgds-KO).
  • FIGURE 4 The mechanism of tumor regression mediated by Hpgds inhibition is driven by macrophages and CD8 + T cells
  • A-B Tumor growth (A), tumor weight (B) of YUMM 1.7 CD90.1 + melanoma tumors injected in CD64- Cre;Hpgds +/+ (“Ctrl”) mice or CD64-Cre;Hpgds i/L (“Hpgds-KO”) mice and treated with an irrelevant IgG antibody (“IgG”) or anti-CD8 antibody/CD8-depleting antibody (“aCD8").
  • IgG irrelevant IgG antibody
  • aCD8 anti-CD8 antibody/CD8-depleting antibody
  • C Quantification of vessel size (based on quantification of CD105 + cells) and pericyte blood vessel coverage (based on quantification of aSMA + cells) in melanoma tumors of Ctrl and Hpgds-KO mice treated with an irrelevant IgG antibody (“IgG”) or anti-CD8 antibody/CD8-depleting antibody (“aCD8").
  • IgG irrelevant IgG antibody
  • aCD8 anti-CD8 antibody/CD8-depleting antibody
  • F-G Tumor growth (F) and weight (G) from Ctrl and Hpgds AM0 tumor-bearing mice treated with IgG or aPDl when the average tumor of the group was 150 mm 3 (tumors from 2 out of 13 Hpgds AM0 mice did not reach 150 mm 3 , thus, excluded from the experiment).
  • FIGURE 5 Pharmacologic inhibition of Hpgds inhibits tumor growth and re-educates macrophages towards an Ml-like phenotype
  • K Calcein area of patient-derived organotypic tumor spheroids (PDOTs) treated with 1 pM HQL-79 or with vehicle control for 72hr.
  • FIGURE 6 HPGDS systemic inhibition improves ICB efficacy.
  • D-E Flow cytometric quantification of CD8 + T cells (D) and PD-1 expressing CD8 + T cells (E) in GEM mice Braf /600E Pten / ' treated with vehicle or with HQL-79.
  • G Left panel: percentage of Ml- like macrophages (CDllc + ) in GEM mice Braf /600E Pten / ' mice treated with vehicle, HQL-79, a-PDl or HQL-79 in combination with and a-PDl.
  • D CD8 + T cells
  • E PD-1 expressing CD8 + T cells
  • FIGURE 7 Expression of HPGDS in the B16 melanoma tumor micro-environment. HPGDS is predominantly expressed (visible as black dots) in malignant cells and alveolar cells 1, with further scattered expression in NK cells, B cells, and monocytes/macrophages.
  • FIGURE 8 Tumor growth inhibition by knocking out PTGDR1 expression in CD8+ T-cells or macrophages.
  • A Tumor growth (top panel) and tumor weight (bottom panel) of YU MM 1.7 melanoma tumors injected in chimeric mice with normal CD8+ T cells (NT), Ptgdrl ("Ptgdr") KO in CD8 + T cells, or Ptgdr2 KO in CD8 + T cells.
  • FIGURE 9 Tumor growth inhibition by DPI inhibitor asapiprant.
  • A Tumor growth (top panel) and tumor weight (bottom panel) of YUMM 1.7 melanoma tumors left untreated (“vehicle”) or treated with increasing doses of asapiprant; 10 mice per group.
  • BID twice a day.
  • HPGDS hematopoietic prostaglandin D2 synthase
  • HPGDS expression in the TME of B16 melanoma was largely in malignant cells (and in other cells), much more than in TAMs (and no co-localization of HPGDS expression with TAMs could be observed by immunohistochemistry).
  • a further tumor model, hepatocellular carcinoma (HCC) was also studied and shown to be sensitive to HPGDS inhibition; expression of HPGDS in the TME was likewise largely restricted to TAMs.
  • the anti-tumoral activity of HPGDS inhibition was shown to be dependent on CD8+ T-cell infiltration.
  • tumors of human cancer patients not responding to immune checkpoint blockers or inhibitors were characterized by maintained high expression of HPGDS in TAMs, whereas tumors of ICB-responsive patients had TAMs with low expression of HPGDS.
  • ICBs or ICIs immune checkpoint blockers or inhibitors
  • predominant expression in TAMs in the TME of a tumor is a criterion for selecting patients having cancer (e.g. naive tumor, or tumor treated with ICI but poorly or non-responding to ICI therapy) for therapy/additional therapy with an inhibitor of HPGDS.
  • HPGDS expression levels in TAMs of a cancer patient responding to ICI therapy was significantly reduced on-treatment versus pre-treatment.
  • HPGDS expression in TAMs therefore can serve as biomarker for early response of a cancer patient to ICI therapy.
  • tumors sensitive to HPGDS inhibition can alternatively be treated with inhibition of DPI such as present in CD8+ T-cells and macrophages, and CD8+ T-cells and macrophages in which DPI expression or function is knocked out or inhibited can be used in an adoptive cell transfer setting for treating such tumors or cancers.
  • Pharmacological inhibition of DPI likewise resulted in anti- tumoral activity, thus replicating the genetic PTGDR1 knock-out data.
  • the prostaglandin D2 receptor 1 is also known as DPI, PTGDR, or PTGDR1.
  • the human PTGDR(l) gene is located on chrl4:52, 267, 698-52,280,914 (GRCh38/hg38; plus strand), on chrl4:52, 734,416-52, 743,442 (GRCh37/hgl9 by NCBI Gene; plus strand), or on chrl4:52, 734,431-52, 743,442 GRCh37/hgl9 by Ensembl; plus strand).
  • the human DPI protein is identified by Uniprot accession No. Q13258 and secondary accession Nos.
  • the NCBI reference mRNA sequence for human PTGDR(1)/DP1 is identified by accession Nos. NM_000953.3 and NM_001281469.2.
  • Small molecule inhibitor of DPI include laropiprant (MK0524; CAS No. 571170-77-9), BW245C (CAS No.
  • HPGDS HPGDS and inhibition of expression or function of HPGDS
  • Hematopoietic prostaglandin D2 synthase or HPGDS is an enzyme (EC:5.3.99.2, EC:2.5.1.18) also known as glutathione-dependent PGD synthase, GSTS, PGDS or PTGDS2.
  • the human HPGDS gene is located on chr4:94, 298, 535-94, 342, 876 (GRCh38/hg38; minus strand), on chr4:95, 219, 686-95, 263, 987 (GRCh37/hgl9 by NCBI Gene; minus strand), or on chr4:95, 219, 686-95, 264, 027 (GRCh37/hgl9 by Ensembl; minus strand).
  • the human HPGDS protein is identified by UniProt accession No. 060760, Ensembl accession No. ENSP00000295256, and NCBI reference sequences with accession Nos. XP_005262989.1, XP_054205671.1, and NP_055300.1.
  • the NCBI reference mRNA sequence for human HPGDS is identified by accession No. NM_014485.3.
  • HPGDS inhibitors are known, including small molecules such as HQL-79 (CAS No 162641-16-9; e.g. Aritake et al. 2006, J Biol Chem 281:15277-15286), TFC-007 (CAS No 927878-49-7; Nabe et al.
  • Origene Catalog No TL315682V HPGDS Human shRNA Lentiviral Particle
  • Santa Cruz Catalog No sc-41638 siRNA
  • Santa Cruz Catalog No sc- 41638-SH plasmid shRNA
  • guide RNAs for use with CRISPR-Cas e.g. ABM Catalog No 23862111as lentiviral set; ABM catalog No 23862151 as AAV set; Origene Catalog No GA109114, Human HPGDS activation kit by CRISPR
  • CRISPR-Cas e.g. ABM Catalog No 23862111as lentiviral set; ABM catalog No 23862151 as AAV set; Origene Catalog No GA109114, Human HPGDS activation kit by CRISPR
  • a first series of aspects relate to the medical use of inhibitors of prostaglandin D2 receptor 1 (PTGDR1) or DPI / PTGDR1 or DPI inhibitors.
  • PTGDR1 prostaglandin D2 receptor 1
  • DPI DPI / PTGDR1 or DPI inhibitors.
  • blocker the terms “antagonist”, “inactivating compound”, “repressor” or “suppressor” can be used.
  • a first aspect of this disclosure relates to an inhibitor of PTGDR1 or DPI for use in treating a tumor or cancer, inhibiting (growth of) a tumor or cancer, or inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth.
  • this aspect relates to an inhibitor of PTGDR1 or DPI for use in the manufacture of a medicament for treating a tumor or cancer, for inhibiting (growth of) a tumor or cancer, or for inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth.
  • a further alternative relates to methods of treating a (subject having) a tumor or cancer, of inhibiting (growth of) a tumor or cancer (in a subject having a tumor or cancer), or of inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth (in a subject having a tumor or cancer), such methods including administering a (therapeutically effective dose of a) PTGDR1 or DPI inhibitor to the subject having a tumor or cancer. With this administering the tumor or cancer is treated or inhibited, or its progression is inhibited.
  • the tumor or cancer is a primary tumor or cancer. In a not mutually exclusive embodiment, the tumor or cancer is a solid tumor or cancer.
  • the inhibitor of PTGDR1 or DPI is combined with a further anticancer therapy (different from a therapy including an inhibitor of PTGDR1 or DPI).
  • the tumor or cancer microenvironment (TME) or TME compartment of the tumor or cancer is characterized in that HPGDS expression is limited to expression in tumor- or cancer-associated macrophages, or in that HPGDS is selectively expressed in tumor- or cancer-associated macrophages (TAMs) compared to in other cells of the TME or TME compartment.
  • the TME or TME compartment of the tumor or cancer is characterized in that the total amount of expression of HPGDS in the TME or TME compartment is mainly/predominantly/primarily/quantitatively selectively contributed by tumor- or cancer-associated macrophages or occurring in tumor- or cancer-associated macrophages.
  • the TME or TME compartment of the tumor or cancer is characterized in that the tumor- or cancer-associated macrophages in the TME or TME compartment are the main/predominant/primary/quantitatively selective contributors to the total amount of expression of HPGDS in the TME or TME compartment of the tumor or cancer.
  • HPGDS is expressed in different cell types present in the TME or TME compartment, all together constituting the total amount of expression of HPGDS in the TME or TME compartment (as could e.g. be determined by bulk RNA sequencing of nucleic acids isolated from an obtained tumor or cancer biopsy sample, or by immunohistochemistry, or by spatial expression detection techniques).
  • the majority of the HPGDS expression should be detected in the TAMs present in the TME or TME compartment /tumor or cancer biopsy sample (as could e.g. be determined by single cell RNA sequencing of nucleic acids isolated from an obtained tumor or cancer biopsy sample, or, at the protein level it could e.g. be determined by immunohistochemical co-localization of HPGDS protein and of a TAM-specific protein marker, e.g. CD206 or CD163; or by spatial expression detection techniques).
  • the HPGDS expression in the TME or TME compartment is then mainly/predominantly/primarily/quantitatively selectively contributed by TAMs or occurring in TAMs.
  • the quantification of the "majority” or the “main/predominant/primary/quantitatively selective contribution" of the HPGDS expression will depend on the tumor or cancer type (and may even differ from patient to patient within the same tumor or cancer type) and/or will depend on the number of cell types in the TME or TME compartment expressing HPGDS.
  • the "majority" or the “main/predominant/primary/quantitatively selective contribution” of the HPGDS expression in the TME or TME compartment can amount to 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or to at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the total HPGDS expression detected or determined in the TME or TME compartment; this major, main, etc. fraction is thus to be contributed or caused by, is originating from, or is occurring in a single cell type, i.e. the TAMs, present in the TME or TME compartment.
  • the inhibitor of PTGDR1 or DPI is selectively inhibiting function or expression (see further) of PTGDR1 or DPI. More in particular, the inhibitor of PTGDR1 or DPI is a small molecule (e.g. laropiprant, BW245C or asapiprant; see hereinabove), a DNA nuclease specifically knocking out or disrupting PTGDR1 or DPI, an RNase specifically targeting PTGDR1 or DPI, or an inhibitory oligonucleotide specifically targeting PTGDR1 or DPI (see further).
  • a small molecule e.g. laropiprant, BW245C or asapiprant
  • the inhibitor of PTGDR1 or DPI is combined with a further anti-tumor or anti-cancer agent (e.g. immune checkpoint inhibitor (comprising) therapy, or other (see hereinafter)), or is combined with surgery or radiation for purposes of treating a tumor or cancer, inhibiting (growth of) a tumor or cancer, or inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth.
  • a further anti-tumor or anti-cancer agent is an inhibitor of HPGDS. More in particular, the inhibitor of HPGDS is a small molecule (e.g.
  • HPGDS-targeting PROTAC see, e.g. Yokoo et al. 2021, ACS Med Chem Lett 12: 236-241
  • a DNA nuclease specifically knocking out or disrupting HPGDS an RNase specifically targeting HPGDS, or an inhibitory oligonucleotide specifically targeting HPGDS (see further).
  • Functional PTGDR(l) or DPI is defined as PTGDR(l) or DPI that is expressed and to which no "foreign" (in the sense of non-naturally occurring, artificially made, man-made, or any combination thereof) compound such as pharmacological inhibitor is bound or linked, wherein the "foreign" compound is capable of interfering directly (e.g. competing) or indirectly (e.g. by inducing degradation of PTGDR(l) or DPI) with the binding of PTGDR(l) or DPI with any one of its potential natural binding partners (e.g. prostaglandin D2, PGD2).
  • Functional PTGDR(l) or DPI may be exposed on the surface of immune cells (e.g. CD8+ T-cells, macrophages) or may be stored inside immune cells such as stored in a manner allowing quick release to the cell surface (e.g. Labrecque et al. 2013, PloS One 8:e65767).
  • functional PTGDR(l) or DPI can be lacking, or be substantially lacking on and/or in a cell by repressing, inhibiting, or blocking expression of PTGDR(l) or DPI, or by binding of a "foreign" compound (as meant hereinabove) to PTGDR(l) or DPI.
  • functional PTGDR(l) or DPI is lacking, or is substantially lacking, on and/or in an isolated immune cell as described herein (in particular CD8+ T-cells or macrophages).
  • Genetic modification of immune cells isolated from a subject is one means of forcing the immune cells to (substantially) lack functional PTGDR(l) or DPI.
  • Such genetic modification can be aimed at repressing, reducing, or inhibiting ongoing expression of PTGDR(l) or DPI in the isolated (unmodified) immune cells, and/or can be aimed at preventing or inhibiting de novo expression of PTGDR(l) or DPI, e.g. in case expression of PTGDR(l) or DPI is low or non-existing in the isolated (unmodified) immune cells.
  • the isolated immune cells when the isolated immune cells are expanded in vitro or ex vivo, it is understood that the genetic modification may occur prior to expansion, such as in case of stable genetic modification.
  • the isolated immune cells may need to be expanded in the continuous presence of e.g. the RNA interference agent.
  • Shielding (part of the) PTGDR(l) or DPI protein exposed on the surface of immune cells and/or stored within immune cells by means of contacting the immune cells with a pharmacological inhibitor of PTGDR(l) or DPI is another means of causing immune cells to (substantially) lack functional PTGDR(l) or DPI.
  • the said shielding can be envisaged as neutralizing (part of the) PTGDR(l) or DPI protein for interaction with other (natural) binding partners.
  • the contacting with the pharmacological inhibitor is continuous during the expansion of the immune cells, or is occurring after expansion of the immune cells.
  • Such pharmacological inhibitors per se are known in the art, see above, and alternatives are discussed in more detail hereinafter.
  • such pharmacological inhibitors bind to PTGDR(l) or DPI with high specificity and/or, optionally, with high affinity.
  • PTGDR(l) or DPI protein present inside immune cells or on the surface of immune cells can further be the target of pharmacologic knock-down such as by molecules or agents inducing specific proteolytic degradation of PTGDR(l) or DPI protein.
  • pharmacologic knock-down such as by molecules or agents inducing specific proteolytic degradation of PTGDR(l) or DPI protein.
  • the agent causing an immune cell to (substantially) lack functional PTGDR(l) or DPI or causing neutralization of PTGDR(l) or DPI as referred to herein may be part of a larger molecule further comprising a moiety directing the agent to the immune cell.
  • the immune cells are CD8+ T-cells and/or macrophages.
  • this disclosure relates to isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, isolated PTGDR1 or DPI knock-out immune cells, or to isolated immune cells conditionally expressing an inhibitor of PTGDR1 or DPI, or to populations of any thereof.
  • compositions such a pharmaceutical compositions, comprising (a population of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, isolated PTGDR1 or DPI knockout immune cells, or comprising (a population of) isolated immune cells conditionally expressing an inhibitor of PTGDR1 or DPI.
  • a further aspect of this disclosure relates to (populations of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, to (populations of) isolated PTGDR1 or DPI knock-out immune cells, to (populations of) isolated immune cells conditionally expressing an inhibitor of PTGDR1 or DPI, or to pharmaceutical composition comprising any of these, for use as a medicament; more in particular, for use as a medicament for treating a cancer or tumor, for inhibiting (growth of) a cancer or tumor, or for inhibiting progression of a cancer or tumor or of progression of tumor or cancer growth.
  • the (populations of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI are for use in the manufacture of a medicament; more in particular, for use in the manufacture of a medicament for treating a cancer or tumor, for inhibiting (growth of) a cancer or tumor, or for inhibiting progression of a cancer or tumor or of progression of tumor or cancer growth.
  • this aspect relates to methods of treating (as subject having) a cancer or tumor, inhibiting (growth of) a cancer or tumor (in a subject having a tumor or cancer), or inhibiting progression of a cancer or tumor or of progression of tumor or cancer growth (in a subject having a tumor or cancer), such methods including administering a (population of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, a (population of) isolated PTGDR1 or DPI knock-out immune cells, a (population of) isolated immune cells conditionally expressing an inhibitor of PTGDR1 or DPI, or a pharmaceutical composition comprising any of these, to a subject having a cancer or tumor.
  • a therapeutically effective amount of such (populations of) immune cells or a therapeutically effective amount of such pharmaceutical composition is administered to the subject. Further in particular, with this administering, the tumor or cancer is treated or inhibited, or its progression is inhibited.
  • such immune cells are for use in a treatment comprising transfer or adoptive transfer of the immune cells to a subject.
  • any of the isolated macrophage or CD8+ T-cells as described above is suitable for any of: (i) for use as medicament, (ii) for use in (a method of) adoptive cell therapy, (iii) for use in (a method of) treating, inhibiting, or suppressing a tumor or cancer; or for any of (iv) use in the manufacture of a medicament, (v) use in the manufacture of a medicament for adoptive cell therapy, or (vi) use in the manufacture of a medicament for treating, inhibiting, or suppressing a tumor or cancer.
  • any of the isolated macrophage or CD8+ T-cells according to the invention may further be for use in combination with surgery, radiation, chemotherapy, targeted therapy, immunotherapy, or a further anticancer agent.
  • any of the isolated macrophage or CD8+ T-cells according to the invention may also be used in (i) the manufacture of a medicament for use in combination with surgery, radiation, chemotherapy, targeted therapy, immunotherapy, or an anticancer agent, (ii) in the manufacture of a medicament for adoptive cell therapy for use in combination with surgery, radiation, chemotherapy, targeted therapy, immunotherapy, or an anticancer agent, or (iii) in the manufacture of a medicament for treating, inhibiting, or suppressing a tumor or cancer for use in combination with surgery, radiation, chemotherapy, targeted therapy, immunotherapy, or an anticancer agent.
  • any of (i) surgery, (ii) radiation, (iii) chemotherapy, (iv) targeted therapy, (v) immunotherapy, or (vi) a further anticancer agent may further be for use in combination with (i) any of the isolated macrophage or CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), (ii) any of the populations of such macrophage or CD8+ T-cells, or (iii) any of the pharmaceutical compositions comprising any such isolated macrophage or CD8+ T-cells or comprising any such population of isolated macrophage or CD8+ T-cells.
  • any of a chemotherapeutic agent, a targeted therapy agent, an immunotherapeutic agent, or an anticancer agent may be for use in the manufacture of a medicament for treating, inhibiting, or suppressing a tumor or cancer in combination with any of the isolated macrophage or CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), any of the populations of such macrophage or CD8+ T-cells, or any of the pharmaceutical compositions comprising any such isolated macrophage or CD8+ T-cells or comprising any such population of isolated macrophage or CD8+ T-cells.
  • Further medical uses include methods of treating, inhibiting, or suppressing a tumor or cancer in a subject having a tumor or cancer, said methods comprising the step of adoptive cell therapy of any of the isolated macrophage or CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), or of any of the populations of such macrophage or CD8+ T-cells; or of administering (in particular: administering a therapeutically effective dose of) any of the isolated macrophage or CD8+ T-cell according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), any of the populations of such macrophage or CD8+ T-cells, or any of the pharmaceutical compositions comprising any such isolated macrophage or CD8+ T-cells or comprising any such population of isolated macrophage or CD8+ T-cells.
  • Such methods may further comprise (simultaneous, separate or sequential) combination with administration of any of (i) surgery, (ii) radiation, (iii) chemotherapy, (iv) targeted therapy, (v) immunotherapy, or (vi) a further anticancer agent.
  • Further medical uses include methods of treating, inhibiting, or suppressing a tumor or cancer in a subject having a tumor or cancer, said methods comprising the step of administering (in particular: administering a therapeutically effective dose of) any of (i) surgery, (ii) radiation, (iii) chemotherapy, (iv) targeted therapy, (v) immunotherapy, or (vi) an anticancer agent, further in combination with adoptive cell therapy of any of the isolated macrophage or CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), or of any of the populations of such macrophage or CD8+ T-cells; or of administration (in particular: administering a therapeutically effective dose of) of any of the isolated macrophage or CD8+ T-cell according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), any of the populations of such macrophage or CD8+ T-cells, or any of the pharmaceutical compositions comprising any such isolated
  • adoptive cell transfer also known as cellular adoptive immunotherapy or cell transfer therapy
  • ex-vivo expanded cells in particular immune cells
  • the ex-vivo expanded (immune) cells can, prior to their transfer back in the subject, be genetically modified.
  • genetic modifications include genetic engineering such as to cause the (immune) cells to express antitumor T cell receptors (TCRs) or chimeric antigen receptors (CARs) to increase anti-tumor activity of the transferred (immune) cells.
  • TCR-engineered macrophages TCR-Ms
  • TCR-engineered CD8+ T-cells TCR-Ts
  • CAR-engineered macrophages CAR-Ms
  • CD8+ T-cells CD8+ T-cells lacking or substantially lacking functional PTGDR1 or DPI (as defined hereinabove)
  • Pharmaceutical compositions comprising TCR-engineered macrophages or CD8+ T-cells, CAR-engineered macrophages or CD8+ T-cells lacking or substantially lacking functional PTGDR1 or DPI are also part of the invention.
  • T-cells including CAR-Ts or TCR-Ts
  • CAR-Ts or TCR-Ts usually is initiated by enriching lymphocytes from a leukapheresis product.
  • T-cells (CD8+ or CD4+) are then separated by use of e.g. an antibody to a celltype specific marker.
  • the obtained T-cells can be activated and expanded ex vivo by incubation in the presence of anti-CD3 antibodies or anti-CD3/anti-CD28 antibodies (e.g. bound to beads) either alone or in combination with feeder cells or growth factors (e.g. interleukin 2).
  • Culture conditions can be adapted such as to obtain a desired polarization state of the T-cells.
  • the immune cells can be grown in the presence of e.g. a means to suppress PTGDR(l) or DPI expression and, optionally, a means introducing the CAR orTCR; such means can be gene transfer, e.g. effectuated by using lentiviral vectors, the Sleeping Beauty transposon system, or mRNA transfection).
  • a means to suppress PTGDR(l) or DPI expression e.g. a means to suppress PTGDR(l) or DPI expression
  • a means introducing the CAR orTCR can be gene transfer, e.g. effectuated by using lentiviral vectors, the Sleeping Beauty transposon system, or mRNA transfection.
  • the resulting modified T-cells are then concentrated and stored/preserved (e.g. in an infusible medium) (see e.g. Levine et al. 2017, Mol Ther Meth Clin Dev 4:92-101 for more details).
  • Autologous or allogeneic bone marrow derived macrophages can be maintained in culture, optionally in the presence of a PTGDR1 or DPI inhibitor.
  • Such macrophages can be genetically engineered or redirected such as to knock out the PTGDR1 or DPI gene, or by introduction of a vector or other genetic construct comprising an inducible promotor operably linked to a cassette allowing expression of a genetic or nucleotide based PTGDR1 or DPI -inhibitor (e.g. miRNA, shRNA, antisense RNA, ribozyme).
  • a genetic or nucleotide based PTGDR1 or DPI -inhibitor e.g. miRNA, shRNA, antisense RNA, ribozyme.
  • the macrophages are conditionally expressing a PTGDR1 or DPI inhibitor.
  • the engineered macrophages can subsequently be transferred into a subject, such as to treat a cancer or tumor as described hereinabove.
  • the expression inducing compound is administered at an appropriate timepoint to the subject having received the engineered macrophages.
  • the transfer can be autologous or heterologous.
  • Adoptive macrophage transfer has been described in the literature (e.g. Ma et al. 2015, Brain Behaviour Immunity 45:157-170; Parsa et al. 2012, Diabetes 61:2881-2892; Wang et al. 2007, Kidney Int 72:290-299; Zhang et al.
  • modified macrophages can carry a further modification so that they can function as e.g. chimeric antigen receptor macrophages (CAR-M, e.g. Wang et al. 2022, eBioMedicine 76:103873) or as T cell receptor macrophages (TCR-Ms).
  • CAR-M chimeric antigen receptor macrophages
  • TCR-Ms T cell receptor macrophages
  • isolated CAR-Ms or TCR-Ms may be PTGDR1 or DPI knock-out CAR- or TCR-Ms, or isolated CAR- or TCR-Ms conditionally expressing an inhibitor of PTGDR1 or DPI.
  • Such CAR-or TCR-Ms likewise find use as a medicament, are for use in the manufacture of a medicament; or find application in methods of treating a disease or disorder in a subject, comprising administering (an effective dose of) the CAR-or TCR-Ms to a subject in need of being treated.
  • the disease or disorder is not limited to cancer or a tumor, as the nature of the CAR- or TCR-ligand may be determining the targeted disease or disorder and the inhibition of PTGDR1 or DPI may be additionally beneficial in directing the CAR-or TCR-Ms to the Ml phenotype.
  • this disclosure therefore also relates to methods of producing autologous or allogeneic Ml-type macrophages, such as Ml-type CAR-or TCR- M, comprising inhibiting expression of PTGDR1 or DPI in macrophages obtained from a subject or in macrophages differentiated ex-vivo from monocytes obtained from a subject.
  • Ml-type CAR-or TCR- M comprising inhibiting expression of PTGDR1 or DPI in macrophages obtained from a subject or in macrophages differentiated ex-vivo from monocytes obtained from a subject.
  • the invention relates to pharmaceutical compositions comprising an inhibitor of PTGDR(l) or DPI and a carrier.
  • the invention relates to pharmaceutical compositions comprising any isolated macrophage or CD8+ T-cell as described above (in particular lacking or substantially lacking functional PTGDR(l) or DPI), or comprising a population of such macrophage or CD8+ T-cells as described above.
  • such pharmaceutical composition comprises the macrophage or CD8+ T-cells as well as a carrier.
  • a carrier in general is both pharmaceutically acceptable (which can be administered to a subject without in itself causing severe side effects) and suitable for supporting stability, and storage if required, of the inhibitor, macrophages or CD8+ T-cells; and is alternatively defined as a pharmaceutically acceptable carrier.
  • Such pharmaceutical composition can comprise a further anticancer agent (detailed further hereinafter, including chemotherapeutic agent, targeted therapy agent, and immunotherapeutic agent). Diagnostic methods
  • this disclosure relates to diagnostic-type methods or companion diagnostic-type methods.
  • the immune cells are CD8+ T-cells and/or macrophages.
  • One such aspect includes methods of or for selecting, or determining the eligibility of, a subject having cancer for therapy including/with an inhibitor of PTGDR(l) or DPI, or for therapy including/with (a population of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, for therapy including/with (a population of) isolated PTGDR(l) or DPI knock-out immune cells (or CAR- immune cells orTCR-immune cells), or for therapy including/with (a population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) conditionally expressing an inhibitor of PTGDR(l) or DPI, or of predicting the response, the likelihood of response, or responsiveness of a subject having cancer to therapy including/with an inhibitor of PTGDR(l) or DPI, to therapy including/with (a population of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, to therapy including/with (a population of) isolated PTGDR(l) or DPI knock-out immune cells (or CAR-imm
  • the tumor biopsy sample is obtained prior to starting therapy with a PTGDR1 or DPI inhibitor, i.e. the tumor biopsy sample is a pre-therapy tumor biopsy sample, or is a pre- PTGDR1 or DPI inhibitor therapy tumor biopsy sample.
  • This disclosure further relates to either (when referring hereinafter to immune cells these are CD8+ T- cells and/or macrophages): an inhibitor of PTGDR1 or DPI for use in treating a tumor or cancer, inhibiting (growth of) a tumor or cancer, or inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth; or an inhibitor of PTGDR1 or DPI for use in the manufacture of a medicament for treating a tumor or cancer, for inhibiting (growth of) a tumor or cancer, or for inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth; or a method of treating a (subject having) a tumor or cancer, of inhibiting (growth of) a tumor or cancer, or of inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth, such methods including administering a (therapeutically effective dose of a) PTGDR1 or DPI inhibitor to the subject having a tumor or cancer; or a (population of) isolated immune cells (or CAR-immune cells orT
  • kits comprising one or more containers or vials (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising a (labelled) antibody or (labelled) oligonucleotide to detect an Hpgds gene expression product.
  • the (labelled) antibody or (labelled) oligonucleotide is in one embodiment adapted for multiplex in situ analysis or detection (see sections on spatial proteomics/transcriptomics).
  • Further (labeled) antibodies or (labeled) oligonucleotides can be comprised in these kits, e.g. targeting a TAM-specific gene or gene product, e.g. CD206 or CD163 and/or e.g. targeting a CD8+ T-cell specific gene or gene product (e.g. CD8a and/or CD8b).
  • the kit is a kit for use in performing the above-described methods of selecting eligibility of a subject having a tumor or cancer to be treated with an inhibitor of PTGDR1 or DPI, a (population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) (substantially) lacking functional PTGDR1 or DPI, a (population of) isolated PTGDR1 or DPI knock-out immune cells (or CAR- immune cells or TCR-immune cells), or a (population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) conditionally expressing an inhibitor of PTGDR1 or DPI; when referring herein to immune cells these are CD8+ T-cells and/or macrophages.
  • kits include one or more (further) diagnostic agents capable of predicting, prognosing, or determining the success of a therapy comprising one of the therapies according to the current disclosure; use instructions; one or more containers with sterile pharmaceutically acceptable carriers, excipients or diluents; one or more syringes; one or more needles; etc.
  • kits may be diagnostic kits or companion diagnostic kits.
  • kits comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising an inhibitor of PTGDR1 or DPI, a (population of) isolated immune cells (or CAR-immune cells or TCR- immune cells) (substantially) lacking functional PTGDR1 or DPI, a (population of) isolated PTGDR1 or DPI knock-out immune cells (or CAR-immune cells or TCR-immune cells), or a (population of) isolated immune cells (or CAR-immune cells orTCR-immune cells) conditionally expressing an inhibitor of PTGDR1 or DPlas described hereinabove, or comprising a composition comprising any one of these; when referring herein to immune cells these are CD8+ T-cells and/or macrophages.
  • kits include e.g. a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising a further anti-tumor or anti-cancer agent (such as e.g. an immune checkpoint inhibitor).
  • Further optional components of such kit include use instructions (such as e.g. kit insert approved by regulatory instance such as FDA or EMEA); one or more containers with sterile pharmaceutically acceptable carriers, excipients or diluents [such as for producing or formulating a (pharmaceutically acceptable) composition of the current disclosure]; one or more syringes; one or more needles; etc.
  • kit may be pharmaceutical kits.
  • Standards or controls for the expression level, or a reference, standard or control expression level (at transcriptomic level or at proteomic level) of a biomarker gene as listed above can be defined in some alternative ways.
  • such reference, standard or control expression level refers to a pre-determined range of expression levels/standard values. Typically such ranges are defined after collecting a set of expression levels of a gene of interest as determined in a suitable number of cancer patients.
  • the expression level of a gene of interest is determined by normalization relative to expression of e.g. a housekeeping gene or set of housekeeping genes.
  • Any method, diagnostic kit or device designed to operate according to any of the above-listed methods of the current disclosure therefore may include the option/possibility to determine, assess, measure, quantify expression of one or more household genes in addition to the means to determine, assess, measure, quantify expression of a gene of interest.
  • antagonist or "inhibitor” of a target as used interchangeable herein refers to inhibitors of function or to inhibitors of expression of a target of interest. Interchangeable alternatives for “antagonist” include inhibitor, repressor, suppressor, inactivator, and blocker. An “antagonist” thus refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with target expression, activation, function, or activity.
  • Downregulating of expression of a gene encoding a target is feasible through antagonists including entities such as antisense oligonucleotides, gapmers, siRNA, shRNA, zinc-finger nucleases, meganucleases, TAL effector nucleases, CRISPR-Cas effectors, etc. (general description of these compounds included hereinafter).
  • Inactivation or inhibition of a process as envisaged in the current disclosure refers to different possible levels of inactivation or inhibition, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even 100% or more if inactivation or inhibition (compared to a normal situation or compared to the situation prior to starting the inactivation or inhibition).
  • the nature of the inactivating/inhibitory compound is not vital/essential to the invention as long as the process envisaged is inactivated/inhibited such as to treat or inhibit (progression of) the disease or disorder as described herein.
  • agents include entities such as antisense oligonucleotides, gapmers, siRNA, shRNA, zinc-finger nucleases, meganucleases, Argonaute, TAL effector nucleases, CRISPR-Cas effectors, and nucleic acid aptamers.
  • any of these agents is specifically, selectively, or exclusively acting on or antagonizing the target of interest; or any of these agents is designed for specifically, selectively, or exclusively acting on or antagonizing the target of interest.
  • the target of interest in particular is PTGDR1 or DPI.
  • ASO antisense oligonucleotides
  • An antisense oligonucleotide (ASO) is a short strand of nucleotides and/or nucleotide analogues that hybridizes with the complementary mRNA in a sequence-specific or -selective manner. Formation of the ASO-mRNA complex ultimately results in downregulation of target protein expression (Chan et al. 2006, Clin Exp Pharmacol Physiol 33:533-540; this reference also describes some of the software available for assisting in design of ASOs).
  • Modifications to ASOs can be introduced at one or more levels: phosphate linkage modification (e.g. introduction of one or more of phosphodiester, phosphoramidate or phosphorothioate bonds), sugar modification (e.g. introduction of one or more of LNA (locked nucleic acids), 2'-O-methyl, 2'-O-methoxy-ethyl, 2' -fluoro, S-constrained ethyl or tricyclo-DNA and/or non-ribose modifications (e.g. introduction of one or more of phosphorodiamidate morpholinos or peptide nucleic acids).
  • phosphate linkage modification e.g. introduction of one or more of phosphodiester, phosphoramidate or phosphorothioate bonds
  • sugar modification e.g. introduction of one or more of LNA (locked nucleic acids)
  • 2'-O-methyl, 2'-O-methoxy-ethyl, 2' -fluoro S-
  • a gapmer antisense oligonucleotide consists of a central DNA region (usually a minimum of 7 or 8 nucleotides) with (usually 2 or 3) 2'-modified nucleosides flanking both ends of the central DNA region.
  • RNAseH RNAseH
  • Antidote strategies are available as demonstrated by administration of an oligonucleotide fully complementary to the antisense oligonucleotide (Crosby et al. 2015, Nucleic Acid Ther 25:297-305). Uptake of oligonucleotides by cells can be spontaneous or be assisted by e.g. transfection etc..
  • RNA interference Another process to selectively modulate expression of a gene/target gene of interest is based on the natural process of RNA interference. It relies on double-stranded RNA (dsRNA) that is cut by an enzyme called Dicer, resulting in double stranded small interfering RNA (siRNA) molecules which are 20-25 nucleotides long. siRNA then binds to the cellular RNA-lnduced Silencing Complex (RISC) separating the two strands into the passenger and guide strand. While the passenger strand is degraded, RISC is cleaving mRNA specifically or selectively at a site instructed by the guide strand. Destruction of the mRNA prevents production of the protein of interest and the gene is 'silenced'.
  • dsRNA double-stranded RNA
  • siRNA double stranded small interfering RNA
  • siRNAs are dsRNAs with 2 nt 3' end overhangs whereas shRNAs are dsRNAs that contains a loop structure that is processed to siRNA.
  • shRNAs are introduced into the nuclei of target cells using a vector (e.g. bacterial or viral) that optionally can stably integrate into the genome.
  • a vector e.g. bacterial or viral
  • manufacturers of RNAi products provide guidelines for designing siRNA/shRNA.
  • siRNA sequences between 19-29 nt are generally the most effective. Sequences longer than 30 nt can result in nonspecific silencing. Ideal sites to target include AA dinucleotides and the 19 nt 3' of them in the target mRNA sequence.
  • siRNAs with 3' dUdU or dTdT dinucleotide overhangs are more effective. Other dinucleotide overhangs could maintain activity but GG overhangs should be avoided. Also to be avoided are siRNA designs with a 4-6 poly(T) tract (acting as a termination signal for RNA pol III), and the G/C content is advised to be between 35-55%.
  • shRNAs should comprise sense and antisense sequences (advised to each be 19-21 nt in length) separated by loop structure, and a 3' AAAA overhang. Effective loop structures are suggested to be 3-9 nt in length.
  • shRNAs are usually transcribed from vectors, e.g. driven by the Pol III U6 promoter or Hl promoter.
  • Vectors allow for inducible shRNA expression, e.g. relying on the Tet-on and Tet-off inducible systems commercially available, or on a modified U6 promoter that is induced by the insect hormone ecdysone.
  • a Cre-Lox recombination system has been used to achieve controlled expression in mice.
  • Synthetic shRNAs can be chemically modified to affect their activity and stability.
  • Plasmid DNA or dsRNA can be delivered to a cell by means of transfection (lipid transfection, cationic polymer-based nanoparticles, lipid or cellpenetrating peptide conjugation) or electroporation.
  • Vectors include viral vectors such as lentiviral, retroviral, adenoviral and adeno-associated viral vectors.
  • Ribozymes ribonucleic acid enzymes
  • They are RNA molecules capable of catalyzing specific biochemical reactions, in the current context capable of targeted cleavage of nucleotide sequences, in particular targeted cleavage of a RNA/RNA target of interest.
  • ribozymes examples include the hammerhead ribozyme, the Varkud Satellite ribozyme, Leadzyme and the hairpin ribozyme.
  • modulation of expression of a gene of interest can be achieved at DNA level such as by gene therapy to knock-out, knock-down or disrupt the target gene/gene of interest.
  • a "gene knock-out" can be a gene knockdown or the gene can be knocked out, knocked down, disrupted or modified by a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques such as described hereafter, including, but not limited to, retroviral gene transfer.
  • Zinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target a desired DNA sequence/DNA sequence of interest, which enable zinc-finger nucleases to target unique sequence within a complex genome. By taking advantage of the endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.
  • a TALEN® is composed of a TALE DNA binding domain for sequence-specific or sequence-selective recognition fused to the catalytic domain of an endonuclease that introduces double strand breaks (DSB).
  • DSB double strand breaks
  • the DNA binding domain of a TALEN® is capable of targeting with high precision a large recognition site (for instance 17bp).
  • Meganucleases are sequence-specific or sequence-selective endonucleases, naturally occurring "DNA scissors", originating from a variety of single-celled organisms such as bacteria, yeast, algae and some plant organelles. Meganucleases have long recognition sites of between 12 and 30 base pairs. The recognition site of natural meganucleases can be modified in order to target native genomic DNA sequences (such as endogenous genes) or DNA sequences of interest. Another recent genome editing technology is the CRISPR/Cas system, which can be used to achieve RNA- guided genome engineering (including knock-out, knock-down or disruption of a gene of interest).
  • CRISPR interference is a genetic technique which allows for sequence-specific or sequence-selective control of expression of a gene of interest in prokaryotic and eukaryotic cells. It is based on the bacterial immune system-derived CRISPR (clustered regularly interspaced palindromic repeats) pathway. Recently, it was demonstrated that the CRISPR-Cas editing system can also be used to target RNA. It has been shown that the Class 2 type Vl-A CRISPR-Cas effector C2c2 (Casl3a; CRISPR-Casl3a or CRISPR-C2c2) can be programmed to cleave single stranded RNA targets carrying complementary protospacers (Abudayyeh et al. 2016 Science353/science.aaf5573). C2c2 is a single-effector endoRNase mediating ssRNA cleavage once it has been guided by a single crRNA guide toward a target RNA/RNA of interest.
  • Methods for administering nucleic acid-based therapeutic modalities/agents include methods applying non-viral (DNA or RNA) or viral nucleic acids (DNA or RNA viral vectors).
  • Methods for non-viral nucleic acid administration include the injection of naked DNA (circular or linear), electroporation, the gene gun, sonoporation, magnetofection, the use of oligonucleotides, lipoplexes (e.g. complexes of nucleic acid with DOTAP or DOPE or combinations thereof, complexes with other cationic lipids), dendrimers, viral- like particles, inorganic nanoparticles, hydrodynamic delivery, photochemical internalization (Berg et al. 2010, Methods Mol Biol 635:133-145) or combinations thereof.
