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WO2025233366A1 - Applications thérapeutiques et diagnostiques d'allongement de la protéine 1 d'acides gras à très longue chaîne - Google Patents

Applications thérapeutiques et diagnostiques d'allongement de la protéine 1 d'acides gras à très longue chaîne

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Publication number
WO2025233366A1
WO2025233366A1 PCT/EP2025/062403 EP2025062403W WO2025233366A1 WO 2025233366 A1 WO2025233366 A1 WO 2025233366A1 EP 2025062403 W EP2025062403 W EP 2025062403W WO 2025233366 A1 WO2025233366 A1 WO 2025233366A1
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cells
cell
cancer
isolated
elovl1
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Massimiliano Mazzone
Samantha PRETTO
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Katholieke Universiteit Leuven
Vlaams Instituut voor Biotechnologie VIB
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Katholieke Universiteit Leuven
Vlaams Instituut voor Biotechnologie VIB
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • 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
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01199—Very-long-chain 3-oxoacyl-CoA synthase (2.3.1.199)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
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    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]

Definitions

  • the invention relates to modulation of the function or expression of Elongation of very long chain fatty acids protein 1 (ELOVL1) and its therapeutic applications.
  • ELOVL1 very long chain fatty acids protein 1
  • CD8+ T cells lacking or substantially lacking functional ELOVL1 are envisaged, and adoptive transfer of such CD8+ T cells is useful in the treatment of cancer.
  • ELOVL1 expression in CD8+ T cells is a biomarker for response to cancer therapy including an immune checkpoint inhibitor.
  • Elovll has garnered considerable attention in neurological disorders like adrenoleukodystrophy (ALD) and specific cancer types, where it is recognized as an adverse prognostic indicator. Elovll was included in gene signatures associated with response to immunotherapy treatment of melanoma in WO2018209324A2.
  • WO2013144325 discloses fatty acid elongation enzymes as targets for cancer diagnostics and therapeutics. The claimed method comprises the diagnosis of cancer cells in biological samples, as well as the use of fatty acid elongation enzyme inhibitors to treat cancer.
  • Zhang et al. explored the mRNA expression and survival data of ELOVLs in patients with hepatocellular carcinoma via the data of The Cancer Genome Atlas (Zhang et al. 2022, Front Oncol 12:884066). Significant expression alteration was observed in the ELOVLs family at the pan-cancer level.
  • the invention relates to isolated CD8+ T Lymphocyte (CD8+ T cell) or population of isolated CD8+ T cells characterized in that the CD8+ T cell or cells are substantially lacking functional very long chain fatty acid elongase 1 (EL0VL1).
  • EL0VL1 very long chain fatty acid elongase 1
  • the function or expression of EL0VL1 can be inhibited or substantially inhibited.
  • the function of EL0VL1 can be inhibited by a pharmacological compound, or the expression of EL0VL1 can be inhibited by a DNA nuclease specifically knocking out or disrupting EL0VL1, by an RNase specifically targeting EL0VL1 or by an inhibitory oligonucleotide specifically targeting EL0VL1.
  • CD8+ T cells can be polyclonal CD8+ T cells, in vitro amplified or expanded CD8+ T cells, antigen-specific CD8+ T cells, engineered T cell receptor (TCR)-CD8+ T cells, engineered chimeric antigen receptor (CAR) - CD8+ T cells, monospecific CAR CD8+ T cells, dual CAR CD8+ T cells, universal CAR CD8+ T, modular CAR CD8+ T, B-cell-targeting, antibody receptor (BAR) CD8+ T cell, design CD8+ T cell, or chimeric cytokine receptor (CCR) CD8+ T cell.
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • compositions comprising isolated CD8+ T cells or populations of isolated CD8+ T cells as defined above.
  • compositions can be pharmacological compositions.
  • the isolated CD8+ T cells or population of isolated CD8+ T cells or the (pharmaceutical) compositions as defined above can be for use as a medicament; optionally more in particular for use in treating a tumor or cancer, inhibiting a tumor or cancer, or inhibiting progression of a tumor or cancer.
  • Such CD8+ T cells or compositions in such use may be combined with a further anti-tumor or anti-cancer agent, and/or with surgery or radiation.
  • the isolated CD8+ T cells or population of isolated CD8+ T cells or the (pharmaceutical) compositions as defined above can be adoptively transferred in a subject.
  • the CD8+ T cells are autologous CD8+ T cells, allogeneic CD8+ T cells, or induced CD8+ T cells.
  • the invention further relates to methods of producing isolated CD8+ T cells as defined above, such methods comprising a step of isolating CD8+ T cells from including skin, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan.
  • An optional further step in such methods is a step of ex-vivo expanding the isolated CD8+ T cells.
  • Another step in such methods can be a step of ex-vivo manipulation to inhibit the function or expression of the ELOVL1 in the CD8+ T cells by means of pharmacological inhibition or by means of a DNA nuclease specifically knocking out or disrupting ELOVL1, an RNase specifically targeting ELOVL1, or an inhibitory oligonucleotide specifically targeting ELOVL1.
  • the invention also relates to pharmaceutical kits comprising at least one vial comprising isolated CD8+ T cells or populations of CD8+ T cells as defined above, or comprising a composition comprising such CD8+ T cells or populations of CD8+ T cells.
  • This disclosure further relates to methods for selecting a subject having cancer for therapy including an immune checkpoint inhibitor, such methods comprising one or more steps of: assessing the expression of ELOVL1 in CD8+ T cells in a sample obtained from the subject, and selecting a subject having cancer for the therapy, when the expression level of ELOVL1 in the CD8+ T cells corresponds to ELOVL1 expression levels in the same type of cancer of subjects known to respond to the therapy.
  • Alternative methods for selecting a subject having cancer for therapy including CD8+ T cells substantially lacking functional ELVOL1 and an immune checkpoint inhibitor comprising one or more steps of: assessing the expression of ELOVL1 in CD8+ T cells in a sample obtained from the subject, and selecting a subject having cancer for the therapy, when the expression level of ELOVL1 in the CD8+ T cells corresponds to ELOVL1 expression levels in the same type of cancer of subjects known not to respond to the immune checkpoint inhibitor.
  • FIGURE 2 An in vivo single-cell CRISPR screen selects Elovll as a promising metabolic target to sustain CD8+ T cell activity.
  • FIGURE 3 Elovll deficient CD8+ T cells have increased antitumoral activity upon aPD-1 treatment.
  • FIGURE 5 Relevance of ELOVL1 function in human CD8+ T cells.
  • FIGURE 6 Change in chain lengths of sphingomyelins ("SM”, left panels) and ceramides ("Cer”, right panels) upon contacting activated OT-1 CD8+ T-cells with: top left and right panels: a control scramble sgRNA (“sgNT”) or sgELOVLl RNA (“sgELOVLl”), as in Extended Data Fig. 4b of Pretto et al. 2025; bottom left and right panels: DMSO (control) or EL0VL1 inhibitor C3.
  • sgNT control scramble sgRNA
  • sgELOVLl RNA sgELOVLl
  • T cell-based therapies such as adoptive T cell therapies (including those involving the administration of engineered cells expressing recombinant, engineered or chimeric receptors specific for a disease or disorder of interest, such as a recombinant T cell receptor (TCR) or other recombinant, engineered or chimeric receptors) can be effective in the treatment of cancer and other diseases and disorders.
  • adoptive T cell therapies including those involving the administration of engineered cells expressing recombinant, engineered or chimeric receptors specific for a disease or disorder of interest, such as a recombinant T cell receptor (TCR) or other recombinant, engineered or chimeric receptors
  • adoptive T cell therapies including those involving the administration of engineered cells expressing recombinant, engineered or chimeric receptors specific for a disease or disorder of interest, such as a recombinant T cell receptor (TCR) or other recombinant, engineered or chimeric receptors
  • TCR recombin
  • efficacy or potency of the engineered cells can depend on one or more of various factors, including T cell exhaustion, immunosuppressive tumor microenvironment (TME), poor cell infiltration into the target, e.g., tumor, lack of endogenous anti-tumor immune response, and poor expression of the recombinant receptor, mispairing or competition with endogenously expressed TCRs.