  • adenovirus or adeno-associated virus vectors retrovirus vectors , naked or plasmid DNA, and lentivirus vectors. Combinations are also possible, e.g. naked or plasmid DNA combined with adenovirus, or RNA combined with naked or plasmid DNA to list just a few.
  • Other viruses e.g. alphaviruses, vaccinia viruses such as vaccinia virus Ankara
  • alphaviruses, vaccinia viruses such as vaccinia virus Ankara
  • nucleic acid e.g. in liposomes (lipoplexes) or polymersomes (synthetic variants of liposomes), as polyplexes (nucleic acid complexed with polymers), carried on dendrimers, in inorganic (nano)particles (e.g. containing iron oxide in case of magnetofection), or combined with a cell penetrating peptide (CPP) to increase cellular uptake.
  • Organ- or cellular-targeting strategies may also be applied to the nucleic acid (nucleic acid combined with organ- or cell-targeting moiety); these include passive targeting (mostly achieved by adapted formulation) or active targeting (e.g.
  • nucleic acid-comprising nanoparticle by coupling a nucleic acid-comprising nanoparticle with any compound (e.g. an aptamer or antibody or antigen binding molecule) binding to a target organ- or cell-specific antigen) (e.g. Steichen et al. 2013, Eur J Pharm Sci 48:416-427).
  • any compound e.g. an aptamer or antibody or antigen binding molecule binding to a target organ- or cell-specific antigen
  • CPPs enable translocation of their payload of interest across the plasma membrane.
  • CPPs are alternatively termed Protein Transduction Domains (TPDs), usually comprise 30 or less (e.g. 5 to 30, or 5 to 20) amino acids, and usually are rich in basic residues, and are derived from naturally occurring CPPs (usually longer than 20 amino acids), or are the result of modelling or design.
  • TPDs Protein Transduction Domains
  • CPPs include the TAT peptide (derived from HIV-1 Tat protein), penetratin (derived from Drosophila Antennapedia - Antp), pVEC (derived from murine vascular endothelial cadherin), signal-sequence based peptides or membrane translocating sequences, model amphipathic peptide (MAP), transportan, MPG, polyarginines; more information on these peptides can be found in Torchilin 2008 (Adv Drug Deliv Rev 60:548-558) and references cited therein.
  • CPPs can be coupled to carriers such as nanoparticles, liposomes, micelles, or generally any hydrophobic particle.
  • Coupling can be by absorption or chemical bonding, such as via a spacer between the CPP and the carrier.
  • an antibody binding to a target-specific antigen can further be coupled to the carrier (Torchilin 2008, Adv Drug Deliv Rev 60:548-558).
  • CPPs have already been used to deliver payloads as diverse as plasmid DNA, oligonucleotides, siRNA, peptide nucleic acids (PNA), proteins and peptides, small molecules and nanoparticles inside the cell (Stalmans et al. 2013, PloS One 8:e71752).
  • any other modification of the DNA or RNA to enhance efficacy of nucleic acid therapy is likewise envisaged to be useful in the context of the applications as outlined herein.
  • the enhanced efficacy can reside in enhanced expression, enhanced delivery properties, enhanced stability and the like.
  • a specific or selective inhibitor of a target of interest may exert the desired level of inhibition of the target of interest with an IC50 of 1000 nM or less, with an IC50 of 500 nM or less, with an IC50 of 100 nM or less, with an IC50 of 50 nM or less, with an IC50 of 10 nM or less, with an IC50 of 1 nM or less, with an IC50 between 1 pM and InM, or with an IC50 between 0.1 pM and 10 nM.
  • Cross-inhibition of more than one target is possible; for clinical development it can e.g. be desired to be able to test an inhibitor in a suitable in vitro model or in vivo animal model before starting clinical testing with the same inhibitor in a human population, which may require the inhibitor to cross-inhibit the animal (or other non-human) target and the orthologous human target.
  • Specificity or selectivity of inhibition refers to the situation in which an inhibitor is, at a certain concentration (sufficient to inhibit the target of interest) inhibiting the target gene or protein with higher efficacy (e.g. with an at least 2-fold, 5-fold, or 10-fold lower IC50, e.g. at least 20-, 50- or 100-fold or more lower IC50) than the efficacy with which it is possibly (if at all) inhibiting other targets (targets not of interest).
  • Such specificity or selectivity of inhibition is in particular determined within the setting of the target subject (e.g. human patient, or animal model) and thus can encompass/does not exclude inhibition of (at least one) orthologous target.
  • Exclusivity of inhibition refers to the situation in which an inhibitor is inhibiting only the target of interest.
  • Specificity or selectivity of (immune) cell targeting refers to the situation in which a composition, at a certain concentration, is interacting with the intended target cell (such as binding to, or such as causing inhibition of function or expression of PTGDR1 or DPI in the intended target cell) with higher efficacy (e.g. with an at least 2-fold, 5-fold, or 10-fold higher efficacy, or e.g. with at least 20-, 50- or 100-fold higher efficacy) than the efficacy with which the composition is interacting with other cells (not intended as target cell).
  • Exclusivity of cell targeting refers to the situation in which a composition is interacting only with the intended target cell.
  • the target cell is an immune cell, a CAR-immune cell or TCR immune cell; more particularly the immune cell is a (tumor-associated) macrophage and/or a CD8+ T-cell. Treatment / therapeutically effective amount
  • therapeutic modality therapeutic agent, agent, and drug are used interchangeably herein, and likewise relate to the immune cells (in particular macrophages and CD8+ T-cells) as described herein (with inhibited function or expression of PTGDR1 or DPI or (substantially) lacking functional PTGDR1 or DPI). All refer to a therapeutically active compound, or to a therapeutically active composition (comprising one or more therapeutically active compounds).
  • Treatment refers to any rate of reduction, delaying or retardation of the progress of the disease or disorder, or a single symptom thereof, compared to the progress or expected progress of the disease or disorder, or single symptom thereof, when left untreated.
  • a therapeutic modality on its own may not result in a complete or partial response (or may even not result in any response), but may, in particular when combined with other therapeutic modalities (such as other immunosuppressants or therapeutic modalities for treating or suppressing cancer or a tumor (or possibly other disease or disorder in case of CAR-Ms, TCR-Ms, CAR-Ts, or TCR-Ts in which PTGDR1 or DPI expression or function is inhibited), contribute to a complete or partial response. More desirable, the treatment results in no/zero progress of the disease or disorder, or single symptom thereof (i.e. "inhibition” or "inhibition of progression”), or even in any rate of regression of the already developed disease or disorder, or single symptom thereof.
  • Treatment/treating can in this context be understood to be comprised within the meaning of the term “treatment/treating”. Treatment/treating also refers to achieving a significant amelioration of one or more clinical symptoms associated with a disease or disorder, or of any single symptom thereof. Depending on the situation, the significant amelioration may be scored quantitatively or qualitatively. Qualitative criteria may e.g. by patient wellbeing.
  • the significant amelioration is typically a 10% or more, a 20% or more, a 25% or more, a 30% or more, a 40% or more, a 50% or more, a 60% or more, a 70% or more, a 75% or more, a 80% or more, a 95% or more, or a 100% improvement over the situation prior to treatment.
  • the time-frame over which the improvement is evaluated will depend on the type of criteria/disease observed and can be determined by the person skilled in the art.
  • a “therapeutically effective amount” refers to an amount of a therapeutic agent to treat, inhibit or prevent a disease or disorder in a subject (such as a mammal). Efficacy in vivo can, e.g., be measured by assessing the duration of survival (e.g. overall survival), time to disease progression (TTP), response rates (e.g., complete response and partial response, stable disease), length of progression-free survival (PFS), duration of response, and/or quality of life.
  • duration of survival e.g. overall survival
  • TTP time to disease progression
  • response rates e.g., complete response and partial response, stable disease
  • PFS length of progression-free survival
  • the term "effective amount” or “therapeutically effective amount” may depend on the dosing regimen of the agent/therapeutic agent or composition comprising the agent/therapeutic agent (e.g. medicament or pharmaceutical composition).
  • the effective amount will generally depend on and/or will need adjustment to the mode of contacting or administration.
  • the effective amount of the agent or composition comprising the agent is the amount required to obtain the desired clinical outcome or therapeutic effect without causing significant or unnecessary toxic effects (often expressed as maximum tolerable dose, MTD).
  • MTD maximum tolerable dose
  • the agent or composition comprising the agent may be administered as a single dose or in multiple doses.
  • the effective amount may further vary depending on the severity of the condition that needs to be treated; this may depend on the overall health and physical condition of the subject or patient and usually the treating doctor's or physician's assessment will be required to establish what is the effective amount.
  • the effective amount may further be obtained by a combination of different types of contacting or administration.
  • the aspects and embodiments described above in general may comprise the administration of one or more therapeutic compounds to a subject (such as a mammal) in need thereof or in need of treatment.
  • a (therapeutically) effective amount of (a) therapeutic compound(s) is administered to the mammal in need thereof in order to obtain the described clinical response(s).
  • administering means any mode of contacting that results in interaction between an agent or composition comprising the agent (such as a medicament or pharmaceutical composition) and an object (e.g. cell, tissue, organ, body lumen) with which said agent or composition is contacted.
  • agent or composition comprising the agent
  • object e.g. cell, tissue, organ, body lumen
  • the interaction between the agent or composition and the object can occur starting immediately or nearly immediately with the administration of the agent or composition, can occur over an extended time period (starting immediately or nearly immediately with the administration of the agent or composition), or can be delayed relative to the time of administration of the agent or composition. More specifically the "contacting" results in delivering an effective amount of the agent or composition comprising the agent to the object.
  • antitumor, anticancer or antineoplastic agents include alkylating agents (nitrogen mustards: melphalan, cyclophosphamide, ifosfamide; nitrosoureas; alkylsulfonates; ethyleneimines; triazene; methyl hydrazines; platinum coordination complexes: cisplatin, carboplatin, oxaliplatin), antimetabolites (folate antagonists: methotrexate; purine antagonists; pyrimidine antagonists: 5-fluorouracil, cytarabibe), natural plant products (Vinca alkaloids: vincristine, vinblastine; taxanes: paclitaxel, docetaxel; epipodophyllotoxins: etoposide; camptothecins: irinotecan), natural microorganism products (antibiotics: doxorubicin, bleomycin; enzymes
  • alkylating agents nitrogen mustards: melphalan, cycl
  • antineoplastic or antitumor agents include hydroxyurea, imatinib mesylate, epirubicin, bortezomib, zoledronic acid, geftinib, leucovorin, pamidronate, and gemcitabine.
  • antitumor, anticancer or antineoplastic antibodies include rituximab, bevacizumab, ibritumomab tiuxetan, tositumomab, brentuximab vedotin, gemtuzumab ozogamicin, alemtuzumab, adecatumumab, labetuzumab, pemtumomab, oregovomab, minretumomab, farletuzumab, etaracizumab, volociximab, cetuximab, panitumumab, nimotuzumab, trastuzumab, pertuzumab, mapatumumab, denosumab, and sibrotuzumab.
  • a particular class of antitumor, anticancer or antineoplastic agents are designed to stimulate the immune system (immune checkpoint or other immunostimulating therapy). These include so-called immune checkpoint inhibitors or inhibitors of co-inhibitory receptors and include PD-1 (Programmed cell death 1) inhibitors (e.g. pembrolizumab, nivolumab, pidilizumab), PD-L1 (Programmed cell death 1 ligand) inhibitors (e.g. atezolizumab, avelumab, durvalumab), CTLA-4 (Cytotoxic T-lymphocyte associated protein 4; CD152) inhibitors (e.g.
  • ipilimumab, tremelimumab e.g. Sharon et al. 2014, Chin J Cane 33:434-444.
  • Inhibition of other co-inhibitory receptors under evaluation as antitumor, anticancer or antineoplastic agents include inhibitors of Lag-3 (lymphocyte activation gene 3), Tim-3 (T cell immunoglobulin 3) and TIG IT (T cell immunoglobulin and ITM domain) (Anderson et al. 2016, Immunity 44:989-1004).
  • Stimulation of members of the TNFR superfamily of co-receptors expressed on T-cells is also evaluated for antitumor, anticancer or antineoplastic therapy (Peggs et al. 2009, Clin Exp Immunol 157:9-19).
  • anticancer or antineoplastic agents include immune-stimulating agents such as - or neo-epitope cancer vaccines (neo-antigen or neo-epitope vaccination; based on the patient's sequencing data to look for tumor-specific mutations, thus leading to a form of personalized immunotherapy; Kaiser 2017, Science 356:112; Sahin et al. 2017, Nature 547:222-226) and some Toll-like receptor (TLR) ligands (Kaczanowska et al. 2013, J Leukoc Biol 93:847-863).
  • immune-stimulating agents such as - or neo-epitope cancer vaccines (neo-antigen or neo-epitope vaccination; based on the patient's sequencing data to look for tumor-specific mutations, thus leading to a form of personalized immunotherapy; Kaiser 2017, Science 356:112; Sahin et al. 2017, Nature 547:222-226) and some Toll-like receptor (TLR
  • anticancer or antineoplastic agents include oncolytic viruses (oncolytic virus therapy) such as employed in oncolytic virus immunotherapy (Kaufman et al. 2015, Nat Rev Drug Discov 14:642-662), any other cancer vaccine (cancer vaccine administration; Guo et al. 2013, Adv Cancer Res 119:421-475), and any other anticancer nucleic acid therapy.
  • oncolytic viruses oncolytic virus therapy
  • oncolytic virus immunotherapy Kerman et al. 2015, Nat Rev Drug Discov 14:642-662
  • cancer vaccine cancer vaccine administration; Guo et al. 2013, Adv Cancer Res 119:421-475
  • nucleic acid therapy any other anticancer nucleic acid therapy.
  • level of expression or “expression level” generally refers to the amount of an expressed gene or (bio)marker in a biological sample.
  • expression may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide modifications (e.g. alternative splicing) and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide).
  • Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications are also regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis.
  • Expressed genes include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs, long non-coding RNA, microRNA or miRNA).
  • “Increased/higher expression,” “increased/higher expression level,” “increased/higher levels,” “elevated expression,” “elevated expression levels,” or “elevated levels” refers to an increased/higher expression or to increased/higher levels of a (bio)marker in an individual relative to a suitable control or standard.
  • detection includes any means of detecting, including direct and indirect detection.
  • the term “marker” or “biomarker” as used herein refers to an indicator molecule or set of indicator molecules (e.g., predictive, diagnostic, and/or prognostic indicator), which can be detected in a sample.
  • the biomarker may be a predictive biomarker and serve as an indicator of the likelihood of sensitivity or benefit to therapeutic treatment of a patient having a particular disease or disorder (e.g., a proliferative cell disorder (e.g., cancer)) to treatment (e.g. with an inhibitor of PTGDR1 or DPI).
  • a particular disease or disorder e.g., a proliferative cell disorder (e.g., cancer)
  • treatment e.g. with an inhibitor of PTGDR1 or DPI.
  • Biomarkers in general include, but are not limited to, polynucleotides (e.g., DNA and/or RNA (e.g., mRNA)), polynucleotide copy number alterations (e.g., DNA copy numbers), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications, nucleotide substitutions, nucleotide insertions or deletions (indels)), carbohydrates, and/or glycolipid-based molecular markers.
  • a biomarker is a gene.
  • the "amount" or "level” of a biomarker, as used herein, is a detectable level in a biological sample. These can be measured by methods known to one skilled in the art and also disclosed herein.
  • transcriptome analysis or analysis of the transcriptome methodologies for determining gene expression by means of determining transcript levels, also referred to as transcriptome analysis or analysis of the transcriptome, is described in more detail. Any such gene detection or gene expression detection method is starting from an analyte nucleic acid (i.e.
  • the nucleic acid of interest (which does not necessarily need to be the whole nucleic acid of interest, parts of such nucleic acids can suffice for determining expression) and of which the amount is to be determined) and may be defined as comprising one or more steps of, for instance, a step of isolating RNA from a (biological) sample (wherein a fraction of the isolated RNA is the analyte strand); a step of reverse transcribing the RNA obtained from the biological sample into DNA; a step of amplifying the isolated DNA; and/or a step of quantifying the isolated RNA, the DNA obtained after reverse transcription, or the amplified DNA.
  • a step of isolating RNA from a (biological) sample wherein a fraction of the isolated RNA is the analyte strand
  • a step of reverse transcribing the RNA obtained from the biological sample into DNA a step of amplifying the isolated DNA
  • this quantification step can be performed concurrent with the amplification of the DNA, or is performed after the amplification of the DNA.
  • the quantification of gene expression or the determination of gene expression levels may be based on at least one of an amplification reaction, a sequencing reaction, a melting reaction, a hybridization reaction or a reverse hybridization reaction. Quantification of gene expression can further involve a normalization step, wherein levels of expression of a gene of interest are normalized to e.g. levels of expression of a housekeeping gene or of a gene of which the expression is relatively constant under different conditions.
  • the detection can comprise a step such as a nucleic acid amplification reaction, a nucleic acid sequencing reaction, a melting reaction, a hybridization reaction to a nucleic acid, or a reverse hybridization reaction to a nucleic acid, or a combination of such steps.
  • oligonucleotides can comprise besides ribonucleic acid monomers or deoxyribonucleic acid monomers: one or more modified nucleotide bases, one or more modified nucleotide sugars, one or more labelled nucleotides, one or more peptide nucleic acid monomers, one or more locked nucleic acid monomers, the backbone of such oligonucleotide can be modified, and/or non-glycosidic bonds may link two adjacent nucleotides.
  • Such oligonucleotides may further comprise a modification for attachment to a solid support, e.g., an amine-, thiol-, 3-'propanolamine or acrydite- modification of the oligonucleotide, or may comprise the addition of a homopolymeric tail (for instance an oligo(dT)-tail added enzymatically via a terminal transferase enzyme or added synthetically) to the oligonucleotide.
  • a homopolymeric tail for instance an oligo(dT)-tail added enzymatically via a terminal transferase enzyme or added synthetically
  • oligonucleotide may also comprise a hairpin structure at either end. Terminal extension of such oligonucleotide may be useful for, e.g., specifically hybridizing with another nucleic acid molecule (e.g.
  • oligonucleotides when functioning as capture probe), and/or for facilitating attachment of said oligonucleotide to a solid support, and/or for modification of said tailed oligonucleotide by an enzyme, ribozyme or DNAzyme and/or for purposes of sequencing (e.g. addition of adaptor oligonucleotides in the preparation of a nextgeneration sequencing library).
  • Such oligonucleotides may be modified in order to detect (the levels of) a target nucleotide sequence and/or to facilitate in any way such detection.
  • Such modifications include labelling with a single label, with two different labels (for instance two fluorophores or one fluorophore and one quencher), the attachment of a different 'universal' tail to two probes or primers hybridizing adjacent or in close proximity to each other with the target nucleotide sequence, the incorporation of a target-specific sequence in a hairpin oligonucleotide (for instance Molecular Beacon-type primer), the tailing of such a hairpin oligonucleotide with a 'universal' tail (for instance Sunrise-type probe and Amplifluor TM -type primer).
  • two different labels for instance two fluorophores or one fluorophore and one quencher
  • a target-specific sequence in a hairpin oligonucleotide for instance Molecular Beacon-type primer
  • a special type of hairpin oligonucleotide incorporates in the hairpin a sequence capable of hybridizing to part of the newly amplified target DNA. Amplification of the hairpin is prevented by the incorporation of a blocking non-amplifiable monomer (such as hexethylene glycol). A fluorescent signal is generated after opening of the hairpin due to hybridization of the hairpin loop with the amplified target DNA.
  • This type of hairpin oligonucleotide is known as scorpion primers (Whitcombe et al. 1999, Nat Biotechnol 17:804-807).
  • oligonucleotide is a padlock oligonucleotide (or circularizable, open circle, or C-oligonucleotide) that are used in RCA (rolling circle amplification).
  • oligonucleotides may also comprise a 3'-terminal mismatching nucleotide and/or, optionally, a 3'-proximal mismatching nucleotide, which can be particularly useful for performing polymorphism-specific PCR and LCR (ligase chain reaction) or any modification of PCR or LCR.
  • LCR ligase chain reaction
  • Such oligonucleotide may can comprise or consist of at least and/or comprise or consist of up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more contiguous nucleotides.
  • the analyte nucleic acid in particular the analyte nucleic acid of a biomarker of interest can be any type of nucleic acid, which will be dependent on the manipulation steps (such as isolation and/or purification and/or duplication, multiplication or amplification) applied to the nucleic acid of the gene of interest in the biological sample; as such it can be DNA, RNA, cDNA, may comprise modified nucleotides, or may be hybrids of DNA and/or RNA and/or modified nucleotides, and can be single- or double-stranded or may be a triplex-forming nucleic acid.
  • the artificial, man-made, non-naturally occurring oligonucleotide(s) as applied in the above detection methods can be probe(s) or a primer(s), or a combination of both.
  • a probe capable of specifically hybridizing with a target nucleic acid is an oligonucleotide mainly hybridizing to one specific nucleic acid sequence in a mixture of many different nucleic acid sequences.
  • Specific hybridization is meant to result, upon detection of the specifically formed hybrids, in a signal-to- noise ratio (wherein the signal represents specific hybridization and the noise represents unspecific hybridization) sufficiently high to enable unambiguous detection of said specific hybrids.
  • signal-to- noise ratio wherein the signal represents specific hybridization and the noise represents unspecific hybridization
  • specific hybridization allows discrimination of up to a single nucleotide mismatch between the probe and the target nucleic acids.
  • Conditions allowing specific hybridization generally are stringent but can obviously be varied depending on the complexity (size, GC-content, overall identity, etc.) of the probe(s) and/or target nucleic acid molecules. Specificity of a probe in hybridizing with a nucleic acid can be improved by introducing modified nucleotides in said probe.
  • a primer capable of directing specific amplification of a target nucleic acid is the at least one oligonucleotide in a nucleic acid amplification reaction mixture that is required to obtain specific amplification of a target nucleic acid.
  • Nucleic acid amplification can be linear or exponential and can result in an amplified single nucleic acid of a single- or double-stranded nucleic acid or can result in both strands of a double-stranded nucleic acid.
  • Specificity of a primer in directing amplification of a nucleic acid can be improved by introducing modified nucleotides in said primer.
  • a nucleotide is meant to include any naturally occurring nucleotide as well as any modified nucleotide wherein said modification can occur in the structure of the nucleotide base (modification relative to A, T, G, C, or U) and/or in the structure of the nucleotide sugar (modification relative to ribose or deoxyribose). Any of the modifications can be introduced in a nucleic acid or oligonucleotide to increase/decrease stability and/or reactivity of the nucleic acid or oligonucleotide and/or for other purposes such as labelling of the nucleic acid or oligonucleotide.
  • Modified nucleotides include phosphorothioates, alkylphosphorothioates, methylphosphonate, phosphoramidate, peptide nucleic acid monomers and locked nucleic acid monomers, cyclic nucleotides, and labelled nucleotides (i.e. nucleotides conjugated to a label which can be isotopic ( ⁇ 32>P, ⁇ 35>S, etc.) or non-isotopic (biotin, digoxigenin, phosphorescent labels, fluorescent labels, fluorescence quenching moiety, etc.)). Other modifications are described higher (see description on oligonucleotides).
  • Nucleotide acid amplification is meant to include all methods resulting in multiplication of the number of a target nucleic acid.
  • Nucleotide sequence amplification methods include the polymerase chain reaction (PCR; DNA amplification), strand displacement amplification (SDA; DNA amplification), transcription-based amplification system (TAS; RNA amplification), self-sustained sequence replication (3SR; RNA amplification), nucleic acid sequence-based amplification (NASBA; RNA amplification), transcription-mediated amplification (TMA; RNA amplification), Qbeta-replicase-mediated amplification and run-off transcription.
  • PCR polymerase chain reaction
  • SDA DNA amplification
  • TAS transcription-based amplification system
  • NASBA nucleic acid sequence-based amplification
  • TMA transcription-mediated amplification
  • Qbeta-replicase-mediated amplification Qbeta-replicase-mediated amplification and run-off transcription.
  • PCR nucleotide sequence amplification technique
  • the target DNA is exponentially amplified.
  • Many methods rely on PCR including AFLP (amplified fragment length polymorphism), IRS-PCR (interspersed repetitive sequence PCR), iPCR (inverse PCR), RAPD (rapid amplification of polymorphic DNA), RT-PCR (reverse transcription PCR) and real-time PCR.
  • RT-PCR can be performed with a single thermostable enzyme having both reverse transcriptase and DNA polymerase activity (Myers et al. 1991, Biochem 30:7661-7666).
  • a single tube-reaction with two enzymes reverse transcriptase and thermostable DNA polymerase
  • Cusi et al. 1994, Biotechniques 17:1034-1036 is possible (Cusi et al. 1994, Biotechniques 17:1034-1036).
  • Solid phases, solid matrices or solid supports on which molecules, e.g., nucleic acids, analyte nucleic acids and/or oligonucleotides as described hereinabove, may be bound (or captured, absorbed, adsorbed, linked, coated, immobilized; covalently or non-covalently) comprise beads or the wells or cups of microtiter plates, or may be in other forms, such as solid or hollow rods or pipettes, particles, e.g., from 0.1 pm to 5 mm in diameter (e.g. "latex" particles, protein particles, or any other synthetic or natural particulate material), microspheres or beads (e.g. protein A beads, magnetic beads).
  • a solid phase may be of a plastic or polymeric material such as nitrocellulose, polyvinyl chloride, polystyrene, polyamide, polyvinylidene fluoride or other synthetic polymers.
  • Other solid phases include membranes, sheets, strips, films and coatings of any porous, fibrous or bibulous material such as nylon, polyvinyl chloride or another synthetic polymer, a natural polymer (or a derivative thereof) such as cellulose (or a derivative thereof such as cellulose acetate or nitrocellulose). Fibers or slides of glass, fused silica or quartz are other examples of solid supports. Paper, e.g., diazotized paper may also be applied as solid phase.
  • molecules such as nucleic acids, analyte nucleic acids and/or oligonucleotides as described hereinabove may be bound, captured, absorbed, adsorbed, linked or coated to any solid phase suitable for use in hybridization assay (irrespective of the format, for instance capture assay, reverse hybridization assay, or dynamic allele-specific hybridization (DASH)).
  • Said molecules, such as nucleic acids, analyte nucleic acids and/or oligonucleotides as described hereinabove can be present on a solid phase in defined zones such as spots or lines.
  • Such solid phases may be incorporated in a component such as a cartridge of e.g. an assay device. Any of the solid phases described above can be developed, e.g. automatically developed in an assay device.
  • Quantification of amplified DNA can be performed concurrent with or during the amplification.
  • Techniques include real-time PCR or (semi-)quantitative polymerase chain reaction (qPCR).
  • One common method includes measurement of a non-sequence specific fluorescent dye (e.g. SYBR Green) intercalating in any double-stranded DNA.
  • Quantification of multiple amplicons with different melting points can be followed simultaneously by means of following or analyzing the melting reaction (melting curve analysis or melt curve analysis; which can be performed at high resolution, see, e.g. Wittwer et al. 2003, Clin Chem 843-860; an alternative method is denaturing gel gradient electrophoresis, DGGE; both methods were compared in e.g. Tindall et al.
  • Another common method includes measurement of sequence-specific labelled probe bound to its complementary sequence; such probe also carries a quencher and the label is only measurable upon exonucleolytic release from the probe (hydrolysis probes such as TaqMan probes) or upon hybridization with the target sequence (hairpin probes such as molecular beacons which carry an internally quenched fluorophore whose fluorescence is restored upon unfolding the hairpin).
  • hydrolysis probes such as TaqMan probes
  • hairpin probes such as molecular beacons which carry an internally quenched fluorophore whose fluorescence is restored upon unfolding the hairpin.
  • This latter method allows for multiplexing by e.g. using mixtures of probes each tagged with a different label e.g. fluorescing at a different wavelength.
  • Exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes also allow for multiplexing.
  • the hybridization-sensitive fluorescence emission of ECHO probes and the further modification of probes have made possible multicolor RNA imaging in living cells and facile detection of gene polymorphisms (Okamoto 2011, Chem Soc Rev, 40:5815-5828).
  • SAGE Serial Analysis of Gene Expression
  • MPSS Massively Parallel Signature Sequencing
  • a biological sample suspected of comprising a target nucleic acid (such as a nucleic acid of a biomarker of interest as described herein), is processed as to generate a readable signal in case the target nucleic acid is actually present in the biological sample.
  • processing may include, as described above, a step of producing an analyte nucleic acid.
  • Simple detection of a produced readable signal indicates the presence of a target or analyte nucleic acid in the biological sample.
  • the amplitude of the produced readable signal is determined, this allows for quantification of levels of a target or analyte nucleic acid as present in a biological sample.
  • the readable signal may be a signal-to-noise ratio (wherein the signal represents specific detection and the noise represents unspecific detection) of an assay optimized to yield signal-to-noise ratios sufficiently high to enable unambiguous detection and/or quantification of the target nucleic acid.
  • the noise signal, or background signal can be determined e.g. on biological samples not comprising the target or analyte nucleic acid of interest, e.g. control samples, or comprising the required reference level of the target or analyte nucleic acid of interest, e.g. reference samples.
  • Such noise or background signal may also serve as comparator value for determining an increase or decrease of the level of a target or analyte nucleic acid in the biological sample, e.g. in a biological sample taken from a subject suffering from a disease or disorder, further e.g. before start of a treatment and during treatment.
  • the readable signal may be produced with all required components in solution or may be produced with some of the required components in solution and some bound to a solid support.
  • Said signals include, e.g., fluorescent signals, (chemi)luminescent signals, phosphorescence signals, radiation signals, light or color signals, optical density signals, hybridization signals, mass spectrometric signals, spectrometric signals, chromatographic signals, electric signals, electronic signals, electrophoretic signals, real-time PCR signals, PCR signals, LCR signals, Invader-assay signals, sequencing signals (by any method such as Sanger dideoxy sequencing, pyrosequencing, 454 sequencing, single-base extension sequencing, sequencing by ligation, sequencing by synthesis, "next-generation” sequencing (NGS) (van Dijk et al.
  • an assay may be run automatically or semi-automatically in an assay device.
  • NGS is finding its way to routine clinical care (Ratner 2018, Nature Biotechnol 36:484).
  • oligonucleotide whether or not comprising one or more modified nucleotides
  • target sequence e.g. Sambrook et al. 1989. Molecular Cloning. A laboratory manual. CSHL Press.
  • SSC hybridization solution
  • SSPE SSPE
  • oligonucleotides should be hybridized at their appropriate temperature in order to attain sufficient specificity.
  • the target nucleic acid molecules are generally thermally, chemically (e.g.
  • the stringency of hybridization is influenced by conditions such as temperature, salt concentration and hybridization buffer composition.
  • High stringency conditions for hybridization include high temperature and/or low salt concentration (salts include NaCI and Na3-citrate) and/or the inclusion of formamide in the hybridization buffer and/or lowering the concentration of compounds such as SDS (detergent) in the hybridization buffer and/or exclusion of compounds such as dextran sulfate or polyethylene glycol (promoting molecular crowding) from the hybridization buffer.
  • Salts include NaCI and Na3-citrate
  • SDS detergent
  • exclusion of compounds such as dextran sulfate or polyethylene glycol (promoting molecular crowding) from the hybridization buffer.
  • Conventional hybridization conditions are described in e.g.
  • optimal hybridization for oligonucleotides of about 10 to 50 bases in length occurs approximately 5 DEG C below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched sequences to hybridize and can therefor result in reduced specificity.
  • RNA oligonucleotides with formamide (50% v/v) it is recommend to use a hybridization temperature of 68 DEG C for detection of target RNA and of 50 DEG C for detection of target DNA.
  • a high SDS hybridization solution can be utilized (Church et al. 1984, Proc Natl Acad Sci USA 81:1991-1995).
  • the specificity of hybridization can furthermore be ensured through the presence of a crosslinking moiety on the oligonucleotide (e.g. Huan et al. 2000, Biotechniques 28: 254-255; WOOO/14281).
  • Said crosslinking moiety enables covalent linking of the oligonucleotide with the target nucleotide sequence and hence allows stringent washing conditions.
  • Such a crosslinking oligonucleotide can furthermore comprise another label suitable for detection/quantification of the oligonucleotide hybridized to the target.
  • RPKM Reads Per Kilobase Million
  • FPKM Frragments Per Kilobase Million
  • RPKM was designed for single-end RNA-seq (every read corresponded to a single sequenced fragment)
  • FPKM was designed for paired-end RNA-seq.
  • paired-end RNA-seq two reads can correspond to a single fragment, or, if one read in the pair did not map, one read can correspond to a single fragment.
  • FPKM takes into account that two reads can map to one fragment (and so it doesn't count this fragment twice).
  • RNA-seq When using RNA-seq, reporting or results often is in RPKM (Reads Per Kilobase Million) or FPKM (Fragments Per Kilobase Million). Whatever metric used (another alternative for example is TPM (Transcripts Per Kilobase Million)), such metric is attempting to normalize for sequencing depth and gene length and provide a measure for quantifying transcript levels/gene expression/expression units.
  • proteomic analysis or analysis of the proteome.
  • Classical proteomic analysis methods include ELISA, western blotting, mass spectrometry, chromatographic separation, immunohistochemistry, cell sorting (based on cell surface marker(s)) etc.
  • FFPE Form-Fixed Paraffin-Embedded
  • FF fresh frozen
  • Multiplexed cytometry methods as well as some predictive cancer biomarkers identified using such methodology, have been reviewed by e.g. Fan et al. 2020 (Cancer Communications 40:135-153) and have emerged with the advent of more sophisticated imaging techniques (e.g. cyclic immunofluorescence, tyramide-based immunofluorescence, epitope-targeted mass spectrometry, RNA detection) and standardized quantification methodologies.
  • imaging techniques e.g. cyclic immunofluorescence, tyramide-based immunofluorescence, epitope-targeted mass spectrometry, RNA detection
  • multiplexed cytometry methods include multiplex immunocytochemistry (mICH), imaging mass spectrometry, multiplexed ion beam imaging, chipcytometry, nucleotide (DNA/RNA)-barcoding-based mICH, and digital spacing profiling.
  • Another technique involving proteomic analysis is Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-se
  • sequencing methods may include, but are not limited to: high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), Next generation sequencing, Single Molecule Sequencing by Synthesis (SMSS) (Helicos), massively- parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxam- Gilbert or Sanger sequencing, primer walking, sequencing using PacBio, SOLID, Ion Torrent, or Nanopore platforms, short read sequencing, long read sequencing, and any other sequencing methods known in the art.
  • the sequencing method can be massively parallel sequencing, that is, simultaneously (or in rapid succession) sequencing any of at least 100, 1000, 10,000, 100,000, 1 million, 10 million, 100 million, 1 billion, or 10 billion
  • Certain DNA sequencing methods may rely on the capture of polynucleotides of interest such as to enrich for these sequences of interest.
  • Polynucleotide or sequence capture typically involves the use of oligonucleotide probes that hybridize to the polynucleotide or sequence of interest.
  • a probe set strategy can involve tiling the probes across a region of interest (complete or partial tiling of the target sequence with probes).
  • Such probes can be, e.g., 10 to 400 or about 400 bases long, 10 to 300 or about 300 bases long, 10 to 200 or about 200 bases long, 10 to 100 or about 100 bases long, 10 to 80 or about 80 bases long, 10 to 60 or about 60 bases long, Such probes may comprise at least one or a set of oligonucleotides of 10 to 60 bases or nucleotides long and/or comprise at least one or a set of oligonucleotides of 15 to 120 bases or nucleotides long.
  • any set of such oligonucleotide probes can have a depth of about O.lx, 0.2x, 0.3x, 0.4 x, 0.5x, O.lx to 0.5x, lx 2x, 3x, 4x, 5x, 6x, 8x, 9x, lOx, 15x, 20x, 50x or more.
  • Enriched nucleic acid molecules can be representative of a nucleic acid features of interest such as, but not necessarily limited to the HPGDS gene markers and macrophage markers as described herein.
  • Sequencing depth refers to the number of times a locus is covered by a sequence read aligned to the locus.
  • a locus can be as small as a nucleotide, as large as a chromosome arm, or as large as the entire genome.
  • Sequencing depth can be expressed as e.g. lOx, 50x, lOOx, where "x" refers to the number of times a locus is covered by a sequence read.
  • Sequencing depth can also be applied to multiple loci, or to the whole genome, in which case "x" can refer to the mean number of times the loci, or whole genome, is sequenced.
  • Ultra-deep sequencing refers to a sequencing depth of at least lOOx. Sequencing depth usually increases with decreasing amounts of analyte strand; the sequencing depth is therefore expected to be higher in case of analyzing cell-free DNA compared to analyzing nucleic acids isolated from a tumor biopsy.
  • Shallow whole genome sequencing, low coverage whole genome sequencing, or ultra-low pass whole genome sequencing in general refers to short-read sequencing of genomes at low coverage, typically less than 3x coverage, less than 2x coverage, less than lx coverage, such as O.lx to lx coverage, such as O.lx to 0.8x coverage, such as O.lx to 0.6x coverage, such as O.lx to 0.5x coverage, such as O.lx to 0.4x coverage, such as O.lx to 0.3x coverage, such as 0.9x coverage, 0.8x coverage, 0.7x coverage, 0.6x coverage, 0.5x coverage, 0.4x coverage, 0.3x coverage, 0.2x coverage or O.lx coverage, such as O.lx coverage or less.
  • Sequencing coverage can also be expressed as average sequencing coverage.
  • Low coverage in the context of sequencing thus can also refer to typically on average less than 3x coverage, on average less than 2x coverage, on average less than lx coverage, such as on average O.lx to lx coverage, such as on average O.lx to 0.8x coverage, such as on average O.lx to 0.6x coverage, such as on average O.lx to 0.5x coverage, such as on average O.lx to 0.4x coverage, such as on average O.lx to 0.3x coverage, such as on average 0.9x coverage, on average 0.8x coverage, on average 0.7x coverage, on average 0.6x coverage, on average 0.5x coverage, on average 0.4x coverage, on average 0.3x coverage, on average 0.2x coverage or on average O.lx coverage, such as on average O.lx coverage or less.
  • each sample is subjected to a small amount of sequencing, allowing application of whole genome sequencing to many samples at low cost per sample.
  • a sequence read is a string of nucleotides sequenced from a part or all of a nucleic acid molecule.
  • a sequence read may be a short string of nucleotides (e.g. 20 to 150 nucleotides, around 50 nucleotides) sequenced from a nucleic acid (fragment).
  • Sequence reads may be obtained at one end of a nucleic acid (fragment) or from both ends of a nucleic acid (fragment).
  • Sequence reads may be obtained by e.g. applying a sequencing technique to the nucleic acid (fragment), by hybridization arrays or capture probes, by amplification techniques (e.g. PCR, linear amplification, isothermal amplification) such as amplification techniques using a single primer.
  • amplification techniques e.g. PCR, linear amplification, isothermal amplification
  • obtaining information from the nucleic acid molecules present in a biological sample may include a step of preparing a sequencing library using the nucleic acid molecules isolated from the biological sample.
  • the preparation of such sequencing library may include a step of DNA amplification, or may, alternatively, not include a step of DNA amplification.
  • Obtaining information from the nucleic acid molecules present in a biological sample may include obtaining DNA sequence reads.
  • Obtaining information from the nucleic acid molecules (e.g. cfDNA molecules) present in a biological sample may include the step of aligning the plurality of (DNA) sequence reads to a reference genome to determine the genomic positions of each (individual) sequence read of the plurality of sequence reads. In view of the size of the reference genome and the number of sequence reads in a plurality of sequence reads, the sequence reads are optionally received at a computer system. Spatial detection methods
  • IHC staining involves binding of antibodies to target proteins of interest, usually these (primary) antibodies are unlabelled and (primary) antibodies bound to its target in e.g. a tissue section are subsequently detected by binding of a labelled, e.g. fluorescently labelled, (secondary) antibody that binds to the (primary) antibody bound to the target protein of interest.
  • mIHC multiplexed IHC
  • Non-iterative methods of target protein of interest detection involve binding of metal isotope-labelled antibodies that are subsequently detected by mass spectrometry upon release from a sample by means of tissue ablation with a laser beam (IMC: imaging mass cytometry) or tissue ionization with an ion beam (MIBI: multiplexed ion beam imaging). These techniques can detect up to 40 different proteins can be detected in a single tissue sample. IMC also allows for detection of an RNA target of interest.
  • IMC imaging mass cytometry
  • MIBI multiplexed ion beam imaging
  • DSP digital spatial profiling
  • FISH fluorescent in situ hybridization
  • LCM laser capture microdissection
  • microfluidic-based methods microfluidic-based methods - all have been reported to allow for detection of 10000 or more targets.
  • In situ methods include in situ sequencing and fluorescence in situ sequencing methods. The sequencing technique can rely on sequence-by-ligation or sequence-by- hybridization methodologies. Again, some of these methods have been reported to allow for detection of 10000 or more targets (incompletely summarized in e.g. Figure 5 of Lewis et al. 2021 (Nature Methods 18:997-1012).
  • a tumor refers to "a mass" which can be benign (more or less harmless) or malignant (cancerous).
  • a cancer is a threatening type of tumor.
  • a tumor is sometimes referred to as a neoplasm: an abnormal cell growth, usually faster compared to growth of normal cells. Benign tumors or neoplasms are nonmalignant/non-cancerous, are usually localized and usually do not spread/metastasize to other locations. Because of their size, they can affect neighboring organs and may therefore need removal and/or treatment.
  • a cancer, malignant tumor or malignant neoplasm is cancerous in nature, can metastasize, and sometimes re-occurs at the site from which it was removed (relapse).
  • the initial site where a cancer starts to develop gives rise to the primary cancer.
  • cancer cells break away from the primary cancer ("seed"), they can move (via blood or lymph fluid) to another site even remote from the initial site. If the other site allows settlement and growth of these moving cancer cells, a new cancer, called secondary cancer, can emerge (“soil").
  • the process leading to secondary cancer is also termed metastasis, and secondary cancers are also termed metastases.
  • liver cancer can arise as primary cancer, but can also be a secondary cancer originating from a primary breast cancer, bowel cancer or lung cancer; some types of cancer show an organ-specific pattern of metastasis. Most cancer deaths are in fact caused by metastases, rather than by primary tumors (Chambers et al. 2002, Nature Rev Cancer2:563-572).
  • the cancer or tumor is a primary cancer or tumor. Not mutually exclusive, the cancer or tumor more in particular is a solid cancer or tumor. More specifically, the cancer is melanoma or hepatocellular carcinoma (HCC), such as advanced HCC (aHCC).
  • HCC hepatocellular carcinoma
  • Immune checkpoints antagonists or inhibitors as referred to herein include the cell surface protein cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death protein-1 (PD-1) and their respective ligands.
  • CTLA-4 binds to its co-receptor B7-1 (CD80) or B7-2 (CD86);
  • PD-1 binds to its ligands PD-L1 (B7- H10) and PD-L2 (B7-DC).
  • immune checkpoint inhibitors include the adenosine A2A receptor (A2AR), B7-H3 (or CD276), B7-H4 (or VTCN1), BTLA (or CD272), IDO (indoleamine 2,3-10 dioxygenase), KIR (killer-cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene-3), NOX2 (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoform 2), TIM3 (T-cell immunoglobulin domain and mucin domain 3), VISTA (V-domain Ig suppressor of T cell activation), SIGLEC7 (sialic acid-binding immunoglobulin-type lectin 7, or CD328) and SIGLEC9 (sialic acid-binding immunoglobulin-type lectin 9, or CD329).
  • A2AR adenosine A2A receptor
  • B7-H3 or CD276
  • the therapy comprising an ICI or therapy with an ICI can in particular be a therapy comprising a combination in any way of two immune checkpoint inhibitors.
  • these are each inhibiting a different immune checkpoint or a different immune checkpoint-ligand interaction.
  • the second immune checkpoint inhibitor could be an inhibitor of PDL1 or an inhibitor of PDL2.
  • Such first and second immune checkpoint inhibitor are each inhibiting a different immune checkpoint protein.
  • an inhibitor of PD1 is selected as a first immune checkpoint inhibitor
  • an inhibitor different from an inhibitor of PDL1 and different from an inhibitor of PDL2 is selected, e.g. an inhibitor of CTLA-4 is selected.
  • the first and second immune checkpoint inhibitor are not only each inhibiting a different immune checkpoint, but also each inhibiting a different immune checkpoint-ligand interaction.
  • Immune checkpoint inhibitors include, but are not limited to anti-PD- 1, anti-PD-Ll or anti-CTLA-4 antibodies.
  • Aliases of PD1 provided in GeneCards® include PDCD1; Programmed Cell Death 1; Systemic Lupus Erythematosus Susceptibility 2; PD-1; CD279; HPD-1; SLEB2; and HPD-L.
  • the genomic locations for the PDCD1 gene are chr2:241, 849, 881-241, 858, 908 (in GRCh38/hg38) and chr2:242, 792, 033-242, 801, 060 (in GRCh37/hgl9).
  • GenBank reference PD1 mRNA sequence is known under accession no. NM_005018.3.
  • Approved PDl-inhibiting antibodies include nivolumab, pembrolizumab, and cemiplimab; PDl-inhibiting antibodies under development include CT-011 (pidilizumab) and therapy with PDl-inhibiting antibodies is referred to herein as a-PD-1 therapy or a-PDl therapy.
  • PD1 siRNA and shRNA products are available through e.g. Origene.
  • Aliases of PD-L1 provided in GeneCards® include CD274, Programmed Cell Death 1 Ligand 1, B7 Homolog 1, B7H1, PDL1, PDCD1 Ligand 1, PDCD1LG1, PDCD1L1, HPD-L1, B7-H1, B7-H, and Programmed Death Ligand 1.
  • the genomic locations for the PDCD1 gene are chr9:5, 450, 503-5, 470, 567 (in GRCh38/hg38) and chr9:5, 450, 503-5, 470, 567 (in GRCh37/hgl9).
  • GenBank reference PD1 mRNA sequence is known under accession no.
  • Approved PD-Ll-inhibiting antibodies include atezolizumab, avelumab, and durvalumab.
  • PD-L1 siRNA and shRNA products are available through e.g. Origene.
  • Aliases of CTLA4 provided in GeneCards® include Cytotoxic T-Lymphocyte Associated Protein 4; CTLA-4; CD152; Insulin-Dependent Diabetes Mellitus 12; Cytotoxic T-Lymphocyte Protein 4; Celiac Disease 3; GSE; Ligand And Transmembrane Spliced Cytotoxic T Lymphocyte Associated Antigen 4; Cytotoxic T Lymphocyte Associated Antigen 4 Short Spliced Form; Cytotoxic T-Lymphocyte-Associated Serine Esterase-4; Cytotoxic T-Lymphocyte-Associated Antigen 4; CELIAC3; IDDM12; ALPS5; and GRD4.
  • CTLA4 The genomic locations for the CTLA4 gene are chr2:203, 867, 771-203, 873, 965 (in GRCh38/hg38) and chr2:204, 732, 509-204, 738, 683 (in GRCh37/hgl9).
  • GenBank reference CTLA4 mRNA sequences are known under accession nos. NM_001037631.3 and NM_005214.5.
  • Approved CTLA4-inhibiting antibodies include ipilumab; CTLA4-inhibiting antibodies under development include tremelimumab; therapy with CTLA4-inhibiting antibodies is referred to herein as a-CTLA4 therapy.
  • CTLA4 siRNA and shRNA products are available through e.g. Origene.
  • “Combination”, “combination in any way” or “combination in any appropriate way” as referred to herein is meant to refer to any sequence of administration of two (or more) therapeutic modalities, i.e. the administration of the two (or more) therapeutic modalities can occur concurrently in time or separated from each other by any amount of time; and/or "combination”, “combination in any way” or “combination in any appropriate way” as referred to herein can refer to the combined or separate formulation of the two (or more) therapeutic modalities, i.e. the two (or more) therapeutic modalities can be individually provided in separate vials or (other suitable) containers, or can be provided combined in the same vial or (other suitable) container.
  • the two (or more) therapeutic modalities can each be provided in the same vial/container chamber of a single-chamber vial/container or in the same vial/container chamber of a multi-chamber vial/container; or can each be provided in a separate vial/container chamber of a multi-chamber vial/container.
  • a "subject" in general is a mammalian species having (or having been diagnosed to have) a disease or disorder, in particular a cancer or a tumor.
  • the mammalian species in general is a higher species including primates, cattle (e.g. cows, sheep, goats, pigs), horses, and pets (e.g. dogs, cats).
  • the patient is a human subject or human patient.
  • RNA-Seq RNA- sequencing datasets
  • HPGDS Hematopoietic Prostaglandin D 2 Synthase
  • HPGDS as a potential marker to identify and thus target the pro- tumoral M2-like macrophages.
  • EXAMPLE 2 Hpgds inhibition re-educates tumor-associated macrophages (TAMs) toward an Ml-like phenotype
  • Hpgds is expressed on T follicular helper (Tfh) cells but not on other CD4 + T cell subsets.
  • Tfh T follicular helper
  • Hpgds expression was nearly absent in in vitro Ml-polarized BMDMs but very high in M2-like BMDMs ( Figure 2C); HPGDS protein could not be detected in other tumor compartments or tissue macrophages from healthy organs. Strikingly, the same expression level of Hpgds observed in both, sorted TAMs and M2-like macrophages was suggestive of a pro-tumoral role of Hpgds-expressing macrophages.
  • Hpgds expression in B16 melanoma was predominant in malignant cells and alveolar cells 1, with further scattered expression in NK cells, B cells, and monocytes/macrophages (Figure 7); when checking several tumor slides by immunohistochemistry no co-localization of HPGDS with TAMs could be observed (whereas such co-localization was observed in other tumors indicating that the HPGDS antibody did work).
  • Hpgds represents an anti-inflammatory macrophage marker and its targeting can have a strong impact on the tumor progression by engaging the immune-system and impeding angiogenesis.
  • TAMs tumor-associated macrophages
  • TME tumor micro-environment
  • Hpgds expression was selectively deleted in the hematopoietic cell lineage of mice.
  • Inducible macrophage-specific Hpgds KO mice were generated by intercrossing the tamoxifen-inducible Csflr.Cre-ERT (a kind gift of J. W. Pollard from the University of Edinburgh, Scotland) with LoxP-STOP-LoxP Cas9 (LSL-CRISPR/Cas9) mice (B6J.129(B6N)- Gt(ROSA) 26Sortml(CAG-cas9*,-EGFP)Fezh/J).
  • mice lineage negative HSCs, Lin HSCs
  • hematopoietic/progenitor cells of these mice were transduced with a validated gRNA against Hpgds or with non-targeting control gRNA.
  • Lethally irradiated wild-type (WT) mice were reconstituted with either of the two transduced hematopoietic/progenitor cells (Lin HSCs), thus generating inducible Hpgds knockout (KO) WT and WT WT chimeras, respectively.
  • KO inducible Hpgds knockout
  • Hpgds genetic ablation was confirmed at the RNA level, by differentiating in vitro HSC into macrophages and in vivo at the protein level (Figure S3A).
  • YUMM 1.7 melanoma cancer cells were intradermally injected in both WT WT and Hpgds KO WT mice 5 to 6 weeks after immune reconstitution.
  • Hpgds deletion in the macrophage compartment led to a significant reduction in the tumor growth and tumor weight (Figure 3A). Consistent with our in vitro observation that Hpgds inhibition promotes an inflammatory phenotype in macrophages, the TAM compartment of Hpgds KO WT melanoma-bearing mice was strongly affected: although Hpgds knockout did not influence overall TAM infiltration (Figure 3B), tumors in Hpgds KO WT chimera mice displayed more anti-tumoral macrophages, defined as positive for MHC-II, CDllc and CD86 ( Figure 3C) and less pro-tumoral M2-like macrophages, expressing lower level of MHC-II and positive for CD206 and CD204 ( Figure 3D), suggesting that Hpgds is crucial for determining their differentiation state into M2 macrophages.
  • Hpgds floxed mice with CD64:Cre mice we generated a murine model where Hpgds is specifically depleted in macrophages (CD64-Cre;Hpgds i/i ; Hpgds-KO, HpgdsTM® or Hpgds AMa> in short).
  • the chimeric macrophage model as used herein plausibly supports use of adoptive transfer of macrophages in which HPDGDS expression is knocked-out or inhibited in the treatment of cancer.
  • Ctrl and Hpgds &M0 mice were orthotopically injected with YUMM 1.7 melanoma cancer cells and administered with aPDl when the average tumor size of both groups was 150 mm 3 .
  • Ctrl mice displayed resistance to aPDl, Hpgds deletion in macrophages, sensitized tumors to aPDl and inhibited tumor relapse in Hpgds &M0 mice ( Figure 4F and 4G).
  • EXAMPLE 5 Pharmacologic inhibition of Hpgds inhibits tumor growth and re-educates macrophages towards an Ml-like phenotype
  • HQL-79 strongly affected the tumor vasculature by decreasing vessel density and size, and by improving morphologic and functional parameters such as pericyte coverage and perfusion, together with reduced hypoxia, cancer cell intravasation into the bloodstream (Figure 5G) and lung colonization (Figure 5G).
  • Hpgds floxed mice were intercrossing with CD64:Cre-ER T2 mice (CD64- Cre-ERT2; Hpgds L/L ; Hpgds ⁇ M0 ⁇ ERT2 in short), where Hpgds was deleted in TAMs upon intraperitoneal injection of tamoxifen.
  • the effect of either systemic inhibition or acute deletion in macrophages was comparable, and the inhibitor did not have any additional effect when administered to Hpgds ⁇ M0 ⁇ ERT2 mice (Figure 5H).
  • TAS-205 (CAS No 1584160-52-0; Inchi Key NFXCKTKGAJDCBA- UHFFFAOYSA-N; Aoyagi et al. 2020, Eur J Pharmacol 875:173030), also showed a robust therapeutic effect at 30 mg/kg BID ( Figure 5J).
  • HPGDS Hepatocellular carcinoma
  • HCC Hepatocellular carcinoma
  • Pharmacological inhibition of HPGDS affected the number and the size of both, macro and micronodules ( Figure 5D-E) and led to an increased number of total macrophages (F4/80 + ) most likely due to more Ml-like macrophages ( Figure 5F).
  • melanoma tumor model we observed that HPGDS targeting resulted in a significantly higher infiltration of CD8 + T cells, whereas the total number was not affected by the treatment.
  • the monotherapy (/.e., HQL-79 or a-PD-1 alone) was not sufficient to reduce the tumor, whereas their combination showed a persistent and synergic effect (Figure 6F).
  • the combination with a-PD-1 treatment did not add anything to the effect on macrophages, nevertheless, to affect tumor progression it requires the infiltration of CD8 + T cells (Figure 6G).
  • HPGDS systemic inhibition sensitizes tumors to immunotherapy, probably by up-regulating PD-1 expression on CD8 + T cells and thus promoting immunotherapy response.
  • EXAMPLE 7 Targeting DPI expression on CD8 + T-cells and in macrophages is affecting tumor progression
  • TAMs tumor-associated macrophages
  • Downstream actions of HPGDS-PGDj axis are mediated through DPI (encoded by the PTGDR1 gene) and DP2 (encoded by the PTGDR2 gene), 2 G-protein coupled receptors.
  • DPI encoded by the PTGDR1 gene
  • DP2 encoded by the PTGDR2 gene
  • 2 G-protein coupled receptors 2 G-protein coupled receptors.
  • Hpgds deletion in tumor-associated macrophages is leading to inhibition of tumor progression and to reshaping the tumor micro-environment (TME) (Example 3). Similar effects can thus be obtained with deletion of PTGDR1 either in macrophages or in CD8+ T-cells. Plausibly, those tumors sensitive to Hpgds inhibition will likewise be sensible to DPI inhibition.
  • the chimeric macrophage model as used herein plausibly supports use of adoptive transfer of macrophages in which DPI expression is knocked-out or inhibited in the treatment of cancer.
  • adoptive transfer of CD8+ T-cells in which DPI expression is knocked-out or inhibited is herewith coming in reach as the treatment of cancer.
  • EXAMPLE 8 Targeting DPI function systemically is affecting tumor progression
  • the YUMM1.7 melanoma model was used to test whether tumor growth inhibition as obtained by knocking out expression of DPI in macrophages could be replicated by systemic inhibition of DPI function by means of the small molecule pharmacological DPI inhibitor asapiprant.
  • the results are depicted in Figure 9A and indeed indicated that systemic inhibition of DPI function was inhibiting tumor growth.
  • HCC hepatocellular carcinoma
  • Macrophage-specific Hpgds KO mice were generated by intercrossing the tamoxifen-inducible Csflr.Cre- ERT (a kind gift of J. W. Pollard from the University of Edinburgh, Scotland) with LoxP-STOP-LoxP Cas9 mice (B6J.129(B6N)- Gt(ROSA) 26Sortml(CAG-cas9*,-EGFP)Fezh/J) (Shang et al. 2020, Nature 587:626- 631), purchased from Jackson Laboratory.
  • Braf 6007 Pter 7 ' mice (a kind gift of J-C. Marine from VIB-Center of Cancer Biology, Leuven, Belgium) were generated by intercrossing Braf A , Tyr::Cre-ER 72 mice with Pten lox4 ⁇ 5 mice.
  • the Cre-mediated conversion of Braf A to Bra E and the deletion of exons 4 and 5 was obtained by topical administration of 2 mg/ml (5mM) of 4-hydroxytamoxifen (4-HT) on the back skin of adult mice.
  • the mouse line C57BL/6N-Hpgds/Tcp was made as part of the KOMP2-DTCC project from KOMP ES cells (Bradley et al. 2012, Mamm Genome 23:580-586) at The Centre for Phenogenomics. It was obtained from the Canadian Mouse Mutant Repository.
  • the NEO cassette was deleted in vivo by using FLP- mediated recombination.
  • the final transgenic line carries 2 LoxP-sites in each Hpgds allele.
  • Fcyrl-Cre mice were a gift from Bernard Malissen (French National Centre for Scientific Research, CNRS). We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders for providing the mutant mouse line C57BL/6NTac-Fcyrl lm(EGFP/Cre/ERT2)wls '/WtsiH, and the European Mouse Mutant Archive (www.infrafrontier.eu; Repository number EM: 11125) partner at the Mary Lyon Centre at MRC Harwell from which the mouse line was received.
  • CD8a-specific Ptgdr and Ptgdr2 KO were generated by intercrossing CD8a mice with LoxP-STOP-LoxP Cas9 mice (B6J.129(B6N)- Gt(ROSA) 26Sortml(CAG-cas9*,-EGFP)Fezh/J mice, generating CD8-specific Ptgdr and Ptgdr2-KO mice.
  • LoxP-STOP-LoxP Cas9 mice (B6J.129(B6N)- Gt(ROSA) 26Sortml(CAG-cas9*,-EGFP)Fezh/J mice were intercrossed with NKp46 mice (Proc Natl Acad Sci USA 108:18324-18329) to generate NK-specific Ptgdr and Ptgdr2-K0 mice.
  • mice used were females between 7-10 weeks old. Wild type C57BL/6N mice were obtained from the KU Leuven breeding facility.
  • the melanoma YUMM 1.7 cell line was a kind gift from Prof. R. Marais (Manchester, UK) and cultured in DMEM/F-12 medium (Gibco) supplemented with 10% FBS, 1% (v/v) Pen/Strep (Gibco) 2 mM glutamine (Gibco), 0.1 mM nonessential amino acid (NEAA, Gibco).
  • the /Vros-driven melanoma cell line (kindly provided by Prof. J-C. Marine, VIB-CCB KU Leuven) was generated by isolation of Tyr::Cre-ER(T2); Tyr::lnk4a-/-;R62R Confetti tumors and cultured in DMEM/F- 12 medium (Gibco) supplemented with 10% FBS, 1% (v/v) Pen/Strep (Gibco) and 2 mM glutamine (Gibco).
  • the cells were incubated at 37°C in a 5% CO2 humidified atmosphere and subcultured approximately every three days to maintain a log growth phase.
  • the KPC pancreatic pancreatic cell line (FC1245) was a kind gift from Prof. D. Tuveson and was derived from spontaneous tumors arising KPC (Kras LSLG12D/+ ; p53 R172H/+ ; Pdx: Cre Tg/+ ) pancreatic cancer mouse model.
  • KPC cells were cultured in DMEM medium (Gibco) supplemented with 10% FBS, 1% (v/v) Pen/Strep (Gibco), ImM sodium pyruvate (Gibco).
  • BMDMs Murine Bone Marrow-Derived Macrophages
  • Murine BMDMs were derived from bone marrow precursors as described before (Casazza et al. 2013, Cancer Cell 24:695-709; Bieniasz-Krzywiec et al. 2019, Cell Metab 30:917-936). Briefly, bone marrow cells were cultured in a 10 cm Petri dish (non-tissue culture treated, bacterial grade) in a volume of 6 ml of DMEM supplemented with 20% FBS and 30% L929-conditioned medium. After 3 days of culture, an additional 3 ml of differentiation medium was added. At day 7, macrophages were detached with ice cold PBS and characterized by FACS, using the pan-macrophage marker F4/80.
  • BMDMs were stimulated for additional 48h with 20 ng/ml I FN-y + 100 ng/ml LPS or lOng/ml IL-4, respectively. 9.3.2. CD8 isolation and activation
  • Murine naive T cells were isolated from the spleen by processing the cells through a 40-pm pore cell strainer in sterile PBS and cells were centrifuged for 10 min at 300x g. Red blood cell lysis buffer (Sigma- Aldrich) was used in order to lysis red blood cells.
  • CD8 + T cells were isolated by using MagniSort Mouse CD8 + T Cell Negative Selection Kit (Thermo Fisher Scientific), according to the manufacturer's instructions.
  • CD8 + T cells were cultured in T cell medium (RPMI supplemented with 10% FBS, 1% P/S, 1% MEM non- essential amino acids NEAA (Gibco), 25 pmol/L P-mercaptoethanol, and 1 mmol/L sodium pyruvate (Gibco)) at 37°C in a humidified atmosphere containing 5% CO2. If needed, T cells were activated for 3 days with CD3/CD28 Dynabeads (Thermo Fisher Scientific) at a 1:1 bead-to-cell ratio and 30 U/ml rlL-2 (PeproTech). 3 days after the activation, the beads were magnetically removed and activated CD8 + T cells were further expanded for maximum 3 days in the presence of rlL-2.
  • T cell medium RPMI supplemented with 10% FBS, 1% P/S, 1% MEM non- essential amino acids NEAA (Gibco), 25 pmol/L P-mercaptoethanol, and 1 mmol/L sodium
  • Murine NK cells were isolated from the spleen by processing the cells through a 40-pm pore cell strainer in sterile PBS and cells were centrifuged for 10 min at 300x g. Red blood cell lysis buffer (Sigma-Aldrich) was used in order to lyse red blood cells.
  • NK cells were expanded in T cell medium (RPMI supplemented with 10% FBS, 1% P/S, 1% MEM non-essential amino acids NEAA (Gibco), 25 pmol/L P-mercaptoethanol, and 1 mmol/L sodium pyruvate (Gibco)) in the presence of rlL-2.
  • FMO Fluorescence Minus One
  • Human monocytes were obtained from healthy donor buffy coats and isolated with CD14-conjugated MicroBeads (Miltenyi Biotec) as described before (Bieniasz-Krzywiec et al. 2019, Cell Metab 30:917-936). Briefly, monocytes were cultured in a 6-well plates (1x10 s cells/well in 2 ml media) in RPMI 10 % FBS, 2 mM L-Glutamine, 1% P/S and 25 ng/ml recombinant human M-CSF for five days. The third day of the differentiation, cytokines were refreshed.
  • macrophages were detached with ice cold PBS or polarized for additional 48h towards an Ml or M2 phenotype with 10 ng/ml IFN-y + 100 ng/ml LPS or 20 ng/ml IL-4, respectively.
  • HUVECs were freshly isolated from umbilical cords obtained from multiple donors (with approval from the Ethics Committee Research UZ/KU Leuven and informed consent obtained from all subjects) as previously described (Bieniasz-Krzywiec et al. 2019, Cell Metab 30:917-936; Schoors et al. 2015, Nature 520: 192-197) and regularly tested for mycoplasma. They were maintained in M199 medium (Invitrogen) supplemented with 20% FBS, 2nM glutamine, 100 U/mL penicillin, 100 pg/mL streptomycin, 0.15% heparin and 20 pg/mL ECGS (Sigma-Aldrich). 0.1% pork gelatin (Sigma-Aldrich) was used to stimulate the adhesion of HUVECs to the flask bottom.
  • mice were randomized and treated intraperitoneally (ip) with 5 mg/kg a-CD8 (BioXcell) or control IgG from rat serum.
  • mice were treated by oral gavage with HQL-79 30 mg/kg BID or control vehicle (methylcellulose). Treatment started when the average tumor reached 100 mm 3 .
  • mice were hydrodynamically cotransfected with 0.1 ug of PB_h-RasG12V and PB_c-Myc, in conjunction with a plasmid encoding a hyperactive PB transposase (Serra et al. 2022, Cell Mol Gastroenterol Hepatol 14:609-624; Tipanee et al. 2020, Mol Ther Nucleic Acids 19:1309-1329; Di Matteo et al. 2014, Mol Ther 22:1614-1624).
  • mice were treated by oral gavage with HQL-79 30 mg/kg BID or control vehicle (methylcellulose).
  • mice Treatment started 9 weeks after hydrodynamic injection. Mice were weighted at least three times per week. Mice showing symptoms of illness, losing 20% of initial body weight, displaying peritoneal leakage or ulcerated tumors were sacrificed and excluded from the experiments. At the end stage (13 weeks after hydrodynamic injection), nodules were counted as measured by using a caliper. Moreover, one lobe was collected for histological examination.
  • mice For the genetically engineered mouse metastatic melanoma model (Braf 600E Pten '), topical administration with 2 mg/ml (5mM) of 4-hydroxytamoxifen (4-HT) on the back skin of adult mice was performed. 4 weeks after tumor induction, mice were randomized and treated intraperitoneally with 10 mg/kg a-PD-1 (BioXcell), control IgG from rat serum. For the pharmacological inhibition of HPGDS, mice were treated for 13 days by oral gavage with HQL-79 30 mg/kg BID or control vehicle (methylcellulose).
  • the stop-floxed Cas9 knockin line (Platt et al. 2014, Cell 159:440-455) was intercrossed with Csflr:Cre-ERT mice allowing the inducible expression of the Cas9 nuclease in monocytes and macrophages only (donor mice). Seven-to-eight-week-old recipient mice were lethally irradiated with a dose of 9.2 Gy using the Small Animal Radiation Research Platform (SARRP, XSTRAHL). After cervical dislocation, femur, tibia and humerus were collected from donor mice of the appropriate genotype.
  • SARRP Small Animal Radiation Research Platform
  • Bone marrow was obtained by flushing the bones with a syringe filled with DMEM supplemented with 10% FBS. The cells were subsequently filtered two times by using a 40-pM- pore-sized mesh and centrifuged for 10 minutes at 300x g. BM cells were counted and resuspended 1x10 s cells/ml.
  • the EasySepTM Mouse Hematopoietic Progenitor Cell Isolation Kit (19856, STEMCELL Technologies) was used to isolate lineage-negative hematopoietic stem cells (Lin HSCs) according to the manufacturer's instructions.
  • Cells were stimulated for 4h in StemSpanTM serum-free medium (09650, STEMCELL Technologies)with 20 ng/ml IL-3, 100 ng/ml SCF, 100 ng/ml TPO and 100 ng/ml FLT-3L and then transduced with a specific gRNA of interest (targeting or, as control, non-targeting). A multiplicity of infection reaching approximately 30% of transduction was used. After a double spin filtration, cells were counted and 1x10 s cells were injected intravenously (iv) via tail vein in the irradiated recipient mice. Tumor experiments were initiated 5 to 6 weeks after immune reconstitution.
  • StemSpanTM serum-free medium 9600, STEMCELL Technologies
  • Hpgds expression in macrophages was induced by intraperitoneal injections of tamoxifen (Sigma-Aldrich, T5648) (lmg/mouse/day) for 5 consecutive days. Red and white blood cell count was determined using hematocytometer and flow cytometry on peripheral blood collected in heparin with capillary pipettes by retro-orbital bleeding. Tumor experiments were initiated 5 to 6 weeks after immune reconstitution.
  • YUMM 1.7 and /Vros-driven melanoma-bearing mice were sacrificed by cervical dislocation. Tumors were harvested and minced in aMEM medium (Lonza) supplemented with 5% FBS, 1% Pen/Strep, 50 pM p- mercaptoethanol (Gibco), 5 U/ml DNase I (Qiagen), 0.85 mg/ml Collagenase V (Collagenase from Clostridium histolyticum, Sigma-Aldrich), 1.25 mg/ml Collagenase D (Collagenase from Clostridium histolyticum, Roche) and 1 mg/ml Dispase II (Gibco) and digested for 30 minutes at 37°C. Digested tissues were filtered using first a 70-pm and then a 40-pm pore mesh strainers.
  • TCM Tumor-conditioned media
  • YUMM 1.7 tumor explanted from WT mice was minced in 12 ml DMEM/F-12 medium, supplemented with 1% Pen/Strep and incubated at 37°C for 72 hours. After that, the medium was filtered, and the cell- free supernatant was supplemented with 20mM HEPES and 2mM of L-glutamine and stored at -20°C.
  • TAF Tumor interstitial fluid
  • YUMM 1.7 tumor explanted from Hpgds WT and KO mice were cut in small pieces which were inserted in collection tubes (2 ml eppendorfs with 5 holes at the bottom in a 15 ml falcon tube). 20 to 40 pl of 9 g/l NaCI pH 7.4 was added on top of the tumor samples prior to a 10 min centrifugation at llOx g at 4°C. The interstitial fluid was collected in new vials and further used to assess lipid metabolites and mediators by using the liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) on a Nexera X2 UHPLC system (Shimadzu).
  • LC-MS/MS liquid chromatography electrospray ionization tandem mass spectrometry
  • 8xl0 4 YUMM 1.7 melanoma cancer cells were seeded on 8 pm polycarbonate membranes (Transwell; Costar).
  • the bottom chamber contained DMEM without PGD2 or with 10 nM, 100 nM, 1 pM or 10 pM PGD2, or alternatively 2xl0 5 BMDMs Hpgds-WT or Hpgds-KO.
  • the bottom chamber contained 2xl0 5 IL-4 polarized macrophages (Ctrl or Hpgds-KO).
  • YUMM 1.7 and BMDMs were cultured in DMEM F-12 with 2% FBS, 1% P/S and 1% L-Glut. Subsequently, cells were seeded and incubated for 12hr. The nonmigrated cells were removed from the top of each membrane by using a cotton stick. The migrated cells were fixed in 4% PFA, washed in PBS, stained with crystal violet (2.5 g/l), and mounted on glass slides with Pro ong Gold mounting medium without DAPL Images were acquired with Olympus BX41 or Leica DM16000 microscope and analyzed using the CellSense imaging software.
  • Ptgdrand Ptgdr2 was achieved by electroporation with specific siRNAs (commercially available). Briefly, 8xl0 6 BMDMs were resuspended in 500 pl of Opti-MEM and electroporated (250V, 950 mF, 00 O) with 100 pmol of the targeting or control siRNA of interest. After electroporation, medium was replaced with DMEM supplemented with 10% FBS, 1% Pen/Strep and 2mM Glutamine (Gibco). Upon 48h of incubation at 37°C in a 5% CO2 humidified atmosphere, qPCR and flow cytometric analysis were performed.
  • siRNAs Silencing of HPGDS was achieved by transfecting hMDMs with specific siRNAs. Briefly, 1x10 s hMDMs were transfected by using LipofectamineTM RNAiMAX Transfection Reagent (Thermo Fisher Scientific). 10 pmol of of the targering or control siRNA of interestwere resuspended in 100 pL of Opti-MEM supplemented with 2 pL of Lipofectamine and incubated for 20 minutes at RT. 6hr after lipofection, medium was replaced with RPMI supplemented with 10% FBS, 1% Pen/Strep and 2 mM Glutamine. Upon 48h of incubation at 37°C in a 5% CO2 humidified atmosphere, qPCR and flow cytometric analysis were performed.
  • BMDMs For the in vitro polarization assays, 1x10 s BMDMs were seeded 24h prior to the addition of lpM of PGD2. After 48h of stimulation, the polarization was assessed by flow cytometry. To differentiate BMDMs towards TAMs-like cells, 4xl0 6 BMDMs were seeded in 6-well plates in DMEM supplemented with 10% FBS, 1% P/S and 20% TCM for 12h at 37 °C in a 5% CO2 humidified atmosphere, as previously described. 9.13. Sprouting assay
  • Hybrid multicellular microspheres were generated by mixing HUVECS and BMDMs (wild-type or silenced for Hpgds) in a 1:1 ratio and incubated overnight in hanging drops in EGM-2 medium supplemented with 20% methylcellulose 4000 cP (Sigma-Aldrich). After harvesting the spheroids, they were embedded in Collagen I gel as described (Heiss et al. 2015, FASEB J 29:3076-3084) and maintained at 37°C for 18 hours to induce sprouting. Cultures were then fixed with 4% PFA at room temperature. Images were taken with a Leica DM16000 microscope. The CellSense imaging software was used to analyze the number of sprouts per spheroid and the total sprout length (represented by the cumulative length of primary sprouts and branches per spheroid).
  • HUVECs were pre-treated for 48hr with PGDj or with DMSO and seeded for 12hr on 8 pm polycarbonate membranes (Transwell; Costar).
  • the bottom chamber contained 200.000 BMDMs.
  • the non-migrated cells were removed from the top of each membrane by using a cotton stick.
  • the migrated cells were fixed in 4% PFA, washed in PBS, stained with crystal violet (2.5 g/l), and mounted on glass slides with Pro ong Gold mounting medium without DAPI. Images were acquired with Olympus BX41 microscope and CellSense imaging software.
  • Reverse transcription to cDNA was performed with the Superscript III and Superscript IV First Strand cDNA Synthesis System (Invitrogen) according to manufacturer's protocol.
  • cDNA, primer/probe mix and PowerUp SYBR Green Mix (Applied Biosystems) or TaqMan Fast Universal PCR Master Mix (Applied Biosystems) were prepared in a volume of 20 pl or 10 pl, according to manufacturer's instructions (Applied Biosystems) and pipetted into an optical 96-well Fast Thermal Cycling plate (Applied Biosystems). Analysis was performed by using the Quantstudio 12K Flex Real-Time PCR System (Applied Biosystems). Gene transcription was presented as number of gene mRNA copies relative to the housekeeping gene. All reactions were run in duplicate.
  • fixable viability dye eF450 or eF506
  • anti-CD45 clone 30-F11
  • anti-CDllb clone MI/70
  • anti-F4/80 BM8
  • anti-MHC-ll clone M5/114.15-12
  • anti-CD80 clone B7-1
  • anti-CD86 clone GL-1
  • anti-CDllc clone N4/18
  • anti-CD206 clone MR5D3
  • anti-CD204 clone 7C9C20
  • anti-CD274/PD-Ll clone 10F.9G2
  • anti-CD8a clone 53-6.7
  • anti-CD4 clone RM4-5
  • anti-CD69 clone H1.2F3
  • anti-CD279/PD-l clone 29F.1A12
  • anti-CD335/NKp46 clone 29A1.4
  • anti-CD69 clone
  • TNF-a tumor necrosis factor-alpha
  • IFN-y interferon-y
  • GZMB granzyme B
  • CD45 enrichment was done by following manufacturer's instructions (CD45 MicroBeads).
  • activated CD8 + T cells (at day 3 of stimulation) were counted and 800 x 10 5 cells were labelled with 3.5 pmol/L Violet Cell Tracer (Thermo Fisher Scientific) at 37°C for 20 minutes. T cells were washed with FACS buffer and cultured for 48 hours the after stimulation.
  • the extraction protocol was adapted from an already described method (Dumlao et al. 2011, Biochim Biophys Acta - Mol Cell Biol Lipids 1811:724-736). Tissue samples were mixed with methanol containing 100 picogram deuterated internal standards and 10 pL antioxidant mix (100 pM indomethacin, 0.2 mg/ml BHT, 100 pM trans-4-(-4-(3-adamantan-l-yl-ureido9-cyclohexyloxy)-benzoic acid (t-AUCB) in MeOH) and were homogenized using a Precellys system at 4°C.
  • the homogenized samples were stored at -80°C for 30 minutes and then centrifuged at 16,000g for 10 minutes at 4°C. The supernatant was transferred to a new tube and diluted with water to achieve a methanol percentage ⁇ 10%. The remaining pellet was redissolved in TBS buffer for protein concentration determination (PierceTM BCA Protein Assay, Thermo Fisher Scientific) or TE buffer for DNA concentration determination (Hoechst assay). Lipids were extracted using StrataTM-X 33 pm Polymeric Reversed Phase extraction columns (Phenomenex, 8B-S100-ECH) as instructed by the manufacturer. Briefly, the columns were preconditioned with 3 mL methanol, followed by 3 mL water.
  • the sample (containing ⁇ 10% methanol) was eluted dropwise, followed by a wash step with 3 mL of 10% methanol and elution with 100% methanol into a glass collection tube containing 6 pL of a 30% glycerol in methanol solution. Samples were evaporated in a vacuum centrifuge, redissolved in a 1:1 solution of water/methanol and transferred to an LC vial.
  • Lipid species were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with a hybrid triple quadrupole/linear ion trap mass spectrometer (Q.TRAP 6500+ system; SCIEX).
  • LC-MS/MS liquid chromatography electrospray ionization tandem mass spectrometry
  • Chromatographic separation was performed on a Polar C18 column (2.6 pm, 3.0 x 100 mm; Phenomenex) maintained at 50°C, using mobile phase A [0.1% formic acid in water] and mobile phase B [0.1% formic acid in methanol] in the following gradient: (0-2 min: 45% B; 2-16.5 min: 45% B 80% B; 16.5-18.5 min: 98% B; 18.5-20.5 min: 10% B) at a flow rate of 0.5 mL/min.
  • PBMCs Peripheral blood mononuclear cells
  • PBS PBS containing 1 mM EDTA.
  • the ring at the interface was collected, washed with PBS and counted.
  • Monocytes were isolated by using magnetic CD14- conjugated Microbeads (Miltenyi Biotec) and differentiated into human monocyte-derived macrophages (MDMs).
  • monocytes were cultured in a 6-well plates (1x10 s cells/well in 2 ml media) in RPMI 10 % FBS, 2 mM L-Glutamine, 1% P/S and 25 ng/ml recombinant human M-CSF (PeproTech) for five days. The third day of the differentiation, cytokines were refreshed. The polarization towards the Ml or the M2- like phenotype was started at day 6.
  • MDMs were polarized towards the Ml-like phenotype by adding 10 ng/ml INF-y (PeproTech) + 100 ng/ml LPS (Sigma-Aldrich) while, for the M2-like phenotype MDMs were polarized by adding 20 ng/ml IL-4 (PeproTech).
  • MO or M2-like macrophages were silenced for HPGDS and the phenotype was checked by qPCR and flow cytometry.
  • macrophages were harvested, re-suspended in FACS buffer (PBS, FBS 2% and EDTA 2mM) and incubated for 15 min at 4°C with human Fc blocking (eBioscience).
  • anti-human antibodies were used: anti-CD80 (clone L307.4), anti-HLA-DR (clone LN3), anti-CD163 (clone GHI/61), anti-CD206 (clone 19.2) and fixable viability dye (ThermoFisher, 65-0865-18). After staining, cells were washed, fixed and re-suspended in cold FACS buffer. Samples were analyzed using a LSRFortessa (BD Biosciences) flow cytometer. FMO controls were utilized in order to ensure proper gating of positive populations.
  • Murine tumors were collected and fixed in 4% paraformaldehyde (PFA) overnight at 4°C, dehydrated and embedded in paraffin.
  • Serial sections of 7-pm thickness were cut using a Microm HM360 Microtome. Human melanoma, hepatocellular carcinoma and pancreatic cancer samples were obtained as 7 pm thick paraffin-embedded slides. Paraffin slides were rehydrated to further proceed with antigen retrieval solution (Dako) for 20 minutes at 100°C. After 20 minutes of cooling down, the sections were washed with TBS and kept in methanol. If necessary, 0.3% H2O2 was added to methanol to inactivate endogenous peroxidases.
  • PFA paraformaldehyde
  • tumors were collected lh after intraperitoneal injection of 60 mg/kg pimonidazole hydrochloride into melanoma-bearing mice.
  • To detect the formation of pimonidazole adducts 7- .m sections were stained with Rabbit anti-hypoxyprobe monoclonal (Hypoxyprobe Kit, Chemicon) following the manufacturer's instructions.
  • To analyze vessel perfusion tumors were collected 15 minutes after retro-orbital injection of 0.05 mg of FITC-conjugated lectin (Lycopersicon esculentum; Vector Laboratories, B-1175-1).
  • the resulting raw count matrices were further processed using the BIOMEX browser-based software platform (vl.0.5) (Taverna et al. 2020, Nucl Acids Res 48:W385-W394) with default parameters and TMM normalization.
  • vl.0.5 BIOMEX browser-based software platform
  • a differential gene expression analysis between responsive and non-responsive tumors was performed including BRAF, NRAS and NF1 mutational status as a covariate.
  • the arachidonic acid metabolism associated ranked gene list was subsequently visualized using the R package ggplot2 (V3.1.1). 9.27. Bioinformatics
  • RNA-seq Single cell RNA-seq
  • Cutaneous, subcutaneous or lymph node metastases were biopsied before initiation of therapy (before treatment; BT). Subsequently, a second tumour biopsy was collected right before the administration of the second ICB treatment cycle (early on-treatment; OT). Response of the patients to the treatment was stratified based on RECISTvl.l. Immune cells were identified based on unsupervised clustering and differentially expressed genes per cluster.
  • the scRNA-seq data for the YUMM1.7 tumor model and melanoma patients were sourced from two GEO datasets with the following accession numbers: GSE146613, GSE115978, and GSE120575. These datasets contained a total of 4,665 cells from 6 mice and 4,861 cells from 31 patients, respectively. Further downstream analysis was performed in R v.4.2.3 using the Seurat package (v4.3.0) (Stuart et al. 2019, Cell 177:1888-1902). Cells were filtered out if they had fewer than 200 or more than 6,000 detected genes or if mitochondrial transcripts constituted more than 10% of the reads. Similarly, genes were excluded if they were detected in fewer than three cells.
  • mice were randomized when the average of the tumor volumes was 150 mm 3 and treated i.p. two times per week with 10 mg/kg aPDl or control IgG from rat serum (Sigma-Aldrich, 14131, RRID: AB_1163627).
  • mice were treated three days before tumor injection (500 pg/mouse), then two times per week (250 pg/mouse) until the end of the experiment with aCD4 (BioXCell, BE0003-1, RRID: AB_1107636). The efficiency of CD4 depletion was assessed by FACS in the blood.
  • mice were treated i.p. with 5 mg/kg aCD8 (BioXcell, BE0117, RRID: AB_10950145) or control IgG from rat serum. Treatment with aCD8 was performed three days before tumor injection, then one time per week until the end of the experiment. The efficiency of CD8 depletion was assessed by FACS in the tumor.
  • aCD8 BioXcell, BE0117, RRID: AB_10950145
  • mice were treated by oral gavage with HQL-79 (Cayman Chemical Company, 10134, RRID: AB_3662692) (15 mg/kg BID), 1,8-naphthyridine ly (in short, Cmpdly) (GSK) (0.1 - 0.3 - 1 - 3 mg/kg BID), TAS-205 MedChemExpress, HY-109134A, RRID: AB_3662693) (30 mg/kg BID) or control vehicle (methylcellulose).
  • mice were randomized when the average tumor reached 100 mm 3 and treated two times per day with the indicated compound.
  • mice For the genetically engineered mouse melanoma model, treatment with HQL-79 15 mg/kg BID, aPDl 10 mg/kg, the combination of both or with Vehicle and IgG started when the average tumor volume was 500 mm 3 ).
  • HQL-79 15 mg/kg BID For the KPC model, 6 days after tumor injection, mice were treated intraperitoneally with 10 mg/kg aPDl, control IgG from rat serum.
  • HPGDS For the pharmacological inhibition of HPGDS, mice were treated for 9 days by oral gavage with HQL-79 15 mg/kg BID or control vehicle (methylcellulose).
  • BMDMs To differentiate BMDMs towards TAM-like cells, 4xl0 6 BMDMs were seeded in 6-well plates in DMEM supplemented with 10% FBS, 1% P/S and 20% TCM for 12h at 37°C in a 5% CO 2 humidified atmosphere, as previously described.
  • BMDMs were isolated as described before. At day 6, were stimulated for 24h with 20 ng/ml IFN-y and 100 ng/ml LPS, 10 ng/ml IL-4, 10 ng/ml IL-6 or 5 ng/ml IL-10. After 24hr, IL-4 polarized macrophages were treated for an additional 24hr with 20 ng/ml IFN-y, 10 ng/ml TNFa, or 100 ng/ml LPS, alone or in combination. Unstimulated BMDMs were used as controls. Hpgds expression level was checked by qPCR.
  • T cell CM T cell-conditioned media
  • CD4 + T cells or CD8 + T cells were cultured in a 6-well plate 24hr in T cell medium (RPMI supplemented with 10% FBS, 1% P/S, 1% MEM non-essential amino acids NEAA, 25 pmol/L p- mercaptoethanol, and 1 mmol/L sodium pyruvate).
  • IL-4 polarized or Ctrl macrophages were then cultured with 250 pL T cell CM for 24hr at 37°C in a 5% CO2 humidified atmosphere.
  • macrophages were co-cultured with activated CD4 + T cell or CD8 + T cells in a ratio 1:1 and incubated 24h at 37° C in a 5% CO2 humidified atmosphere.
  • 25 pg/ml aTNFa, 20 pg/ml alFNy or Isotype Ctrl were added to the medium.
  • qPCR to assess Hpgds expression in macrophages was performed.
  • CD8 + T cells were pre-treated with 1 pM PGD2 for 4hr.
  • the bottom chamber contained T cell medium with or without 20ng/mL CXCL10 or 2xl0 5 IL-4 polarized BMDMs Hpgds- ⁇ N ⁇ or Hpgds-KO pre-treated or not with 1 pM PGD2 for 24hr.
  • CD8 + T cells and macrophages were cultured in T cell medium (RPMI supplemented with 2% FBS, 1% P/S, 1% MEM non- essential amino acids NEAA, 25 pmol/L p-mercaptoethanol, and 1 mmol/L sodium pyruvate). Subsequently, 2xl0 5 CD8 + T cells were seeded and incubated for 2hr. The migrated cells were collected and counted under the microscope.
  • Human melanoma sample was received in cold RPMI complete (10% FBS, 1% P/S) on ice and minced in 13.5 mL RPMI supplemented with 10% FBS, 0.5% P/S, 1.1 mg/ml Collagenase I (Thermo Fisher Scientific), 2.3 mg/ml Dispase II (Gibco) and 2.2 pl DNase I (Quiagen) and digested for 20 minutes at 37°C. Digested tissue was centrifuged 5 minutes at 1500 rpm, resuspended in RPMI complete and filtered using first a 100-pm and then a 40-pm pore mesh strainer to generate S2 fraction (40-100 pm) spheroid fractions.
  • WOBI Plant Eukaryotic Strand Specific mRNA