  • optimal activity or outcome can depend on the ability of the administered cells to express the recombinant receptor, e.g., recombinant TCR, on the surface, recognize and bind to a target, e.g., target antigen, to traffic, localize to and/or successfully enter appropriate sites within the subject, tumors, and environments thereof.
  • optimal activity or outcome can depend on the ability of the administered cells to uniformly and/or continuously express the recombinant receptor, become activated, expand, to exert various effector functions, including cytotoxic killing and secretion of various factors such as cytokines, to persist, including long-term, to differentiate, transition or engage in reprogramming into certain phenotypic states (such as long-lived memory, less-differentiated, and effector states), to avoid or reduce immunosuppressive conditions in the local microenvironment of a disease, to provide effective and robust recall responses following clearance and re-exposure to target ligand or antigen, and avoid or reduce exhaustion, anergy, peripheral tolerance, terminal differentiation, and/or differentiation into a suppressive state.
  • cytotoxic killing and secretion of various factors such as cytokines
  • Reprogramming T cell metabolism can improve intratumoral fitness of the resulting T cells.
  • a CRISPR/Cas9 metabolic survey in CD8+ T cells was performed.
  • 83 targets enriched at the primary and/or metastatic niche of pancreatic cancer were identified.
  • a single-cell RNA sequencing was applied to disclose transcriptome changes associated with each metabolic perturbation. This revealed the Elongation of very long chain fatty acids protein 1 (ELOVL1) as a metabolic target to sustain proliferation and both effector and memory phenotypes in CD8+ T cells.
  • ELOVL1 very long chain fatty acids protein 1
  • ELOVL1 inactivation in adoptively transferred cells showed therapeutic efficacy in pancreatic tumors, especially when combined with an immune checkpoint inhibitor (to which the pancreatic tumor is not or barely responding, is resistant).
  • ELOVL1 in CD8+ T cells correlated with aPD-1 response in melanoma patients.
  • the invention therefore in the first aspect relates an isolated CD8+ T Lymphocyte (CD8+ T cell) or to a population of isolated CD8+ T cells characterized in that the CD8+ T cell or cells are lacking or substantially lacking functional very long chain fatty acid elongase 1 (ELOVL1). in one embodiment thereto, the isolated CD8+ T cells or a population of isolated CD8+ T cells characterized in that the function or expression of the ELOVL1 is inhibited, or substantially inhibited, in the CD8+ T cells cell or cells.
  • CD8+ T cell or to a population of isolated CD8+ T cells characterized in that the CD8+ T cell or cells are lacking or substantially lacking functional very long chain fatty acid elongase 1 (ELOVL1).
  • ELOVL1 very long chain fatty acid elongase 1
  • the CD8+ T cells are genetically engineered cells, expressing a recombinant receptor, such as a recombinant T cell receptor (TCR), that binds or recognizes a peptide epitope associated with a disease-associated antigen, such as a cancer antigen or a tumor antigen.
  • a recombinant receptor such as a recombinant T cell receptor (TCR)
  • TCR recombinant T cell receptor
  • CD8+ T cells with such modification resides in their increased or enhanced antitumoral action when transferred into subjects having or suffering from cancer or a tumor. Also provided are methods for engineering, preparing, and producing the engineered cells, kits and articles of manufacture for generating or producing the engineered cells as will be described in more detail hereinafter.
  • Very long chain fatty acid elongase 1 or ELOVL1 is an enzyme (EC:2.3.1.199) also known as 3-keto acyl- CoA synthase ELOVL1, ELOVL fatty acid elongase (ELOVL FA elongase 1), Elongation of very long chain fatty acids protein 1, Very long chain 3-ketoacyl-CoA synthase 1, Very long chain 3-oxoacyl-CoA synthase 1, CGI-88, IKSHD, Sscl.
  • the human ELOVL1 protein is identified by UniProt accession No. Q9BW560, Ensembl accession No. ENSG00000066322, and NCBI reference sequences with accession Nos. NP_073732.1, NP_001243328.1, NP_001243330.1 and NP_001243331.1.
  • the NCBI reference RNA sequence for human ELOVL1 is identified by accession NR_046117.2.
  • ELOVL1 inhibitors are known, including small molecules such as ELOVL1-IN-1 (CAS No. 2227482-41-7), ELOVL1-IN-2 (CAS No. 2761063-79-8), ELOVL1-IN-3 (Compound 22, CAS No.
  • ELOVL1-27 (CAS No. 2227482-41-7).
  • the experimental work outlined herein further supports that inhibition of EL0VL1 expression is obtainable by means of targeted inhibition or genetic knock-out of EL0VL1 or by means of siRNAs targeting EL0VL1 in CD8+ T cells.
  • the genetic knonk out can be achieved using CRISPR technology.
  • Inhibitory RNAs e.g. Thermo Fisher Scientific Catalog no AM51331, Biorbyt. Catalog no orbl856152, MedChem Express Cat. No HY-RS04321
  • guide RNAs for use with CRISPR-Cas e.g. Santa Cruz Biotechnolgy Catalog No sc-417698, OriGene Catalog No KN400027) are furthermore commercially available.
  • Functional EL0VL1 is defined as EL0VL1 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 ELOVL1) with the binding of ELOVL1 with any one of its potential natural binding .
  • functional ELOVL1 can be lacking, or be substantially lacking on and/or in a cell by repressing, inhibiting, or blocking expression of ELOVL1, or by binding of a "foreign" compound (as meant hereinabove) to ELOVL1.
  • functional ELOVL1 is lacking, or is substantially lacking, on and/or in an isolated immune cell as described herein (in particular CD8+ T-cells).
  • Genetic modification of immune cells isolated from a subject is one means of forcing the immune cells to (substantially) lack functional ELOVL1.
  • Such genetic modification can be aimed at repressing, reducing, or inhibiting ongoing expression of ELOVL1 in the isolated (unmodified) immune cells, and/or can be aimed at preventing or inhibiting de novo expression of ELOVL1, e.g. in case expression of ELOVL1 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) ELOVL1 protein in immune cells by means of contacting the immune cells with a pharmacological inhibitor of ELOVL1 is another means of causing immune cells to (substantially) lack functional ELOVL1.
  • the said shielding can be envisaged as neutralizing (part of the) ELOVL1 protein for interaction with other (natural) binding partners, or for interaction with substrates or reaction intermediates or products.
  • 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 ELOVL1 with high specificity and/or, optionally, with high affinity.
  • Such pharmacological inhibitors can be administered to a subject in need thereto such as to obtain a sufficient level of in vivo CD8+ T-cells in which functional ELOVL1 is lacking, or is substantially lacking.
  • Such administration can be targeted (increasing the specificity or selectivity for CD8+ T-cells) or untargeted.
  • ELOVL1 protein present inside immune cells can further be the target of pharmacologic knock-down such as by molecules or agents inducing specific proteolytic degradation of ELOVL1 protein.
  • pharmacologic knock-down such as by molecules or agents inducing specific proteolytic degradation of ELOVL1 protein.
  • the agent causing an immune cell to (substantially) lack functional ELOVL1 or causing neutralization of ELOVL1 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.
  • such CD8+ T-cells are intermediates in a process of obtaining CD8+ T-cells comprising a further (genetic) modification, e.g. CAR-T cells or TCR-T cells.
  • a further (genetic) modification e.g. CAR-T cells or TCR-T cells.
  • such CD8+ T-cells are concurrently subjected to more than one modification including (substantially) inhibition or expression of ELOVL1.