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Urology & Nephrology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Developmental Biology & Embryology (AREA)
  • General Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Food Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The disclosure relates to the medical use of inhibitors of expression or function of the prostaglandin D2 receptor 1 in the treatment of cancer in general or more in particular in the treatment of cancer in which HPGDS is predominantly expressed by tumor-associated macrophages in the tumor micro-environment. The disclosure further relates to isolated immune cells in which prostaglandin D2 receptor 1 expression or function is inhibited, such as for medical use.

Description

Inhibition of prostaglandin D2 receptor 1 as means for treating cancer
FIELD OF THE INVENTION
The disclosure relates to the medical use of inhibitors of expression or function of the prostaglandin D2 receptor 1 in the treatment of cancer in general or more in particular in the treatment of cancer in which HPGDS is predominantly expressed by tumor-associated macrophages in the tumor micro-environment. The disclosure further relates to isolated immune cells in which prostaglandin D2 receptor 1 expression or function is inhibited, such as for medical use.
BACKGROUND TO THE INVENTION
Prostaglandin D2 (PGD2; CAS No 41598-07-6), an arachidonic acid metabolite produced by hematopoietic PGD synthase (H-PGDS or HPGDS) and by lipocalin-type PGD Synthase (L-PGDS or LPGDS), is mainly interacting with two receptors: DPI (DP, PTGDR1) and DP2 (CRTH2, PTGDR2, CD294).
About 90% of PGD2 is believed to be produced through HPGDS. Hematopoietic Prostaglandin D2 signaling has been explored as a therapeutic target for treating allergic diseases including allergic asthma, rhinitis, atopic dermatitis, food allergy, gastrointestinal allergic disorder, and anaphylaxis; and several small molecule HPGDS inhibitors have been reported (reviewed in e.g. Beura and Chetti 2022, J Mol Structure 1259:132704).
The effect on tumor growth of meddling with the HPGDS - PGD2 -DP1/DP2 signaling pathway has mostly been documented for Lewis lung cancer (LLC) and B16 melanoma (B16). Growth of subcutaneous implanted LLC and B16 was reported to be promoted by knock-down or inhibition of HPGDS and to be inhibited by DPI agonism whereas DP2 knock-down or inhibition had no effect (Murata et al. 2011, PNAS 108 (49):19802-19807; WO2017/209272).
On the other-hand, in a metastasis model of either LLC or B16 (via intravenous injection of tumor cells), inhibition of metastasis was reported by knock-down or inhibition of HPGDS and by knock-down or inhibition of DP2 whereas DPI knock-down had no effect (data only for B16) (Murata et al. 2008, PNAS 105(50):20009-14; Omori et al. 2018, J Pathol 244 (l):84-96; WO2017/209272; Mary et al. 2022, Cancer Immunol Res 10:900-916).
PGD2 itself was reported to inhibit gastric cancer whereas DP2 knock-down promoted growth (CN106619651; Zhang et al. 2018, Stem Cells 36:990-1003; subcutaneous cancer model). Knock-down of HPGDS was further reported to promote intestinal adenomas (Park et al. 2007, Cancer Res 67:881), whereas treatment of leukemia with an agonist of a prostaglandin D receptor (DPI or DP2) was claimed in US9623031. Mary et al. 2022 (Cancer Immunol Res 10:900-916) reported on the possible involvement of T follicular helper (Tfh) cells in HPGDS/CRTH2-mediated tumor metastasis, and on the combination of anti-IL4 antibodies with inhibition of HPGDS.
In the setting of acute promyelocytic leukemia, Trabanelli et al. 2017 (Nat Commun 8:593) described an immunosuppressive mechanism implying in part activation of group 2 innate lymphoid cells (I LC2s) by PGD2 via CRTH2 on ILC2s, in turn activating monocytic myeloid-derived suppressor cells (M-MDSCs).
SUMMARY OF THE INVENTION
The current disclosure relates to inhibitors of prostaglandin D2 receptor 1 (DPI) for use in treating a tumor or cancer, inhibiting a tumor or cancer, or inhibiting progression of a tumor or cancer. More in particular, the tumor or cancer can be a primary tumor or cancer.
In one embodiment, the tumor or cancer micro-environment of the tumor or cancer is characterized in that the total amount of expression of HPGDS in the tumor micro-environment compartment is predominantly contributed by tumor- or cancer-associated macrophages.
In a further embodiment, the inhibitor of DPI is selectively inhibiting function or expression of DPI. In a further embodiment, the inhibitor of DPI is for use in treating a tumor or cancer, inhibiting a tumor or cancer, or inhibiting progression of a tumor or cancer in combination with a further anti-tumor or anti-cancer agent, and/or in combination with surgery or radiation.
The current disclosure further relates to isolated macrophages characterized by substantially lacking functional DPI, as well as their use as a medicament, as well as to pharmaceutical compositions comprising such macrophages.
The current disclosure further relates to isolated CD8+ T-cells characterized by substantially lacking functional DPI, as well as their use as a medicament, as well as to pharmaceutical compositions comprising such CD8+ T-cells.
The current disclosure further relates to methods for selecting a subject having cancer for therapy including an inhibitor of DPI, an isolated macrophage substantially lacking functional DPI or an isolated CD8+ T-cell substantially lacking functional DPI, comprising: assessing the expression of HPGDS in a tumor biopsy sample obtained from the subject, and selecting a subject having cancer for therapy, when the total amount of expression of HPGDS in the tumor micro-environment compartment of the tumor biopsy is predominantly contributed by or occurring in tumor- or cancer- associated macrophages. BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1. Identification of HPGDS expression on immunosuppressive TAMs
(A) HPGDS expression in the immune cells from an in-house single-cell RNA-Seq dataset of melanoma patients.
(B) Violin plot from an in house single-cell RNA-Seq dataset showing the expression of HPGDS in different macrophage subsets.
(C) Violin plot from an in house single-cell RNA-Seq dataset of HPGDS expression in Ml-and M2-like macrophages of melanoma patients.
(D) Correlation analysis of HPGDS high M2-like macrophages with activated CD8+ T cells of human melanoma tumors.
(E) Co-localization of the monocyte/macrophage-specific CD68 marker and HPGDS in primary tumor, lymph node and liver metastatic site.
P value was assessed by two sided Wilcoxon test (C) and Spearman's rank correlation test (D).
FIGURE 2. Hpgds inhibition re-educates macrophages towards an anti-tumoral Ml-like phenotype
(A) qRT-PCR analysis of the expression of Hpgds in cultured YUMM 1.7 CD90.1+ melanoma cancer cells ("YUMM (in vitro)"), or in cells sorted from a YUMM 1.7 tumor: YUMM 1.7 CD90.1+ cancer cells (CD45-, CD90.T; "YUMM (in vivo)"), CAFs (CD45 CD90.2+), ECs (CD45 CD31+), CD8+ T cells (CD45+, CDllb CD4‘ , CD8+), CD4+ T cells (CD45+, CDllb CD8‘, CD4+), TAMs (CD45+, CDllb+, F4/80+), TANs (CD45+, F4/80 Ly6G+), DCs (CD45+, F480 MHC-llhigh, CDllc+) and Mast cells (CD45+, CD117+, FceRl+).
(B) Representative images of YUMM 1.7 tumor sections stained for Hpgds and F480; CD31; CD8 or CD4. Nuclei are stained with Hoechst.
(C) qRT-PCR analysis of the expression of Hpgds in in vitro polarized BMDMs and sorted TAMs (CD45+, CDllb+, F4/80+).
(D) FACS analysis of the expression of Ml (CDllc) or M2 (CD206) polarization markers in M2-like macrophages in which siHpgds was introduced (Hpgds expression silenced), or in which a scramble siRNA was introduced (Hpgds expression not silenced).
(E) HPGDS expression assessed by qRT-PCR in human monocyte derived macrophages (hMDMs) differentiated into MO, Ml or M2-like macrophages.
(F) Expression assessed by qRT-PCR of Ml (CXCL10) and M2 (CD206) polarization markers in M2-like hMDMs in which siHpgds was introduced (Hpgds expression silenced), or in which a scramble siRNA was introduced (Hpgds expression not silenced).
(G) Intracellular abundance of PGD2 measured by LC/MS in macrophages in which siHpgds was introduced (Hpgds expression silenced), or in which a scramble siRNA was introduced (Hpgds expression not silenced). (H) FACS analysis of the expression of Ml (CD80) or M2 (CD206) polarization markers in bone marrow derived macrophages (BMDMs) treated for 48hr with 1 pM PGDj.
(I-K) Quantification of the total sprout length (I), number of sprouts (J), and representative images (K) of HUVEC spheroids embedded in collagen I ("HUVEC") and of HUVEC spheroids co-incubated with BMDMs in which siHpgds was introduced (Hpgds expression silenced; "siHpgds"), or in which a scramble siRNA was introduced (Hpgds expression not silenced; "scramble").
(L) VEGFC and VEGFA expression over time assessed by qRT-PCR in HUVECs treated with 1 pM PGDj.
(M) Quantification of HUVEC migration through BMDMs (seeded on 8 pm pore-transwell). Prior to the assay, HUVECs were incubated for 48hr with 1 pM PGDj.
P value was assessed by unpaired, two-tailed Student's t-test (D, F; G, H, M) or one-way ANOVA (C, I, J; L). All graphs show mean ± SEM.
(N) FACS analysis of the expression of Ml (CDllc) or M2 (CD206) polarization markers in MO-like macrophages in which siHpgds was introduced (Hpgds expression silenced), or in which a scramble siRNA was introduced (Hpgds expression not silenced).
(O) qRT-PCR analysis of the analysis of the expression of Ml (CXCL10) or M2 (CD206) polarization markers in human macrophages in which siHpgds was introduced (Hpgds expression silenced), or in which a scramble siRNA was introduced (Hpgds expression silenced).
(P) Arachidonic acid and thromboxane B2 abundance measured by LC/MS in scramble or siHpgds BMDMs.
(Q) qRT-PCR analysis of the expression of Lpgds (lipocalin-type prostaglandin D synthase) in cultured YUMM 1.7 CD90.1+ melanoma cancer cells ("YUMM (in vitro)"), or in cells sorted from a YUMM 1.7 tumor: YUMM 1.7 CD90.1+ cancer cells (CD45 CD90.T; "YUMM (in vivo)"), CAFs (CD45 CD90.2+), ECs (CD45‘, CD31+), CD8+ T cells (CD45+, CDllb CD4’, CD8+), CD4+ T cells (CD45+, CDllb CD8‘, CD4+), TAMs (CD45+, CDllb+, F4/80+), TANs (CD45+, F4/80 Ly6G+), DCs (CD45+, F480 MHC-llhigh, CDllc+) and Mast cells (CD45+, CD117+, FceRl+).
P value was assessed by unpaired, two-tailed Student's t-test (N-P). All graphs show mean ± SEM.
FIGURE 3. Hpgds deletion in TAMs potently inhibits tumor growth and reshapes the TME
(A) Tumor growth and tumor weight in WT->WT and Hpgds KO-> WT chimeras (see Example 3).
(B-D) FACS analysis of total TAMs (B), Ml- (MHC-llhigh- CDllc and CD86) (C) and M2- (MHC-Illow, CD206, CD204) (D) polarization markers in WT->WT and Hpgds KO-> WT chimeras (see Example 3).
(E) Quantification of F480, CD80 or CD206 staining in WT->WT and Hpgds KO-> WT chimeras (see Example 3).
(F-G) Flow cytometric quantification of CD8+T cells (F) and activated CD8+T cells (CD8+IFNy+; CD8+GZMB+) (G) in WT->WT and Hpgds KO-> WT chimeras (see Example 3). (H) Quantification of CD8+ T cells infiltration in the tumor core in WT->WT and Hpgds KO-> WT chimeras (see Example 3).
(I) qRT-PCR analysis of the expression of Hpgds in cells sorted from a YUMM 1.7 tumor: YUMM 1.7 CD90.T cancer cells (CD45 CD90.T; "YUMM (in vivo)"), CAFs (CD45 CD90.2+), ECs (CD45 CD31+), CD8+ T cells (CD45+, CDllb CD4’, CD8+), CD4+ T cells (CD45+, CDllb CD8', CD4+), TAMs (CD45+, CDllb+, F4/80+), TANs (CD45+, F4/80 Ly6G+), DCs (CD45+, F480 MHC-llhigh, CDllc+) and Mast cells (CD45+, CD117+, FceRl+) from control mice ("Ctrl"; left bars for each cell type on the X-axis) and Hpgds KO mice (right bars for each cell type on the X-axis).
P value was assessed by unpaired, two-tailed Student's t-test (A, right panel; B-G), two-way ANOVA (A, left panel) or paired, two-tailed Student's t-test (H). All graphs show mean ± SEM.
(J) Tumor growth and tumor weight of YUMM 1.7-melanoma bearing mice implanted in CD64- Cre;Hpgds+/+ or CD64-Cre;Hpgdsi/L mice.
(K-M) FACS analysis of the percentage of TAMs (CD45+, CDllb+, F480+) (K) and of the expression of different Ml (MHC-llhigh, CDllc, CD86) (L) or M2 (MHC-lllow, CD206, CD204) (M) polarization markers from CD64-Cre; Hpgds+/+ or CD64-Cre; HpgdsL/L YU M M 1.7 melanoma bearing mice.
(N) Quantification of melanoma tumors of Ctrl and Hpgds-KO sections stained for F480, CD80 (Ml- polarization marker) or CD206 (M2-polarization marker).
(O-P) FACS analysis of the percentage (O) and activation (CD69, I FNy and GZMB) (P) of CD8+ T cells in melanoma tumors of Ctrl and Hpgds-KO mice.
(Q) Quantification of vessel size (based on quantification of CD105+ cells), perfused vessels (based on quantification of lectin-FITC+ cells), pericyte blood vessel coverage (based on quantification of aSMA+ cells), and of hypoxic region in (based on quantification of PIMO+ cells) in YUMM 1.7 melanoma tumors of Ctrl and Hpgds-KO mice.
(R) Extracellular levels of PGDj, Arachidonic Acid (AA) and PGEj in the interstitial fluid of melanoma tumors of Ctrl and Hpgds-KO mice.
(S) Intratumoral concentration of PGDj measured by LC/MS in melanoma bearing mice Hpgds WT and KO.
(T), quantification of the number of YUMM 1.7 melanoma cancer cells migrated towards macrophages (Ctrl or Hpgds-KO).
(U) FACS analysis (left panel) and qRT-PCR analysis (right panel) of the percentage of CD90.1+ cells (out of viable) in the blood collected from Ctrl and Hpgds&M0 (Hpgds-KO ) YUMM 1.7-melanoma bearing mice (n = 3 for FACS; n = 5 for qRT-PCR).
P value was assessed by unpaired, two-tailed Student's t-test (J, right panel; K-N, O-S, U) or two-way ANOVA (J, left panel). All graphs show mean ± SEM. FIGURE 4. The mechanism of tumor regression mediated by Hpgds inhibition is driven by macrophages and CD8+T cells
(A-B) Tumor growth (A), tumor weight (B) of YUMM 1.7 CD90.1+ melanoma tumors injected in CD64- Cre;Hpgds+/+ ("Ctrl") mice or CD64-Cre;Hpgdsi/L ("Hpgds-KO") mice and treated with an irrelevant IgG antibody ("IgG") or anti-CD8 antibody/CD8-depleting antibody ("aCD8").
(C) Quantification of vessel size (based on quantification of CD105+ cells) and pericyte blood vessel coverage (based on quantification of aSMA+ cells) in melanoma tumors of Ctrl and Hpgds-KO mice treated with an irrelevant IgG antibody ("IgG") or anti-CD8 antibody/CD8-depleting antibody ("aCD8").
(D) FACS analysis of IFNg+ CD8+ T cells and TNFa+ CD8+ T cells upon co-culture with Ctrl or Hpgds-KO BMDMs.
(E) qRT-PCR analysis for CD90.1 in lungs collected from Ctrl and Hpgds-KO (HpgdsAM<i) melanoma-bearing mice treated with IgG or aCD8 antibody (n = 3 - 4).
(F-G) Tumor growth (F) and weight (G) from Ctrl and HpgdsAM0 tumor-bearing mice treated with IgG or aPDl when the average tumor of the group was 150 mm3 (tumors from 2 out of 13 HpgdsAM0 mice did not reach 150 mm3, thus, excluded from the experiment).
P value was assessed by unpaired, two-tailed Student's t-test (C, D), two-way ANOVA (A) , or one-way ANOVA (B), two-way repeated measures ANOVA (F), or two-way ANOVA with Turkey's multiple comparison test (E, G). All graphs show mean ± SEM.
FIGURE 5: Pharmacologic inhibition of Hpgds inhibits tumor growth and re-educates macrophages towards an Ml-like phenotype
(A) Tumor growth (left panel) and tumor weight (right panel) of YUMM 1.7 CD90.1+ melanoma tumor bearing mice treated by oral gavage with HQL-79, 30 mg/kg BID or with vehicle control.
(B) Left panel: fold change of the expression of Ml (CD80 and CXCL10) and M2 (Argl and CD206) markers in TAMs sorted from mice treated with HQL-79 versus vehicle treated mice. Right panel: Quantification of CD80 or CD206 staining in melanoma tumors of vehicle or HQL-79 treated mice.
(C) Tumor growth (left panel) and tumor weight (right panel) of NrasQ61K; lnk4A~/~ melanoma tumor bearing mice treated by oral gavage with vehicle or with HQL-79 30 mg/kg BID.
(D-E) Quantification of the number (D) and size (E) of the micro-and macro-nodules of HCC bearing mice treated by oral gavage with HQL-79 30 mg/kg BID or with vehicle.
(F) Quantification of F4/80 and CD206 staining in HCC tumors of vehicle or HQL-79 treated mice.
(G) FACS analysis (n = 6; left panel) and qRT-PCR analysis (n = 5 - 6; right panel) of YUMM 1.7 CD90.1+ cancer cell intravasation into the bloodstream (left panel) or in lungs (right panel) in melanoma-bearing mice treated by oral gavage with HQL-79 or with vehicle control (n = 6).
(H) Tumor growth (left panel) and tumor weight (right panel) of YUMM 1.7 CD90.1+ melanoma-bearing Ctrl or HpgdsAM0'ERT2 mice treated with vehicle or HQL-79 (n = 4). (I) Tumor growth (left panel) and tumor weight (right panel) of YUMM 1.7 CD90.1+ melanoma-bearing mice treated by oral gavage with Cmpdly 0.1 - 0.3 - 1 - 3 mg/kg BID or with vehicle (n = 7 - 9). Data shows a pool of two independent experiments.
(J) Tumor growth of YUMM 1.7 CD90.1+ melanoma-bearing mice treated by oral gavage with TAS-205 30 mg/kg BID or vehicle (n = 9).
(K) Calcein area of patient-derived organotypic tumor spheroids (PDOTs) treated with 1 pM HQL-79 or with vehicle control for 72hr.
P value was assessed by unpaired, two-tailed Student's t-test (A-E), one-way ANOVA (F-G), unpaired, two-tailed Student's t-test (H), two-way repeated measures ANOVA (L), multiple unpaired Student's t- test (left panel of H; left panel of I; J), or two-way ANOVA with Turkey's multiple comparison test (right panel of H), one-way ANOVA with Turkey's multiple comparison test (right panel of H). All graphs show mean ± SEM.
FIGURE 6: HPGDS systemic inhibition improves ICB efficacy.
(A) Violin plot from an in house single-cell RNA-Seq dataset showing the expression of HPGDS in melanoma patients before and during the treatment with a-PDl stratified in responders and nonresponders.
(B) Tumor volume (left panel) and tumor weight (right panel) of GEM mice Braf/600EPten/' in mice treated with vehicle as control or with HQL-79 30 mg/kg BID.
(C) FACS analysis of the expression of CDllc+ macrophages (Ml-like) in GEM mice Braf/600EPten /' treated with HQL-79 or with vehicle.
(D-E) Flow cytometric quantification of CD8+ T cells (D) and PD-1 expressing CD8+ T cells (E) in GEM mice Braf/600EPten /' treated with vehicle or with HQL-79.
(F) Tumor volume (left panel) and tumor weight (right panel) of GEM Braf/600EPten/' mice treated with vehicle, HQL-79 30 mg/kg BID, a-PDl or the combination of HQL-79 and a-PDl.
(G) Left panel: percentage of Ml- like macrophages (CDllc+) in GEM mice Braf/600EPten/' mice treated with vehicle, HQL-79, a-PDl or HQL-79 in combination with and a-PDl. Right panel: Flow cytometric quantification of CD8+ T cells (D) and PD-1 expressing CD8+ T cells (E) in GEM mice Braf''600EPten /^ treated with vehicle, HQL-79, a-PDl or HQL-79 in combination with and a-PDl.
(H) Tumor weight of KPC FC1245 PDAC-bearing mice treated with vehicle/IgG, HQL-79, aPDl, or HQL-79 in combination with aPDl (n = 11 - 16).
P value was assessed by unpaired, two-tailed Student's t-test (A-E), one-way ANOVA (F-G), or two-way repeated measures ANOVA (H). All graphs show mean ± SEM.
FIGURE 7. Expression of HPGDS in the B16 melanoma tumor micro-environment. HPGDS is predominantly expressed (visible as black dots) in malignant cells and alveolar cells 1, with further scattered expression in NK cells, B cells, and monocytes/macrophages. FIGURE 8. Tumor growth inhibition by knocking out PTGDR1 expression in CD8+ T-cells or macrophages.
(A) Tumor growth (top panel) and tumor weight (bottom panel) of YU MM 1.7 melanoma tumors injected in chimeric mice with normal CD8+ T cells (NT), Ptgdrl ("Ptgdr") KO in CD8+ T cells, or Ptgdr2 KO in CD8+ T cells.
(B) Tumor growth (top panel) and tumor weight (bottom panel) of YUMM 1.7 melanoma tumors in chimeric mice with normal macrophages cells (NT) or Ptgdrl ("Ptgdr") KO in macrophages.
P value was assessed by two-way ANOVA (A, bottom panel) or one-way ANOVA (A, top panel). All graphs show mean ± SEM.
FIGURE 9. Tumor growth inhibition by DPI inhibitor asapiprant.
(A) Tumor growth (top panel) and tumor weight (bottom panel) of YUMM 1.7 melanoma tumors left untreated ("vehicle") or treated with increasing doses of asapiprant; 10 mice per group. BID: twice a day.
(B) Quantification of the number of nodules of HCC bearing mice left untreated ("vehicle") or treated with a dose of 3 mg/kg BID asapiprant.
DETAILED DESCRIPTION
In work leading to the current disclosure, it was observed that inhibition of the expression or activity of hematopoietic prostaglandin D2 synthase (HPGDS) led to inhibition of tumor growth. As initial model, the YUMM1.7 melanoma tumor was studied. These results contradict literature reporting promotion of B16 melanoma tumor growth following inhibition of HPGDS. An explanation for these contradicting was subsequently found in the observation that HPGDS expression in the tumor micro-environment (TME) of YUMM1.7 melanoma tumors is largely restricted to tumor-associated macrophages (TAMs). HPGDS expression in the TME of B16 melanoma, however, was largely in malignant cells (and in other cells), much more than in TAMs (and no co-localization of HPGDS expression with TAMs could be observed by immunohistochemistry). A further tumor model, hepatocellular carcinoma (HCC), was also studied and shown to be sensitive to HPGDS inhibition; expression of HPGDS in the TME was likewise largely restricted to TAMs. The anti-tumoral activity of HPGDS inhibition was shown to be dependent on CD8+ T-cell infiltration. It was further found that tumors of human cancer patients not responding to immune checkpoint blockers or inhibitors (ICBs or ICIs) were characterized by maintained high expression of HPGDS in TAMs, whereas tumors of ICB-responsive patients had TAMs with low expression of HPGDS. In mouse tumor models non-responsive to either HPGDS inhibitor or ICB therapy, such HPGDS inhibition did sensitize the tumor to ICBs and synergistic tumor growth inhibition was observed. Thus, predominant expression in TAMs in the TME of a tumor is a criterion for selecting patients having cancer (e.g. naive tumor, or tumor treated with ICI but poorly or non-responding to ICI therapy) for therapy/additional therapy with an inhibitor of HPGDS. Furthermore, HPGDS expression levels in TAMs of a cancer patient responding to ICI therapy was significantly reduced on-treatment versus pre-treatment. On-treatment HPGDS expression levels in TAMs of a cancer patient not or poorly responding to ICI therapy in contrast was similar to pre-treatment HPGDS expression levels in TAMs. HPGDS expression in TAMs therefore can serve as biomarker for early response of a cancer patient to ICI therapy.
Working further on the above, it was found that PGD2 produced by TAMs is hindering anti-tumoral activity of both CD8+ T-cells and of TAMs via the DPI receptor on these immune cells. When knocking out expression of the DPI expression in either CD8+ T-cells or macrophages in a wild-type HPGDS background, potent anti-tumor activity was observed, fitting with the data observed relating to inhibition of HPGDS. Based on these data, tumors sensitive to HPGDS inhibition can alternatively be treated with inhibition of DPI such as present in CD8+ T-cells and macrophages, and CD8+ T-cells and macrophages in which DPI expression or function is knocked out or inhibited can be used in an adoptive cell transfer setting for treating such tumors or cancers. Pharmacological inhibition of DPI likewise resulted in anti- tumoral activity, thus replicating the genetic PTGDR1 knock-out data.
PTGDR(1)/DP1 and inhibition of expression or function of PTGDR(1)/DP1
The prostaglandin D2 receptor 1 is also known as DPI, PTGDR, or PTGDR1. The human PTGDR(l) gene is located on chrl4:52, 267, 698-52,280,914 (GRCh38/hg38; plus strand), on chrl4:52, 734,416-52, 743,442 (GRCh37/hgl9 by NCBI Gene; plus strand), or on chrl4:52, 734,431-52, 743,442 GRCh37/hgl9 by Ensembl; plus strand). The human DPI protein is identified by Uniprot accession No. Q13258 and secondary accession Nos. G3V5L3, Q13250, Q13251, and Q1ZZ52; by Ensembl accession Nos ENSP00000303424 and ENSP00000452408; and by NCBI reference sequences with accession Nos. XP_005267948.1, XP_054232419.1, NP_000944.1, and NP_001268398.1. The NCBI reference mRNA sequence for human PTGDR(1)/DP1 is identified by accession Nos. NM_000953.3 and NM_001281469.2. Small molecule inhibitor of DPI include laropiprant (MK0524; CAS No. 571170-77-9), BW245C (CAS No. 72814-32-5), and asapiprant (S-555739; CAS No. 932372-01-5). The experimental work outlined herein further supports that inhibition of DPI expression is obtainable by means of targeted inhibition or genetic knock-out of PTGDR(l), or by means of siRNAs targeting PTGDR(l). A dual inhibitor, inhibiting PTGDR1/DP1 and PTGDR2/DP2, is also available: vidupiprant (AMG853; CAS No. 1169483-24-2).
HPGDS and inhibition of expression or function of HPGDS
Hematopoietic prostaglandin D2 synthase or HPGDS is an enzyme (EC:5.3.99.2, EC:2.5.1.18) also known as glutathione-dependent PGD synthase, GSTS, PGDS or PTGDS2. The human HPGDS gene is located on chr4:94, 298, 535-94, 342, 876 (GRCh38/hg38; minus strand), on chr4:95, 219, 686-95, 263, 987 (GRCh37/hgl9 by NCBI Gene; minus strand), or on chr4:95, 219, 686-95, 264, 027 (GRCh37/hgl9 by Ensembl; minus strand). The human HPGDS protein is identified by UniProt accession No. 060760, Ensembl accession No. ENSP00000295256, and NCBI reference sequences with accession Nos. XP_005262989.1, XP_054205671.1, and NP_055300.1. The NCBI reference mRNA sequence for human HPGDS is identified by accession No. NM_014485.3. Several HPGDS inhibitors are known, including small molecules such as HQL-79 (CAS No 162641-16-9; e.g. Aritake et al. 2006, J Biol Chem 281:15277-15286), TFC-007 (CAS No 927878-49-7; Nabe et al. 2011, Prostaglandins & Other Lipid Mediators 95: 27-34), hPGDS inhibitor I (CAS No 1033836-12-2; Pellefigues et al. 2018, Nat Commun 9 :725), TAS-204 (Kajiwara et al. 2011, Eur J Pharmacol 667:389-395, ZL-2102 (classic.clinicaltrials.gov/ct2/show/NCT02397005), TAS-205 (TAS-205 free-base: pizuglanstat, CAS No 1244967-98-3; Takeshita et al. 2018, Ann Clin Transl Neurol 5:1338-1134), KMN-698 (Marie et al. 2018, J Allergy Clin Immunol 143:2202-2214), and SP1, SP2, SP5 and SP10 (Beura and Chetti 2022, J Mol Structure 1259:132704). A HPGDS degradation inducing proteolysis targeting chimera (PROTAC) has been reported recently by Yokoo et al. 2021 (ACS Med Chem Lett 12: 236-241). The experimental work outlined herein further supports that inhibition of HPGDS expression is obtainable by means of targeted inhibition or genetic knock-out of HPGDS in macrophages, or by means of siRNAs targeting HPGDS. Inhibitory RNAs (e.g. Origene Catalog No TL315682V, HPGDS Human shRNA Lentiviral Particle; Santa Cruz Catalog No sc-41638, siRNA; Santa Cruz Catalog No sc- 41638-SH, plasmid shRNA) and guide RNAs for use with CRISPR-Cas (e.g. ABM Catalog No 23862111as lentiviral set; ABM catalog No 23862151 as AAV set; Origene Catalog No GA109114, Human HPGDS activation kit by CRISPR) are furthermore commercially available.
Medical uses, methods and products
In view of the above, the present disclosure defines a number of aspects and embodiments thereto.
A first series of aspects relate to the medical use of inhibitors of prostaglandin D2 receptor 1 (PTGDR1) or DPI / PTGDR1 or DPI inhibitors. In general, as an alternative for "inhibitor", the terms "blocker", "antagonist", "inactivating compound", "repressor" or "suppressor" can be used.
More in particular, a first aspect of this disclosure relates to an inhibitor of PTGDR1 or DPI for use in treating a tumor or cancer, inhibiting (growth of) a tumor or cancer, or inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth. Alternatively, this aspect relates to an inhibitor of PTGDR1 or DPI for use in the manufacture of a medicament for treating a tumor or cancer, for inhibiting (growth of) a tumor or cancer, or for inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth. A further alternative relates to methods of treating a (subject having) a tumor or cancer, of inhibiting (growth of) a tumor or cancer (in a subject having a tumor or cancer), or of inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth (in a subject having a tumor or cancer), such methods including administering a (therapeutically effective dose of a) PTGDR1 or DPI inhibitor to the subject having a tumor or cancer. With this administering the tumor or cancer is treated or inhibited, or its progression is inhibited.
In one embodiment to this alternatives, the tumor or cancer is a primary tumor or cancer. In a not mutually exclusive embodiment, the tumor or cancer is a solid tumor or cancer.
In another, not mutually exclusive embodiment to this alternatives, the inhibitor of PTGDR1 or DPI is combined with a further anticancer therapy (different from a therapy including an inhibitor of PTGDR1 or DPI).
In one (further) embodiment to the above aspects and embodiments, the tumor or cancer microenvironment (TME) or TME compartment of the tumor or cancer is characterized in that HPGDS expression is limited to expression in tumor- or cancer-associated macrophages, or in that HPGDS is selectively expressed in tumor- or cancer-associated macrophages (TAMs) compared to in other cells of the TME or TME compartment. Alternatively, the TME or TME compartment of the tumor or cancer is characterized in that the total amount of expression of HPGDS in the TME or TME compartment is mainly/predominantly/primarily/quantitatively selectively contributed by tumor- or cancer-associated macrophages or occurring in tumor- or cancer-associated macrophages. Alternatively, the TME or TME compartment of the tumor or cancer is characterized in that the tumor- or cancer-associated macrophages in the TME or TME compartment are the main/predominant/primary/quantitatively selective contributors to the total amount of expression of HPGDS in the TME or TME compartment of the tumor or cancer. In further explanation hereof, it may be that HPGDS is expressed in different cell types present in the TME or TME compartment, all together constituting the total amount of expression of HPGDS in the TME or TME compartment (as could e.g. be determined by bulk RNA sequencing of nucleic acids isolated from an obtained tumor or cancer biopsy sample, or by immunohistochemistry, or by spatial expression detection techniques). In this case and in the current context, the majority of the HPGDS expression should be detected in the TAMs present in the TME or TME compartment /tumor or cancer biopsy sample (as could e.g. be determined by single cell RNA sequencing of nucleic acids isolated from an obtained tumor or cancer biopsy sample, or, at the protein level it could e.g. be determined by immunohistochemical co-localization of HPGDS protein and of a TAM-specific protein marker, e.g. CD206 or CD163; or by spatial expression detection techniques). In other words, the HPGDS expression in the TME or TME compartment is then mainly/predominantly/primarily/quantitatively selectively contributed by TAMs or occurring in TAMs. The quantification of the "majority" or the "main/predominant/primary/quantitatively selective contribution" of the HPGDS expression will depend on the tumor or cancer type (and may even differ from patient to patient within the same tumor or cancer type) and/or will depend on the number of cell types in the TME or TME compartment expressing HPGDS. In general, the "majority" or the "main/predominant/primary/quantitatively selective contribution" of the HPGDS expression in the TME or TME compartment can amount to 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or to at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the total HPGDS expression detected or determined in the TME or TME compartment; this major, main, etc. fraction is thus to be contributed or caused by, is originating from, or is occurring in a single cell type, i.e. the TAMs, present in the TME or TME compartment.
In one (further) embodiment to the above aspects and embodiments, the inhibitor of PTGDR1 or DPI is selectively inhibiting function or expression (see further) of PTGDR1 or DPI. More in particular, the inhibitor of PTGDR1 or DPI is a small molecule (e.g. laropiprant, BW245C or asapiprant; see hereinabove), a DNA nuclease specifically knocking out or disrupting PTGDR1 or DPI, an RNase specifically targeting PTGDR1 or DPI, or an inhibitory oligonucleotide specifically targeting PTGDR1 or DPI (see further).
In one (further) embodiment to the above aspects and embodiments, the inhibitor of PTGDR1 or DPI is combined with a further anti-tumor or anti-cancer agent (e.g. immune checkpoint inhibitor (comprising) therapy, or other (see hereinafter)), or is combined with surgery or radiation for purposes of treating a tumor or cancer, inhibiting (growth of) a tumor or cancer, or inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth. In particular the further anti-tumor or anti-cancer agent is an inhibitor of HPGDS. More in particular, the inhibitor of HPGDS is a small molecule (e.g. HQL-79, TFC- 007, hPGDS inhibitor I, TAS-204, ZL-2102, TAS-205, KMN-698, SP1, SP2, SP5 and SP10; see hereinabove), a HPGDS degradation inducing proteolysis targeting chimera (HPGDS-targeting PROTAC, see, e.g. Yokoo et al. 2021, ACS Med Chem Lett 12: 236-241), a DNA nuclease specifically knocking out or disrupting HPGDS, an RNase specifically targeting HPGDS, or an inhibitory oligonucleotide specifically targeting HPGDS (see further).
Functional PTGDR(1)/DP1
Functional PTGDR(l) or DPI, as referred to herein, is defined as PTGDR(l) or DPI that is expressed and to which no "foreign" (in the sense of non-naturally occurring, artificially made, man-made, or any combination thereof) compound such as pharmacological inhibitor is bound or linked, wherein the "foreign" compound is capable of interfering directly (e.g. competing) or indirectly (e.g. by inducing degradation of PTGDR(l) or DPI) with the binding of PTGDR(l) or DPI with any one of its potential natural binding partners (e.g. prostaglandin D2, PGD2). Functional PTGDR(l) or DPI may be exposed on the surface of immune cells (e.g. CD8+ T-cells, macrophages) or may be stored inside immune cells such as stored in a manner allowing quick release to the cell surface (e.g. Labrecque et al. 2013, PloS One 8:e65767).
As such, functional PTGDR(l) or DPI can be lacking, or be substantially lacking on and/or in a cell by repressing, inhibiting, or blocking expression of PTGDR(l) or DPI, or by binding of a "foreign" compound (as meant hereinabove) to PTGDR(l) or DPI. In particular, functional PTGDR(l) or DPI is lacking, or is substantially lacking, on and/or in an isolated immune cell as described herein (in particular CD8+ T-cells or macrophages).
Genetic modification of immune cells isolated from a subject is one means of forcing the immune cells to (substantially) lack functional PTGDR(l) or DPI. Such genetic modification can be aimed at repressing, reducing, or inhibiting ongoing expression of PTGDR(l) or DPI in the isolated (unmodified) immune cells, and/or can be aimed at preventing or inhibiting de novo expression of PTGDR(l) or DPI, e.g. in case expression of PTGDR(l) or DPI is low or non-existing in the isolated (unmodified) immune cells. In this context, when the isolated immune cells are expanded in vitro or ex vivo, it is understood that the genetic modification may occur prior to expansion, such as in case of stable genetic modification. When the genetic modification is relying on e.g. RNA interference mechanisms, the isolated immune cells may need to be expanded in the continuous presence of e.g. the RNA interference agent.
Shielding (part of the) PTGDR(l) or DPI protein exposed on the surface of immune cells and/or stored within immune cells by means of contacting the immune cells with a pharmacological inhibitor of PTGDR(l) or DPI is another means of causing immune cells to (substantially) lack functional PTGDR(l) or DPI. The said shielding can be envisaged as neutralizing (part of the) PTGDR(l) or DPI protein for interaction with other (natural) binding partners. In this context, when the isolated immune cells are expanded in vitro or ex vivo, it is understood that the contacting with the pharmacological inhibitor is continuous during the expansion of the immune cells, or is occurring after expansion of the immune cells. Such pharmacological inhibitors per se are known in the art, see above, and alternatives are discussed in more detail hereinafter. In particular, such pharmacological inhibitors bind to PTGDR(l) or DPI with high specificity and/or, optionally, with high affinity.
PTGDR(l) or DPI protein present inside immune cells or on the surface of immune cells can further be the target of pharmacologic knock-down such as by molecules or agents inducing specific proteolytic degradation of PTGDR(l) or DPI protein. In this context, when the isolated immune cells are expanded in vitro or ex vivo, it is understood that the contacting with the pharmacological knock-down agent is continuous during the expansion of the immune cells, or is occurring after expansion of the immune cells.
The agent causing an immune cell to (substantially) lack functional PTGDR(l) or DPI or causing neutralization of PTGDR(l) or DPI as referred to herein may be part of a larger molecule further comprising a moiety directing the agent to the immune cell. Immune cells in which PTGDR(l) or DPI expression or function is inhibited, and medical uses
When referring hereinafter to immune cells in which PTGDR(l) or DPI expression or function is inhibited or (substantially) lacking, the immune cells are CD8+ T-cells and/or macrophages.
In a further aspect, this disclosure relates to isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, isolated PTGDR1 or DPI knock-out immune cells, or to isolated immune cells conditionally expressing an inhibitor of PTGDR1 or DPI, or to populations of any thereof. Furthermore included are compositions, such a pharmaceutical compositions, comprising (a population of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, isolated PTGDR1 or DPI knockout immune cells, or comprising (a population of) isolated immune cells conditionally expressing an inhibitor of PTGDR1 or DPI.
A further aspect of this disclosure relates to (populations of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, to (populations of) isolated PTGDR1 or DPI knock-out immune cells, to (populations of) isolated immune cells conditionally expressing an inhibitor of PTGDR1 or DPI, or to pharmaceutical composition comprising any of these, for use as a medicament; more in particular, for use as a medicament for treating a cancer or tumor, for inhibiting (growth of) a cancer or tumor, or for inhibiting progression of a cancer or tumor or of progression of tumor or cancer growth. Alternatively, the (populations of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, the (populations of) isolated PTGDR1 or DPI knock-out immune cells, the (populations of) isolated immune cells conditionally expressing an inhibitor of PTGDR1 or DPI, or pharmaceutical compositions comprising any of these, are for use in the manufacture of a medicament; more in particular, for use in the manufacture of a medicament for treating a cancer or tumor, for inhibiting (growth of) a cancer or tumor, or for inhibiting progression of a cancer or tumor or of progression of tumor or cancer growth. Further alternatively, this aspect relates to methods of treating (as subject having) a cancer or tumor, inhibiting (growth of) a cancer or tumor (in a subject having a tumor or cancer), or inhibiting progression of a cancer or tumor or of progression of tumor or cancer growth (in a subject having a tumor or cancer), such methods including administering a (population of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, a (population of) isolated PTGDR1 or DPI knock-out immune cells, a (population of) isolated immune cells conditionally expressing an inhibitor of PTGDR1 or DPI, or a pharmaceutical composition comprising any of these, to a subject having a cancer or tumor. In particular, a therapeutically effective amount of such (populations of) immune cells or a therapeutically effective amount of such pharmaceutical composition is administered to the subject. Further in particular, with this administering, the tumor or cancer is treated or inhibited, or its progression is inhibited. In particular to this aspect, such immune cells are for use in a treatment comprising transfer or adoptive transfer of the immune cells to a subject.
Any of the isolated macrophage or CD8+ T-cells as described above (thus at least modified to (substantially) lack functional PTGDR1 or DPI), any of the populations of such macrophage or CD8+ T- cells, or any of the pharmaceutical compositions comprising any such isolated macrophage or CD8+ T- cell or comprising any such population of isolated macrophage or CD8+ T-cells, is suitable for any of: (i) for use as medicament, (ii) for use in (a method of) adoptive cell therapy, (iii) for use in (a method of) treating, inhibiting, or suppressing a tumor or cancer; or for any of (iv) use in the manufacture of a medicament, (v) use in the manufacture of a medicament for adoptive cell therapy, or (vi) use in the manufacture of a medicament for treating, inhibiting, or suppressing a tumor or cancer.
When for use in treating, inhibiting, or suppressing a tumor or cancer, (i) any of the isolated macrophage or CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), (ii) any of the populations of such macrophage or CD8+ T-cells, or (iii) any of the pharmaceutical compositions comprising any such isolated macrophage or CD8+ T-cells or comprising any such population of isolated macrophage or CD8+ T-cells, may further be for use in combination with surgery, radiation, chemotherapy, targeted therapy, immunotherapy, or a further anticancer agent.
Any of the isolated macrophage or CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), (ii) any of the populations of such macrophage or CD8+ T-cells, or (iii) any of the pharmaceutical compositions comprising any such isolated macrophage or CD8+ T-cells or comprising any such population of isolated macrophage or CD8+ T-cells may also be used in (i) the manufacture of a medicament for use in combination with surgery, radiation, chemotherapy, targeted therapy, immunotherapy, or an anticancer agent, (ii) in the manufacture of a medicament for adoptive cell therapy for use in combination with surgery, radiation, chemotherapy, targeted therapy, immunotherapy, or an anticancer agent, or (iii) in the manufacture of a medicament for treating, inhibiting, or suppressing a tumor or cancer for use in combination with surgery, radiation, chemotherapy, targeted therapy, immunotherapy, or an anticancer agent.
When for use in treating, inhibiting, or suppressing a tumor or cancer, any of (i) surgery, (ii) radiation, (iii) chemotherapy, (iv) targeted therapy, (v) immunotherapy, or (vi) a further anticancer agent may further be for use in combination with (i) any of the isolated macrophage or CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), (ii) any of the populations of such macrophage or CD8+ T-cells, or (iii) any of the pharmaceutical compositions comprising any such isolated macrophage or CD8+ T-cells or comprising any such population of isolated macrophage or CD8+ T-cells.
Any of a chemotherapeutic agent, a targeted therapy agent, an immunotherapeutic agent, or an anticancer agent may be for use in the manufacture of a medicament for treating, inhibiting, or suppressing a tumor or cancer in combination with any of the isolated macrophage or CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), any of the populations of such macrophage or CD8+ T-cells, or any of the pharmaceutical compositions comprising any such isolated macrophage or CD8+ T-cells or comprising any such population of isolated macrophage or CD8+ T-cells.
Further medical uses include methods of treating, inhibiting, or suppressing a tumor or cancer in a subject having a tumor or cancer, said methods comprising the step of adoptive cell therapy of any of the isolated macrophage or CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), or of any of the populations of such macrophage or CD8+ T-cells; or of administering (in particular: administering a therapeutically effective dose of) any of the isolated macrophage or CD8+ T-cell according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), any of the populations of such macrophage or CD8+ T-cells, or any of the pharmaceutical compositions comprising any such isolated macrophage or CD8+ T-cells or comprising any such population of isolated macrophage or CD8+ T-cells. Such methods may further comprise (simultaneous, separate or sequential) combination with administration of any of (i) surgery, (ii) radiation, (iii) chemotherapy, (iv) targeted therapy, (v) immunotherapy, or (vi) a further anticancer agent.
Further medical uses include methods of treating, inhibiting, or suppressing a tumor or cancer in a subject having a tumor or cancer, said methods comprising the step of administering (in particular: administering a therapeutically effective dose of) any of (i) surgery, (ii) radiation, (iii) chemotherapy, (iv) targeted therapy, (v) immunotherapy, or (vi) an anticancer agent, further in combination with adoptive cell therapy of any of the isolated macrophage or CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), or of any of the populations of such macrophage or CD8+ T-cells; or of administration (in particular: administering a therapeutically effective dose of) of any of the isolated macrophage or CD8+ T-cell according to the invention (thus at least modified to (substantially) lack functional PTGDR1 or DPI), any of the populations of such macrophage or CD8+ T-cells, or any of the pharmaceutical compositions comprising any such isolated macrophage or CD8+ T-cell or comprising any such population of isolated macrophage or CD8+ T-cells.
Adoptive cell transfer
In general, adoptive cell transfer (also known as cellular adoptive immunotherapy or cell transfer therapy) refers to the administration of ex-vivo expanded cells, in particular immune cells, to a subject in need of such adoptive cell transfer, wherein the original (immune) cell is obtained from the subject (in case of autologous cell transfer therapy) prior to its expansion. The ex-vivo expanded (immune) cells can, prior to their transfer back in the subject, be genetically modified. Well-known genetic modifications include genetic engineering such as to cause the (immune) cells to express antitumor T cell receptors (TCRs) or chimeric antigen receptors (CARs) to increase anti-tumor activity of the transferred (immune) cells. In the context of the present invention, these well-known genetic modifications are not excluded and can be combined with genetic modifications aimed at forcing the macrophage or CD8+ T-cells to lack or to substantially lack functional PTGDR1 or DPI (as described above). In particular, in view of the experimental data presented herein, it is plausible that omission of PTGDR1 or DPI in macrophages or CD8+ T-cells employed in TCR-engineered or CAR-engineered adoptive cell transfer will further increase their anti-tumor activity. Thus, TCR-engineered macrophages (TCR-Ms), TCR-engineered CD8+ T-cells (TCR-Ts), CAR-engineered macrophages (CAR-Ms) or CD8+ T-cells (CAR-Ts) lacking or substantially lacking functional PTGDR1 or DPI (as defined hereinabove) are part of the invention. Pharmaceutical compositions comprising TCR-engineered macrophages or CD8+ T-cells, CAR-engineered macrophages or CD8+ T-cells lacking or substantially lacking functional PTGDR1 or DPI are also part of the invention. It can also be envisaged to take this a step further, i.e., to engineer the cells produced for adoptive cell transfer to express an inhibitor of PTGDR1 or DPI, or to load expanded cells with an inhibitor of PTGDR1 or DPI prior to adoptive transfer.
Production of CD8+ T-cells, including CAR-Ts or TCR-Ts, usually is initiated by enriching lymphocytes from a leukapheresis product. T-cells (CD8+ or CD4+) are then separated by use of e.g. an antibody to a celltype specific marker. The obtained T-cells can be activated and expanded ex vivo by incubation in the presence of anti-CD3 antibodies or anti-CD3/anti-CD28 antibodies (e.g. bound to beads) either alone or in combination with feeder cells or growth factors (e.g. interleukin 2). Culture conditions can be adapted such as to obtain a desired polarization state of the T-cells. For producing CD8+ T-cells or CAR/TCR-Ts as described herein (i.e. lacking or substantially lacking PTGDR(l) or DPI), the immune cells can be grown in the presence of e.g. a means to suppress PTGDR(l) or DPI expression and, optionally, a means introducing the CAR orTCR; such means can be gene transfer, e.g. effectuated by using lentiviral vectors, the Sleeping Beauty transposon system, or mRNA transfection). The resulting modified T-cells are then concentrated and stored/preserved (e.g. in an infusible medium) (see e.g. Levine et al. 2017, Mol Ther Meth Clin Dev 4:92-101 for more details).
Autologous or allogeneic bone marrow derived macrophages can be maintained in culture, optionally in the presence of a PTGDR1 or DPI inhibitor. Such macrophages can be genetically engineered or redirected such as to knock out the PTGDR1 or DPI gene, or by introduction of a vector or other genetic construct comprising an inducible promotor operably linked to a cassette allowing expression of a genetic or nucleotide based PTGDR1 or DPI -inhibitor (e.g. miRNA, shRNA, antisense RNA, ribozyme). In the latter case the macrophages are conditionally expressing a PTGDR1 or DPI inhibitor. The engineered macrophages can subsequently be transferred into a subject, such as to treat a cancer or tumor as described hereinabove. In case of macrophages engineered towards inducible PTGDR1 or DPI - inhibition, the expression inducing compound is administered at an appropriate timepoint to the subject having received the engineered macrophages. The transfer (adoptive cell transfer) can be autologous or heterologous. Adoptive macrophage transfer has been described in the literature (e.g. Ma et al. 2015, Brain Behaviour Immunity 45:157-170; Parsa et al. 2012, Diabetes 61:2881-2892; Wang et al. 2007, Kidney Int 72:290-299; Zhang et al. 2014, Glia 62:804-817). In addition, such modified macrophages can carry a further modification so that they can function as e.g. chimeric antigen receptor macrophages (CAR-M, e.g. Wang et al. 2022, eBioMedicine 76:103873) or as T cell receptor macrophages (TCR-Ms). Or, vice versa, isolated CAR-Ms or TCR-Ms may be PTGDR1 or DPI knock-out CAR- or TCR-Ms, or isolated CAR- or TCR-Ms conditionally expressing an inhibitor of PTGDR1 or DPI. Such CAR-or TCR-Ms likewise find use as a medicament, are for use in the manufacture of a medicament; or find application in methods of treating a disease or disorder in a subject, comprising administering (an effective dose of) the CAR-or TCR-Ms to a subject in need of being treated. More in particular, the disease or disorder is not limited to cancer or a tumor, as the nature of the CAR- or TCR-ligand may be determining the targeted disease or disorder and the inhibition of PTGDR1 or DPI may be additionally beneficial in directing the CAR-or TCR-Ms to the Ml phenotype. In this context, this disclosure therefore also relates to methods of producing autologous or allogeneic Ml-type macrophages, such as Ml-type CAR-or TCR- M, comprising inhibiting expression of PTGDR1 or DPI in macrophages obtained from a subject or in macrophages differentiated ex-vivo from monocytes obtained from a subject.
Pharmaceutical compositions
In yet a further aspect, the invention relates to pharmaceutical compositions comprising an inhibitor of PTGDR(l) or DPI and a carrier.
In yet a further aspect, the invention relates to pharmaceutical compositions comprising any isolated macrophage or CD8+ T-cell as described above (in particular lacking or substantially lacking functional PTGDR(l) or DPI), or comprising a population of such macrophage or CD8+ T-cells as described above. In particular, such pharmaceutical composition comprises the macrophage or CD8+ T-cells as well as a carrier.
A carrier in general is both pharmaceutically acceptable (which can be administered to a subject without in itself causing severe side effects) and suitable for supporting stability, and storage if required, of the inhibitor, macrophages or CD8+ T-cells; and is alternatively defined as a pharmaceutically acceptable carrier.
Such pharmaceutical composition can comprise a further anticancer agent (detailed further hereinafter, including chemotherapeutic agent, targeted therapy agent, and immunotherapeutic agent). Diagnostic methods
In further aspects, this disclosure relates to diagnostic-type methods or companion diagnostic-type methods. When referring hereinafter to immune cells in which PTGDR(l) or DPI expression or function is inhibited or (substantially) lacking, the immune cells are CD8+ T-cells and/or macrophages.
One such aspect includes methods of or for selecting, or determining the eligibility of, a subject having cancer for therapy including/with an inhibitor of PTGDR(l) or DPI, or for therapy including/with (a population of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, for therapy including/with (a population of) isolated PTGDR(l) or DPI knock-out immune cells (or CAR- immune cells orTCR-immune cells), or for therapy including/with (a population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) conditionally expressing an inhibitor of PTGDR(l) or DPI, or of predicting the response, the likelihood of response, or responsiveness of a subject having cancer to therapy including/with an inhibitor of PTGDR(l) or DPI, to therapy including/with (a population of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, to therapy including/with (a population of) isolated PTGDR(l) or DPI knock-out immune cells (or CAR-immune cells or TCR-immune cells), or to therapy including/with (a population of) isolated immune cells (or CAR- immune cells or TCR-immune cells) conditionally expressing an inhibitor of PTGDR(l) or DPI, such methods comprising: measuring, determining, assessing, quantifying, or analyzing the expression of HPGDS in a tumor biopsy sample obtained from the subject, and selecting a subject having cancer or determining a subject having cancer to be eligible for therapy including/with an inhibitor of PTGDR(l) or DPI, for therapy including/with (a population of) isolated immune cells lacking or substantially lacking functional PTGDR1 or DPI, for therapy including/with (a population of) isolated PTGDR1 or DPI knock-out immune cells (or CAR- immune cells orTCR-immune cells), or for therapy including/with (a population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) conditionally expressing an inhibitor of PTGDR(l) or DPI: when the HPGDS expression in the tumor biopsy sample is limited to expression in tumor- or cancer- associated macrophages in the tumor micro-environment (TME) or TME compartment, or when HPGDS is selectively expressed in tumor- or cancer-associated macrophages compared to in other cells in the TME or TME compartment; or when the TME or TME compartment of the tumor or cancer is characterized in that the total amount of expression of HPGDS in the TME or TME compartment is mainly/predominantly/primarily/quantitatively selectively contributed by or occurring in tumor- or cancer-associated macrophages; or when the tumor- or cancer-associated macrophages in the TME or TME compartment are the main/predominant/primary/quantitatively selective contributors to the total amount of expression of HPGDS in the TME or TME compartment of the tumor or cancer.
In one embodiment, the tumor biopsy sample is obtained prior to starting therapy with a PTGDR1 or DPI inhibitor, i.e. the tumor biopsy sample is a pre-therapy tumor biopsy sample, or is a pre- PTGDR1 or DPI inhibitor therapy tumor biopsy sample.
Medical use combined with diagnostic methods
This disclosure further relates to either (when referring hereinafter to immune cells these are CD8+ T- cells and/or macrophages): an inhibitor of PTGDR1 or DPI for use in treating a tumor or cancer, inhibiting (growth of) a tumor or cancer, or inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth; or an inhibitor of PTGDR1 or DPI for use in the manufacture of a medicament for treating a tumor or cancer, for inhibiting (growth of) a tumor or cancer, or for inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth; or a method of treating a (subject having) a tumor or cancer, of inhibiting (growth of) a tumor or cancer, or of inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth, such methods including administering a (therapeutically effective dose of a) PTGDR1 or DPI inhibitor to the subject having a tumor or cancer; or a (population of) isolated immune cells (or CAR-immune cells orTCR-immune cells) (substantially) lacking functional PTGDR1 or DPI, a (population of) isolated PTGDR1 or DPI knock-out immune cells (or CAR- immune cells orTCR-immune cells), a (population of) isolated immune cells (or CAR-immune cells orTCR- immune cells) conditionally expressing an inhibitor of PTGDR1 or DPI, or a pharmaceutical composition comprising any of these, for use in treating a tumor or cancer, inhibiting (growth of) a tumor or cancer, or inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth; or a (population of) isolated immune cells (or CAR-immune cells orTCR-immune cells) (substantially) lacking functional PTGDR1 or DPI, a (population of) isolated PTGDR1 or DPI knock-out immune cells (or CAR- immune cells orTCR-immune cells), a (population of) isolated immune cells (or CAR-immune cells orTCR- immune cells) conditionally expressing an inhibitor of PTGDR1 or DPI, or a pharmaceutical composition comprising any of these, for use in the manufacture of a medicament for treating a tumor or cancer, inhibiting (growth of) a tumor or cancer, or inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth; or a method of treating a (subject having) a tumor or cancer, of inhibiting (growth) a tumor or cancer, or of inhibiting progression of a tumor or cancer or of progression of tumor or cancer growth, such methods including administering a (therapeutically effective dose of a) a (population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) (substantially) lacking functional PTGDR1 or DPI, a (population of) isolated PTGDR1 or DPI knock-out immune cells (or CAR-immune cells or TCR-immune cells), a (population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) conditionally expressing an inhibitor of PTGDR1 or DPI, or a pharmaceutical composition comprising any of these, to the subject having a tumor or cancer; either of these comprising or further comprising selecting or determining the eligibility of a subject having cancer for therapy including/with an inhibitor of PTGDR1 or DPI, including/with a (population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) (substantially) lacking functional PTGDR1 or DPI, including/with (a population of) isolated PTGDR1 or DPI knock-out immune cells (or CAR-immune cells or TCR-immune cells), or including/with (a population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) conditionally expressing an inhibitor of PTGDR1 or DPI according to the appropriate above-described methods.
Diagnostic Kits
This disclosure further relates to (diagnostic) kits comprising one or more containers or vials (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising a (labelled) antibody or (labelled) oligonucleotide to detect an Hpgds gene expression product. In particular the (labelled) antibody or (labelled) oligonucleotide is in one embodiment adapted for multiplex in situ analysis or detection (see sections on spatial proteomics/transcriptomics). Further (labeled) antibodies or (labeled) oligonucleotides can be comprised in these kits, e.g. targeting a TAM-specific gene or gene product, e.g. CD206 or CD163 and/or e.g. targeting a CD8+ T-cell specific gene or gene product (e.g. CD8a and/or CD8b).
Further in particular, the kit is a kit for use in performing the above-described methods of selecting eligibility of a subject having a tumor or cancer to be treated with an inhibitor of PTGDR1 or DPI, a (population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) (substantially) lacking functional PTGDR1 or DPI, a (population of) isolated PTGDR1 or DPI knock-out immune cells (or CAR- immune cells or TCR-immune cells), or a (population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) conditionally expressing an inhibitor of PTGDR1 or DPI; when referring herein to immune cells these are CD8+ T-cells and/or macrophages.
Other optional components of such kit include one or more (further) diagnostic agents capable of predicting, prognosing, or determining the success of a therapy comprising one of the therapies according to the current disclosure; use instructions; one or more containers with sterile pharmaceutically acceptable carriers, excipients or diluents; one or more syringes; one or more needles; etc. In particular, such kits may be diagnostic kits or companion diagnostic kits. Therapeutic or pharmaceutical kits
This disclosure further relates to (therapeutic/pharmaceutical/medicament) kits comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising an inhibitor of PTGDR1 or DPI, a (population of) isolated immune cells (or CAR-immune cells or TCR- immune cells) (substantially) lacking functional PTGDR1 or DPI, a (population of) isolated PTGDR1 or DPI knock-out immune cells (or CAR-immune cells or TCR-immune cells), or a (population of) isolated immune cells (or CAR-immune cells orTCR-immune cells) conditionally expressing an inhibitor of PTGDR1 or DPlas described hereinabove, or comprising a composition comprising any one of these; when referring herein to immune cells these are CD8+ T-cells and/or macrophages. Other optional components of such kits include e.g. a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising a further anti-tumor or anti-cancer agent (such as e.g. an immune checkpoint inhibitor). Further optional components of such kit include use instructions (such as e.g. kit insert approved by regulatory instance such as FDA or EMEA); one or more containers with sterile pharmaceutically acceptable carriers, excipients or diluents [such as for producing or formulating a (pharmaceutically acceptable) composition of the current disclosure]; one or more syringes; one or more needles; etc. In particular, such kits may be pharmaceutical kits.
Reference, standard or control / reference, standard or control expression level
Standards or controls for the expression level, or a reference, standard or control expression level (at transcriptomic level or at proteomic level) of a biomarker gene as listed above (such as HPGDS expression) can be defined in some alternative ways.
In one embodiment, such reference, standard or control expression level refers to a pre-determined range of expression levels/standard values. Typically such ranges are defined after collecting a set of expression levels of a gene of interest as determined in a suitable number of cancer patients.
In particular, the expression level of a gene of interest is determined by normalization relative to expression of e.g. a housekeeping gene or set of housekeeping genes. Any method, diagnostic kit or device designed to operate according to any of the above-listed methods of the current disclosure (see further) therefore may include the option/possibility to determine, assess, measure, quantify expression of one or more household genes in addition to the means to determine, assess, measure, quantify expression of a gene of interest.
Inhibition of a target of interest
The term "antagonist" or "inhibitor" of a target as used interchangeable herein refers to inhibitors of function or to inhibitors of expression of a target of interest. Interchangeable alternatives for "antagonist" include inhibitor, repressor, suppressor, inactivator, and blocker. An "antagonist" thus refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with target expression, activation, function, or activity.
Downregulating of expression of a gene encoding a target is feasible through antagonists including entities such as antisense oligonucleotides, gapmers, siRNA, shRNA, zinc-finger nucleases, meganucleases, TAL effector nucleases, CRISPR-Cas effectors, etc. (general description of these compounds included hereinafter).
Inactivation or inhibition of a process as envisaged in the current disclosure refers to different possible levels of inactivation or inhibition, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even 100% or more if inactivation or inhibition (compared to a normal situation or compared to the situation prior to starting the inactivation or inhibition). The nature of the inactivating/inhibitory compound is not vital/essential to the invention as long as the process envisaged is inactivated/inhibited such as to treat or inhibit (progression of) the disease or disorder as described herein.
Selectively downregulating expression of a gene encoding a target is feasible through agents include entities such as antisense oligonucleotides, gapmers, siRNA, shRNA, zinc-finger nucleases, meganucleases, Argonaute, TAL effector nucleases, CRISPR-Cas effectors, and nucleic acid aptamers. In particular, any of these agents is specifically, selectively, or exclusively acting on or antagonizing the target of interest; or any of these agents is designed for specifically, selectively, or exclusively acting on or antagonizing the target of interest. In the context of the present disclosure, the target of interest in particular is PTGDR1 or DPI.
One process of selectively modulating/downregulating expression of a gene/target gene of interest relies on antisense oligonucleotides (ASOs), or variants thereof such as gapmers. An antisense oligonucleotide (ASO) is a short strand of nucleotides and/or nucleotide analogues that hybridizes with the complementary mRNA in a sequence-specific or -selective manner. Formation of the ASO-mRNA complex ultimately results in downregulation of target protein expression (Chan et al. 2006, Clin Exp Pharmacol Physiol 33:533-540; this reference also describes some of the software available for assisting in design of ASOs). Modifications to ASOs can be introduced at one or more levels: phosphate linkage modification (e.g. introduction of one or more of phosphodiester, phosphoramidate or phosphorothioate bonds), sugar modification (e.g. introduction of one or more of LNA (locked nucleic acids), 2'-O-methyl, 2'-O-methoxy-ethyl, 2' -fluoro, S-constrained ethyl or tricyclo-DNA and/or non-ribose modifications (e.g. introduction of one or more of phosphorodiamidate morpholinos or peptide nucleic acids). The introduction of 2'-modifications has been shown to enhance safety and pharmacologic properties of antisense oligonucleotides. Antisense strategies relying on degradation of mRNA by RNase H requires the presence of nucleotides with a free 2' -oxygen, i.e. not all nucleotides in the antisense molecule should be 2'-modified. The gapmer strategy has been developed to this end. A gapmer antisense oligonucleotide consists of a central DNA region (usually a minimum of 7 or 8 nucleotides) with (usually 2 or 3) 2'-modified nucleosides flanking both ends of the central DNA region. This is sufficient for the protection against exonucleases while allowing RNAseH to act on the (2'-modification free) gap region. Antidote strategies are available as demonstrated by administration of an oligonucleotide fully complementary to the antisense oligonucleotide (Crosby et al. 2015, Nucleic Acid Ther 25:297-305). Uptake of oligonucleotides by cells can be spontaneous or be assisted by e.g. transfection etc..
Another process to selectively modulate expression of a gene/target gene of interest is based on the natural process of RNA interference. It relies on double-stranded RNA (dsRNA) that is cut by an enzyme called Dicer, resulting in double stranded small interfering RNA (siRNA) molecules which are 20-25 nucleotides long. siRNA then binds to the cellular RNA-lnduced Silencing Complex (RISC) separating the two strands into the passenger and guide strand. While the passenger strand is degraded, RISC is cleaving mRNA specifically or selectively at a site instructed by the guide strand. Destruction of the mRNA prevents production of the protein of interest and the gene is 'silenced'. siRNAs are dsRNAs with 2 nt 3' end overhangs whereas shRNAs are dsRNAs that contains a loop structure that is processed to siRNA. shRNAs are introduced into the nuclei of target cells using a vector (e.g. bacterial or viral) that optionally can stably integrate into the genome. Apart from checking for lack of cross-reactivity with non-target genes, manufacturers of RNAi products provide guidelines for designing siRNA/shRNA. siRNA sequences between 19-29 nt are generally the most effective. Sequences longer than 30 nt can result in nonspecific silencing. Ideal sites to target include AA dinucleotides and the 19 nt 3' of them in the target mRNA sequence. Typically, siRNAs with 3' dUdU or dTdT dinucleotide overhangs are more effective. Other dinucleotide overhangs could maintain activity but GG overhangs should be avoided. Also to be avoided are siRNA designs with a 4-6 poly(T) tract (acting as a termination signal for RNA pol III), and the G/C content is advised to be between 35-55%. shRNAs should comprise sense and antisense sequences (advised to each be 19-21 nt in length) separated by loop structure, and a 3' AAAA overhang. Effective loop structures are suggested to be 3-9 nt in length. It is suggested to follow the sense-loop-antisense order in designing the shRNA cassette and to avoid 5' overhangs in the shRNA construct. shRNAs are usually transcribed from vectors, e.g. driven by the Pol III U6 promoter or Hl promoter. Vectors allow for inducible shRNA expression, e.g. relying on the Tet-on and Tet-off inducible systems commercially available, or on a modified U6 promoter that is induced by the insect hormone ecdysone. A Cre-Lox recombination system has been used to achieve controlled expression in mice. Synthetic shRNAs can be chemically modified to affect their activity and stability. Plasmid DNA or dsRNA can be delivered to a cell by means of transfection (lipid transfection, cationic polymer-based nanoparticles, lipid or cellpenetrating peptide conjugation) or electroporation. Vectors include viral vectors such as lentiviral, retroviral, adenoviral and adeno-associated viral vectors. Ribozymes (ribonucleic acid enzymes) are another type of molecules that can be used to selectively modulate expression of a gene/target gene of interest. They are RNA molecules capable of catalyzing specific biochemical reactions, in the current context capable of targeted cleavage of nucleotide sequences, in particular targeted cleavage of a RNA/RNA target of interest. Examples of ribozymes include the hammerhead ribozyme, the Varkud Satellite ribozyme, Leadzyme and the hairpin ribozyme. Besides the use of the inhibitory RNA technology, modulation of expression of a gene of interest can be achieved at DNA level such as by gene therapy to knock-out, knock-down or disrupt the target gene/gene of interest. As used herein, a "gene knock-out" can be a gene knockdown or the gene can be knocked out, knocked down, disrupted or modified by a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques such as described hereafter, including, but not limited to, retroviral gene transfer. One way in which genes can be knocked out, knocked down, disrupted or modified is by the use of zinc finger nucleases. Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target a desired DNA sequence/DNA sequence of interest, which enable zinc-finger nucleases to target unique sequence within a complex genome. By taking advantage of the endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.
Other technologies for genome customization that can be used to specifically or selectively knock out, knock down or disrupt a gene/gene of interest are meganucleases and TAL effector nucleases (TALENs, Cellectis bioresearch). A TALEN® is composed of a TALE DNA binding domain for sequence-specific or sequence-selective recognition fused to the catalytic domain of an endonuclease that introduces double strand breaks (DSB). The DNA binding domain of a TALEN® is capable of targeting with high precision a large recognition site (for instance 17bp). Meganucleases are sequence-specific or sequence-selective endonucleases, naturally occurring "DNA scissors", originating from a variety of single-celled organisms such as bacteria, yeast, algae and some plant organelles. Meganucleases have long recognition sites of between 12 and 30 base pairs. The recognition site of natural meganucleases can be modified in order to target native genomic DNA sequences (such as endogenous genes) or DNA sequences of interest. Another recent genome editing technology is the CRISPR/Cas system, which can be used to achieve RNA- guided genome engineering (including knock-out, knock-down or disruption of a gene of interest). CRISPR interference is a genetic technique which allows for sequence-specific or sequence-selective control of expression of a gene of interest in prokaryotic and eukaryotic cells. It is based on the bacterial immune system-derived CRISPR (clustered regularly interspaced palindromic repeats) pathway. Recently, it was demonstrated that the CRISPR-Cas editing system can also be used to target RNA. It has been shown that the Class 2 type Vl-A CRISPR-Cas effector C2c2 (Casl3a; CRISPR-Casl3a or CRISPR-C2c2) can be programmed to cleave single stranded RNA targets carrying complementary protospacers (Abudayyeh et al. 2016 Science353/science.aaf5573). C2c2 is a single-effector endoRNase mediating ssRNA cleavage once it has been guided by a single crRNA guide toward a target RNA/RNA of interest.
Methods for administering nucleic acid-based therapeutic modalities/agents include methods applying non-viral (DNA or RNA) or viral nucleic acids (DNA or RNA viral vectors). Methods for non-viral nucleic acid administration include the injection of naked DNA (circular or linear), electroporation, the gene gun, sonoporation, magnetofection, the use of oligonucleotides, lipoplexes (e.g. complexes of nucleic acid with DOTAP or DOPE or combinations thereof, complexes with other cationic lipids), dendrimers, viral- like particles, inorganic nanoparticles, hydrodynamic delivery, photochemical internalization (Berg et al. 2010, Methods Mol Biol 635:133-145) or combinations thereof.
Many different vectors have been used in nucleic acid administration. Currently the major groups are adenovirus or adeno-associated virus vectors, retrovirus vectors , naked or plasmid DNA, and lentivirus vectors. Combinations are also possible, e.g. naked or plasmid DNA combined with adenovirus, or RNA combined with naked or plasmid DNA to list just a few. Other viruses (e.g. alphaviruses, vaccinia viruses such as vaccinia virus Ankara) are used in nucleic acid administration and are not excluded in the context of the current disclosure.
Administration may be aided by specific formulation of the nucleic acid e.g. in liposomes (lipoplexes) or polymersomes (synthetic variants of liposomes), as polyplexes (nucleic acid complexed with polymers), carried on dendrimers, in inorganic (nano)particles (e.g. containing iron oxide in case of magnetofection), or combined with a cell penetrating peptide (CPP) to increase cellular uptake. Organ- or cellular-targeting strategies may also be applied to the nucleic acid (nucleic acid combined with organ- or cell-targeting moiety); these include passive targeting (mostly achieved by adapted formulation) or active targeting (e.g. by coupling a nucleic acid-comprising nanoparticle with any compound (e.g. an aptamer or antibody or antigen binding molecule) binding to a target organ- or cell-specific antigen) (e.g. Steichen et al. 2013, Eur J Pharm Sci 48:416-427).
CPPs enable translocation of their payload of interest across the plasma membrane. CPPs are alternatively termed Protein Transduction Domains (TPDs), usually comprise 30 or less (e.g. 5 to 30, or 5 to 20) amino acids, and usually are rich in basic residues, and are derived from naturally occurring CPPs (usually longer than 20 amino acids), or are the result of modelling or design. A non-limiting selection of CPPs includes the TAT peptide (derived from HIV-1 Tat protein), penetratin (derived from Drosophila Antennapedia - Antp), pVEC (derived from murine vascular endothelial cadherin), signal-sequence based peptides or membrane translocating sequences, model amphipathic peptide (MAP), transportan, MPG, polyarginines; more information on these peptides can be found in Torchilin 2008 (Adv Drug Deliv Rev 60:548-558) and references cited therein. CPPs can be coupled to carriers such as nanoparticles, liposomes, micelles, or generally any hydrophobic particle. Coupling can be by absorption or chemical bonding, such as via a spacer between the CPP and the carrier. To increase target specificity or target selectivity, an antibody binding to a target-specific antigen can further be coupled to the carrier (Torchilin 2008, Adv Drug Deliv Rev 60:548-558). CPPs have already been used to deliver payloads as diverse as plasmid DNA, oligonucleotides, siRNA, peptide nucleic acids (PNA), proteins and peptides, small molecules and nanoparticles inside the cell (Stalmans et al. 2013, PloS One 8:e71752).
Any other modification of the DNA or RNA to enhance efficacy of nucleic acid therapy is likewise envisaged to be useful in the context of the applications as outlined herein. The enhanced efficacy can reside in enhanced expression, enhanced delivery properties, enhanced stability and the like.
A specific or selective inhibitor of a target of interest may exert the desired level of inhibition of the target of interest with an IC50 of 1000 nM or less, with an IC50 of 500 nM or less, with an IC50 of 100 nM or less, with an IC50 of 50 nM or less, with an IC50 of 10 nM or less, with an IC50 of 1 nM or less, with an IC50 between 1 pM and InM, or with an IC50 between 0.1 pM and 10 nM.
Cross-inhibition of more than one target is possible; for clinical development it can e.g. be desired to be able to test an inhibitor in a suitable in vitro model or in vivo animal model before starting clinical testing with the same inhibitor in a human population, which may require the inhibitor to cross-inhibit the animal (or other non-human) target and the orthologous human target.
Specificity or selectivity of inhibition refers to the situation in which an inhibitor is, at a certain concentration (sufficient to inhibit the target of interest) inhibiting the target gene or protein with higher efficacy (e.g. with an at least 2-fold, 5-fold, or 10-fold lower IC50, e.g. at least 20-, 50- or 100-fold or more lower IC50) than the efficacy with which it is possibly (if at all) inhibiting other targets (targets not of interest). Such specificity or selectivity of inhibition is in particular determined within the setting of the target subject (e.g. human patient, or animal model) and thus can encompass/does not exclude inhibition of (at least one) orthologous target. Exclusivity of inhibition refers to the situation in which an inhibitor is inhibiting only the target of interest.
Specificity or selectivity of (immune) cell targeting refers to the situation in which a composition, at a certain concentration, is interacting with the intended target cell (such as binding to, or such as causing inhibition of function or expression of PTGDR1 or DPI in the intended target cell) with higher efficacy (e.g. with an at least 2-fold, 5-fold, or 10-fold higher efficacy, or e.g. with at least 20-, 50- or 100-fold higher efficacy) than the efficacy with which the composition is interacting with other cells (not intended as target cell). Exclusivity of cell targeting refers to the situation in which a composition is interacting only with the intended target cell. In particular, the target cell is an immune cell, a CAR-immune cell or TCR immune cell; more particularly the immune cell is a (tumor-associated) macrophage and/or a CD8+ T-cell. Treatment / therapeutically effective amount
The terms therapeutic modality, therapeutic agent, agent, and drug are used interchangeably herein, and likewise relate to the immune cells (in particular macrophages and CD8+ T-cells) as described herein (with inhibited function or expression of PTGDR1 or DPI or (substantially) lacking functional PTGDR1 or DPI). All refer to a therapeutically active compound, or to a therapeutically active composition (comprising one or more therapeutically active compounds).
"Treatment"/"treating" refers to any rate of reduction, delaying or retardation of the progress of the disease or disorder, or a single symptom thereof, compared to the progress or expected progress of the disease or disorder, or single symptom thereof, when left untreated. This implies that a therapeutic modality on its own may not result in a complete or partial response (or may even not result in any response), but may, in particular when combined with other therapeutic modalities (such as other immunosuppressants or therapeutic modalities for treating or suppressing cancer or a tumor (or possibly other disease or disorder in case of CAR-Ms, TCR-Ms, CAR-Ts, or TCR-Ts in which PTGDR1 or DPI expression or function is inhibited), contribute to a complete or partial response. More desirable, the treatment results in no/zero progress of the disease or disorder, or single symptom thereof (i.e. "inhibition" or "inhibition of progression"), or even in any rate of regression of the already developed disease or disorder, or single symptom thereof. "Suppression/suppressing" can in this context be understood to be comprised within the meaning of the term "treatment/treating". Treatment/treating also refers to achieving a significant amelioration of one or more clinical symptoms associated with a disease or disorder, or of any single symptom thereof. Depending on the situation, the significant amelioration may be scored quantitatively or qualitatively. Qualitative criteria may e.g. by patient wellbeing. In the case of quantitative evaluation, the significant amelioration is typically a 10% or more, a 20% or more, a 25% or more, a 30% or more, a 40% or more, a 50% or more, a 60% or more, a 70% or more, a 75% or more, a 80% or more, a 95% or more, or a 100% improvement over the situation prior to treatment. The time-frame over which the improvement is evaluated will depend on the type of criteria/disease observed and can be determined by the person skilled in the art.
A "therapeutically effective amount" refers to an amount of a therapeutic agent to treat, inhibit or prevent a disease or disorder in a subject (such as a mammal). Efficacy in vivo can, e.g., be measured by assessing the duration of survival (e.g. overall survival), time to disease progression (TTP), response rates (e.g., complete response and partial response, stable disease), length of progression-free survival (PFS), duration of response, and/or quality of life.
The term "effective amount" or "therapeutically effective amount" may depend on the dosing regimen of the agent/therapeutic agent or composition comprising the agent/therapeutic agent (e.g. medicament or pharmaceutical composition). The effective amount will generally depend on and/or will need adjustment to the mode of contacting or administration. The effective amount of the agent or composition comprising the agent is the amount required to obtain the desired clinical outcome or therapeutic effect without causing significant or unnecessary toxic effects (often expressed as maximum tolerable dose, MTD). To obtain or maintain the effective amount, the agent or composition comprising the agent may be administered as a single dose or in multiple doses. The effective amount may further vary depending on the severity of the condition that needs to be treated; this may depend on the overall health and physical condition of the subject or patient and usually the treating doctor's or physician's assessment will be required to establish what is the effective amount. The effective amount may further be obtained by a combination of different types of contacting or administration.
The aspects and embodiments described above in general may comprise the administration of one or more therapeutic compounds to a subject (such as a mammal) in need thereof or in need of treatment. In general a (therapeutically) effective amount of (a) therapeutic compound(s) is administered to the mammal in need thereof in order to obtain the described clinical response(s).
"Administering" means any mode of contacting that results in interaction between an agent or composition comprising the agent (such as a medicament or pharmaceutical composition) and an object (e.g. cell, tissue, organ, body lumen) with which said agent or composition is contacted. The interaction between the agent or composition and the object can occur starting immediately or nearly immediately with the administration of the agent or composition, can occur over an extended time period (starting immediately or nearly immediately with the administration of the agent or composition), or can be delayed relative to the time of administration of the agent or composition. More specifically the "contacting" results in delivering an effective amount of the agent or composition comprising the agent to the object.
Further anti-tumor or anti-cancer agent
Without being exhaustive, further antitumor, anticancer or antineoplastic agents (other than inhibitors of PTGDR1 or DPI) include alkylating agents (nitrogen mustards: melphalan, cyclophosphamide, ifosfamide; nitrosoureas; alkylsulfonates; ethyleneimines; triazene; methyl hydrazines; platinum coordination complexes: cisplatin, carboplatin, oxaliplatin), antimetabolites (folate antagonists: methotrexate; purine antagonists; pyrimidine antagonists: 5-fluorouracil, cytarabibe), natural plant products (Vinca alkaloids: vincristine, vinblastine; taxanes: paclitaxel, docetaxel; epipodophyllotoxins: etoposide; camptothecins: irinotecan), natural microorganism products (antibiotics: doxorubicin, bleomycin; enzymes: L-asparaginase), hormones and antagonists (corticosteroids: prednisone, dexamethasone; estrogens: ethinyloestradiol; antiestrogens: tamoxifen; progesterone derivative: megestrol acetate; androgen: testosterone propionate; antiandrogen: flutamide , bicalutamide; aromatase inhibitor: letrozole, anastrazole; 5-alpha reductase inhibitor: finasteride; GnRH analogue: leuprolide, buserelin; growth hormone, glucagon and insulin inhibitor: octreotide). Other antineoplastic or antitumor agents include hydroxyurea, imatinib mesylate, epirubicin, bortezomib, zoledronic acid, geftinib, leucovorin, pamidronate, and gemcitabine.
Without being exhaustive, further antitumor, anticancer or antineoplastic antibodies (antibody therapy) include rituximab, bevacizumab, ibritumomab tiuxetan, tositumomab, brentuximab vedotin, gemtuzumab ozogamicin, alemtuzumab, adecatumumab, labetuzumab, pemtumomab, oregovomab, minretumomab, farletuzumab, etaracizumab, volociximab, cetuximab, panitumumab, nimotuzumab, trastuzumab, pertuzumab, mapatumumab, denosumab, and sibrotuzumab.
A particular class of antitumor, anticancer or antineoplastic agents are designed to stimulate the immune system (immune checkpoint or other immunostimulating therapy). These include so-called immune checkpoint inhibitors or inhibitors of co-inhibitory receptors and include PD-1 (Programmed cell death 1) inhibitors (e.g. pembrolizumab, nivolumab, pidilizumab), PD-L1 (Programmed cell death 1 ligand) inhibitors (e.g. atezolizumab, avelumab, durvalumab), CTLA-4 (Cytotoxic T-lymphocyte associated protein 4; CD152) inhibitors (e.g. ipilimumab, tremelimumab) (e.g. Sharon et al. 2014, Chin J Cane 33:434-444). Inhibition of other co-inhibitory receptors under evaluation as antitumor, anticancer or antineoplastic agents include inhibitors of Lag-3 (lymphocyte activation gene 3), Tim-3 (T cell immunoglobulin 3) and TIG IT (T cell immunoglobulin and ITM domain) (Anderson et al. 2016, Immunity 44:989-1004). Stimulation of members of the TNFR superfamily of co-receptors expressed on T-cells, such as stimulation of 4-1BB (CD137), 0X40 (CD134) or GITR (glucocorticoid-induced TNF receptor family-related gene), is also evaluated for antitumor, anticancer or antineoplastic therapy (Peggs et al. 2009, Clin Exp Immunol 157:9-19).
Further antitumor, anticancer or antineoplastic agents include immune-stimulating agents such as - or neo-epitope cancer vaccines (neo-antigen or neo-epitope vaccination; based on the patient's sequencing data to look for tumor-specific mutations, thus leading to a form of personalized immunotherapy; Kaiser 2017, Science 356:112; Sahin et al. 2017, Nature 547:222-226) and some Toll-like receptor (TLR) ligands (Kaczanowska et al. 2013, J Leukoc Biol 93:847-863).
Yet further antitumor, anticancer or antineoplastic agents include oncolytic viruses (oncolytic virus therapy) such as employed in oncolytic virus immunotherapy (Kaufman et al. 2015, Nat Rev Drug Discov 14:642-662), any other cancer vaccine (cancer vaccine administration; Guo et al. 2013, Adv Cancer Res 119:421-475), and any other anticancer nucleic acid therapy.
Gene expression and quantification of gene expression
The term "level of expression" or "expression level" generally refers to the amount of an expressed gene or (bio)marker in a biological sample. As used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide modifications (e.g. alternative splicing) and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide) are also regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs, long non-coding RNA, microRNA or miRNA).
"Increased/higher expression," "increased/higher expression level," "increased/higher levels," "elevated expression," "elevated expression levels," or "elevated levels" refers to an increased/higher expression or to increased/higher levels of a (bio)marker in an individual relative to a suitable control or standard. The term "detection" includes any means of detecting, including direct and indirect detection. The term "marker" or "biomarker" as used herein refers to an indicator molecule or set of indicator molecules (e.g., predictive, diagnostic, and/or prognostic indicator), which can be detected in a sample. The biomarker may be a predictive biomarker and serve as an indicator of the likelihood of sensitivity or benefit to therapeutic treatment of a patient having a particular disease or disorder (e.g., a proliferative cell disorder (e.g., cancer)) to treatment (e.g. with an inhibitor of PTGDR1 or DPI). Biomarkers in general include, but are not limited to, polynucleotides (e.g., DNA and/or RNA (e.g., mRNA)), polynucleotide copy number alterations (e.g., DNA copy numbers), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications, nucleotide substitutions, nucleotide insertions or deletions (indels)), carbohydrates, and/or glycolipid-based molecular markers. In some embodiments, a biomarker is a gene. The "amount" or "level" of a biomarker, as used herein, is a detectable level in a biological sample. These can be measured by methods known to one skilled in the art and also disclosed herein.
In first instance, methodologies for determining gene expression by means of determining transcript levels, also referred to as transcriptome analysis or analysis of the transcriptome, is described in more detail. Any such gene detection or gene expression detection method is starting from an analyte nucleic acid (i.e. the nucleic acid of interest (which does not necessarily need to be the whole nucleic acid of interest, parts of such nucleic acids can suffice for determining expression) and of which the amount is to be determined) and may be defined as comprising one or more steps of, for instance, a step of isolating RNA from a (biological) sample (wherein a fraction of the isolated RNA is the analyte strand); a step of reverse transcribing the RNA obtained from the biological sample into DNA; a step of amplifying the isolated DNA; and/or a step of quantifying the isolated RNA, the DNA obtained after reverse transcription, or the amplified DNA.
In case an amplified DNA is quantified, this quantification step can be performed concurrent with the amplification of the DNA, or is performed after the amplification of the DNA.
The quantification of gene expression or the determination of gene expression levels may be based on at least one of an amplification reaction, a sequencing reaction, a melting reaction, a hybridization reaction or a reverse hybridization reaction. Quantification of gene expression can further involve a normalization step, wherein levels of expression of a gene of interest are normalized to e.g. levels of expression of a housekeeping gene or of a gene of which the expression is relatively constant under different conditions.
This disclosure covers methods which include measurement, determination, assessment, analysis, detection or quantification of nucleic acids corresponding to one or more gene(s) or (bio)markers as defined herein (more specifically HPGDS). In any of these methods the detection can comprise a step such as a nucleic acid amplification reaction, a nucleic acid sequencing reaction, a melting reaction, a hybridization reaction to a nucleic acid, or a reverse hybridization reaction to a nucleic acid, or a combination of such steps.
Often one or more artificial, man-made, or non-naturally occurring oligonucleotide is used in such method. In particular, such oligonucleotides can comprise besides ribonucleic acid monomers or deoxyribonucleic acid monomers: one or more modified nucleotide bases, one or more modified nucleotide sugars, one or more labelled nucleotides, one or more peptide nucleic acid monomers, one or more locked nucleic acid monomers, the backbone of such oligonucleotide can be modified, and/or non-glycosidic bonds may link two adjacent nucleotides. Such oligonucleotides may further comprise a modification for attachment to a solid support, e.g., an amine-, thiol-, 3-'propanolamine or acrydite- modification of the oligonucleotide, or may comprise the addition of a homopolymeric tail (for instance an oligo(dT)-tail added enzymatically via a terminal transferase enzyme or added synthetically) to the oligonucleotide. If said homopolymeric tail is positioned at the 3'-terminus of the oligonucleotide or if any other 3'-terminal modification preventing enzymatic extension is incorporated in the oligonucleotide, the priming capacity of the oligonucleotide can be decreased or abolished. Such oligonucleotides may also comprise a hairpin structure at either end. Terminal extension of such oligonucleotide may be useful for, e.g., specifically hybridizing with another nucleic acid molecule (e.g. when functioning as capture probe), and/or for facilitating attachment of said oligonucleotide to a solid support, and/or for modification of said tailed oligonucleotide by an enzyme, ribozyme or DNAzyme and/or for purposes of sequencing (e.g. addition of adaptor oligonucleotides in the preparation of a nextgeneration sequencing library). Such oligonucleotides may be modified in order to detect (the levels of) a target nucleotide sequence and/or to facilitate in any way such detection. Such modifications include labelling with a single label, with two different labels (for instance two fluorophores or one fluorophore and one quencher), the attachment of a different 'universal' tail to two probes or primers hybridizing adjacent or in close proximity to each other with the target nucleotide sequence, the incorporation of a target-specific sequence in a hairpin oligonucleotide (for instance Molecular Beacon-type primer), the tailing of such a hairpin oligonucleotide with a 'universal' tail (for instance Sunrise-type probe and Amplifluor TM -type primer). A special type of hairpin oligonucleotide incorporates in the hairpin a sequence capable of hybridizing to part of the newly amplified target DNA. Amplification of the hairpin is prevented by the incorporation of a blocking non-amplifiable monomer (such as hexethylene glycol). A fluorescent signal is generated after opening of the hairpin due to hybridization of the hairpin loop with the amplified target DNA. This type of hairpin oligonucleotide is known as scorpion primers (Whitcombe et al. 1999, Nat Biotechnol 17:804-807). Another special type of oligonucleotide is a padlock oligonucleotide (or circularizable, open circle, or C-oligonucleotide) that are used in RCA (rolling circle amplification). Such oligonucleotides may also comprise a 3'-terminal mismatching nucleotide and/or, optionally, a 3'-proximal mismatching nucleotide, which can be particularly useful for performing polymorphism-specific PCR and LCR (ligase chain reaction) or any modification of PCR or LCR. Such oligonucleotide may can comprise or consist of at least and/or comprise or consist of up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more contiguous nucleotides.
The analyte nucleic acid, in particular the analyte nucleic acid of a biomarker of interest can be any type of nucleic acid, which will be dependent on the manipulation steps (such as isolation and/or purification and/or duplication, multiplication or amplification) applied to the nucleic acid of the gene of interest in the biological sample; as such it can be DNA, RNA, cDNA, may comprise modified nucleotides, or may be hybrids of DNA and/or RNA and/or modified nucleotides, and can be single- or double-stranded or may be a triplex-forming nucleic acid.
The artificial, man-made, non-naturally occurring oligonucleotide(s) as applied in the above detection methods can be probe(s) or a primer(s), or a combination of both.
A probe capable of specifically hybridizing with a target nucleic acid is an oligonucleotide mainly hybridizing to one specific nucleic acid sequence in a mixture of many different nucleic acid sequences. Specific hybridization is meant to result, upon detection of the specifically formed hybrids, in a signal-to- noise ratio (wherein the signal represents specific hybridization and the noise represents unspecific hybridization) sufficiently high to enable unambiguous detection of said specific hybrids. In a specific case specific hybridization allows discrimination of up to a single nucleotide mismatch between the probe and the target nucleic acids. Conditions allowing specific hybridization generally are stringent but can obviously be varied depending on the complexity (size, GC-content, overall identity, etc.) of the probe(s) and/or target nucleic acid molecules. Specificity of a probe in hybridizing with a nucleic acid can be improved by introducing modified nucleotides in said probe.
A primer capable of directing specific amplification of a target nucleic acid is the at least one oligonucleotide in a nucleic acid amplification reaction mixture that is required to obtain specific amplification of a target nucleic acid. Nucleic acid amplification can be linear or exponential and can result in an amplified single nucleic acid of a single- or double-stranded nucleic acid or can result in both strands of a double-stranded nucleic acid. Specificity of a primer in directing amplification of a nucleic acid can be improved by introducing modified nucleotides in said primer. The fact that a primer does not have to match exactly with the corresponding template or target sequence to warrant specific amplification of said template or target sequence is amply documented in literature (for instance: Kwok et al. 1990, Nucl Acids Res 18:999-1005. Primers as short as 8 nucleotides in length have been applied successfully in directing specific amplification of a target nucleic acid molecule (e.g. Majzoub et al. 1983, J Biol Chem 258:14061-14064).
A nucleotide is meant to include any naturally occurring nucleotide as well as any modified nucleotide wherein said modification can occur in the structure of the nucleotide base (modification relative to A, T, G, C, or U) and/or in the structure of the nucleotide sugar (modification relative to ribose or deoxyribose). Any of the modifications can be introduced in a nucleic acid or oligonucleotide to increase/decrease stability and/or reactivity of the nucleic acid or oligonucleotide and/or for other purposes such as labelling of the nucleic acid or oligonucleotide. Modified nucleotides include phosphorothioates, alkylphosphorothioates, methylphosphonate, phosphoramidate, peptide nucleic acid monomers and locked nucleic acid monomers, cyclic nucleotides, and labelled nucleotides (i.e. nucleotides conjugated to a label which can be isotopic (<32>P, <35>S, etc.) or non-isotopic (biotin, digoxigenin, phosphorescent labels, fluorescent labels, fluorescence quenching moiety, etc.)). Other modifications are described higher (see description on oligonucleotides).
Nucleotide acid amplification is meant to include all methods resulting in multiplication of the number of a target nucleic acid. Nucleotide sequence amplification methods include the polymerase chain reaction (PCR; DNA amplification), strand displacement amplification (SDA; DNA amplification), transcription-based amplification system (TAS; RNA amplification), self-sustained sequence replication (3SR; RNA amplification), nucleic acid sequence-based amplification (NASBA; RNA amplification), transcription-mediated amplification (TMA; RNA amplification), Qbeta-replicase-mediated amplification and run-off transcription. During amplification, the amplified products can be conveniently labelled either using labelled primers or by incorporating labelled nucleotides.
The most widely spread nucleotide sequence amplification technique is PCR. The target DNA is exponentially amplified. Many methods rely on PCR including AFLP (amplified fragment length polymorphism), IRS-PCR (interspersed repetitive sequence PCR), iPCR (inverse PCR), RAPD (rapid amplification of polymorphic DNA), RT-PCR (reverse transcription PCR) and real-time PCR. RT-PCR can be performed with a single thermostable enzyme having both reverse transcriptase and DNA polymerase activity (Myers et al. 1991, Biochem 30:7661-7666). Alternatively, a single tube-reaction with two enzymes (reverse transcriptase and thermostable DNA polymerase) is possible (Cusi et al. 1994, Biotechniques 17:1034-1036).
Solid phases, solid matrices or solid supports on which molecules, e.g., nucleic acids, analyte nucleic acids and/or oligonucleotides as described hereinabove, may be bound (or captured, absorbed, adsorbed, linked, coated, immobilized; covalently or non-covalently) comprise beads or the wells or cups of microtiter plates, or may be in other forms, such as solid or hollow rods or pipettes, particles, e.g., from 0.1 pm to 5 mm in diameter (e.g. "latex" particles, protein particles, or any other synthetic or natural particulate material), microspheres or beads (e.g. protein A beads, magnetic beads). A solid phase may be of a plastic or polymeric material such as nitrocellulose, polyvinyl chloride, polystyrene, polyamide, polyvinylidene fluoride or other synthetic polymers. Other solid phases include membranes, sheets, strips, films and coatings of any porous, fibrous or bibulous material such as nylon, polyvinyl chloride or another synthetic polymer, a natural polymer (or a derivative thereof) such as cellulose (or a derivative thereof such as cellulose acetate or nitrocellulose). Fibers or slides of glass, fused silica or quartz are other examples of solid supports. Paper, e.g., diazotized paper may also be applied as solid phase. Clearly, molecules such as nucleic acids, analyte nucleic acids and/or oligonucleotides as described hereinabove, may be bound, captured, absorbed, adsorbed, linked or coated to any solid phase suitable for use in hybridization assay (irrespective of the format, for instance capture assay, reverse hybridization assay, or dynamic allele-specific hybridization (DASH)). Said molecules, such as nucleic acids, analyte nucleic acids and/or oligonucleotides as described hereinabove, can be present on a solid phase in defined zones such as spots or lines. Such solid phases may be incorporated in a component such as a cartridge of e.g. an assay device. Any of the solid phases described above can be developed, e.g. automatically developed in an assay device.