  • the CD8+ T cells are polyclonal CD8+ T cells, in vitro amplified or expanded CD8+ T cells, antigen-specific CD8+ T cells, engineered T cell receptor (TCR)-CD8+ T cells, engineered chimeric antigen receptor (CAR) - CD8+ T cells, monospecific CAR CD8+ T cells, dual CAR CD8+ T cells, universal CAR CD8+ T cells, modular CAR CD8+ T cells, B-cell-targeting antibody receptor (BAR) CD8+ T cells, design CD8+ T cells, or chimeric cytokine receptor (CCR) CD8+ T cells.
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • T cells Prior to genetic modification, CD8+ T cells are obtained from a subject.
  • T cells can be obtained from a number of sources, including skin, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan.
  • this disclosure relates to isolated immune cells lacking or substantially lacking functional ELOVL1, isolated ELOVL1 knock-out immune cells, or to isolated immune cells expressing or conditionally expressing an inhibitor of ELOVL1, or to populations of any thereof.
  • compositions such a pharmaceutical compositions, comprising (a population of) isolated immune cells lacking or substantially lacking functional ELOVL1, isolated ELOVL1 knockout immune cells, or comprising (a population of) isolated immune cells expressing or conditionally expressing an inhibitor of ELOVL1.
  • a further aspect of this disclosure relates to (populations of) isolated immune cells lacking or substantially lacking functional ELOVL1, to (populations of) isolated ELOVL1 knock-out immune cells, to (populations of) isolated immune cells expressing or conditionally expressing an inhibitor of ELOVL1, or to a 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 a cancer or tumor, or for inhibiting progression of a cancer or tumor.
  • the (populations of) isolated immune cells lacking or substantially lacking functional ELOVL1, the (populations of) isolated ELOVL1 knock-out immune cells, the (populations of) isolated immune cells expressing or conditionally expressing an inhibitor of ELOVL1, 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 a cancer or tumor, or for inhibiting progression of a cancer or tumor.
  • this aspect relates to methods of treating (a subject having) a cancer or tumor, inhibiting a cancer or tumor (in a subject having a tumor or cancer), or inhibiting progression of a cancer or tumor (in a subject having a tumor or cancer), such methods including administering a (population of) isolated immune cells lacking or substantially lacking functional ELOVL1, a (population of) isolated ELOVL1 knock-out immune cells, a (population of) isolated immune cells expressing or conditionally expressing an inhibitor of ELOVL1, 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.
  • the tumor or cancer is treated or inhibited, or its progression is inhibited.
  • any of the isolated CD8+ T-cells as described above (thus at least modified to (substantially) lack functional ELOVL1), any of the populations of such CD8+ T-cells, or any of the pharmaceutical compositions comprising any such isolated CD8+ T-cell or comprising any such population of isolated 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.
  • any of the isolated CD8+ T-cells according to the invention may further be for use in combination with or combined in any way with surgery, radiation, chemotherapy, targeted therapy, immunotherapy, and/or a further anticancer agent.
  • any of the isolated 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, and/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, and/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 additional treatment.
  • an additional therapeutic agent is administered together with the CD8+ T-cells or cell compositions.
  • the additional therapeutic agent is an immune checkpoint blockade inhibitor.
  • any treatment, therapy and/or anticancer agent may be for use in combination with (i) any of the isolated CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional ELOVL1), (ii) any of the populations of such CD8+ T-cells, or (iii) any of the pharmaceutical compositions comprising any such isolated CD8+ T- cells or comprising any such population of isolated CD8+ T-cells.
  • Examples of such treatments, therapies and/or anticancer agents include but are not limited to, chemotherapy, radiation, surgery, medication, immune checkpoint inhibitors, immune checkpoint blockade (ICB) antibodies, immune checkpoint inhibitors that block CTLA-4 or PD1, anti-CTLA4 monoclonal antibody, anti-PD 1 monoclonal antibody, anti-PD-Ll monoclonal antibody, adoptive cell transfer, human recombinant cytokines, cancer vaccines, immunotherapy, targeted therapy, hormone therapy, stem cell transplant, precision medicine, nonspecific immunotherapy (e g. cytokines and chemokines, such as IL-2, IFNa, IFNb, I FNg), oncolytic virus therapy, T-cell therapy (e.g.
  • TILs adoptive transfer of TILs, CAR-T therapy), cancer vaccines (e.g. conventional DC vaccine), ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, anti-LAG-3, anti-TIMI, anti-TIM3, anti-CSF-R, IDO inhibitor, OX- 40 agonist, GITR agonist, CD80 agonist, CD86 agonist, ICOS agonist, ICOSLG agonist, CD276 agonist, VTCN1 agonist, TNFSF14 agonist, TNFSF9 agonist, TNFSF4 agonist, CD70 agonist, CD40 agonist, LGALS9 agonist, CD80 inhibitor, CD86 inhibitor, ICOS inhibitor, ICOSLG inhibitor, CD276 inhibitor, VTCNl inhibitor, TNFSF14 inhibitor, TNFSF9 inhibitor, TNFSF4 inhibitor, CD70 inhibitor, CD40 inhibitor, LGALS9 inhibitor, TLR9 agonist, CD20
  • 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 CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional EL0VL1), any of the populations of such CD8+ T-cells, or any of the pharmaceutical compositions comprising any such isolated CD8+ T-cells or comprising any such population of isolated 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 CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional ELOVL1), or of any of the populations of such CD8+T-cells; or of administering (in particular: administering a therapeutically effective dose of) any of the isolated CD8+ T-cell according to the invention (thus at least modified to (substantially) lack functional ELOVL1), any of the populations of such CD8+ T-cells, or any of the pharmaceutical compositions comprising any such isolated CD8+ T-cells or comprising any such population of isolated CD8+ T-cells.
  • Such methods may further comprise (simultaneous, separate or sequential) combination with administration of any of additional treatments.
  • 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 treatment or therapy, further in combination with adoptive cell therapy of any of the isolated CD8+ T-cells according to the invention (thus at least modified to (substantially) lack functional ELOVL1), or of any of the populations of such CD8+ T-cells; or of administration (in particular: administering a therapeutically effective dose of) of any of the isolated CD8+ T-cell according to the invention (thus at least modified to (substantially) lack functional ELOVL1), any of the populations of such CD8+ T-cells, or any of the pharmaceutical compositions comprising any such isolated CD8+ T-cell or comprising any such population of isolated CD8+ T-cells.
  • the subject or patient to which a (composition comprising) CD8+ T-cell (substantially) lacking functional ELOVL1 is administered is a subject or patient not or poorly responding to therapy with or including an immune checkpoint inhibitor.
  • a therapy with a (composition comprising) CD8+ T-cell (substantially) lacking functional ELOVL1 is for use in/for use in the manufacture of a medicament for potentiating or breaking resistance to a therapy with or including an immune checkpoint inhibitor.
  • methods of stimulating or improving fitness of CD8+ T cells comprising the step of inhibiting, blocking or suppress ELOVL1 function or expression in the CD8+ T cells.
  • such method is an in vitro method.
  • the resulting CD8+ T cells are lacking or substantially lacking functional ELOVL1, such as for application in any of the medical uses described hereinabove.
  • adoptive cell transfer also known as cellular adoptive immunotherapy or cell transfer therapy
  • adoptive cell transfer 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 immune cells can be from an allogeneic origin.
  • 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.
  • TCRs antitumor T cell receptors
  • CARs chimeric antigen receptors
  • these well-known genetic modifications are not excluded and can be combined with genetic modifications aimed at forcing the CD8+ T-cells to lack or to substantially lack functional ELOVL1 (as described above; the step of forcing the CD8+ T-cells to lack or to substantially lack functional ELOVL1 can be in intermediate step).