Quantification of amplified DNA can be performed concurrent with or during the amplification. Techniques include real-time PCR or (semi-)quantitative polymerase chain reaction (qPCR). One common method includes measurement of a non-sequence specific fluorescent dye (e.g. SYBR Green) intercalating in any double-stranded DNA. Quantification of multiple amplicons with different melting points can be followed simultaneously by means of following or analyzing the melting reaction (melting curve analysis or melt curve analysis; which can be performed at high resolution, see, e.g. Wittwer et al. 2003, Clin Chem 843-860; an alternative method is denaturing gel gradient electrophoresis, DGGE; both methods were compared in e.g. Tindall et al. 2009, Hum Mutat 30:857-859). Another common method includes measurement of sequence-specific labelled probe bound to its complementary sequence; such probe also carries a quencher and the label is only measurable upon exonucleolytic release from the probe (hydrolysis probes such as TaqMan probes) or upon hybridization with the target sequence (hairpin probes such as molecular beacons which carry an internally quenched fluorophore whose fluorescence is restored upon unfolding the hairpin). This latter method allows for multiplexing by e.g. using mixtures of probes each tagged with a different label e.g. fluorescing at a different wavelength.
Exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes also allow for multiplexing. The hybridization-sensitive fluorescence emission of ECHO probes and the further modification of probes have made possible multicolor RNA imaging in living cells and facile detection of gene polymorphisms (Okamoto 2011, Chem Soc Rev, 40:5815-5828).
Other methods of quantifying expression include SAGE (Serial Analysis of Gene Expression) and MPSS (Massively Parallel Signature Sequencing), each involving reverse-transcription of RNA.
With "assaying" or "determining" or "detecting" and the like (e.g. assessing, measuring) is meant that a biological sample, suspected of comprising a target nucleic acid (such as a nucleic acid of a biomarker of interest as described herein), is processed as to generate a readable signal in case the target nucleic acid is actually present in the biological sample. Such processing may include, as described above, a step of producing an analyte nucleic acid. Simple detection of a produced readable signal indicates the presence of a target or analyte nucleic acid in the biological sample. When in addition the amplitude of the produced readable signal is determined, this allows for quantification of levels of a target or analyte nucleic acid as present in a biological sample.
In particular, the readable signal may be a signal-to-noise ratio (wherein the signal represents specific detection and the noise represents unspecific detection) of an assay optimized to yield signal-to-noise ratios sufficiently high to enable unambiguous detection and/or quantification of the target nucleic acid. The noise signal, or background signal, can be determined e.g. on biological samples not comprising the target or analyte nucleic acid of interest, e.g. control samples, or comprising the required reference level of the target or analyte nucleic acid of interest, e.g. reference samples. Such noise or background signal may also serve as comparator value for determining an increase or decrease of the level of a target or analyte nucleic acid in the biological sample, e.g. in a biological sample taken from a subject suffering from a disease or disorder, further e.g. before start of a treatment and during treatment.
The readable signal may be produced with all required components in solution or may be produced with some of the required components in solution and some bound to a solid support. Said signals include, e.g., fluorescent signals, (chemi)luminescent signals, phosphorescence signals, radiation signals, light or color signals, optical density signals, hybridization signals, mass spectrometric signals, spectrometric signals, chromatographic signals, electric signals, electronic signals, electrophoretic signals, real-time PCR signals, PCR signals, LCR signals, Invader-assay signals, sequencing signals (by any method such as Sanger dideoxy sequencing, pyrosequencing, 454 sequencing, single-base extension sequencing, sequencing by ligation, sequencing by synthesis, "next-generation" sequencing (NGS) (van Dijk et al. 2014, Trends Genet 30:418-426)), nanopore sequencing, melting curve signals etc. An assay may be run automatically or semi-automatically in an assay device. In view of its relatively low costs compared to e.g. very costly cancer therapies, NGS is finding its way to routine clinical care (Ratner 2018, Nature Biotechnol 36:484).
Specific hybridization of an oligonucleotide (whether or not comprising one or more modified nucleotides) to its target sequence is to be understood to occur under stringent conditions as generally known in the art (e.g. Sambrook et al. 1989. Molecular Cloning. A laboratory manual. CSHL Press). However, depending to the hybridization solution (SSC, SSPE, etc.), oligonucleotides should be hybridized at their appropriate temperature in order to attain sufficient specificity. In order to allow hybridization to occur, the target nucleic acid molecules are generally thermally, chemically (e.g. by NaOH) or electrochemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids. The stringency of hybridization is influenced by conditions such as temperature, salt concentration and hybridization buffer composition. High stringency conditions for hybridization include high temperature and/or low salt concentration (salts include NaCI and Na3-citrate) and/or the inclusion of formamide in the hybridization buffer and/or lowering the concentration of compounds such as SDS (detergent) in the hybridization buffer and/or exclusion of compounds such as dextran sulfate or polyethylene glycol (promoting molecular crowding) from the hybridization buffer. Conventional hybridization conditions are described in e.g. Sambrook et al. 1989 (Molecular Cloning. A laboratory manual. CSHL Press) but the skilled craftsman will appreciate that numerous different hybridization conditions can be designed in function of the known or the expected homology and/or length of the nucleic acid sequence. Generally, for hybridizations with DNA oligonucleotides without formamide, a temperature of 68 DEG C, and for hybridization with formamide, 50% (v/v), a temperature of 42 DEG C is recommended. For hybridizations with oligonucleotides, the optimal conditions (formamide concentration and/or temperature) depend on the length and base composition of the probe and must be determined individually. In general, optimal hybridization for oligonucleotides of about 10 to 50 bases in length occurs approximately 5 DEG C below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched sequences to hybridize and can therefor result in reduced specificity. When using RNA oligonucleotides with formamide (50% v/v) it is recommend to use a hybridization temperature of 68 DEG C for detection of target RNA and of 50 DEG C for detection of target DNA. Alternatively, a high SDS hybridization solution can be utilized (Church et al. 1984, Proc Natl Acad Sci USA 81:1991-1995). The specificity of hybridization can furthermore be ensured through the presence of a crosslinking moiety on the oligonucleotide (e.g. Huan et al. 2000, Biotechniques 28: 254-255; WOOO/14281). Said crosslinking moiety enables covalent linking of the oligonucleotide with the target nucleotide sequence and hence allows stringent washing conditions. Such a crosslinking oligonucleotide can furthermore comprise another label suitable for detection/quantification of the oligonucleotide hybridized to the target.
RPKM (Reads Per Kilobase Million) is often used as measure for expression. FPKM (Fragments Per Kilobase Million) is very similar to RPKM; whereas RPKM was designed for single-end RNA-seq (every read corresponded to a single sequenced fragment), FPKM was designed for paired-end RNA-seq. With paired-end RNA-seq, two reads can correspond to a single fragment, or, if one read in the pair did not map, one read can correspond to a single fragment. The only difference between RPKM and FPKM is that FPKM takes into account that two reads can map to one fragment (and so it doesn't count this fragment twice). When using RNA-seq, reporting or results often is in RPKM (Reads Per Kilobase Million) or FPKM (Fragments Per Kilobase Million). Whatever metric used (another alternative for example is TPM (Transcripts Per Kilobase Million)), such metric is attempting to normalize for sequencing depth and gene length and provide a measure for quantifying transcript levels/gene expression/expression units.
Next to methodologies for determining gene expression by means of determining transcript levels (transcriptome analysis), it is also possible to quantify gene expression by means of proteomic analysis (proteome analysis or analysis of the proteome). Classical proteomic analysis methods include ELISA, western blotting, mass spectrometry, chromatographic separation, immunohistochemistry, cell sorting (based on cell surface marker(s)) etc. Although not necessarily required, it can be advantageous to rely on multiplexed cytometry methods that can be performed directly on, e.g., a section of a cancer tissue biopsy (Formalin-Fixed Paraffin-Embedded (FFPE), fresh frozen (FF), ...). Multiplexed cytometry methods, as well as some predictive cancer biomarkers identified using such methodology, have been reviewed by e.g. Fan et al. 2020 (Cancer Communications 40:135-153) and have emerged with the advent of more sophisticated imaging techniques (e.g. cyclic immunofluorescence, tyramide-based immunofluorescence, epitope-targeted mass spectrometry, RNA detection) and standardized quantification methodologies. Such multiplexed cytometry methods include multiplex immunocytochemistry (mICH), imaging mass spectrometry, multiplexed ion beam imaging, chipcytometry, nucleotide (DNA/RNA)-barcoding-based mICH, and digital spacing profiling. Another technique involving proteomic analysis is Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq).
Polynucleotide sequence determination is a common step in some of the methods as applied in the current disclosure. In general, sequencing methods may include, but are not limited to: high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), Next generation sequencing, Single Molecule Sequencing by Synthesis (SMSS) (Helicos), massively- parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxam- Gilbert or Sanger sequencing, primer walking, sequencing using PacBio, SOLID, Ion Torrent, or Nanopore platforms, short read sequencing, long read sequencing, and any other sequencing methods known in the art. The sequencing method can be massively parallel sequencing, that is, simultaneously (or in rapid succession) sequencing any of at least 100, 1000, 10,000, 100,000, 1 million, 10 million, 100 million, 1 billion, or 10 billion polynucleotide molecules.
Certain DNA sequencing methods may rely on the capture of polynucleotides of interest such as to enrich for these sequences of interest. Polynucleotide or sequence capture typically involves the use of oligonucleotide probes that hybridize to the polynucleotide or sequence of interest. A probe set strategy can involve tiling the probes across a region of interest (complete or partial tiling of the target sequence with probes). Such probes can be, e.g., 10 to 400 or about 400 bases long, 10 to 300 or about 300 bases long, 10 to 200 or about 200 bases long, 10 to 100 or about 100 bases long, 10 to 80 or about 80 bases long, 10 to 60 or about 60 bases long, Such probes may comprise at least one or a set of oligonucleotides of 10 to 60 bases or nucleotides long and/or comprise at least one or a set of oligonucleotides of 15 to 120 bases or nucleotides long. Any set of such oligonucleotide probes can have a depth of about O.lx, 0.2x, 0.3x, 0.4 x, 0.5x, O.lx to 0.5x, lx 2x, 3x, 4x, 5x, 6x, 8x, 9x, lOx, 15x, 20x, 50x or more. Enriched nucleic acid molecules can be representative of a nucleic acid features of interest such as, but not necessarily limited to the HPGDS gene markers and macrophage markers as described herein.
Sequencing depth refers to the number of times a locus is covered by a sequence read aligned to the locus. A locus can be as small as a nucleotide, as large as a chromosome arm, or as large as the entire genome. Sequencing depth can be expressed as e.g. lOx, 50x, lOOx, where "x" refers to the number of times a locus is covered by a sequence read. Sequencing depth can also be applied to multiple loci, or to the whole genome, in which case "x" can refer to the mean number of times the loci, or whole genome, is sequenced. Ultra-deep sequencing refers to a sequencing depth of at least lOOx. Sequencing depth usually increases with decreasing amounts of analyte strand; the sequencing depth is therefore expected to be higher in case of analyzing cell-free DNA compared to analyzing nucleic acids isolated from a tumor biopsy.
Shallow whole genome sequencing, low coverage whole genome sequencing, or ultra-low pass whole genome sequencing in general refers to short-read sequencing of genomes at low coverage, typically less than 3x coverage, less than 2x coverage, less than lx coverage, such as O.lx to lx coverage, such as O.lx to 0.8x coverage, such as O.lx to 0.6x coverage, such as O.lx to 0.5x coverage, such as O.lx to 0.4x coverage, such as O.lx to 0.3x coverage, such as 0.9x coverage, 0.8x coverage, 0.7x coverage, 0.6x coverage, 0.5x coverage, 0.4x coverage, 0.3x coverage, 0.2x coverage or O.lx coverage, such as O.lx coverage or less. Sequencing coverage can also be expressed as average sequencing coverage. Low coverage in the context of sequencing thus can also refer to typically on average less than 3x coverage, on average less than 2x coverage, on average less than lx coverage, such as on average O.lx to lx coverage, such as on average O.lx to 0.8x coverage, such as on average O.lx to 0.6x coverage, such as on average O.lx to 0.5x coverage, such as on average O.lx to 0.4x coverage, such as on average O.lx to 0.3x coverage, such as on average 0.9x coverage, on average 0.8x coverage, on average 0.7x coverage, on average 0.6x coverage, on average 0.5x coverage, on average 0.4x coverage, on average 0.3x coverage, on average 0.2x coverage or on average O.lx coverage, such as on average O.lx coverage or less.
By performing shallow whole genome sequencing, low coverage whole genome sequencing, or ultra-low pass whole genome sequencing, each sample is subjected to a small amount of sequencing, allowing application of whole genome sequencing to many samples at low cost per sample.
A sequence read is a string of nucleotides sequenced from a part or all of a nucleic acid molecule. A sequence read may be a short string of nucleotides (e.g. 20 to 150 nucleotides, around 50 nucleotides) sequenced from a nucleic acid (fragment). Sequence reads may be obtained at one end of a nucleic acid (fragment) or from both ends of a nucleic acid (fragment). Sequence reads may be obtained by e.g. applying a sequencing technique to the nucleic acid (fragment), by hybridization arrays or capture probes, by amplification techniques (e.g. PCR, linear amplification, isothermal amplification) such as amplification techniques using a single primer.
Thus, obtaining information from the nucleic acid molecules present in a biological sample may include a step of preparing a sequencing library using the nucleic acid molecules isolated from the biological sample. The preparation of such sequencing library may include a step of DNA amplification, or may, alternatively, not include a step of DNA amplification. Obtaining information from the nucleic acid molecules present in a biological sample may include obtaining DNA sequence reads. Obtaining information from the nucleic acid molecules (e.g. cfDNA molecules) present in a biological sample may include the step of aligning the plurality of (DNA) sequence reads to a reference genome to determine the genomic positions of each (individual) sequence read of the plurality of sequence reads. In view of the size of the reference genome and the number of sequence reads in a plurality of sequence reads, the sequence reads are optionally received at a computer system. Spatial detection methods
Spatial proteomics
A good overview of spatial detection methods is provided by Lewis et al. 2021 (Nature Methods 18:997- 1012) with Figures 3 to 5 therein summarizing the described methods and their performance.
The most classical spatial detection methods are histopathological staining (e.g. haematoxylin and eosin staining) and immunohistochemical staining. Histopathological staining provides information on different tissue structures and possible abnormalities therein. Immunohistochemical (IHC) staining involves binding of antibodies to target proteins of interest, usually these (primary) antibodies are unlabelled and (primary) antibodies bound to its target in e.g. a tissue section are subsequently detected by binding of a labelled, e.g. fluorescently labelled, (secondary) antibody that binds to the (primary) antibody bound to the target protein of interest. In multiplexed IHC (mIHC) usually up to 4 or 5 target proteins of interest can be detected simultaneously. In modern mIHC, iterative cycles of target protein of interest detection are applied. This involves successive cycles of antibody binding and stripping of the antibody or stripping or bleaching of the antibody-labels. Alternatively, a pool of DNA-barcoded antibodies is applied and iterative hybridization with differently labelled oligonucleotides is performed. As a result, some of these techniques can detect up to 100 different proteins can be detected in a single tissue sample.
Non-iterative methods of target protein of interest detection involve binding of metal isotope-labelled antibodies that are subsequently detected by mass spectrometry upon release from a sample by means of tissue ablation with a laser beam (IMC: imaging mass cytometry) or tissue ionization with an ion beam (MIBI: multiplexed ion beam imaging). These techniques can detect up to 40 different proteins can be detected in a single tissue sample. IMC also allows for detection of an RNA target of interest.
Concurrent quantitation of more than 40 proteins of interest is furthermore possible by quantitative analysis (involving sequencing) of oligonucleotides cleaved off from oligo-nucleotide labelled primary antibodies, this in a technique called digital spatial profiling (DSP). DSP also allows for detection of an RNA target of interest. These spatial proteomic techniques have been summarized in e.g. Figure 3 of Lewis et al. 2021 (Nature Methods 18:997-1012).
Spatial transcriptomics
Spatial transcriptomics rely on direct detection of transcripts of interest with fluorescently labelled probes, the technology called fluorescent in situ hybridization (FISH). Many different FISH-based spatial transcriptomic methods have been developed and include iterative hybridization or bleaching or destruction of the labelled probes. Some of the FISH-based spatial transcriptomic methods allow for detection of up to 10000 different transcripts (summarized in e.g. Figure 4 of Lewis et al. 2021 (Nature Methods 18:997-1012). Another series of spatial transcriptomic methods involve sequencing. In vitro methods (e.g. on a tissue sample) include laser capture microdissection (LCM) methods, methods including an mRNA capture step, and microfluidic-based methods - all have been reported to allow for detection of 10000 or more targets. In situ methods include in situ sequencing and fluorescence in situ sequencing methods. The sequencing technique can rely on sequence-by-ligation or sequence-by- hybridization methodologies. Again, some of these methods have been reported to allow for detection of 10000 or more targets (incompletely summarized in e.g. Figure 5 of Lewis et al. 2021 (Nature Methods 18:997-1012).
Tumor, cancer, neoplasm
The terms tumor and cancer are sometimes used interchangeably but can be distinguished from each other. A tumor refers to "a mass" which can be benign (more or less harmless) or malignant (cancerous). A cancer is a threatening type of tumor. A tumor is sometimes referred to as a neoplasm: an abnormal cell growth, usually faster compared to growth of normal cells. Benign tumors or neoplasms are nonmalignant/non-cancerous, are usually localized and usually do not spread/metastasize to other locations. Because of their size, they can affect neighboring organs and may therefore need removal and/or treatment. A cancer, malignant tumor or malignant neoplasm is cancerous in nature, can metastasize, and sometimes re-occurs at the site from which it was removed (relapse). The initial site where a cancer starts to develop gives rise to the primary cancer. When cancer cells break away from the primary cancer ("seed"), they can move (via blood or lymph fluid) to another site even remote from the initial site. If the other site allows settlement and growth of these moving cancer cells, a new cancer, called secondary cancer, can emerge ("soil"). The process leading to secondary cancer is also termed metastasis, and secondary cancers are also termed metastases. For instance, liver cancer can arise as primary cancer, but can also be a secondary cancer originating from a primary breast cancer, bowel cancer or lung cancer; some types of cancer show an organ-specific pattern of metastasis. Most cancer deaths are in fact caused by metastases, rather than by primary tumors (Chambers et al. 2002, Nature Rev Cancer2:563-572).
Cancer is referred to in general terms herein. More in particular, the cancer or tumor is a primary cancer or tumor. Not mutually exclusive, the cancer or tumor more in particular is a solid cancer or tumor. More specifically, the cancer is melanoma or hepatocellular carcinoma (HCC), such as advanced HCC (aHCC).
Immune checkpoint inhibitors/blockers (ICIs/ICBs/CPIs)
Immune checkpoints antagonists or inhibitors as referred to herein include the cell surface protein cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death protein-1 (PD-1) and their respective ligands. CTLA-4 binds to its co-receptor B7-1 (CD80) or B7-2 (CD86); PD-1 binds to its ligands PD-L1 (B7- H10) and PD-L2 (B7-DC). Other immune checkpoint inhibitors include the adenosine A2A receptor (A2AR), B7-H3 (or CD276), B7-H4 (or VTCN1), BTLA (or CD272), IDO (indoleamine 2,3-10 dioxygenase), KIR (killer-cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene-3), NOX2 (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoform 2), TIM3 (T-cell immunoglobulin domain and mucin domain 3), VISTA (V-domain Ig suppressor of T cell activation), SIGLEC7 (sialic acid-binding immunoglobulin-type lectin 7, or CD328) and SIGLEC9 (sialic acid-binding immunoglobulin-type lectin 9, or CD329).
In any of the above aspects, embodiments, and kits, the therapy comprising an ICI or therapy with an ICI can in particular be a therapy comprising a combination in any way of two immune checkpoint inhibitors. In one embodiment these are each inhibiting a different immune checkpoint or a different immune checkpoint-ligand interaction. For instance, when an inhibitor of PD1 is selected as a first immune checkpoint inhibitor, the second immune checkpoint inhibitor could be an inhibitor of PDL1 or an inhibitor of PDL2. Such first and second immune checkpoint inhibitor are each inhibiting a different immune checkpoint protein. In a further non-limiting example, an inhibitor of PD1 is selected as a first immune checkpoint inhibitor, and as second immune checkpoint inhibitor an inhibitor different from an inhibitor of PDL1 and different from an inhibitor of PDL2 is selected, e.g. an inhibitor of CTLA-4 is selected. In this latter example, the first and second immune checkpoint inhibitor are not only each inhibiting a different immune checkpoint, but also each inhibiting a different immune checkpoint-ligand interaction.
An overview of clinical developments in the field of immune checkpoint therapy is given by Fan et al. 2019 (Oncology Reports 41:3-14). Immune checkpoint inhibitors include, but are not limited to anti-PD- 1, anti-PD-Ll or anti-CTLA-4 antibodies.
PD1
Aliases of PD1 provided in GeneCards® include PDCD1; Programmed Cell Death 1; Systemic Lupus Erythematosus Susceptibility 2; PD-1; CD279; HPD-1; SLEB2; and HPD-L. The genomic locations for the PDCD1 gene are chr2:241, 849, 881-241, 858, 908 (in GRCh38/hg38) and chr2:242, 792, 033-242, 801, 060 (in GRCh37/hgl9). The GenBank reference PD1 mRNA sequence is known under accession no. NM_005018.3. Approved PDl-inhibiting antibodies include nivolumab, pembrolizumab, and cemiplimab; PDl-inhibiting antibodies under development include CT-011 (pidilizumab) and therapy with PDl-inhibiting antibodies is referred to herein as a-PD-1 therapy or a-PDl therapy. PD1 siRNA and shRNA products are available through e.g. Origene.
PD-L1
Aliases of PD-L1 provided in GeneCards® include CD274, Programmed Cell Death 1 Ligand 1, B7 Homolog 1, B7H1, PDL1, PDCD1 Ligand 1, PDCD1LG1, PDCD1L1, HPD-L1, B7-H1, B7-H, and Programmed Death Ligand 1. The genomic locations for the PDCD1 gene are chr9:5, 450, 503-5, 470, 567 (in GRCh38/hg38) and chr9:5, 450, 503-5, 470, 567 (in GRCh37/hgl9). The GenBank reference PD1 mRNA sequence is known under accession no. NM_001267706.1, NM_001314029.2 and NM_014143.4. Approved PD-Ll-inhibiting antibodies include atezolizumab, avelumab, and durvalumab. PD-L1 siRNA and shRNA products are available through e.g. Origene.
CTLA4
Aliases of CTLA4 provided in GeneCards® include Cytotoxic T-Lymphocyte Associated Protein 4; CTLA-4; CD152; Insulin-Dependent Diabetes Mellitus 12; Cytotoxic T-Lymphocyte Protein 4; Celiac Disease 3; GSE; Ligand And Transmembrane Spliced Cytotoxic T Lymphocyte Associated Antigen 4; Cytotoxic T Lymphocyte Associated Antigen 4 Short Spliced Form; Cytotoxic T-Lymphocyte-Associated Serine Esterase-4; Cytotoxic T-Lymphocyte-Associated Antigen 4; CELIAC3; IDDM12; ALPS5; and GRD4.
The genomic locations for the CTLA4 gene are chr2:203, 867, 771-203, 873, 965 (in GRCh38/hg38) and chr2:204, 732, 509-204, 738, 683 (in GRCh37/hgl9). The GenBank reference CTLA4 mRNA sequences are known under accession nos. NM_001037631.3 and NM_005214.5. Approved CTLA4-inhibiting antibodies include ipilumab; CTLA4-inhibiting antibodies under development include tremelimumab; therapy with CTLA4-inhibiting antibodies is referred to herein as a-CTLA4 therapy. CTLA4 siRNA and shRNA products are available through e.g. Origene.
Combination, combination in any wav
"Combination", "combination in any way" or "combination in any appropriate way" as referred to herein is meant to refer to any sequence of administration of two (or more) therapeutic modalities, i.e. the administration of the two (or more) therapeutic modalities can occur concurrently in time or separated from each other by any amount of time; and/or "combination", "combination in any way" or "combination in any appropriate way" as referred to herein can refer to the combined or separate formulation of the two (or more) therapeutic modalities, i.e. the two (or more) therapeutic modalities can be individually provided in separate vials or (other suitable) containers, or can be provided combined in the same vial or (other suitable) container. When combined in the same vial or (other suitable) container, the two (or more) therapeutic modalities can each be provided in the same vial/container chamber of a single-chamber vial/container or in the same vial/container chamber of a multi-chamber vial/container; or can each be provided in a separate vial/container chamber of a multi-chamber vial/container.
Other Definitions
The present disclosure is described with respect to particular embodiments and with reference to certain drawings but the disclosure is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are nonlimiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the current disclosure described herein are capable of operation in other sequences than described or illustrated herein. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the current disclosure. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.
In any of the above, a "subject" in general is a mammalian species having (or having been diagnosed to have) a disease or disorder, in particular a cancer or a tumor. The mammalian species in general is a higher species including primates, cattle (e.g. cows, sheep, goats, pigs), horses, and pets (e.g. dogs, cats). In one embodiment the patient is a human subject or human patient.
It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for cells and methods according to the present disclosure, various changes or modifications in form and detail may be made without departing from the scope and spirit of this disclosure. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.
In referring to genes or proteins herein, distinction between their respective annotation is not always made hereinabove or hereinafter.
The content of the documents cited herein are incorporated by reference. EXAMPLES
EXAMPLE 1. Identification of HPGDS expression on immunosuppressive tumor-associated macrophages (TAMs)
A meta-analysis was performed by combining two publicly available pre- and post-treatment bulk RNA- sequencing datasets (RNA-Seq) of tumors responding and resistant to a-PDl treatment (Hugo et al. 2016, Cell 165:35-44; Riaz et al. 2017, Cell 171:934-949). For each individual dataset, a differential gene expression analysis between tumors responsive to a-PDl treatment and non-responsive to a-PDl treatment was performed including BRAF, NRAS and NF1 mutational status as a covariate. We then ranked genes by their Iog2 fold change and combined the resulting rank by calculating the median rank product to identify metabolic genes that were consistently associated with PD1 immunotherapy response. The rank-product meta-analysis on metabolic genes specifically identified Hematopoietic Prostaglandin D2 Synthase (HPGDS) as a potential top target. When we filtered the ranked gene list for genes associated with arachidonic acid metabolism, HPGDS was again among the top candidates. The arachidonic acid metabolism associated ranked gene list was subsequently visualized using the R package ggplot2 (v3.1.1).
The analysis of a public single-cell RNA-Seq dataset (Jerby-Arnon et al. 2018, Cell 175:984-997) of melanoma patients revealed that HPGDS expression is exclusively restricted to the TAM compartment, and is not expressed in the tumor stroma. This finding was further confirmed by analyzing an in-house single-cell RNA-Seq dataset where we found that HPGDS was highly expressed in macrophages but also in melanophages and mast cells (Figure 1A). Given the limiting number of mast cells infiltrating the tumor and the higher expression of HPGDS in macrophages compared to melanophages (Figure IB), we decided to focus our attention only on macrophages. Interestingly, the expression of HPGDS was restricted to the pro-tumoral M2-like macrophages (Figure 1C) and negatively correlated with activated CD8+ T cells (Figure ID). Our findings were further corroborated by immunohistochemistry analysis on primary tumor as well as lymph nodes and liver metastases of melanoma patients, where a selective co-localization of HPGDS with TAMs can be appreciated (Figure IE).
Altogether, these observations pointed HPGDS as a potential marker to identify and thus target the pro- tumoral M2-like macrophages.
EXAMPLE 2. Hpgds inhibition re-educates tumor-associated macrophages (TAMs) toward an Ml-like phenotype
Among all the cells of the immune system, we found that in orthotopic YUMM 1.7 melanoma Hpgds was almost exclusively expressed in TAMs, as assessed by measuring the transcript of Hpgds in different cell types sorted from the tumor micro-environment (TME) (Figure 2A). According to Mary et al. 2022 (Cancer Immunology Research 10:900-916), Hpgds is expressed on T follicular helper (Tfh) cells but not on other CD4+ T cell subsets. We confirmed the expression of Hpgds in this subset but its expression on lymph nodes was 80-fold lower than the expression detected on macrophages. In addition, no expression of Hpgds on intratumoral CD4+ T cells was detected.
Then, we confirmed the selective colocalization of Hpgds with TAMs by immunofluorescence (Figure 2B). In line with this, Hpgds expression was nearly absent in in vitro Ml-polarized BMDMs but very high in M2-like BMDMs (Figure 2C); HPGDS protein could not be detected in other tumor compartments or tissue macrophages from healthy organs. Strikingly, the same expression level of Hpgds observed in both, sorted TAMs and M2-like macrophages was suggestive of a pro-tumoral role of Hpgds-expressing macrophages. In contrast to YUMM1.7 melanoma, Hpgds expression in B16 melanoma was predominant in malignant cells and alveolar cells 1, with further scattered expression in NK cells, B cells, and monocytes/macrophages (Figure 7); when checking several tumor slides by immunohistochemistry no co-localization of HPGDS with TAMs could be observed (whereas such co-localization was observed in other tumors indicating that the HPGDS antibody did work).
We then inquired about the biological role of Hpgds in macrophages. Thus, when characterizing Hpgds- silenced macrophages, we observed that its deletion in MO or M2-like macrophages, strongly affects the expression of main Ml and M2 macrophage polarization markers, by re-educating macrophages towards an Ml-like phenotype (Figure 2D and 2N).
The same was true in human monocyte-derived macrophages (hMDMs) where M2-like polarization (stimulation with IL-4) resulted in a higher expression of HPGDS compared to Ml-like hMDMs (stimulated with LPS and I FNy) (Figure 2E). Supporting the murine data, HPGDS-silencing in both MO and M2-like macrophages, skews their pro-tumoral, anti-inflammatory phenotype towards an anti-tumoral, immunostimulatory Ml-like phenotype (Figure 2F and 20).
In further elucidating the functional role of Hpgds, we measured the abundance of Prostaglandin D2 (PGDj) and we confirmed a reduction of this lipid mediator upon Hpgds inhibition (Figure 2G). As expected, the lipidomic analysis did not reveal any modification of other pro-tumoral lipid and lipid mediators, like Arachidonic Acid (AA) and Prostaglandin E2 (Figure 2P). However, the decreased levels of PGD2 and its downstream active mediators was associated with a statistically significant increase of some pro-inflammatory mediators, like Thromboxane B2 (Figure 2 P).
Considering that PGD2 can be synthetized by the action of two enzymes, Lpgds and Hpgds and only this latter is expressed in macrophages, while Lpgds is predominantly expressed in endothelial cells (Figure 2Q), we hypothesized that the decrease of PGD2 is a determinant factor to induce a pro-tumoral phenotype of these cells. Thus, we assessed macrophage polarization upon exposure to 1 pM of PGD2. Flow cytometric analysis revealed that PGD2 affects their polarization by promoting an anti-inflammatory M2-like phenotype (Figure 2H). Consistent with the notion that macrophages can define blood vessel's structure and function, we conducted a 3D spheroid sprouting assay in a type I collagen gel by coculturing Hpgds-silenced macrophages with HUVECs. In these conditions we demonstrated that the silencing for Hpgds decreased the number and the length of sprouts formed by HUVECs (Figure 21-K). Of note, we found that PGD2 treatment enhanced the expression in HUVECs of key regulators of angiogenesis and lymphangiogenesis (/.e., VEGFA and VEGFC) in a time-dependent manner (Figure 2L), pointing at the Hpgds-PGDj signaling as key driving force for tumor angiogenesis. In line with this, the pre-treatment of HUVECs with PGD2 for 48 hr strongly increase their migratory capacity towards macrophages (Figure 2M).
Together, these data suggest that Hpgds represents an anti-inflammatory macrophage marker and its targeting can have a strong impact on the tumor progression by engaging the immune-system and impeding angiogenesis.
EXAMPLE 3. Hpgds deletion in tumor-associated macrophages (TAMs) inhibits tumor progression and reshapes the tumor micro-environment (TME)
To investigate the functional role of Hpgds in TAMs, Hpgds expression was selectively deleted in the hematopoietic cell lineage of mice. Inducible macrophage-specific Hpgds KO mice were generated by intercrossing the tamoxifen-inducible Csflr.Cre-ERT (a kind gift of J. W. Pollard from the University of Edinburgh, Scotland) with LoxP-STOP-LoxP Cas9 (LSL-CRISPR/Cas9) mice (B6J.129(B6N)- Gt(ROSA) 26Sortml(CAG-cas9*,-EGFP)Fezh/J). hematopoietic/progenitor cells of these mice (lineage negative HSCs, Lin HSCs) were transduced with a validated gRNA against Hpgds or with non-targeting control gRNA. Lethally irradiated wild-type (WT) mice were reconstituted with either of the two transduced hematopoietic/progenitor cells (Lin HSCs), thus generating inducible Hpgds knockout (KO)
Figure imgf000049_0001
WT and WT WT chimeras, respectively. Four weeks after the generation of the chimera, the immune reconstitution was confirmed and mice were treated with tamoxifen for five days. Hpgds genetic ablation was confirmed at the RNA level, by differentiating in vitro HSC into macrophages and in vivo at the protein level (Figure S3A). YUMM 1.7 melanoma cancer cells were intradermally injected in both WT WT and Hpgds KO
Figure imgf000049_0002
WT mice 5 to 6 weeks after immune reconstitution.
Hpgds deletion in the macrophage compartment led to a significant reduction in the tumor growth and tumor weight (Figure 3A). Consistent with our in vitro observation that Hpgds inhibition promotes an inflammatory phenotype in macrophages, the TAM compartment of Hpgds KO
Figure imgf000049_0003
WT melanoma-bearing mice was strongly affected: although Hpgds knockout did not influence overall TAM infiltration (Figure 3B), tumors in Hpgds KO
Figure imgf000049_0004
WT chimera mice displayed more anti-tumoral macrophages, defined as positive for MHC-II, CDllc and CD86 (Figure 3C) and less pro-tumoral M2-like macrophages, expressing lower level of MHC-II and positive for CD206 and CD204 (Figure 3D), suggesting that Hpgds is crucial for determining their differentiation state into M2 macrophages. By histological analysis we confirmed the pro-inflammatory phenotype of macrophages, characterized by more anti-tumoral Ml-like macrophages, defined as positive for both F4/80 and CD80 and less pro-tumoral M2-like macrophages, defined as positive for both F4/80 and CD206 (Figure 3E). In addition, flow cytometry analysis of the immune landscape revealed that tumors in Hpgds KO
Figure imgf000050_0001
WT chimera mice displayed a slight increase in CD8+ T cell infiltration (Figure 3F) with augmented secretion of Interferon y (IFNy) and granzyme B (GZMB) (Figure 3G). By histological analysis we could consistently observe an increase in CD8+ T cell infiltration at the tumor core (Figure 3H), suggesting a transition from an immune-excluded phenotype, characterized by an high number of CD8+T cells at the tumor border to the inflamed phenotype, in which immune cells are abundant and localized at the tumor core (Galluzi et al. 2018, Sci Transl Med 10:eaat7807).
These in vivo evidences were further validated using a more conventional approach, where the specificity of the KO was assessed by checking the expression of Hpgds (Figure 31). Thus, by intercrossing Hpgds floxed mice with CD64:Cre mice we generated a murine model where Hpgds is specifically depleted in macrophages (CD64-Cre;Hpgdsi/i; Hpgds-KO, Hpgds™® or HpgdsAMa> in short). When YUMM 1.7 melanoma cancer cells where intradermally injected in the Hpgds-KO mice and in the littermate controls (CD64-Cre;Hpgds+/+ or Hpgdswt/wt; Ctrl in short) the antitumoral effect of Hpgds was even more pronounced, with a reduction in tumor growth of almost 80% and tumor weight (Figures 3J). The reduced tumor size was associated with a pro-inflammatory phenotype in macrophages, with an increased number of Ml-like macrophages expressing MHC-II, CDllc and CD86 and lower number of M2-like macrophages, expressing CD206 and CD204 (Figures 3K-O). The results obtained by flow cytometry were confirmed by histological analysis. In addition, as in the Hpgds KO
Figure imgf000050_0002
WT model, the number of CD8+ T cells (Figure 30) and their activation status (Figure 3P) was augmented in Hpgds-KO tumors. Moving to the vessel compartment we found that tumor blood vessel perimeter as well as pericyte coverage, perfusion vessels and hypoxia (parameters linked to cancer cell intravasation into the bloodstream) were strongly affected by Hpgds-KO, with an overt functional and structural improvement which usually correlates with a better disease outcome in patients (Mazzone et al. 2009, Cell 136:839-851) (Figures 3Q). Finally, when checking the interstitial fluid we confirmed that the depletion of Hpgds in macrophages resulted in reduced levels of PGDj (Figure 3R) while other bio-active lipid mediators, like AA and PGEj were not affected by its inhibition (Figure 3R). This data where further confirmed by measuring the intratumoral level of PGDj of Hpgds-KO tumors compared to the Ctrl tumors (Figure 3S). Hence, to further investigate the effect of a change in PGDj released by macrophages, firstly we proved that YUMM 1.7 were migrating towards a gradient of PGDj , then we performed a co-culture of Hpgds- KO BMDMs with YUMM 1.7-melanoma cancer cells. Interestingly, cancer cell migration towards BMDMs Hpgds-KO was lower than towards Hpgds-WT (Figure 3T). Thus, tumor blood vessel density, perimeter, size, pericyte coverage, vessel perfusion, and tumor hypoxia were strongly affected by Hpgds deletion in TAMs, resulting in fewer vessels but with overt functional and structural improvements. We have shown before that increased vessel coverage and reduced tumor hypoxia prevent cancer cell dissemination from the primary tumor to distant organs (Mazzone et al. 2009, Cell 136:839-851). Consistently, cancer cell intravasation into the bloodstream and lodging to the lungs in tumor-bearing Hpgds&M0 mice was lower than in controls (Figure 3U).
All these data indicate that the metabolic editing of the TME via inhibition of HPGDS reshapes the immunosuppressive TAMs and boosts an anti-tumor immune response.
The chimeric macrophage model as used herein plausibly supports use of adoptive transfer of macrophages in which HPDGDS expression is knocked-out or inhibited in the treatment of cancer.
EXAMPLE 4. Targeting HPGDS in tumor-associated macrophages (TAMs) requires the presence of CD8+ T cells to affect tumor progression
Based on the modified tumor micro-environment (TME) upon Hpgds depletion in macrophages, we proceeded with assessing the interaction of Hpgds-KO macrophages (Hpgds^M0) with other tumorinfiltrating immune cells. To this end, we treated Ctrl and Hpgds-KO melanoma-bearing mice with an a- CD8 depleting antibody. Of note, the depletion of CD8+ T cells was checked in the blood before tumor injection and at the end stage of the experiment and in the tumor. Tumors in CD8-depleted mice in general showed bigger tumors and the anti-tumoral effect of Hpgds-KO in macrophages was completely abolished upon CD8 depletion (Figure 4A-B), with the macrophages being re-educated toward an Ml- like phenotype. Interestingly, the absence of tumor infiltrating CD8+ T cells was not affecting the anti- tumoral role of TAMs in promoting vascular normalization (Figure 4C), and was not affecting metastasis inhibition by Hpgds-KO (Figure 4E). Taken together, these data argue that macrophages perse are able to normalize the tumor vasculature which is not sufficient to inhibit tumor growth as the presence of CD8+ T cells is required to dampen tumor progression. Confirming the relevance CD8+ T cells in the observed phenotype, flow cytometry analysis performed on Hpgds-KO and Ctrl BMDMs co-cultured with CD8+T cells revealed that the interaction between these two cells is sufficient to increase their activation (IFNy+; TNF-a+) (Figure 4D).
Finally, Ctrl and Hpgds&M0 mice were orthotopically injected with YUMM 1.7 melanoma cancer cells and administered with aPDl when the average tumor size of both groups was 150 mm3. Although Ctrl mice displayed resistance to aPDl, Hpgds deletion in macrophages, sensitized tumors to aPDl and inhibited tumor relapse in Hpgds&M0 mice (Figure 4F and 4G). EXAMPLE 5. Pharmacologic inhibition of Hpgds inhibits tumor growth and re-educates macrophages towards an Ml-like phenotype
To translate the results obtained by using the genetic approach in a clinically relevant setting, we used the HPGDS-specific inhibitor HQL-79 [4-(Diphenylmethoxy)-l-(3-2H-tetrazol-5-yl)propyl]-piperidine; CAS No. 162641-16-9; InChi key TZQGXAHOROZEKN-UHFFFAOYSA-N] to pharmacologically target HPGDS in YUMM 1.7 melanoma bearing mice. Strikingly, by using a systemic approach we were able to recapitulate the anti-tumor phenotype induced by genetic deletion of Hpgds (Figure 5A). Indeed, the impairment in tumor growth was driven by a re-education of tumor-associated macrophages (TAMs) toward an Ml- like, pro-inflammatory, anti-angiogenic phenotype (Figure 5D-F) a robust infiltration of CD8+T cells with reinvigorated effector functions as assessed by the augmented secretion of effector cytokines such as IFNy and GZMB, pointing to a shift toward an inflamed, anti-tumor phenotype. Similar results were obtained by taking advantage of a tumor cell line derived from an NrasQ61K; lnk4A~/~ spontaneous melanoma model (Figure 5C).
Like macrophage-specific Hpgds deletion, HQL-79 strongly affected the tumor vasculature by decreasing vessel density and size, and by improving morphologic and functional parameters such as pericyte coverage and perfusion, together with reduced hypoxia, cancer cell intravasation into the bloodstream (Figure 5G) and lung colonization (Figure 5G).
The specificity of HQL-79 was proved by intercrossing Hpgds floxed mice with CD64:Cre-ERT2 mice (CD64- Cre-ERT2; HpgdsL/L; Hpgds^M0~ERT2 in short), where Hpgds was deleted in TAMs upon intraperitoneal injection of tamoxifen. The effect of either systemic inhibition or acute deletion in macrophages was comparable, and the inhibitor did not have any additional effect when administered to Hpgds^M0~ERT2 mice (Figure 5H).
Comparable results were obtained with the use of a second HPGDS inhibitor, Cmpdly (7-Cyclopropyl-N- ((trans)-4-(2-hydroxypropan-2-yl)cyclohexyl)-l,8- naphthyridine-3-carboxamide; Cadilla et al. 2020, Bioorganic & Medical Chemistry 28:115791) showing a dose-response effect on tumor inhibition with the highest efficacy at 3 mg/kg BID (/.e., 55% of tumor growth and weight inhibition versus vehicle) (Figure 51). A less potent HPGDS inhibitor, TAS-205 (CAS No 1584160-52-0; Inchi Key NFXCKTKGAJDCBA- UHFFFAOYSA-N; Aoyagi et al. 2020, Eur J Pharmacol 875:173030), also showed a robust therapeutic effect at 30 mg/kg BID (Figure 5J).
Taken together, these data offer proof that pharmacological inhibition of HPGDS can sculpt TAMs away from their immunosuppressive state, providing a promising therapeutic option against melanoma.
Furthermore, the involvement of HPGDS in cancer progression was studied in an high metastatic and lethal cancer indication. Hepatocellular carcinoma (HCC) was chosen. Firstly, we verified the selective expression of HPGDS in macrophages (murine and human data). In the HCC model used, HPGDS was systemically inhibited. Pharmacological inhibition of HPGDS affected the number and the size of both, macro and micronodules (Figure 5D-E) and led to an increased number of total macrophages (F4/80+) most likely due to more Ml-like macrophages (Figure 5F). As in the melanoma tumor model, we observed that HPGDS targeting resulted in a significantly higher infiltration of CD8+T cells, whereas the total number was not affected by the treatment.
Taken together, these data offer a proof-of-concept that pharmacological inhibition of HPGDS can sculpt TAMs away from their immunosuppressive state and increase the fitness of CD8+ T cells.
The therapeutic effect of pharmacologic HPGDS inhibition was further tested in patient-derived organotypic tumor spheroids (PDOTs)(Jenkins et al. 2018, Cancer Discovery 8:196-215). The treatment with lpM HQL-79 for 72hr decreased the viability of the tumoroid (Figure 5K), and this decrease was associated with an accumulation of total and TNFa-producing CD8+ T cells. HQL-79 did not affect total TAMs; however, these macrophages were re-educated towards an immunostimulatory phenotype.
EXAMPLE 6. HPGDS systemic inhibition improves efficacy of immune checkpoint blockers
Despite the strong anti-tumor effect mediated by the inhibition of HPGDS, we wondered if HPGDS targeting can enhance the response to immunotherapy. Thus, we referred back to human datasets and we investigated the expression of HPGDS in immunotherapy treated patients. Strikingly, we found that HPGDS expression remains high in TAMs from non-responding patients, whereas it is "turned-off" in those patients that are responsive to a-PD-1 therapy (Figure 6A).
Our in vitro findings show that the inhibition of HPGDS induces Pdcdl (gene encoding for PD-1) upregulation in BMDMs.
Of note, when treating YUMM 1.7 melanoma-bearing mice either with HPGDS pharmacological inhibitor alone or in concert with a-PD-1, the effect of HPGDS inhibition in combination with a-PD-1 administration could not be analyzed due to total regression of the tumors. We then took advantage of a genetically engineered mouse (GEM) metastatic melanoma model (Dankort et al. 2009, Nat Genet 41:544-552) carrying the most common mutations identified in human melanomas, Braf/600EPten/'. In contrast to the orthotopic YUMM 1.7 model, HPGDS targeting did not affect tumor progression (Figure 6B) albeit macrophages were re-educated towards an Ml-like phenotype (Figure 6C). Flow cytometry analysis revealed no differences in the number of CD8+T cells (Figure 6D), but augmented PD1 expression (Figure 6E). This data suggested that although the total number of CD8+ T cells did not change, these cells were better suited to induce immunotherapy-response. Based on these observations, we hypothesized that, in this model, the mechanism of HPGDS-resistance is mediated by PD-1 up-regulation. Accordingly, the monotherapy (/.e., HQL-79 or a-PD-1 alone) was not sufficient to reduce the tumor, whereas their combination showed a persistent and synergic effect (Figure 6F). From a mechanistic point of view, the combination with a-PD-1 treatment did not add anything to the effect on macrophages, nevertheless, to affect tumor progression it requires the infiltration of CD8+T cells (Figure 6G). Based on these data we speculated that HPGDS systemic inhibition sensitizes tumors to immunotherapy, probably by up-regulating PD-1 expression on CD8+ T cells and thus promoting immunotherapy response. Congruently, we observed PD1 upregulation on CD8+ T cells upon co-culture with Hpgds-KO BMDMs.
Lastly, we extended our findings to one of the most lethally and aggressive tumor types, like pancreatic ductal adenocarcinoma (PDAC). Thus, we investigated the expression of HPGDS in PDAC. Consistent with the pro-tumoral role of HPGDS, its expression was high in the tumor tissue compared to the adjacent healthy tissue. We then confirmed the co-localization of HPGDS with macrophages in both, mouse and human tumors. To confirm the relevance of HPGDS in pancreatic tumors, we systemically inhibited HPGDS in orthotopically injected KPC FC1245. Like in the GEM model of melanoma, also here CD8+ T cells are excluded (Scolaro et al. 2024, Nat Cancer 5:1206-1226). No differences in body weight were observed. Congruently, HPGDS systemic inhibition slightly decreased tumor weight while aPDl did not have any effect. However, their combination was greatly effective (Figure 6H).
Overall, these data show that inhibition of HPGDS in combination with a-PD-1 reduces tumor growth, abates immunosuppression and can pave the way toward a possible therapeutic strategy to overcome resistance to immunotherapy.
EXAMPLE 7. Targeting DPI expression on CD8+ T-cells and in macrophages is affecting tumor progression
As indicated in Example 4, targeting HPGDS in tumor-associated macrophages (TAMs) requires the presence of CD8+ T cells to affect tumor progression. This was investigated further.
Downstream actions of HPGDS-PGDj axis are mediated through DPI (encoded by the PTGDR1 gene) and DP2 (encoded by the PTGDR2 gene), 2 G-protein coupled receptors. Expression analysis on YUMM 1.7 melanoma tumors highlighted that Ptgdrl is highly expressed in CD8+ T cells, CD4+ T cells, neutrophils and macrophages, while Ptgdr2 expression was high in CD4+T cells, neutrophils and macrophages.
Although both receptors were highly expressed in CD4+ T cells, PGDj supplementation was not sufficient to alter their phenotype. Overall, this expression pattern strongly suggested that both autocrine and paracrine loops arising from Hpgds expressing cells connects TAMs with the other cell compartments. Of note, PGDj binds with a high affinity DPI, while many ligands have been described for DP2 (Pettipher et al. 2007, Nat Rev Drug Discov 6:313-325). Therefore, we hypothesized that PGDj secreted by macrophages could promote a pro-tumoral and angiogenic phenotype on the cells expressing DPI and/or DP2 (/.e., macrophages, endothelial cells and CD8+ T cells).
To elucidate the role of the immune compartment in promoting PGDj-mediated immunosuppression, we assessed T cell activation/proliferation upon exposure to different concentrations of PGDj, with or without BMDMs (Hpgds-KO and WT). Like BMDMs, where increasing concentrations of PGDj resulted in an increase of their anti-inflammatory activity in a dose-dependent manner, CD8+ T cell stimulation with PGDj resulted in a decreased cytotoxicity. Overall, these data shed light on a new mechanism, where macrophages, by releasing PGD2 in the TME sustain their immunosuppression and dampen CD8+ T cell activation.
Hence, considering the major role of CD8+ T cells in mediating the immune response, the higher cytotoxic activity of these cells in Hpgds-KO mice and the high expression of the two PGD2 receptors, we genetically targeted DPI and DP2 in CD8+ T cells by employing a constitutive CRISPR-Cas9 strategy. Thus, two gRNAs targeting Ptgdrl and Ptgdr2 (each of them alone) and a non-targeting gRNA (NT) as control was delivered in the Lin HSCs from LSL-CRISPR/Cas9 mice intercrossed with CD8a:Cre (same strategy used to generate Hpgds KO chimeras). Ptgdrl and Ptgdr2 deletion was confirmed by isolating CD8+T cells from the spleen. After confirming the immune reconstitution (data not shown), mice were intradermally injected with YUMM 1.7 melanoma cancer cells. In accordance with our hypothesis, targeting of DPI in CD8+ T cells decreased tumor growth and weight (Figure 8A). By contrast, the KO of DP2, did not achieve any tumor growth inhibition (Figure 8A). In line with this, when analyzing the proliferation status of CD8+ T cells isolated from spleen of NT, DPI and DP2-KO mice we found that DPI and DP2 KO CD8+ T cells showed a higher proliferation than NT cells.
To further elucidate the role of macrophages in mounting PGDj-mediated immunosuppression, we intercrossed LSL-CRISPR/Cas9 mice with CD64:Cre mice. Thus, by employing the strategy described before we genetically targeted DPI specifically in CD64 macrophages. Ptgdrl KO was confirmed by differentiating monocytes into BMDMs. In brief, when implanting in immune-reconstituted mice YUMM 1.7 melanoma cancer cells, we observed that knocking out Ptgdrl in the macrophage compartment led to a reduction in the tumor growth and tumor weight (Figure 8B). Together, these data further support the existence of an immunosuppressive and autocrine loop mediated by PGD2 in Hpgds-DPl macrophages.
Hpgds deletion in tumor-associated macrophages (TAMs) is leading to inhibition of tumor progression and to reshaping the tumor micro-environment (TME) (Example 3). Similar effects can thus be obtained with deletion of PTGDR1 either in macrophages or in CD8+ T-cells. Plausibly, those tumors sensitive to Hpgds inhibition will likewise be sensible to DPI inhibition.
Furthermore, the chimeric macrophage model as used herein plausibly supports use of adoptive transfer of macrophages in which DPI expression is knocked-out or inhibited in the treatment of cancer. Similarly, adoptive transfer of CD8+ T-cells in which DPI expression is knocked-out or inhibited is herewith coming in reach as the treatment of cancer.
EXAMPLE 8. Targeting DPI function systemically is affecting tumor progression The YUMM1.7 melanoma model was used to test whether tumor growth inhibition as obtained by knocking out expression of DPI in macrophages could be replicated by systemic inhibition of DPI function by means of the small molecule pharmacological DPI inhibitor asapiprant. The results are depicted in Figure 9A and indeed indicated that systemic inhibition of DPI function was inhibiting tumor growth. These results were extended to hepatocellular carcinoma (HCC), as indicated in Figure 9B.
EXAMPLE 9.
9.1. Animal strains
All experimental animal procedures were approved by the Institutional Animal Care and Research Advisory Committee of the KU Leuven (P226/2017 and P012/2022). Mice were maintained under pathogen-free and temperature- and humidity-controlled conditions with a 12-h light/12-h dark cycle and received normal chow (sniff, R/M-H). Animals were removed from the study and killed if any signs of pain and distress were detected or if the tumor volume reached 1000mm3. The maximal tumor size was not exceeded in all reported studies.
Macrophage-specific Hpgds KO mice were generated by intercrossing the tamoxifen-inducible Csflr.Cre- ERT (a kind gift of J. W. Pollard from the University of Edinburgh, Scotland) with LoxP-STOP-LoxP Cas9 mice (B6J.129(B6N)- Gt(ROSA) 26Sortml(CAG-cas9*,-EGFP)Fezh/J) (Shang et al. 2020, Nature 587:626- 631), purchased from Jackson Laboratory.
Braf 6007 Pter 7' mice (a kind gift of J-C. Marine from VIB-Center of Cancer Biology, Leuven, Belgium) were generated by intercrossing BrafA, Tyr::Cre-ER72 mice with Ptenlox4~5 mice. The Cre-mediated conversion of BrafA to Bra E and the deletion of exons 4 and 5 was obtained by topical administration of 2 mg/ml (5mM) of 4-hydroxytamoxifen (4-HT) on the back skin of adult mice.
The mouse line C57BL/6N-Hpgds/Tcp was made as part of the KOMP2-DTCC project from KOMP ES cells (Bradley et al. 2012, Mamm Genome 23:580-586) at The Centre for Phenogenomics. It was obtained from the Canadian Mouse Mutant Repository. The NEO cassette was deleted in vivo by using FLP- mediated recombination. The final transgenic line carries 2 LoxP-sites in each Hpgds allele. Finally, we generated Fcyrl.Cre x Hpgds'mflm mice in a C57BL/6N background by intercrossing Hpgds'mflm mice with the macrophage specific Fcyrl.Cre delete mouse line.
Fcyrl-Cre mice were a gift from Bernard Malissen (French National Centre for Scientific Research, CNRS). We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders for providing the mutant mouse line C57BL/6NTac-Fcyrllm(EGFP/Cre/ERT2)wls'/WtsiH, and the European Mouse Mutant Archive (www.infrafrontier.eu; Repository number EM: 11125) partner at the Mary Lyon Centre at MRC Harwell from which the mouse line was received. Thus, we intercrossed Fcyrl-Cre-ERT2 with Hpgds'mflm mice to generate the conditional Hpgds-KO mouse. Macrophage-specific Ptgdr and Ptgdr2 KO were generated by intercrossing Hpgdslox/lox Fcyrl.Cre mice with LoxP-STOP-LoxP Cas9 mice (B6J.129(B6N)- Gt(ROSA) 26Sortml(CAG-cas9*,-EGFP)Fezh/J mice.
CD8a-specific Ptgdr and Ptgdr2 KO were generated by intercrossing CD8a mice with LoxP-STOP-LoxP Cas9 mice (B6J.129(B6N)- Gt(ROSA) 26Sortml(CAG-cas9*,-EGFP)Fezh/J mice, generating CD8-specific Ptgdr and Ptgdr2-KO mice.
LoxP-STOP-LoxP Cas9 mice (B6J.129(B6N)- Gt(ROSA) 26Sortml(CAG-cas9*,-EGFP)Fezh/J mice were intercrossed with NKp46 mice (Proc Natl Acad Sci USA 108:18324-18329) to generate NK-specific Ptgdr and Ptgdr2-K0 mice.
All mice used were females between 7-10 weeks old. Wild type C57BL/6N mice were obtained from the KU Leuven breeding facility.
9.2. Cell lines
The melanoma YUMM 1.7 cell line was a kind gift from Prof. R. Marais (Manchester, UK) and cultured in DMEM/F-12 medium (Gibco) supplemented with 10% FBS, 1% (v/v) Pen/Strep (Gibco) 2 mM glutamine (Gibco), 0.1 mM nonessential amino acid (NEAA, Gibco).
The /Vros-driven melanoma cell line (kindly provided by Prof. J-C. Marine, VIB-CCB KU Leuven) was generated by isolation of Tyr::Cre-ER(T2); Tyr::lnk4a-/-;R62R Confetti tumors and cultured in DMEM/F- 12 medium (Gibco) supplemented with 10% FBS, 1% (v/v) Pen/Strep (Gibco) and 2 mM glutamine (Gibco).
The cells were incubated at 37°C in a 5% CO2 humidified atmosphere and subcultured approximately every three days to maintain a log growth phase.
The KPC pancreatic pancreatic cell line (FC1245) was a kind gift from Prof. D. Tuveson and was derived from spontaneous tumors arising KPC (KrasLSLG12D/+; p53R172H/+; Pdx: CreTg/+) pancreatic cancer mouse model. KPC cells were cultured in DMEM medium (Gibco) supplemented with 10% FBS, 1% (v/v) Pen/Strep (Gibco), ImM sodium pyruvate (Gibco).
All the cancer cell lines used were authenticated by Idexx Bioresearch.
9.3. Cell Isolation and Culture
9.3.1. Murine Bone Marrow-Derived Macrophages (BMDMs)
Murine BMDMs were derived from bone marrow precursors as described before (Casazza et al. 2013, Cancer Cell 24:695-709; Bieniasz-Krzywiec et al. 2019, Cell Metab 30:917-936). Briefly, bone marrow cells were cultured in a 10 cm Petri dish (non-tissue culture treated, bacterial grade) in a volume of 6 ml of DMEM supplemented with 20% FBS and 30% L929-conditioned medium. After 3 days of culture, an additional 3 ml of differentiation medium was added. At day 7, macrophages were detached with ice cold PBS and characterized by FACS, using the pan-macrophage marker F4/80. For the in vitro Ml or M2 polarization, BMDMs were stimulated for additional 48h with 20 ng/ml I FN-y + 100 ng/ml LPS or lOng/ml IL-4, respectively. 9.3.2. CD8 isolation and activation
Murine naive T cells were isolated from the spleen by processing the cells through a 40-pm pore cell strainer in sterile PBS and cells were centrifuged for 10 min at 300x g. Red blood cell lysis buffer (Sigma- Aldrich) was used in order to lysis red blood cells. CD8+T cells were isolated by using MagniSort Mouse CD8+T Cell Negative Selection Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. CD8+T cells were cultured in T cell medium (RPMI supplemented with 10% FBS, 1% P/S, 1% MEM non- essential amino acids NEAA (Gibco), 25 pmol/L P-mercaptoethanol, and 1 mmol/L sodium pyruvate (Gibco)) at 37°C in a humidified atmosphere containing 5% CO2. If needed, T cells were activated for 3 days with CD3/CD28 Dynabeads (Thermo Fisher Scientific) at a 1:1 bead-to-cell ratio and 30 U/ml rlL-2 (PeproTech). 3 days after the activation, the beads were magnetically removed and activated CD8+T cells were further expanded for maximum 3 days in the presence of rlL-2.
9.3.3. NK cells isolation
Murine NK cells were isolated from the spleen by processing the cells through a 40-pm pore cell strainer in sterile PBS and cells were centrifuged for 10 min at 300x g. Red blood cell lysis buffer (Sigma-Aldrich) was used in order to lyse red blood cells. Cells were re-suspended in FACS buffer and incubated form 15 minutes with BD Fc Block purified Rat Anti-Mouse CD16/CD32 mAb (BD Pharmingen) and stained with the following antibodies for 30 minutes at 4°C protected from light: viability dye (eF506), anti-CD45 (clone 30-F11), anti-CDllb (clone MI/70), anti-F4/80 (clone BM8), anti-CD8 (clone 53-6.7), anti- CD161/NK1.1 (clone PK136), anti-CD335/NKp46 (clone 29A1.4). Cells were then washed and resuspended in cold FACS buffer before flow sorting by FACS Aria (BD Biosciences). FMO (Fluorescence Minus One) control was used in order to ensure proper gating of positive population. Subsequently, NK cells were expanded in T cell medium (RPMI supplemented with 10% FBS, 1% P/S, 1% MEM non-essential amino acids NEAA (Gibco), 25 pmol/L P-mercaptoethanol, and 1 mmol/L sodium pyruvate (Gibco)) in the presence of rlL-2.
9.3.4. Human Monocyte-Derived Macrophages (hMDMs)
Human monocytes were obtained from healthy donor buffy coats and isolated with CD14-conjugated MicroBeads (Miltenyi Biotec) as described before (Bieniasz-Krzywiec et al. 2019, Cell Metab 30:917-936). Briefly, monocytes were cultured in a 6-well plates (1x10s cells/well in 2 ml media) in RPMI 10 % FBS, 2 mM L-Glutamine, 1% P/S and 25 ng/ml recombinant human M-CSF for five days. The third day of the differentiation, cytokines were refreshed. At day 6, macrophages were detached with ice cold PBS or polarized for additional 48h towards an Ml or M2 phenotype with 10 ng/ml IFN-y + 100 ng/ml LPS or 20 ng/ml IL-4, respectively.
9.3.5. Human Umbilical Endothelial Cells (HUVECs)
HUVECs were freshly isolated from umbilical cords obtained from multiple donors (with approval from the Ethics Committee Research UZ/KU Leuven and informed consent obtained from all subjects) as previously described (Bieniasz-Krzywiec et al. 2019, Cell Metab 30:917-936; Schoors et al. 2015, Nature 520: 192-197) and regularly tested for mycoplasma. They were maintained in M199 medium (Invitrogen) supplemented with 20% FBS, 2nM glutamine, 100 U/mL penicillin, 100 pg/mL streptomycin, 0.15% heparin and 20 pg/mL ECGS (Sigma-Aldrich). 0.1% pork gelatin (Sigma-Aldrich) was used to stimulate the adhesion of HUVECs to the flask bottom.
9.4. Tumor models
1x10s YUMM1.7 or 1x10s /Vros-driven melanoma cell lines, genetically engineered by lentiviral transduction to express CD90.1, were injected intradermally in the right flank of the mouse in a final suspension of 50 pl PBS. Tumor volumes were measured three times a week with a caliper and calculated using the formula: V = K x [d2 x D] / 6, where d is the minor tumor axis and D is the major tumor axis. At the indicated time points, mice were randomized and treated intraperitoneally (ip) with 5 mg/kg a-CD8 (BioXcell) or control IgG from rat serum. For the pharmacological inhibition of Hpgds, mice were treated by oral gavage with HQL-79 30 mg/kg BID or control vehicle (methylcellulose). Treatment started when the average tumor reached 100 mm3.
For the hepatocellular carcinoma spontaneous model, mice were hydrodynamically cotransfected with 0.1 ug of PB_h-RasG12V and PB_c-Myc, in conjunction with a plasmid encoding a hyperactive PB transposase (Serra et al. 2022, Cell Mol Gastroenterol Hepatol 14:609-624; Tipanee et al. 2020, Mol Ther Nucleic Acids 19:1309-1329; Di Matteo et al. 2014, Mol Ther 22:1614-1624). For the pharmacological inhibition of Hpgds, mice were treated by oral gavage with HQL-79 30 mg/kg BID or control vehicle (methylcellulose). Treatment started 9 weeks after hydrodynamic injection. Mice were weighted at least three times per week. Mice showing symptoms of illness, losing 20% of initial body weight, displaying peritoneal leakage or ulcerated tumors were sacrificed and excluded from the experiments. At the end stage (13 weeks after hydrodynamic injection), nodules were counted as measured by using a caliper. Moreover, one lobe was collected for histological examination.
For the genetically engineered mouse metastatic melanoma model (Braf 600E Pten '), topical administration with 2 mg/ml (5mM) of 4-hydroxytamoxifen (4-HT) on the back skin of adult mice was performed. 4 weeks after tumor induction, mice were randomized and treated intraperitoneally with 10 mg/kg a-PD-1 (BioXcell), control IgG from rat serum. For the pharmacological inhibition of HPGDS, mice were treated for 13 days by oral gavage with HQL-79 30 mg/kg BID or control vehicle (methylcellulose).
9.5. Bone marrow transplantation
For the generation of Hpgds
Figure imgf000059_0001
WT mice, the stop-floxed Cas9 knockin line (Platt et al. 2014, Cell 159:440-455) was intercrossed with Csflr:Cre-ERT mice allowing the inducible expression of the Cas9 nuclease in monocytes and macrophages only (donor mice). Seven-to-eight-week-old recipient mice were lethally irradiated with a dose of 9.2 Gy using the Small Animal Radiation Research Platform (SARRP, XSTRAHL). After cervical dislocation, femur, tibia and humerus were collected from donor mice of the appropriate genotype. Bone marrow (BM) was obtained by flushing the bones with a syringe filled with DMEM supplemented with 10% FBS. The cells were subsequently filtered two times by using a 40-pM- pore-sized mesh and centrifuged for 10 minutes at 300x g. BM cells were counted and resuspended 1x10s cells/ml. The EasySep™ Mouse Hematopoietic Progenitor Cell Isolation Kit (19856, STEMCELL Technologies) was used to isolate lineage-negative hematopoietic stem cells (Lin HSCs) according to the manufacturer's instructions. Cells were stimulated for 4h in StemSpan™ serum-free medium (09650, STEMCELL Technologies)with 20 ng/ml IL-3, 100 ng/ml SCF, 100 ng/ml TPO and 100 ng/ml FLT-3L and then transduced with a specific gRNA of interest (targeting or, as control, non-targeting). A multiplicity of infection reaching approximately 30% of transduction was used. After a double spin filtration, cells were counted and 1x10s cells were injected intravenously (iv) via tail vein in the irradiated recipient mice. Tumor experiments were initiated 5 to 6 weeks after immune reconstitution. Deletion of Hpgds expression in macrophages was induced by intraperitoneal injections of tamoxifen (Sigma-Aldrich, T5648) (lmg/mouse/day) for 5 consecutive days. Red and white blood cell count was determined using hematocytometer and flow cytometry on peripheral blood collected in heparin with capillary pipettes by retro-orbital bleeding. Tumor experiments were initiated 5 to 6 weeks after immune reconstitution.
9.6. Tissue dissociation
YUMM 1.7 and /Vros-driven melanoma-bearing mice were sacrificed by cervical dislocation. Tumors were harvested and minced in aMEM medium (Lonza) supplemented with 5% FBS, 1% Pen/Strep, 50 pM p- mercaptoethanol (Gibco), 5 U/ml DNase I (Qiagen), 0.85 mg/ml Collagenase V (Collagenase from Clostridium histolyticum, Sigma-Aldrich), 1.25 mg/ml Collagenase D (Collagenase from Clostridium histolyticum, Roche) and 1 mg/ml Dispase II (Gibco) and digested for 30 minutes at 37°C. Digested tissues were filtered using first a 70-pm and then a 40-pm pore mesh strainers.
Bra 60^ Pten ^ mice were sacrificed by cervical dislocation. Spontaneous tumors were harvested and minced in RPMI supplemented with 5% FBS, 300 pg/ml Liberase (Sigma Aldrich) and 1 pg/ml DNase I (Qiagen) and digested for 45 minutes at 37°C. The single cell suspension was then passed through a 40 pm strainer and red blood cells were lysed by using a Hybri-Max solution (Sigma Aldrich).
9.7. Tumor-conditioned media (TCM)
YUMM 1.7 tumor explanted from WT mice was minced in 12 ml DMEM/F-12 medium, supplemented with 1% Pen/Strep and incubated at 37°C for 72 hours. After that, the medium was filtered, and the cell- free supernatant was supplemented with 20mM HEPES and 2mM of L-glutamine and stored at -20°C.
9.8. Tumor interstitial fluid (TIF)
YUMM 1.7 tumor explanted from Hpgds WT and KO mice were cut in small pieces which were inserted in collection tubes (2 ml eppendorfs with 5 holes at the bottom in a 15 ml falcon tube). 20 to 40 pl of 9 g/l NaCI pH 7.4 was added on top of the tumor samples prior to a 10 min centrifugation at llOx g at 4°C. The interstitial fluid was collected in new vials and further used to assess lipid metabolites and mediators by using the liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) on a Nexera X2 UHPLC system (Shimadzu).
9.9. YUMM 1.7 migration assay
In the cancer migration assays, 8xl04 YUMM 1.7 melanoma cancer cells were seeded on 8 pm polycarbonate membranes (Transwell; Costar). The bottom chamber contained DMEM without PGD2 or with 10 nM, 100 nM, 1 pM or 10 pM PGD2, or alternatively 2xl05 BMDMs Hpgds-WT or Hpgds-KO. For the co-culture with macrophages, the bottom chamber contained 2xl05 IL-4 polarized macrophages (Ctrl or Hpgds-KO). 12hr before starting the assay, YUMM 1.7 and BMDMs were cultured in DMEM F-12 with 2% FBS, 1% P/S and 1% L-Glut. Subsequently, cells were seeded and incubated for 12hr. The nonmigrated cells were removed from the top of each membrane by using a cotton stick. The migrated cells were fixed in 4% PFA, washed in PBS, stained with crystal violet (2.5 g/l), and mounted on glass slides with Pro ong Gold mounting medium without DAPL Images were acquired with Olympus BX41 or Leica DM16000 microscope and analyzed using the CellSense imaging software.
9.10. BMDM electroporation
Silencing of Hpgds, Ptgdrand Ptgdr2 was achieved by electroporation with specific siRNAs (commercially available). Briefly, 8xl06 BMDMs were resuspended in 500 pl of Opti-MEM and electroporated (250V, 950 mF, 00 O) with 100 pmol of the targeting or control siRNA of interest. After electroporation, medium was replaced with DMEM supplemented with 10% FBS, 1% Pen/Strep and 2mM Glutamine (Gibco). Upon 48h of incubation at 37°C in a 5% CO2 humidified atmosphere, qPCR and flow cytometric analysis were performed.
9.11. hMDMs transfection
Silencing of HPGDS was achieved by transfecting hMDMs with specific siRNAs. Briefly, 1x10s hMDMs were transfected by using Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific). 10 pmol of of the targering or control siRNA of interestwere resuspended in 100 pL of Opti-MEM supplemented with 2 pL of Lipofectamine and incubated for 20 minutes at RT. 6hr after lipofection, medium was replaced with RPMI supplemented with 10% FBS, 1% Pen/Strep and 2 mM Glutamine. Upon 48h of incubation at 37°C in a 5% CO2 humidified atmosphere, qPCR and flow cytometric analysis were performed.
9.12. BMDMs in vitro
For the in vitro polarization assays, 1x10s BMDMs were seeded 24h prior to the addition of lpM of PGD2. After 48h of stimulation, the polarization was assessed by flow cytometry. To differentiate BMDMs towards TAMs-like cells, 4xl06 BMDMs were seeded in 6-well plates in DMEM supplemented with 10% FBS, 1% P/S and 20% TCM for 12h at 37 °C in a 5% CO2 humidified atmosphere, as previously described. 9.13. Sprouting assay
Hybrid multicellular microspheres were generated by mixing HUVECS and BMDMs (wild-type or silenced for Hpgds) in a 1:1 ratio and incubated overnight in hanging drops in EGM-2 medium supplemented with 20% methylcellulose 4000 cP (Sigma-Aldrich). After harvesting the spheroids, they were embedded in Collagen I gel as described (Heiss et al. 2015, FASEB J 29:3076-3084) and maintained at 37°C for 18 hours to induce sprouting. Cultures were then fixed with 4% PFA at room temperature. Images were taken with a Leica DM16000 microscope. The CellSense imaging software was used to analyze the number of sprouts per spheroid and the total sprout length (represented by the cumulative length of primary sprouts and branches per spheroid).
9.14. HUVECs in vitro
100.000 HUVECs were seeded 24h prior to the addition of lpM of PGDj. After 12-24-48hr of stimulation with PGDj, the expression of VEGFA and VEGFC was assessed by qPCR.
9.15. HUVEC migration assay
100.000 HUVECs were pre-treated for 48hr with PGDj or with DMSO and seeded for 12hr on 8 pm polycarbonate membranes (Transwell; Costar). The bottom chamber contained 200.000 BMDMs. The non-migrated cells were removed from the top of each membrane by using a cotton stick. The migrated cells were fixed in 4% PFA, washed in PBS, stained with crystal violet (2.5 g/l), and mounted on glass slides with Pro ong Gold mounting medium without DAPI. Images were acquired with Olympus BX41 microscope and CellSense imaging software.
9.16. Gene expression analysis
To assess gene expression, cells were washed in PBS, collected in RLT buffer (Qiagen) and stored at - 80°C. RNA was extracted with the RNeasy Minikit (Qiagen) according to the manufacturer's instructions. Reverse transcription to cDNA was performed with the Superscript III and Superscript IV First Strand cDNA Synthesis System (Invitrogen) according to manufacturer's protocol. cDNA, primer/probe mix and PowerUp SYBR Green Mix (Applied Biosystems) or TaqMan Fast Universal PCR Master Mix (Applied Biosystems) were prepared in a volume of 20 pl or 10 pl, according to manufacturer's instructions (Applied Biosystems) and pipetted into an optical 96-well Fast Thermal Cycling plate (Applied Biosystems). Analysis was performed by using the Quantstudio 12K Flex Real-Time PCR System (Applied Biosystems). Gene transcription was presented as number of gene mRNA copies relative to the housekeeping gene. All reactions were run in duplicate.
9.17. Flow cytometry
Cells were counted and blocked by using BD Fc Block purified Rat Anti-Mouse CD16/CD32 mAb (BD Pharmingen).
For the extracellular staining, cells were stained for 30 minutes on ice with the following antibodies: fixable viability dye (eF450 or eF506), anti-CD45 (clone 30-F11), anti-CDllb (clone MI/70), anti-F4/80 (BM8), anti-MHC-ll (clone M5/114.15-12), anti-CD80 (clone B7-1), anti-CD86 (clone GL-1), anti-CDllc (clone N4/18), anti-CD206 (clone MR5D3), anti-CD204 (clone 7C9C20), anti-CD274/PD-Ll (clone 10F.9G2), anti-CD8a (clone 53-6.7), anti-CD4 (clone RM4-5), anti-CD69 (clone H1.2F3), anti-CD279/PD-l (clone 29F.1A12), anti-CD335/NKp46 (clone 29A1.4), anti-CD49b (clone DX5), anti-NKl.l (clone PK136) from Cell Signaling, BD Bioscience, BioLegencl, eBioscience and Miltenyi Biotec.
For the intracellular measurements of tumor necrosis factor-alpha (TNF-a), interferon-y (IFN-y) and granzyme B (GZMB), single-cell suspensions were stimulated with phorbol 12-myristate 13- acetate/ionomycin cell stimulation cocktail (eBioscience) in presence of Brefeldin A (BioLegend) and Monensin (eBioscience) for 4h at 37°C in RPMI supplemented with 10% FBS and 1% Pen/Strep. Afterward, cells were stained for surface markers, followed by 30 min incubation in Fix/Perm (eBioscience) at 4°C. Cells were then washed with permeabilization buffer and stained overnight with the following antibodies: anti-TNF-a (clone MP6-XT22), anti-IFN-y (clone XMG1.2) and anti-GZMB (clone GB11). Cells were subsequently washed and resuspended in cold FACS buffer. FACS data were acquired by a FACS Fortessa (BD Biosciences). Fluorescence minus one (FMO) controls and unstained control were used to ensure proper gating of positive populations. Data were analyzed by using FlowJo (TreeStar).
9.18. Cell sorting
1x10s YUMM1.7 CD90.1+ were intradermally injected in the right flank of the mouse. Tumors were harvested for FACS as previously mentioned. After obtaining single cell suspension, CD45 enrichment was done by following manufacturer's instructions (CD45 MicroBeads). Cells (CD45+ and CD45 ) were then incubated with Mouse BD Fc Block purified Rat-Anti-Mouse CD16/CD32 mAb (BD Pharmingen) for 15 minutes at 4°C and stained with the following antibodies for 30 minutes at 4°C: fixable viability dye (eF450 or eF506), anti-CD45 (clone 30-F11), anti-CDllb (clone MI/70), anti-F4/80 (clone BM8), anti-TCR- P chain (clone H57-597), anti-CD8a (clone 53-6.7), anti-CD4 (clone RM4-5), anti-CD90.1 (clone OX-7), anti-CD90.2 (clone 53-2.1) anti-CD31 (clone MEC 13.3), anti-Ly6G (clone 1A8), anti-FceRla (clone MAR- 1), anti-CD117 (clone 2B8), anti-CD49b (clone DX5), anti-NKp46 (clone 29A1.4), anti-NKl.l (clone PK136), anti-MHC-ll (clone M5/114.15.2), anti-CDllc (clone N418), anti-CD279/PDl, or anti-CXCR5. Cells were washed and resuspended in cold FACS buffer before flow sorting. FMOs controls and unstained control were used to ensure proper gating of positive populations. Data were analyzed by FlowJo (TreeStar).
9.19. T-cell proliferation assay
To assess T cell proliferation, activated CD8+ T cells (at day 3 of stimulation) were counted and 800 x 105 cells were labelled with 3.5 pmol/L Violet Cell Tracer (Thermo Fisher Scientific) at 37°C for 20 minutes. T cells were washed with FACS buffer and cultured for 48 hours the after stimulation.
9.20. Lipid extraction
The extraction protocol was adapted from an already described method (Dumlao et al. 2011, Biochim Biophys Acta - Mol Cell Biol Lipids 1811:724-736). Tissue samples were mixed with methanol containing 100 picogram deuterated internal standards and 10 pL antioxidant mix (100 pM indomethacin, 0.2 mg/ml BHT, 100 pM trans-4-(-4-(3-adamantan-l-yl-ureido9-cyclohexyloxy)-benzoic acid (t-AUCB) in MeOH) and were homogenized using a Precellys system at 4°C. The homogenized samples were stored at -80°C for 30 minutes and then centrifuged at 16,000g for 10 minutes at 4°C. The supernatant was transferred to a new tube and diluted with water to achieve a methanol percentage < 10%. The remaining pellet was redissolved in TBS buffer for protein concentration determination (Pierce™ BCA Protein Assay, Thermo Fisher Scientific) or TE buffer for DNA concentration determination (Hoechst assay). Lipids were extracted using Strata™-X 33 pm Polymeric Reversed Phase extraction columns (Phenomenex, 8B-S100-ECH) as instructed by the manufacturer. Briefly, the columns were preconditioned with 3 mL methanol, followed by 3 mL water. The sample (containing < 10% methanol) was eluted dropwise, followed by a wash step with 3 mL of 10% methanol and elution with 100% methanol into a glass collection tube containing 6 pL of a 30% glycerol in methanol solution. Samples were evaporated in a vacuum centrifuge, redissolved in a 1:1 solution of water/methanol and transferred to an LC vial.
9.21. LC-MS/MS
Lipid species were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with a hybrid triple quadrupole/linear ion trap mass spectrometer (Q.TRAP 6500+ system; SCIEX). Chromatographic separation was performed on a Polar C18 column (2.6 pm, 3.0 x 100 mm; Phenomenex) maintained at 50°C, using mobile phase A [0.1% formic acid in water] and mobile phase B [0.1% formic acid in methanol] in the following gradient: (0-2 min: 45% B; 2-16.5 min: 45% B
Figure imgf000064_0001
80% B; 16.5-18.5 min: 98% B; 18.5-20.5 min: 10% B) at a flow rate of 0.5 mL/min. The instrument parameters were as follows: Curtain Gas = 30 psi; Collision Gas = 8 a.u. (medium); lonSpray Voltage = 5200 V and -4,200 V; Temperature = 520°C; Ion Source Gas 1 = 50 psi; Ion Source Gas 2 = 70 psi. The endocannabinoids, LTC4, LTD4, LTD4, 2-AG, MTCR and PTCR compounds were detected in positive ion mode and all other compounds were detected in negative ion mode. MRM transitions were sourced from Murphy 2014 (Tandem Mass Spectrometry of Lipids (Robert C Murphy) 10.1039/9781782626350) and Stassburg et al. 2015 (Targeted Lipidomics of Oxylipins (Oxygenated Fatty Acids). 10.13140/RG.2.1.3943.9207).
9.22. PBMC isolation, MDM differentiation and polarization
Buffy coat samples from healthy donors were obtained from the Red Cross-Flanders (institutional approval RKOV_19015). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation (Axis-Shield, 1114545) and washed in PBS containing 1 mM EDTA. The ring at the interface was collected, washed with PBS and counted. Monocytes were isolated by using magnetic CD14- conjugated Microbeads (Miltenyi Biotec) and differentiated into human monocyte-derived macrophages (MDMs). Thus, monocytes were cultured in a 6-well plates (1x10s cells/well in 2 ml media) in RPMI 10 % FBS, 2 mM L-Glutamine, 1% P/S and 25 ng/ml recombinant human M-CSF (PeproTech) for five days. The third day of the differentiation, cytokines were refreshed. The polarization towards the Ml or the M2- like phenotype was started at day 6. MDMs were polarized towards the Ml-like phenotype by adding 10 ng/ml INF-y (PeproTech) + 100 ng/ml LPS (Sigma-Aldrich) while, for the M2-like phenotype MDMs were polarized by adding 20 ng/ml IL-4 (PeproTech). After 48h polarization, MO or M2-like macrophages were silenced for HPGDS and the phenotype was checked by qPCR and flow cytometry. Thus, macrophages were harvested, re-suspended in FACS buffer (PBS, FBS 2% and EDTA 2mM) and incubated for 15 min at 4°C with human Fc blocking (eBioscience). The following anti-human antibodies were used: anti-CD80 (clone L307.4), anti-HLA-DR (clone LN3), anti-CD163 (clone GHI/61), anti-CD206 (clone 19.2) and fixable viability dye (ThermoFisher, 65-0865-18). After staining, cells were washed, fixed and re-suspended in cold FACS buffer. Samples were analyzed using a LSRFortessa (BD Biosciences) flow cytometer. FMO controls were utilized in order to ensure proper gating of positive populations.
9.23. Histology and immunostainings
Murine tumors were collected and fixed in 4% paraformaldehyde (PFA) overnight at 4°C, dehydrated and embedded in paraffin. Serial sections of 7-pm thickness were cut using a Microm HM360 Microtome. Human melanoma, hepatocellular carcinoma and pancreatic cancer samples were obtained as 7 pm thick paraffin-embedded slides. Paraffin slides were rehydrated to further proceed with antigen retrieval solution (Dako) for 20 minutes at 100°C. After 20 minutes of cooling down, the sections were washed with TBS and kept in methanol. If necessary, 0.3% H2O2 was added to methanol to inactivate endogenous peroxidases. The samples were washed and blocked with the appropriate serum (pre-immune donkey serum or pre-immune goat serum, Sigma-Aldrich) diluted 1:5 in Tris-NaCI-blocking buffer (TNB). Afterwards, the sections were incubated overnight with the following antibodies: rabbit anti-Hpgds (Cayman Chemical Company, 10004349), rat anti-F4/80 (AbD Serotec, MCA497 or Cell Signaling D2S9R), goat-anti-MMR/CD206 (R&D System AF2535), rabbit anti-CD80 (Abeam, ab64116), rabbit anti-mouse CD8 (Cell Signaling, 98941S), rabbit-anti mouse NKp46 (BD Pharmingen, 137601), goat anti-mouse CD105/Endoglin (R&D System, AF1320), rabbit anti-aSMA-Cy3 (Sigma Aldrich, C6198), rabbit anti- hypoxyprobe (Hypoxyprobe kit, Chemicon), rabbit-anti FITC (AbD Serotec, 4510-7604), anti-human CD68 (Dako, M0876), anti-human HPGDS (R&D System, MAB6487). Next, Alexa 488, 568 or 647 conjugated secondary antibodies (Molecular Probes) or biotin-labeled antibodies (Jackson Immunoresearch) were applied. After biotin-labelled antibodies, TSA Cyanine 3, TSA Cyanine 5 or TSA Fluoresceine system amplification kits (Perkin Elmer, Life Sciences) were used according to the manufacturer's instructions. The sections were then washed, stained for 30 min with Hoechst solution and mounted with ProLong Gold mounting medium without DAPI (Invitrogen). Imaging and microscopic analyses were performed with an Olympus BX41 microscope and CellSense imaging software. 9.24. Hypoxia assessment and tumor perfusion
For the detection of tumor hypoxia, tumors were collected lh after intraperitoneal injection of 60 mg/kg pimonidazole hydrochloride into melanoma-bearing mice. To detect the formation of pimonidazole adducts, 7- .m sections were stained with Rabbit anti-hypoxyprobe monoclonal (Hypoxyprobe Kit, Chemicon) following the manufacturer's instructions. To analyze vessel perfusion, tumors were collected 15 minutes after retro-orbital injection of 0.05 mg of FITC-conjugated lectin (Lycopersicon esculentum; Vector Laboratories, B-1175-1).
9.25. Statistics
Data entry were performed in a blinded fashion. All statistical analyses were performed using GraphPad Prism software (Version 10.0.2). Statistical significance was calculated by two-tailed unpaired Student's t-tests on two experimental conditions and one-way or two-way analysis of variance ANOVA when repeated measures were compared. All the results are shown as mean ± standard error of the mean (SEM). Statistics were indicated only when significant. Detection of mathematical outliers was performed using the Grubbs' test in GraphPad. Sample sizes for all experiments were selected based on those reported in previous studies. Independent experiments were pooled and analyzed together whenever possible as detailed in figure legends. Animals were excluded if they died or had to be killed according to protocols approved by the animal experimental committees.
9.26. Meta-analysis
To identify metabolic genes that are associated with the outcome of PD1 immunotherapy on human melanoma, we performed a meta-analysis on two publicly available bulk RNA-sequencing datasets of human melanoma tumors responding or resistant to a-PD-1 immune checkpoint inhibitors (Hugo et al. 2016, Cell 165:35-44; Riaz et al. 2017, Cell 171:934-949). Briefly, raw reads were retrieved using the SRA toolkit (v2.8.0) and aligned to the human reference genome GRCh38.pl3 using STAR (v2.7.3a) with default parameters. Next, featurecounts from the R (v3.6.2) package 'Rsubread' (v2.0.1) was used to assign mapped reads to genomic features. The resulting raw count matrices were further processed using the BIOMEX browser-based software platform (vl.0.5) (Taverna et al. 2020, Nucl Acids Res 48:W385-W394) with default parameters and TMM normalization. For each individual dataset, a differential gene expression analysis between responsive and non-responsive tumors was performed including BRAF, NRAS and NF1 mutational status as a covariate. We then ranked genes by their Iog2 fold change and combined the resulting rank by calculating the median rank product to identify metabolic genes that were consistently associated with a-PDl immunotherapy response. We filtered rank-based meta-analysis results for genes associated with arachidonic acid metabolism. The arachidonic acid metabolism associated ranked gene list was subsequently visualized using the R package ggplot2 (V3.1.1). 9.27. Bioinformatics
For the in-house Single cell RNA-seq (scRNA-seq) data from immune cells was acquired from the Pozniak et al. Study (Pozniak et al. 2022, BioRxiv doi.org/10.1101/2022.08.11.502598). Briefly the study included treatment naive stage 111/IV (AJCC 8th edition) melanoma patients receiving a-PD-1 based therapy (a-PD- 1 monotherapy (nivolumab): n=17; a-PD-1 and a-CTLA-4 combination therapy (ipilimumab + nivolumab): (n=6) SPECIAL trial; UZ/KU Leuven #S62275). Cutaneous, subcutaneous or lymph node metastases were biopsied before initiation of therapy (before treatment; BT). Subsequently, a second tumour biopsy was collected right before the administration of the second ICB treatment cycle (early on-treatment; OT). Response of the patients to the treatment was stratified based on RECISTvl.l. Immune cells were identified based on unsupervised clustering and differentially expressed genes per cluster.
The scRNA-seq data for the YUMM1.7 tumor model and melanoma patients were sourced from two GEO datasets with the following accession numbers: GSE146613, GSE115978, and GSE120575. These datasets contained a total of 4,665 cells from 6 mice and 4,861 cells from 31 patients, respectively. Further downstream analysis was performed in R v.4.2.3 using the Seurat package (v4.3.0) (Stuart et al. 2019, Cell 177:1888-1902). Cells were filtered out if they had fewer than 200 or more than 6,000 detected genes or if mitochondrial transcripts constituted more than 10% of the reads. Similarly, genes were excluded if they were detected in fewer than three cells. These cells underwent scaling after normalization through a linear regression model that utilized the log-normalization method. Cells were clustered using the "FindNeighbors" function with 20 dimensions of reduction and the "FindClusters" function with default settings. Uniform Manifold Approximation and Projection (UMAP) was utilized for visualizing the clusters. Differential gene expression analysis between each cluster was performed using a Wilcoxon rank-sum test. In the murine scRNA-seq dataset, previously reported marker genes were used to annotate different macrophage subpopulations, including Ml-like Macrophages (Clqa, Cd80, Cd86, Itgax, Tlr2, Fcgrl, II lb) and M2-like Macrophages (Clqa, Mrcl, Seppi, Ctsb, Retnla, Cd200rl). In the human scRNA-seq dataset, similar subpopulations were identified as Ml-like Macrophages (C1QA, FCN1, CD80, CXCL9, CXCL10, S0CS3) and M2-like Macrophages (C1QA, SPP1, SEPPI, CD163, CD28, MSR1, SLC40A1). The R package ggplot2 v3.4.1 was used to check HPGDS expression in different clusters of cells.
9.28 In vivo treatments
For the treatment with aPDl (BioLegend, 114122, RRID: AB_2800578) mice were randomized when the average of the tumor volumes was 150 mm3 and treated i.p. two times per week with 10 mg/kg aPDl or control IgG from rat serum (Sigma-Aldrich, 14131, RRID: AB_1163627). For CD4 depletion, mice were treated three days before tumor injection (500 pg/mouse), then two times per week (250 pg/mouse) until the end of the experiment with aCD4 (BioXCell, BE0003-1, RRID: AB_1107636). The efficiency of CD4 depletion was assessed by FACS in the blood. For CD8 depletion, mice were treated i.p. with 5 mg/kg aCD8 (BioXcell, BE0117, RRID: AB_10950145) or control IgG from rat serum. Treatment with aCD8 was performed three days before tumor injection, then one time per week until the end of the experiment. The efficiency of CD8 depletion was assessed by FACS in the tumor. For the pharmacological inhibition of HPGDS, mice were treated by oral gavage with HQL-79 (Cayman Chemical Company, 10134, RRID: AB_3662692) (15 mg/kg BID), 1,8-naphthyridine ly (in short, Cmpdly) (GSK) (0.1 - 0.3 - 1 - 3 mg/kg BID), TAS-205 MedChemExpress, HY-109134A, RRID: AB_3662693) (30 mg/kg BID) or control vehicle (methylcellulose). For the engrafted melanoma mouse models, mice were randomized when the average tumor reached 100 mm3 and treated two times per day with the indicated compound. For the genetically engineered mouse melanoma model, treatment with HQL-79 15 mg/kg BID, aPDl 10 mg/kg, the combination of both or with Vehicle and IgG started when the average tumor volume was 500 mm3). For the KPC model, 6 days after tumor injection, mice were treated intraperitoneally with 10 mg/kg aPDl, control IgG from rat serum. For the pharmacological inhibition of HPGDS, mice were treated for 9 days by oral gavage with HQL-79 15 mg/kg BID or control vehicle (methylcellulose).
9.29 TAM-like macrophages
To differentiate BMDMs towards TAM-like cells, 4xl06 BMDMs were seeded in 6-well plates in DMEM supplemented with 10% FBS, 1% P/S and 20% TCM for 12h at 37°C in a 5% CO2 humidified atmosphere, as previously described.
9.30 HPGDS regulation in BMDMs
BMDMs were isolated as described before. At day 6, were stimulated for 24h with 20 ng/ml IFN-y and 100 ng/ml LPS, 10 ng/ml IL-4, 10 ng/ml IL-6 or 5 ng/ml IL-10. After 24hr, IL-4 polarized macrophages were treated for an additional 24hr with 20 ng/ml IFN-y, 10 ng/ml TNFa, or 100 ng/ml LPS, alone or in combination. Unstimulated BMDMs were used as controls. Hpgds expression level was checked by qPCR.
9.31 PGD2 release by macrophages
BMDMs were isolated as described before. At day 1x10s macrophages were seeded in a 24-well plated and stimulated or not with for 24hr 10 ng/ml IL-4. IL-4 polarized macrophages were then treated for an additional 24hr 10 ng/ml TNFa. After that, medium was replaced with 200 pL of DMEM complete and conditioned for an additional 48hr. The day of the collection, secreted PGD2 was stabilized by adding Methoxime (MOX) and measured with Prostaglandin D2-MOX ELISA Kit (Cayman Chemical, 512011) in accordance with the manufacturer's protocol.
9.32 HPGDS regulation in hMDMs hMDMs were obtained from healthy donor buffy coats and isolated with CD14-conjugated MicroBeads (Miltenyi Biotec) as described before. At day 6, hMDMs were stimulated for 24hr with 20 ng/ml IL-4 or 40 ng/ml IL-10. Unstimulated hMDMs were used as controls. After 24hr-stimulation, IL-4 polarized macrophages were treated for an additional day with 10 ng/ml TNFa. HPGDS expression level was checked by qPCR. 9.33 In vitro T cells with macrophages
24hr before starting the co-culture, IL-4 polarized or Ctrl macrophages were collected, seeded in 24-well plates, and incubated overnight at 37°C in a 5% CO2 humidified atmosphere. When indicated, macrophages were pre-treated for 24hr with lpM of PGD2. To obtain T cell-conditioned media (T cell CM), 1 x 106 activated CD4+ T cells or CD8+ T cells were cultured in a 6-well plate 24hr in T cell medium (RPMI supplemented with 10% FBS, 1% P/S, 1% MEM non-essential amino acids NEAA, 25 pmol/L p- mercaptoethanol, and 1 mmol/L sodium pyruvate). IL-4 polarized or Ctrl macrophages were then cultured with 250 pL T cell CM for 24hr at 37°C in a 5% CO2 humidified atmosphere. In the co-culture experiment, macrophages were co-cultured with activated CD4 + T cell or CD8+ T cells in a ratio 1:1 and incubated 24h at 37° C in a 5% CO2 humidified atmosphere. When indicated, 25 pg/ml aTNFa, 20 pg/ml alFNy or Isotype Ctrl were added to the medium. Upon the indicated time of incubation, qPCR to assess Hpgds expression in macrophages was performed.
9.34 PGD2 supplementation on CD8+ T cells
1 x 106 activated CD8+ T cells were cultured in a 6-well plate 24hr in T cell medium before the addition of 1 mM of PGD2. Cells were then cultured for an additional 4hr in the presence or not of PGD2. Upon the indicated time of incubation, flow cytometry to assess cytokines production (/.e., GZMB, TN Fa, I FNy), activation status (/.e., CD69) and PD1 expression was performed.
9.35 CD8+ T cell proliferation
Ovalbumin-specific OT-I CD8+ T cells pre-activated with lpg/ml of the OT-I TCR-binding ovalbumin peptide (IBA Lifesciences) cultured with 10 ng/ml IL-2 (Peprotech) were treated for 4hr with ImM PGD2 and then labeled with 5 pM CellTrace Violet (CTV, Invitrogen, C34571) for 15 min at 379C. OT-I expressing cells were then co-cultured with pre-seeded OVA-expressing YUMM 1.7 for 24hr in the presence of ImM PGD2 at a ratio 2:1 (2 CD8+ T cells: 1 cancer cell). Cells were then stained with viability dye. Data were collected and analyzed with BD FACSDiva.
9.36 CD8+ T cell cytotoxicity
Ovalbumin-specific OT-I CD8+ T cells pre-activated with lpg/ml of the OT-I TCR-binding ovalbumin peptide (IBA Lifesciences) cultured with 10 ng/ml IL-2 (Peprotech) were treated for 4hr with ImM PGD2 and then labeled then co-cultured with pre-seeded OVA-expressing YUMM 1.7 in the presence of ImM PGD2 for 24hr at a ratio 2:1 (2 CD8+ T cel Is : 1 cancer cell). CD8+ T cell cytotoxicity activity was determined with the Cytotoxicity Detection LDH Kit (Roche, 11644793001), according to the manufacturer's instruction.
9.37 CD8+ T cell migration
Migration of CD8+ T cells was assessed by using a 5-pm-pore polycarbonate membrane (Transwell; Costar). When indicated CD8+ T cells were pre-treated with 1 pM PGD2 for 4hr. The bottom chamber contained T cell medium with or without 20ng/mL CXCL10 or 2xl05 IL-4 polarized BMDMs Hpgds-\N~ or Hpgds-KO pre-treated or not with 1 pM PGD2 for 24hr. 12hr before starting the assay, CD8+ T cells and macrophages were cultured in T cell medium (RPMI supplemented with 2% FBS, 1% P/S, 1% MEM non- essential amino acids NEAA, 25 pmol/L p-mercaptoethanol, and 1 mmol/L sodium pyruvate). Subsequently, 2xl05 CD8+T cells were seeded and incubated for 2hr. The migrated cells were collected and counted under the microscope.
9.38 Patient derived organotypic spheroid (PDOT)
Human melanoma sample was received in cold RPMI complete (10% FBS, 1% P/S) on ice and minced in 13.5 mL RPMI supplemented with 10% FBS, 0.5% P/S, 1.1 mg/ml Collagenase I (Thermo Fisher Scientific), 2.3 mg/ml Dispase II (Gibco) and 2.2 pl DNase I (Quiagen) and digested for 20 minutes at 37°C. Digested tissue was centrifuged 5 minutes at 1500 rpm, resuspended in RPMI complete and filtered using first a 100-pm and then a 40-pm pore mesh strainer to generate S2 fraction (40-100 pm) spheroid fractions. This fraction was then centrifuged at 1500 rpm for 5 minutes at 4°C and resuspended in the collagen, containing 2.5 mg/ml type I rat tail collagen (Corning) buffered with PBS 10X with phenol red and adjusted the pH to 7.4 using 0.5M NaOH. The mixture containing the spheroid embedded in the collagen was then loaded into the 3D microfluidic culture devices. Spheroids were then incubated for 40 minutes at 37°C to let the collagen solidified and then hydrated with DMEM supplemented with 10% FBS and 10% P/S. After 24hr PDOTS were treated with lpM HQL-79 or with vehicle by refreshing the inhibitior every day for an additional 96hr. After 96hr-treatment, PDOTS were then digested by using the digestion medium, incubated for 15 minutes at 37°C, centrifuged and stained to assess by flow cytometry TAM polarization (i.e., HLA-DR), CD8+ T cell and TN Fa-released by CD8+ T cells.
9.39 RNA-seq on TAMs sorted from YUMM 1.7 Ctrl and HpgdsaM0 mice
Total RNA was isolated from in vivo sorted Hpgds KO and Ctrl TAMs (Viability, CD45+, CDllb+, F4/80+) using the RNeasy Minikit (Qiagen) following the manufacturer's protocol and resuspended in RNase-free water. Frozen RNA samples were shipped to Novogene for the Plant and Animal Eukaryotic Strand Specific mRNA (WOBI) service and the resulting paired-end 150bp reads were sequenced on a Novaseq X Plus instrument. Next, reads were aligned to the mus musculus reference genome GRCm39 using STAR (v2.7.10b) with default parameters resulting in an average of 33.6 million uniquely mapped reads per sample. Aligned reads were quantified using featurecounts (v2.0.1) in R (v4.3.3) with options -t gene and -s 2. Subsequent analyses were performed with DESeq2 package (vl.42.0)(96). Gene Set Enrichment Analysis (GSEA) was conducted with clusterProfiler (v4.10.0) and org.Mm.eg.db (v3.18.0) and visualized using DOSE (v3.28.1) and enrichplot (vl.22.0) (Wu et al. 2021, Innovation 2:100141; Yu et al. 2015, Bioinformatics 31:608-609). Marker genes of Mrcl-immunosuppressive macrophages were defined above. The "Angiogenesis related cytokines signature includes Fgf2, Pdgfc, Egf, Hgf, Pdgfa, Tgfa, Angpt2, Vegfc, Angptl, Pdgfb, Vegfa, Vegfb, Igfl and Pigf. 9.40 Analysis of data from TCGA
Gene expression and clinical data for TCGA SKCM were obtained from cBioPortal (https://www.cbioportal.org/datasets). The gene expression values were normalized to Transcripts Per Million (TPM) for subsequent analysis. The infiltration of 22 types of immune cells in each melanoma sample was determined using the CIBERSORT package (vO.l.O) in R software. Gene set permutations were set at 1000 repeats for each analysis. The R package ggpubr (v0.6.0) was employed to elucidate the differences between TME scores and HPGDS levels in these patients. To investigate the expression of HPGDS in human PDAC Normalized RNA-sequencing (RNA-seq) expression data from 181 PDAC tissue samples and 167 normal pancreatic samples were obtained from cBioPortal and the Genome-Tissue Expression (GTEx) project (https://www.genome.gov/Funded-Programs-Proiects/Genotype-Tissue- Expression-Project), respectively. HPGDS expression levels were compared between tumor and adjacent normal/healthy tissues using both the TCGA and GTEx datasets. The Limma package was used to adjust batch effects between the two datasets. Gene expression distributions are presented as box plots, with statistical significance calculated using the Wilcoxon test.