  • TCR-engineered CD8+ T-cells TCR-Ts
  • CAR-engineered CD8+ T-cells CAR-Ts
  • Any pharmaceutical compositions comprising TCR- engineered CD8+ T-cells, and CAR-engineered CD8+ T-cells lacking or substantially lacking functional ELOVL1 are also part of the invention.
  • T-cells including CAR-Ts or TCR-Ts
  • T-cells usually is initiated by enriching lymphocytes from a leukapheresis product (but other origins are feasible, see above).
  • T-cells (CD8+ or CD4+) are then separated by use of e.g. an antibody to a cell type 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 ELOVL1 expression and, optionally, of a means for introducing the CAR or TCR; such means can be gene transfer, e.g. effectuated by using lentiviral vectors, the Sleeping Beauty transposon system, or mRNA transfection).
  • a means for introducing the CAR or TCR 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).
  • methods of producing isolated CD8+ T cells (substantially) lacking functional EL0VL1 or of compositions comprising such cells are part of this disclosure, such methods comprising a step of isolating CD8+ T cells from peripheral blood, umbilical cord blood, thymus or leukapheresis product obtained from a subject.
  • Such methods may further comprise a step of ex-vivo manipulation to inhibit the function or expression of the ELOVL1 in the CD8+ T cells by means of pharmacological inhibition (as described herein) or by means of a DNA nuclease specifically knocking out or disrupting ELOVL1, an RNase specifically targeting ELOVL1, or an inhibitory oligonucleotide specifically targeting ELOVL1.
  • expression of the endogenous ELOVL1 locus is knocked out, reduced or eliminated, in the engineered cell, by virtue of the genetic disruption (such as a knockout (KO)) at the ELOVL1 locus.
  • the term "antagonist” or “inhibitor” of a target 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 EL0VL1.
  • 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 e.g
  • 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 30 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.
  • siRNA double-stranded RNA
  • Dicer double stranded small interfering RNA
  • siRNA RNA-lnduced Silencing Complex
  • 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 RNAguided 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.
  • 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.
  • nucleic acid with DOTAP or DOPE or combinations thereof, complexes with other cationic lipids), dendrimers, virallike 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.
  • 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.
  • inhibition of EL0VL1 such as to obtain a cell or cells lacking or substantially lacking functional EL0VL1
  • a chemical or small molecule inhibitor see above.
  • EL0VL1 is an intracellular protein
  • an intrabody specifically binding to and inhibiting EL0VL1 can be considered as pharmacological inhibitor.
  • Intrabodies are antibodies binding and/or acting to intracellular target; this typically requires the expression of the antibody within the target cell, which can be accomplished by gene therapy/genetic modification involving introduction in a cell of a suitable genetic construct or vector comprising a suitable promoter (e.g. inducible, organ- or cell-specific,...) operably linked to an intrabody coding sequence.
  • 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 EL0VL1 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) a CD8+ T-cell.
  • therapeutic modality therapeutic agent, agent, and drug are used interchangeably herein, and likewise relate to the immune cells (in particular CD8+ T-cells) as described herein (with inhibited function or expression of EL0VL1 or (substantially) lacking functional EL0VL1). 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-Ts orTCR-Ts (substantially) lacking functional EL0VL1, or in which EL0VL1 expression or function is (substantially) 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.
  • 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 15 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.
  • 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 th "contacting" results in delivering an effective amount of the agent or composition comprising the agent to the object.
  • the invention relates to pharmaceutical compositions comprising (a population of) isolated CD8+ T cells (substantially) lacking functional ELOVL1, optionally further comprising 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 CD8+ T cells; and is alternatively defined as a pharmaceutically acceptable carrier.
  • Such pharmaceutical composition can optionally comprise a further anticancer agent (detailed further hereinafter, including chemotherapeutic agent, targeted therapy agent, and immunotherapeutic agent).
  • a further anticancer agent included in a further anticancer agent (detailed further hereinafter, including chemotherapeutic agent, targeted therapy agent, and immunotherapeutic agent).
  • kits comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising an inhibitor of ELOVL1, a (population of) isolated immune cells (or CAR-immune cells or TCR-immune cells) (substantially) lacking functional ELOVL1, a (population of) isolated ELOVL1 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) expressing or conditionally expressing an inhibitor of ELOVL1 as described hereinabove, or comprising a composition comprising any one of these; when referring herein to immune cells these are CD8+ T-cells.
  • 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. Combination, combination in any way
  • “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.
  • this disclosure relates to diagnostic-type methods or companion diagnostic-type methods.
  • One such aspect relates to methods of or for selecting a subject having cancer for therapy with or including an immune checkpoint inhibitor, such methods comprising one or more steps of: assessing, determining, measuring, quantifying or analyzing the expression or level of expression of ELOVL1 in CD8+ T cells in a sample obtained from the subject, and selecting a subject having cancer for the therapy, when the expression level of ELOVL1 in the CD8+ T cells corresponds to ELOVL1 expression levels in the same type of cancer of subjects known to respond to the therapy.
  • such method are methods of or for selecting a subject having cancer for therapy including CD8+ T cells substantially lacking functional ELOVL1 and an immune checkpoint inhibitor, comprising one or more steps of: assessing, determining, measuring, quantifying or analyzing the expression of ELOVL1 in CD8+ T cells in a sample obtained from the subject, and selecting a subject having cancer for the therapy, when the expression level of ELOVL1 in the CD8+ T cells corresponds to ELOVL1 expression levels in the same type of cancer of subjects known not to respond to the (therapy with an) immune checkpoint inhibitor or known to poorly respond to the (therapy with an) immune checkpoint inhibitor.
  • Another such aspect includes methods of predicting the response, the likelihood of response, or responsiveness of a subject having cancer to therapy with or including an immune checkpoint inhibitor, such methods comprising one or more steps of: measuring, determining, assessing, quantifying, or analyzing the expression of ELOVL1 in CD8+ T cells sample obtained from the subject, and selecting a subject having cancer or determining a subject having cancer to be eligible for the therapy when the ELOVL1 expression in the CD8+ T cells corresponds to ELOVL1 expression levels in the CD8+ T cells of the same type of cancer of subjects known to respond to the therapy comprising an immune checkpoint inhibitor.
  • such methods are methods of predicting the response, the likelihood of response, or responsiveness of a subject having cancer to therapy including CD8+ T cells substantially lacking functional ELOVL1 and an immune checkpoint inhibitor, such methods comprising one or more steps of: measuring, determining, assessing, quantifying, or analyzing the expression of Elovll in CD8+ T cells sample obtained from the subject, and selecting a subject having cancer or determining a subject having cancer to be eligible for the therapy when the ELOVL1 expression in the CD8+ T cells corresponds to ELOVL1 expression levels in CD8+ T cells in the same type of cancer of subjects known not to respond to the (therapy with an) immune checkpoint inhibitor or known to poorly respond to the (therapy with an) immune checkpoint inhibitor.
  • the sample referred to hereinabove in particular is a biological sample, more in particular a biological sample comprising CD8+ T cells.
  • a biological sample can be a solid sample (e.g. solid biopsy, or part of an surgically excised or removed tumor) or a fluid sample (e.g. a liquid biopsy, such as non-invasive liquid biopsy or non-invasive sample).
  • a fluid sample e.g. a liquid biopsy, such as non-invasive liquid biopsy or non-invasive sample.
  • the first sample can be a solid biopsy sample whereas the at least one second sample can be a liquid biopsy sample; or vice versa.