Claims

1. An inhibitor of prostaglandin D2 receptor 1 (DPI) for use in treating a tumor or cancer, inhibiting a tumor or cancer, or inhibiting progression of a tumor or cancer.
2. The inhibitor of DPI for use according to claim 1, wherein the tumor or cancer is a primary tumor or cancer.
3. The inhibitor of DPI for use according to claim 1 or 2, wherein the tumor or cancer microenvironment of the tumor or cancer is characterized in that the total amount of expression of HPGDS in the tumor micro-environment is predominantly contributed by tumor- or cancer- associated macrophages.
4. The inhibitor of DPI for use according to any one of claims 1 to 3 wherein the inhibitor of DPI is selectively inhibiting function or expression of DPI.
5. The inhibitor of DPI for use according to any one of claims 1 to 4 in combination with a further antitumor or anti-cancer agent, and/or in combination with surgery or radiation.
6. An isolated macrophage characterized by substantially lacking functional DPI.
7. The isolated macrophage according to claim 6 for use as a medicament.
8. An isolated CD8+ T-cell characterized by substantially lacking functional DPI.
9. The isolated CD8+ T-cell according to claim 8 for use as a medicament.
10. A pharmaceutical composition comprising an isolated macrophage according to claim 6 or comprising an isolated CD8+ T-cell according to claim 8.
11. A method for selecting a subject having cancer for therapy including an inhibitor of DPI, an isolated macrophage substantially lacking functional DPI or an isolated CD8+ T-cell substantially lacking functional DPI, comprising: assessing the expression of HPGDS in a tumor biopsy sample obtained from the subject, and selecting a subject having cancer for therapy, when the total amount of expression of HPGDS in the tumor micro-environment of the tumor biopsy is predominantly contributed by or occurring in tumor- or cancer-associated macrophages.
PCT/EP2024/082474 2023-11-16 2024-11-15 Inhibition of prostaglandin d2 receptor 1 as means for treating cancer Pending WO2025104233A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23210284.8 2023-11-16
EP23210284 2023-11-16

Publications (1)

Publication Number Publication Date
WO2025104233A1 true WO2025104233A1 (en) 2025-05-22

Family

ID=88837398

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/082474 Pending WO2025104233A1 (en) 2023-11-16 2024-11-15 Inhibition of prostaglandin d2 receptor 1 as means for treating cancer

Country Status (1)

Country Link
WO (1) WO2025104233A1 (en)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000014281A2 (en) 1998-08-21 2000-03-16 Naxcor Assays using crosslinkable immobilized nucleic acids
WO2009061676A2 (en) * 2007-11-06 2009-05-14 Amira Pharmaceuticals, Inc. Antagonists of pgd2 receptors
WO2010008864A2 (en) * 2008-06-24 2010-01-21 Amira Pharmaceuticals, Inc. Cycloalkane[b]indole angtagonists of prostaglandin d2 receptors
WO2011014587A2 (en) * 2009-07-31 2011-02-03 Amira Pharmaceuticals, Inc. Ophthalmic pharmaceutical compositions of dp2 receptor antagonists
WO2011014588A2 (en) * 2009-07-31 2011-02-03 Amira Pharmaceuticals, Inc. Dermal formulations of dp2 receptor antagonists
US9623031B2 (en) 2011-06-29 2017-04-18 The Penn State Research Foundation Compositions, methods and kits for treating cancer
CN106619651A (en) 2016-12-23 2017-05-10 江苏大学 Application of PGD2 (prostaglandin D2) in preparation of drug for treating stomach cancer
JP6168520B2 (en) * 2013-09-17 2017-07-26 国立大学法人 千葉大学 Indole compounds, DP prostanoid receptor antagonists, drugs using the same, and use of DP prostanoid receptor antagonists.
WO2017209272A1 (en) 2016-06-03 2017-12-07 国立大学法人東京大学 Cancer metastasis inhibitor

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000014281A2 (en) 1998-08-21 2000-03-16 Naxcor Assays using crosslinkable immobilized nucleic acids
WO2009061676A2 (en) * 2007-11-06 2009-05-14 Amira Pharmaceuticals, Inc. Antagonists of pgd2 receptors
WO2010008864A2 (en) * 2008-06-24 2010-01-21 Amira Pharmaceuticals, Inc. Cycloalkane[b]indole angtagonists of prostaglandin d2 receptors
WO2011014587A2 (en) * 2009-07-31 2011-02-03 Amira Pharmaceuticals, Inc. Ophthalmic pharmaceutical compositions of dp2 receptor antagonists
WO2011014588A2 (en) * 2009-07-31 2011-02-03 Amira Pharmaceuticals, Inc. Dermal formulations of dp2 receptor antagonists
US9623031B2 (en) 2011-06-29 2017-04-18 The Penn State Research Foundation Compositions, methods and kits for treating cancer
JP6168520B2 (en) * 2013-09-17 2017-07-26 国立大学法人 千葉大学 Indole compounds, DP prostanoid receptor antagonists, drugs using the same, and use of DP prostanoid receptor antagonists.
WO2017209272A1 (en) 2016-06-03 2017-12-07 国立大学法人東京大学 Cancer metastasis inhibitor
CN106619651A (en) 2016-12-23 2017-05-10 江苏大学 Application of PGD2 (prostaglandin D2) in preparation of drug for treating stomach cancer

Non-Patent Citations (89)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. NM_001037631.3
"Uniprot", Database accession no. Q13258
ABUDAYYEH ET AL., SCIENCE, 2016
ANDERSON ET AL., IMMUNITY, vol. 44, 2016, pages 989 - 1004
AOYAGI ET AL., EUR J PHARMACOL, vol. 875, no. 1584160-52-0, 2020, pages 173030
ARITAKE ET AL., J BIOL CHEM, vol. 281, no. 1169483-24-2, 2006, pages 15277 - 15286
BERG ET AL., METHODS MOL BIOL, vol. 635, 2010, pages 133 - 145
BEURACHETTI, J MOL STRUCTURE, vol. 1259, no. 41598-07-6, 2022, pages 132704
BIENIASZ-KRZYWIEC ET AL., CELL METAB, vol. 30, 2019, pages 917 - 936
BRADLEY ET AL., MAMM GENOME, vol. 23, 2012, pages 580 - 586
CADILLA ET AL., BIOORGANIC & MEDICAL CHEMISTRY, vol. 28, 2020, pages 115791
CASAZZA ET AL., CANCER CELL, vol. 24, 2013, pages 695 - 709
CHAMBERS ET AL., NATURE REV CANCER, vol. 2, 2002, pages 563 - 572
CHAN ET AL., CLIN EXP PHARMACOL PHYSIOL, vol. 33, 2006, pages 533 - 540
CHURCH ET AL., PROC NATL ACAD SCI USA, vol. 81, 1984, pages 1991 - 1995
CROSBY ET AL., NUCLEIC ACID THER, vol. 25, 2015, pages 297 - 305
CUSI ET AL., BIOTECHNIQUES, vol. 17, 1994, pages 1034 - 1036
DANKORT ET AL., NAT GENET, vol. 41, 2009, pages 544 - 552
DASH PUJARINI ET AL: "High PGD2 receptor 2 levels are associated with poor prognosis in colorectal cancer patients and induce VEGF expression in colon cancer cells and migration in a zebrafish xenograft model", BRITISH JOURNAL OF CANCER, NATURE PUBLISHING GROUP UK, LONDON, vol. 126, no. 4, 8 November 2021 (2021-11-08), pages 586 - 597, XP037695079, ISSN: 0007-0920, [retrieved on 20211108], DOI: 10.1038/S41416-021-01595-4 *
DI MATTEO ET AL., MOL THER, vol. 22, 2014, pages 1614 - 1624
DUMLAO ET AL., BIOCHIM BIOPHYS ACTA - MOL CELL BIOL LIPIDS, vol. 1811, 2011, pages 724 - 736
FAN ET AL., CANCER COMMUNICATIONS, vol. 40, 2020, pages 135 - 153
FAN ET AL., ONCOLOGY REPORTS, vol. 41, 2019, pages 3 - 14
GALLUZI ET AL., SCI TRANSL MED, 2018
GUO ET AL., ADV CANCER RES, vol. 119, 2013, pages 421 - 475
HEISS ET AL., FASEB J, vol. 29, 2015, pages 3076 - 3084
HUAN ET AL., BIOTECHNIQUES, vol. 28, 2000, pages 254 - 255
HUGO ET AL., CELL, vol. 165, 2016, pages 35 - 44
JENKINS ET AL., CANCER DISCOVERY, vol. 8, 2018, pages 196 - 215
JERBY-ARNON ET AL., CELL, vol. 175, 2018, pages 984 - 997
KACZANOWSKA ET AL., J LEUKOC BIOL, vol. 93, 2013, pages 847 - 863
KAISER, SCIENCE, vol. 356, 2017, pages 112
KAJIWARA ET AL., EUR J PHARMACOL, vol. 667, 2011, pages 389 - 395
KAUFMAN ET AL., NAT REV DRUG DISCOV, vol. 14, 2015, pages 642 - 662
KWOK ET AL., NUCL ACIDS RES, vol. 18, 1990, pages 999 - 1005
LABRECQUE ET AL., PLOS ONE, vol. 8, 2013, pages 71752
LEVINE ET AL., MOL THER METH CLIN DEV, vol. 4, 2017, pages 92 - 101
LEWIS ET AL., NATURE METHODS, vol. 18, 2021, pages 997 - 1012
MA ET AL., BRAIN BEHAVIOUR IMMUNITY, vol. 45, 2015, pages 157 - 170
MAJZOUB ET AL., J BIOL CHEM, vol. 258, 1983, pages 14061 - 14064
MARIC ET AL., J ALLERGY CLIN IMMUNOL, vol. 143, 2018, pages 2202 - 2214
MARY ET AL., CANCER IMMUNOL RES, vol. 10, 2022, pages 900 - 916
MARY ET AL., CANCER IMMUNOLOGY RESEARCH, vol. 10, 2022, pages 900 - 916
MAZZONE ET AL., CELL, vol. 136, 2009, pages 839 - 851
MURATA ET AL., PNAS, vol. 105, no. 50, 2008, pages 20009 - 14
MURATA ET AL., PNAS, vol. 108, no. 49, 2011, pages 19802 - 19807
MYERS ET AL., BIOCHEM, vol. 30, 1991, pages 7661 - 7666
NABE ET AL., PROSTAGLANDINS & OTHER LIPID MEDIATORS, vol. 95, no. 927878-49-7, 2011, pages 27 - 34
OKAMOTO, CHEM SOC REV, vol. 40, 2011, pages 5815 - 5828
OMORI ET AL., J PATHOL, vol. 244, no. 1, 2018, pages 84 - 96
OYAMA SATOMI ET AL: "A novel indole compound, AWT-489, inhibits prostaglandin D2-induced CD55 expression by acting on DP prostanoid receptors as an antagonist in LS174T human colon cancer cells", vol. 541, 1 January 2014 (2014-01-01), US, pages 21 - 29, XP093246927, ISSN: 0003-9861, Retrieved from the Internet <URL:https://pdf.sciencedirectassets.com/272380/1-s2.0-S0003986113X00190/1-s2.0-S0003986113003433/main.pdf?hash=1e8e3faab8119bd246b2cb1654e23a318b9cc07a81d64efb534548c05840b17f&host=68042c943591013ac2b2430a89b270f6af2c76d8dfd086a07176afe7c76c2c61&pii=S0003986113003433&tid=spdf-8560362b-59b6-492c-a040-430> DOI: 10.1016/j.abb.2013.10.023 *
PARK ET AL., CANCER RES, vol. 67, 2007, pages 881
PARSA ET AL., DIABETES, vol. 61, 2012, pages 2881 - 2892
PEGGS ET AL., CLIN EXP IMMUNOL, vol. 157, 2009, pages 9 - 19
PELLEFIGUES ET AL., NAT COMMUN, vol. 9, no. 1033836-12-2, pages 725
PETTIPHER ET AL., NAT REV DRUG DISCOV, vol. 6, 2007, pages 313 - 325
PLATT ET AL., CELL, vol. 159, 2014, pages 440 - 455
POZNIAK ET AL., STUDY, 2022
PROC NATL ACAD SCI USA, vol. 108, pages 18324 - 18329
RATNER, NATURE BIOTECHNOL, vol. 36, 2018, pages 484
RIAZ ET AL., CELL, vol. 171, 2017, pages 934 - 949
ROBERT C MURPHY, TANDEM MASS SPECTROMETRY OF LIPIDS, 2014
SAHIN ET AL., NATURE, vol. 547, 2017, pages 222 - 226
SAMBROOK ET AL.: "Molecular Cloning. A laboratory manual", 1989, CSHL PRESS
SCHOORS ET AL., NATURE, vol. 520, 2015, pages 192 - 197
SCOLARO ET AL., NAT CANCER, vol. 5, 2024, pages 1206 - 1226
SERRA ET AL., CELL MOL GASTROENTEROL HEPATOL, vol. 14, 2022, pages 609 - 624
SHANG ET AL., NATURE, vol. 587, 2020, pages 626 - 631
SHARON ET AL., CHIN J CANC, vol. 33, 2014, pages 434 - 444
STASSBURG ET AL., TARGETED LIPIDOMICS OF OXYLIPINS (OXYGENATED FATTY ACIDS, 2015
STEICHEN ET AL., EUR J PHARM SCI, vol. 48, 2013, pages 416 - 427
STUART ET AL., CELL, vol. 177, 2019, pages 1888 - 1902
TAKAHASHI GO ET AL: "Effect of the potent and selective DP1 receptor antagonist, asapiprant (S-555739), in animal models of allergic rhinitis and allergic asthma", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 765, 1 October 2015 (2015-10-01), NL, pages 15 - 23, XP055863753, ISSN: 0014-2999, DOI: 10.1016/j.ejphar.2015.08.003 *
TAKESHITA ET AL., ANN CLIN TRANSL NEUROL, vol. 5, no. 1244967-98-3, 2018, pages 1338 - 1134
TAVERNA ET AL., NUCL ACIDS RES, vol. 48, 2020, pages 385 - 394
TINDALL ET AL., HUM MUTAT, vol. 30, 2009, pages 857 - 859
TIPANEE ET AL., MOL THER NUCLEIC ACIDS, vol. 19, 2020, pages 1309 - 1329
TORCHILIN, ADV DRUG DELIV REV, vol. 60, 2008, pages 548 - 558
TRABANELLI ET AL., NAT COMMUN, vol. 8, 2017, pages 593
VAN DIJK ET AL., TRENDS GENET, vol. 30, 2014, pages 418 - 426
WANG ET AL., EBIOMEDICINE, vol. 76, 2022, pages 103873
WANG ET AL., KIDNEY INT, vol. 72, 2007, pages 290 - 299
WHITCOMBE ET AL., NAT BIOTECHNOL, vol. 17, 1999, pages 804 - 807
WITTWER ET AL., CLIN CHEM, 2003, pages 843 - 860
WU ET AL., INNOVATION, vol. 2, 2021, pages 100141
YOKOO ET AL., ACS MED CHEM LETT, vol. 12, 2021, pages 236 - 241
YU ET AL., BIOINFORMATICS, vol. 31, 2015, pages 608 - 609
ZHANG ET AL., GLIA, vol. 62, 2014, pages 804 - 817
ZHANG ET AL., STEM CELLS, vol. 36, 2018, pages 990 - 1003

Similar Documents

Publication Publication Date Title
Neviani et al. Natural killer–derived exosomal miR-186 inhibits neuroblastoma growth and immune escape mechanisms
Zhang et al. Knockdown of NEAT1 induces tolerogenic phenotype in dendritic cells by inhibiting activation of NLRP3 inflammasome
EP3500244A1 (en) Methods of suppressing delivery of exosomes to liver and spleen
Yang et al. Focus on exosomes: novel pathogenic components of leukemia
JP2021522188A (en) Compositions and Methods for Multiple Tumor Vaccination with Endogenous Gene Activation
CN110997902A (en) SUV39H1-deficient immune cells
CN120239746A (en) Compositions, systems and methods for modulating T cell function
WO2021030627A1 (en) Methods for predicting outcomes of checkpoint inhibition and treatment thereof
Zhao et al. MicroRNA‐200a promotes phagocytosis of macrophages and suppresses cell proliferation, migration, and invasion in nasopharyngeal carcinoma by targeting CD47
Hajaj et al. Alternative splicing of the inhibitory immune checkpoint receptor SLAMF6 generates a dominant positive form, boosting T-cell effector functions
Marques et al. MicroRNAs in B-cells: from normal differentiation to treatment of malignancies
JP2023518852A (en) Methods of delivering nucleic acids to immune cells using RBCEV
Trotta et al. Activated T Cells Break Tumor Immunosuppression by Macrophage Reeducation
Song et al. miRNAs in HCC, pathogenesis, and targets
US20220054548A1 (en) Mirna for use in therapy
CN117460541A (en) Methods of enhancing isolated cell therapeutic efficacy for cell therapy
WO2025104233A1 (en) Inhibition of prostaglandin d2 receptor 1 as means for treating cancer
WO2025104238A1 (en) Inhibition of prostaglandin d2 receptor 2 as means for treating cancer
WO2025104229A1 (en) Hematopoietic prostaglandin synthase (hpgds) inhibition for treating cancer
US20200087666A1 (en) Compounds targeting long non coding rna for the treatment of cancer
WO2025233366A1 (en) Therapeutic and diagnostic applications of elongation of very long chain fatty acids protein 1
JP2025503987A (en) Tumor-infiltrating lymphocytes engineered to express a payload
CN117120062A (en) In vivo CRISPR screening system for discovery of therapeutic targets in CD8 T cells
Zhou et al. P21-positive senescent stromal cells promote prostate cancer immune suppression and progression that can be reversed by senolytic therapy
Ohishi et al. A forward genetic screen to study mammalian RNA interference–essential role of RNase IIIa domain of Dicer1 in 3′ strand cleavage of dsRNA in vivo

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24808634

Country of ref document: EP

Kind code of ref document: A1