  • This disclosure further relates to an immune checkpoint inhibitor for use as a medicament; more in particular, for use as a medicament for treating (a subject having) a cancer or tumor, for inhibiting a cancer or tumor (in a subject), or for inhibiting progression of a cancer or tumor (in a subject); all as described above but further comprising one or more steps of:
  • this disclosure relates to a combination of CD8+ T cells substantially lacking functional ELOVL1 and an immune checkpoint inhibitor for use as a medicament; more in particular, for use as a medicament for treating (a subject having) a cancer or tumor, for inhibiting a cancer or tumor (in a subject), or for inhibiting progression of a cancer or tumor (in a subject); all as described above but further comprising one or more steps of:
  • response to (therapy with) the immune checkpoint inhibitor is herein meant as response to (therapy with) the immune checkpoint inhibitor in the absence of CD8+ T cells substantially lacking functional ELOVL1
  • This disclosure further relates to an immune checkpoint inhibitor for use as a medicament; more in particular, for use as a medicament for treating (a subject having) a cancer or tumor, for inhibiting a cancer or tumor (in a subject), or for inhibiting progression of a cancer or tumor (in a subject); all as described above but further comprising predicting the response, the likelihood of response, or responsiveness of a subject having cancer to therapy with or including an immune checkpoint inhibitor.
  • an immune checkpoint inhibitor for use as a medicament; more in particular, for use as a medicament for treating (a subject having) a cancer or tumor, for inhibiting a cancer or tumor (in a subject), or for inhibiting progression of a cancer or tumor (in a subject); all as described above but further comprising predicting the response, the likelihood of response, or responsiveness of a subject having cancer to therapy with or including an immune checkpoint inhibitor.
  • prediction of (likelihood) of response or responsiveness is performed with a method as described hereinabove; alternatively, any other known method to predict (
  • this disclosure relates to a combination of CD8+ T cells substantially lacking functional ELOVL1 and an immune checkpoint inhibitor for use as a medicament; more in particular, for use as a medicament for treating (a subject having) a cancer or tumor, for inhibiting a cancer or tumor (in a subject), or for inhibiting progression of a cancer or tumor (in a subject); all as described above but further comprising predicting the response, the likelihood of response, or responsiveness of a subject having cancer to therapy including CD8+ T cells substantially lacking functional ELOVL1 and an immune checkpoint inhibitor.
  • prediction of (likelihood) of response or responsiveness is performed with a method as described hereinabove; alternatively, any other known method to predict (likelihood) of response or responsiveness to an immune checkpoint inhibitor can be applied.
  • level of expression or “expression level” generally refers to the amount of an expressed biomarker in a biological sample.
  • “Expression” generally refers to the process by which information (e.g., gene- encoded and/or epigenetic information) is converted into the structures present and operating in the cell. Therefore, as used herein, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide 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).
  • biomarker refers to an indicator molecule or set of 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.
  • Biomarkers 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,
  • RNA from a (biological) sample wherein a fraction of the isolated RNA is the analyte strand
  • 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.
  • the invention covers methods which include detection/quantification of nucleic acids corresponding to one or more biomarkers as defined herein.
  • 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.
  • 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 nonamplifiable 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. 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).
  • 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).
  • 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).
  • 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. 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.
  • 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. 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.
  • a temperature of 68 DEG C 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.
  • the optimal conditions 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.
  • RNA oligonucleotides with formamide 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.
  • 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).
  • crosslinking moiety enables covalent linking of the oligonucleotide with the target nucleotide sequence and hence allows stringent washing conditions.
  • 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
  • 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.
  • 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.
  • 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 is an Pancreatic ductal adenocarcinoma.
  • Types of cancer that can be treated include, but are not limited to, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma, AIDS-Related Lymphoma, Primary CNS Lymphoma, Anal Cancer, Appendix Cancer (Gastrointestinal Carcinoid Tumors), Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Brain Cancer, Basal Cell Carcinoma of the Skin, Bile Duct Cancer, Bladder Cancer, Bone Cancer (includes Ewing Sarcoma and Osteosarcoma and Malignant Fibrous Histiocytoma), Brain Tumors, Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Non- Hodgkin Lymphom
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not"isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is"isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • a "subject" in general is a mammalian species.
  • 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 subject is a human subject.
  • treatment and “therapeutic method” refer to both therapeutic treatment and prophylactic/preventative measures.
  • Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventative measures).
  • the KPC pancreatic cell line used in this study (FC1245) was kindly provided by the lab of David A. Tuveson and was derived from spontaneous tumors arising in the KPC (KrasLSL.G12D/+; p53R172H/+; Pdx: CreTg/+ ) pancreatic cancer mouse model.
  • OVA-expressing KPC cell line was established by stable transduction of the parental cell line with a lentiviral vector harboring the expression cassette of the Ovalbumin (OVA)257-264 immunogenic "SIINFEKL" peptide and was maintained in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher) with 10% fetal bovine serum (FBS, Gibco), 1% Penicillinstreptomycin (Pen/Strep Gibco), 1% Sodium Pyruvate (Gibco) and Geneticin (G418, InvivoGen).
  • DMEM Dulbecco's Modified Eagle Medium
  • HEK293 cells were provided from ATCC and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin-streptomycin, and 2 mmol/L glutamine (Gibco). Cells were cultured at 37°C and 5% CO2. All the cell lines were passaged in the laboratory for no longer than 10 passages after receipt and tested for Mycoplasma by PlasmoTestMycoplasma Detection kit (InvivoGen) every 6 months.
  • T cells were freshly isolated from spleens of both male and female mice between 6 and 10 weeks of age.
  • T cells were isolated from buffy coats of healthy male and female volunteers aged between 25 and 65 years provided by Red Cross Donor Center Mechelen, Belgium (institutional approval S68611) (anonymized). Donors provided written consent.
  • OT-l:Rosa26-Cas9 mice were generated by intercrossing Rag2/OT-I mice with Rosa26-Cas9 knockin mice - which constitutively express the Cas9 nuclease.
  • OT-I mice express an ap TCR recognizing ovalbumin peptide residues 257-264 (OVA257-264) in the context of H2-Kb.
  • Rosa26-Cas9 immunocompetent mice C57BL/6 background
  • C57BL/6 WT were used as recipient mice and were inoculated with KPC_OVA or KPC cells.
  • mice used were between 6 and 12 weeks old, without specific gender selection. In all experiments, mice were randomly assigned to the different experimental groups, to have a similar weight average and standard deviation. Euthanasia was performed by cervical dislocation or CO2. Housing conditions and all experimental animal procedures were approved by the Animal Ethics Committee of the KU Leuven.
  • CD8+ T cells were prepared as follows: spleen and lymph nodes (4 superficial cervicals, 2 axillary and 2 branchial, 2 inguinal and 2 lumbar) were isolated from OT- l:Rosa26-Cas9 mice. The organs were then mechanically dissociated in a 70 pm Cell Strainer. Red blood cells were lysed in Red Blood Cell Lysing Buffer (Sigma-Aldrich), incubated for 2 minutes at 37°C, washed, and filtered through a 40 pm Cell Strainer. CD8+ T cells were isolated using the mouse CD8+ T cells Isolation Kit (MojoSort) according to the manufacturer's guidelines.
  • T cell medium RPMI 1640 (Thermofisher), 10% of Fetal Bovine Serum (FBS, Gibco), 1% Penicillin-Streptomycin (Pen/Strep Gibco), 0.1% 2-Mercaptoethanol (Gibco), 1% Non-Essential Amino Acids Solution (NEAA, Gibco) and 1% Sodium Pyruvate (Gibco) with 1:1 ratio of Mouse T-Activator CD3/CD28 Dynabeads (Thermofisher Scientific).
  • activated CD8+ T cells were expanded in T cell medium supplemented only with lOng/ml mlL-2, 5ng/ml mlL-7, 5ng/ml mlL-15 (all from Prepotech), and used for lentiviral transduction.
  • OT-I T cells were prepared as follows: spleen and lymph nodes were isolated from OT- I mice and processed as described above. Total splenocytes and lymphocytes were resuspended in T cells medium added with l pg/ml OVA257-264 peptide in the presence of lOng/ml mlL-2, 5ng/ml mlL-7, 5ng/ml mlL-15 for 3 days, then used for nucleofection.
  • CD8+ T cells were transduced with the vector of choice, on day 2 post-isolation, by adding to the medium lOpg/mL of Protamine Sulfate (Sigma-Aldrich), lOng/ml mlL-2, 5ng/ml mlL-7, 5ng/ml mlL15 and the proper volume of concentrated lentivirus to have a Multiplicity of infection (MOI) of 80-100.
  • the cells were then expanded for 5 days in T cells medium supplemented with lOng/ml mlL-2, 5ng/ml mlL-7, 5ng/ml mlL-15 to provide time for sgRNA expression. Transduction with the libraries was performed on a total of > 2xl0 7 Cas9 OT-I T cells to achieve an initial library coverage of > 2000x.
  • CD90.1+ OT-I T cells were selected via magnetic positive selection using CD90.1 MicroBeads (Miltenyi Biotech) and LS magnetic columns (Miltenyi Biotech), according to the manufacturer's instructions.
  • CD90.1 isolation was performed 4 days post-transduction, right before Adoptive T cell transfer (ACT). An aliquot of the cells was taken before and after the isolation for FACS analysis to determine the efficiency of transduction and the purity of the isolated CD90.1+ OTI T cell population.
  • the validation of the screening was performed with the use of electroporation (Schumann et. al. 2015, Proc Natl Acad Sci USA 112:10437-10442). Splenocytes and lymphocytes were isolated, activated, and cultured as described above. The nucleofection was performed 3 days after OT-I T T cells isolation.
  • Alt-R CRISPR-Cas9 RNA (Alt-R crRNA, IDT) of choice and the Alt-R trans-activating crRNA (Alt-R tracRNA, IDT) were mixed in equimolar concentrations to have a final duplex concentration of 50 pM and the annealing was performed as follows: 95°C 5min; 90°C 2min; 85°C 2min; 80°C 2min; 75°C 2min; 70°C 2min; 65°C 2min; 60°C 2min; 55°C 2min; 50°C 2min; 45°C 2min; 40°C 2min; 35°C 2min; 30°C 2min; 25°C inf.
  • RNP complexes were then generated by incubating duplex RNA with the Cas9 enzyme in a 3:1 ratio at RT for 20 minutes.
  • OT-I T cells were harvested, washed twice in PBS, and resuspended at a concentration of 1x10 s /ml in P4 Nucleofector solution (P4 Primary Cell 4DNucleofector X kit L, Lonza).
  • lxlO 7 OT-I T cells were then incubated with the RNP complex RT for 2 minutes, transferred to the cuvette (P4 Primary Cell 4D-Nucleofector X kit L, Lonza), and electroporated with the program CM137 on a 4D- Nucleofector System (Lonza).
  • the cells were then collected and maintained in culture at a concentration of 0.5-2xl0 s T cell medium added with lOng/ml mlL-2, 5ng/ml mlL-7, 5ng/ml mlL-15 for the next 3 days, when they were used for ACT or in vitro assays.
  • KPC_OVA murine cells were detached with 0,25% Trypsin-EDTA (Gibco), harvested in PBS (Gibco), and counted. 1x10 s KPC_OVA cells were resuspended in 20pl and injected orthotopically in the pancreas head of recipient Rosa26-Cas9 mice. ACT was performed with 2x10 s CD90.1+ OT-I T cells 5 days post KPC_OVA injection. For the in vivo metabolic screen an aliquot of CD90.1+ T cells was pelleted and frozen at -20°C for NGS analysis (TO).
  • TO NGS analysis
  • mice were sacrificed 7 days after ACT and spleen, draining and non-draining lymph nodes, primary tumor, liver, lungs, and peritoneal metastasis were collected and processed for sorting of OT-I T cells. Samples collected were sequenced in 3 independent runs and pooled in the following analysis. For the CROP-seq, only the primary tumor was collected and processed for CD90.1+ OT-I T cells sorting.
  • KPC_OVA were detached as previously described. 4xl0 4 cells were resuspended in 20 pl of PBS and injected orthotopically in the pancreas head of recipient mice. ACT of 5xl0 6 engineered OT-I T cells was performed 7 days post KPC_OVA injection. Mice were sacrificed 7 days post ACT and relevant organs were collected and processed for FACS analysis.
  • ICB treatment aPD-1 antibody (Biolegend) and the control Immunoglobulin G from rat serum (IgG, Sigma-Aldrich) were administered at a dosage of 10 mg/kg, through intraperitoneal injection. aPD-1 and IgG were diluted in PBS. The treatment was given from the day of ACT every 2 days in the screenings and from the day after ACT every 2 days for the target validation experiments.
  • Tumors were collected, weighed, and kept in ice-cold PBS. The tumor mass was then mechanically dissociated in digestion buffer (Minimum Essential Medium - Alpha (aMEM, Lonza) supplemented with 1% Pen/Strep, 50pM p-mercaptoethanol, 5% FBS, 5U/mL DNase I (Sigma-Aldrich), 0,85 mg/mL Collagenase V (Sigma-Aldrich), 1,25 mg/ml Collagenase D (Sigma-Aldrich) and 1 mg/mL Dispase (Gibco).
  • digestion buffer Minimum Essential Medium - Alpha (aMEM, Lonza) supplemented with 1% Pen/Strep, 50pM p-mercaptoethanol, 5% FBS, 5U/mL DNase I (Sigma-Aldrich), 0,85 mg/mL Collagenase V (Sigma-Aldrich), 1,25 mg/ml Collagenase D (S
  • Tumor pieces were collected into gentleMACS C tubes (Miltenyi Biotec) and dissociated by using first the h_cord_l program of an automatic tissue gentleMACS Dissociator (Miltenyi Biotec) and then incubated for 40 minutes at 37°C.
  • Peritoneal metastases were collected and mechanically dissociated in 5 ml of the same digestion buffer used for the primary tumor. The pieces were then collected into gentleMACS C tubes (Miltenyi Biotec) and dissociated by using the 37C_m_TDK_l program.
  • Lungs and livers were collected and dissociated with 10 ml of lung and liver Digestion Buffer (RPMI supplemented with 1% Pen/Strep, 5% FBS, 40U/mL DNase I (Sigma-Aldrich), 1 mg/mL Collagenase I (Sigma-Aldrich) and 2 mg/mL Dispase (Gibco) in C tubes (Miltenyi Biotec) using the 37C_m_LDK_l program. The digestion was then stopped with FACS buffer and the sample was filtered through a 70 pm cell strainer. Red blood cell lysis was performed by using Hybri-Max (Sigma-Aldrich, R7757).
  • the sample was then passed through a 40 pm cell strainer to have a single cell suspension. Spleens were recovered from mice and weighted. The dissociation into single cell suspension was performed as previously described. Single cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mmol/L EDTA) and incubated for 15 minutes with Mouse BD Fc Block purified anti-mouse CD16/CD32 (BD Pharmingen). Extracellular staining was performed for 30 minutes at 4°C.
  • FACS buffer PBS containing 2% FBS and 2 mmol/L EDTA
  • single-cell suspensions were resuspended in RPMI (10% FBS and 1% pen/strep) and stimulated with phorbol 12-myristate 13-acetate/ionomycin cell stimulation cocktail (Invitrogen, 1:500) in the presence of brefeldin A (BioLegend; 1:1,000) and monensin (Invitrogen; 1:1,000) for 4 h (37 °C). The cells were then washed in FACS buffer and stained with a viability dye and extracellular markers.
  • the cells were permeabilized by using the Foxp3/Transcription Factor Fixation/Permeabilization Kit (Invitrogen) according to the manufacturer's instructions and incubated overnight at 4°C with the specific intracellular antibodies.
  • purified Rabbit anti-mouse SREBP2 was incubated overnight at 4°C in Permeabilization buffer (Invitrogen), then the cells were washed and incubated for lh with a donkey anti- Rabbit-A488 and 7-AAD, for DNA staining.
  • the library used for the in vivo metabolic screening on CD8+ T cells was synthetized as previously described by Pinioti et. al. (2023, Cancer Immunol Res, 11:1611-1629).
  • the library used for in vivo CROP-seq made of 246 sgRNA targeting the 83 distilled candidate genes and 34 non-targeting sequences was synthesized and cloned into CROPseqGuide-Thyl.l.
  • Genomic DNA was isolated using DNeasy blood and tissue Kit (QIAGEN) following the manufacturer's guidelines. PCR of gDNA was performed to attach sequencing adaptors and barcode samples. For each sample, the gDNA was split into multiple 25 pl PCR reactions (total volume) containing a maximum of 1 pg gDNA. PCR mixture per reaction: 12,5 pL KAPA HIFI HOT START MIX (2X), 1 pl of P5 stagger primer mix (stock at 10 pM concentration), 1 pl of a uniquely barcoded P7 primer (stock at 10 pM concentration), adding mQ water and gDNA input (max 1 pg per reaction) to 25 pl.
  • PCR cycling conditions an initial 2 min at 98 °C; followed by 30 s at 98 °C, 30 s at 60 °C, 30 s at 72 °C, for 5 cycles + additional 20-25 cycles of 30 s at 98 °C, 30 s at 65 °C, 30 s at 72 °C, and a final 5 min extension at 72 °C.
  • P5 and P7 primers were synthesized at Integrated DNA Technologies (IDT).
  • IDTT Integrated DNA Technologies
  • PCR products were purified with Agencourt AMPure XP SPRI beads according to the manufacturer's instructions (Beckman Coulter). DNA concentrations were measured, and samples were equimolarly pooled and subjected to Illumina next-generation sequencing. Mapped read counts were subsequently used as input for the MAGeCK analysis software package (Li et al. 2014, Genome Biology 15: 554) and STARS (Doench et al. 2016, Nat Biotechnol 34:184-191).
  • Single cell libraries were prepared using the Chromium Next GEM Single Cell 3' _v3.1 kit (10X Genomics). Briefly, the single-cell suspensions were loaded onto the Chromium Controller according to their respective cell counts to generate 9,000 single-cell gel beads in emulsion (GEMs) per sample. Each sample was loaded into a separate channel. The complementary DNA content of each sample after cDNA amplification of 11 cycles was quantified and quality checked using a high-sensitivity DNA chip in a tapestation (Agilent). 25% of cDNA from the previous step was used for fragmentation, end repair and A- tailing followed by adaptor ligation and PCR indexing. After library quantification and quality checking by tapestation (Agilent), samples were diluted and loaded onto the NovaSeq (Illumina) to a sequencing depth of 500 million reads per sample (approximately 50,000 reads per cell).
  • OT-I T cells were isolated and activated in presence of 10 ng/ml mlL-2. From day 4 the cells were cultured in the presence of 5 ng/ml mlL-7 and 5 ng/ml mlL-15 to induce memory differentiation. On day 7 the cells were collected and stained for FACS to assess the expression of CD62L and CD44 as a readout of memory differentiation.
  • TCR signalling was assessed by immunoblotting of proximal and downstream proteins of the TCR.
  • naive OT-I T cells were isolated and treated with 5 pM EL0VL1 inhibitor (C3) or DMSO for 6 h and then stimulated with 2 pg/ml soluble aCD3 and 5 pg/ml aCD28 for the indicated time. T cells were then collected and Immunoblotting on whole-cell lysate was performed as previously described (Virga et al.2021, Sci Adv 7:eabf0466).
  • the following antibodies were used: rabbit anti-LCK (1:2000), rabbit anti- pLCK (1:2000), rabbit anti-ZAP70 (1:2000), rabbit anti-pZAP70 (1:2000), rabbit anti-ERKl/2 (1:2000), rabbit antipERKl/2 (1:2000), anti-loading control HRP (1:2000; Abeam, ab21058), mouse anti-vinculin (1:2000) and appropriate HRP-conjugated secondary antibodies (1:3000).
  • the signal was visualized by enhanced chemiluminescent reagents (ECL, Invitrogen) or West Femto (Thermo Scientific), according to the manufacturer's instructions, and images were acquired by a LAS-4000-CCD camera with ImageQuant software (GE Healthcare).
  • PBMCs Peripheral blood mononuclear cells
  • Isolated T cells were cultured for 24h in T cell medium with 1:1 ratio of Human T-Activator CD3/CD28 Dynabeads (Thermofisher Scientific). For the following 2 days activated CD8+ T cells were expanded in T cell medium supplemented with 20 ng/mL hlL-2. For Elovll inhibition, hCD8+ T cells were treated for 3 days with 5pM of EL0VL1 inhibitor (Medchem) and used for in vitro experiments.
  • EL0VL1 inhibitor Medchem
  • CD8+ T cells were isolated from C57/B6 mice or from human buffyeoat as described above. 2xl0 5 CD8+ T cells were then seeded in T cell medium with 1:1 ratio of Mouse or Human T-Activator CD3/CD28 Dynabeads (Thermofisher Scientific) on non-treated 48-well plates (corning) coated with RetroNectin (Takara Bio) in presence of 5pM EL0VL1 inhibitor (medchem) or DMSO as a control.
  • the multi-organ CRISPR/Cas9 screen of 2,078 genes involved in cellular metabolism was performed using a lentiviral library encoding 10,390 specific single guide RNA (sgRNAs) and 250 non-targeting control sgRNAs.
  • MAGeCK-VISPR version 0.5.3 was used to process CRISPR/Cas9 screen sequencing data.
  • MAGeCK 'count' module generated raw count table with sgRNA as rows and samples as columns. This table was normalized by 250 non-targeting control sgRNAs and corrected for batches effects between different organs by combat. Two samples were excluded after initial quality control.
  • MAGeCK 'mle' module calculated beta score for each targeted gene to measure positive or negative selection. STARS method was performed via pinapl-py.ucsd.edu/. Gene ontology (GO) was performed via geneontology.org/docs/go-enrichment-analysis/.
  • CROP-seq was performed on a lOx platform.
  • scRNA sequencing generated data in two separate libraries: Gene Expression library and CRISPR Guide Capture library.
  • Raw reads (.fastq format) from Gene Expression library were mapped to mouse genome (mmlO) by CellRanger (v3.1.0).
  • CellRanger feature barcoding analysis pipeline was applied to processes reads from CRISPR Guide Capture library. This pipeline searches reads against designed guide protospacer sequences and returns sgRNA assignment together with cell barcode using automatic number of UMI thresholds.
  • EXAMPLE 2 An in vivo CRISPR screen identifies metabolic genes regulating CD8+ T cell fitness in the tumor and metastatic organs.
  • This library was cloned into a lentiviral CRISPR vector that additionally contains a CD90.1 (Thyl.l) expression cassette to mark transduced T cells.
  • CD90.1 Thyl.l
  • To perform the screen we transduced OT-I Cas9 knock-in T cells with the lentiviral metabolic library and adoptively transferred them to KPC_OVA tumor-bearing mice. The same day we also started the treatment with aPD-1 or control antibody ( Figure la). Seven days post adoptive T cell transfer (ACT), we sacrificed the mice to sort sgRNA- transduced CD90.1 + OT-I T cells. Importantly, we observed no difference in tumor size of mice that received aPD-1 or control treatment.
  • the sgRNA representation in the sorted T cells was determined by high-throughput sequencing and data were analyzed with MAGeCK (Li et al. 2014, Genome Biology 15:554). This enabled us to identify metabolic targets enriched in the different niches under a specific treatment condition. Among them, we retrieved sgRNAs targeting metabolic genes known to sustain T cells fitness and antitumoral activity such as Dgkz (Riese et al. 2016, Front Cell Dev Biol 4:108; Jing et al. 2017, Cancer Research 77:5676-5686; Wichroski et al. 2023, Sci TransI Med 15:eadhl892). Pi3k family members (Aragoneses-Fenoll et al.
  • EXAMPLE 3 An in vivo single-cell CRISPR screen selects Elovll as a promising metabolic target to sustain CD8+ T cell activity.
  • the corresponding lentiviral library was transduced in OT-I T cells, which were used for adoptive cell transfer in mice bearing KPC_OVA pancreatic tumors and treated with aPD- 1 or an IgG control antibody (Figure 2a). Strikingly, we observed significantly smaller tumors in mice receiving OT-I T cells transduced with the CROP-seq metabolic library and treated with aPD-1, a first indication that, among the distilled 83 genes, we successfully enriched candidate genes whose inhibition synergizes with aPD-1.
  • T cells from aPD- 1-treated animals differed in phenotype from control-treated animals, with more T cells having an effector phenotype (cluster 0, 2 and 5) and fewer showing a precursor exhausted phenotype (cluster 4), in line with previous studies (Miller et al. 2019, Nat Immunol 20:326-336).
  • Elovll resulted enriched particularly in the liver of mice treated with aPD-1. thus highlighting the efficiency of our approach in identifying putative targets having a systemic relevance. Moreover, the role of Elovll in the antitumoral activity of CD8+ T cells is unknown.
  • EXAMPLE 4 Elovll deficient CD8+ T cells have increased antitumoral activity upon aPD-1 treatment.
  • sgElovIl OT-I T cells To assess the in vivo functionality of sgElovIl OT-I T cells, we analyzed the expression of effector and exhaustion markers. Compared to control OT-I T cells, sgElovIl cells were more polyfunctional, producing higher levels of IL-2, I FNy, and TNFa, in the presence of aPD-1 treatment ( Figure 3e, f). This improved effector function was associated with higher expression of co-inhibitory molecules such as PD-1 and TIM3, suggesting stronger activation (Figure 3g). Additionally, sgElovIl OT-I cells expressed higher Ki67, validating the increase in proliferative capacity suggested by CROP-seq (Figure 3h) .
  • E/ov/l-deficient CD8+ T cells are more functional and prone to memory differentiation.
  • Elovll inhibition induces a stronger TCR signaling, leading to heightened activation and increased proliferation, while at the same time priming T cells to differentiate toward a more memory-like phenotype.
  • EXAMPLE 6 Relevance of ELOVL1 function in human CD8+ T cells.
  • T cell-based therapies including T and CAR-T cell transfer, have great therapeutic potential but are still confined in their use.
  • CRISPR loss-of-function screens have been used to identify genes involved in memory/effector differentiation, driving T cell exhaustion, and enhancing CAR T cell fitness in the TME (Huang et al. 2021, Cell 184:1245-1261.e21; Wang et al. 2021, Cancer Discovery 11:1192-1211; Trefny et al. 2023, Nat Commun 14:86).
  • These studies mostly focused on models of melanoma, breast cancer, and glioblastoma, disregarding other cancer types and metastatic niches.
  • Immune-checkpoint inhibitors have revolutionized cancer therapy.
  • aPD-1 is used to reinvigorate T cell function in solid tumors, where constant antigen exposure and an unfavorable microenvironment induce T cell progression into exhausted states (Trefny et al. 2023, Nat Commun 14: 86; Dolina et al. 2021, Front Immunol 12:715234).
  • PD1 blockade also rewires T cell metabolism, inducing glycolysis to sustain fast proliferation and differentiation into short-lived effector CD8+ T cells (Patsoukis et al.2015, Nat Commun 6:6692).
  • T cell phenotypic states rely on distinct metabolic programs, the metabolic pressure imposed by specific TMEs (Reina-Campos et al. 2021, Nat Rev Immunol 21:718-738,) and concomitant therapies need to be considered when investigating ways to improve T cell fitness (Bacigalupa et al. 2024, Cell Metabolism 36:10-20). For these reasons, we performed the in vivo screens in the presence of aPD-1 therapy to identify possible synergism and achieve greater results.
  • Elovll is a particularly appealing target to favor proliferation, effector, and memory functions in synergy with aPD-1 treatment.
  • Elovll which was enriched in the liver in the initial screening, proved to be a highly efficient target to combat primary tumor and peritoneal metastasis, supporting the relevance of our multi-dimensional approach in identifying promising targets with enhanced systemic fitness.
  • Elovll has been widely studied in brain diseases such as adrenoleukodystrophy (ALD) and certain tumor types as an unfavorable prognostic marker (Ofman et al. 2010, EM BO Mol Med 2:90-97; Hama et al. 2021, Sci Rep 11:6163; Zhang et al. 2022, Front Oncol 12:884066).
  • ALD adrenoleukodystrophy
  • EM BO Mol Med 2:90-97 Hama et al. 2021, Sci Rep 11:6163; Zhang et al. 2022, Front Oncol 12:884066
  • pharmacological inhibition of EL0VL1 during CD8+ T cell priming mediated stronger TCR signaling, leading to enhanced activation and proliferation. Higher activation was accompanied by increased expression of activation markers.
  • Elovll-deficient CD8+ T cells due to their stronger activation, expressed higher levels of PD-1.
  • solid tumors are characterized by a high infiltration of immunosuppressive cells expressing checkpoint molecules such PD-L1/2 (Kaunitz et al. 2017, Lab Investig 97:1063-1071; Cha et al. 2019, Molecular Cell 76:359-370; Yi et al. 2021, J Hematol Oncol 14:10), we showed that Elovll- deficient CD8+ T cells particularly benefit from aPD-1 treatment and synergize with it to unleash their potentiated antitumoral activity, thus mediating tumor reduction.
  • ACT approaches including CAR T cell therapy, showed poor efficacy in solid tumors due to their limited persistence and fast differentiation into dysfunctional states. It has been proposed that the persistence of infused T cells is higher when cells retain memory-like phenotypes and can sustain proliferation in the harsh TME (Wang et al. 2021, Cancer Discovery 11:1192-1211; Lopez-Cantillo et al. 2022, Front Immunol 13:878209; Tang et al. 2020, JCI Insight 5:el33977; Kumar et al. 2021, J Immunother Cancer 9:e001688; Liu et al. 2022, Biomark Res 10:86).
  • Pretto et al. 2025 in addition extended the data as provided herein for PDAC to a second tumor type, i.e. melanoma (BF16 mouse model), see Figures 2 j-k and Figures 3 k-o) and mechanistically linked EL0VL1- deficiency in CD8+ T cells to rewiring of the lipid profile in the T cells, including an increase in cholesterol that is mediated by INSIGI degradation and SREBP2 activation ( Figure 4 and Extended Data Figure 4 in Pretto et al. 2025, by means of sgELOVLl). Not reported by Pretto et al.
  • a second tumor type i.e. melanoma (BF16 mouse model)
  • Figures 2 j-k and Figures 3 k-o mechanistically linked EL0VL1- deficiency in CD8+ T cells to rewiring of the lipid profile in the T cells, including an increase in cholesterol that is mediated by INSI

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Abstract

L'invention concerne la modulation de la fonction ou de l'expression de l'allongement de la protéine 1 d'acides gras à très longue chaîne (ELOVL1) et ses applications thérapeutiques. En particulier, des lymphocytes T CD8+ dépourvus ou sensiblement dépourvus de ELOVL1 fonctionnelle sont envisagés, et le transfert adoptif de tels lymphocytes T CD8+ est utile dans le traitement du cancer. En outre, l'expression de ELOVL1 dans les lymphocytes T CD8+ est un biomarqueur pour la réponse à une thérapie anticancéreuse comprenant un inhibiteur de point de contrôle immunitaire.
PCT/EP2025/062403 2024-05-07 2025-05-06 Applications thérapeutiques et diagnostiques d'allongement de la protéine 1 d'acides gras à très longue chaîne Pending WO2025233366A1 (fr)

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