[go: up one dir, main page]

WO2024068010A1 - Cibler snx9 sauve les lymphocytes t recombinés dans le cadre d'une thérapie adoptive - Google Patents

Cibler snx9 sauve les lymphocytes t recombinés dans le cadre d'une thérapie adoptive Download PDF

Info

Publication number
WO2024068010A1
WO2024068010A1 PCT/EP2022/077391 EP2022077391W WO2024068010A1 WO 2024068010 A1 WO2024068010 A1 WO 2024068010A1 EP 2022077391 W EP2022077391 W EP 2022077391W WO 2024068010 A1 WO2024068010 A1 WO 2024068010A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
snx9
cell
nucleic acid
acid agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2022/077391
Other languages
English (en)
Inventor
Marcel TREFNY
Alfred Zippelius
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaet Basel
Original Assignee
Universitaet Basel
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universitaet Basel filed Critical Universitaet Basel
Priority to PCT/EP2022/077391 priority Critical patent/WO2024068010A1/fr
Publication of WO2024068010A1 publication Critical patent/WO2024068010A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4267—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K40/4269—NY-ESO
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • 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
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to recombinant T cell therapy, particularly the field of recombinant Chimeric Antigen Receptor (CAR) T cells and other adoptive T cell therapies.
  • the invention provides agents and methods to facilitate preventing recombinant T cells from being exhausted.
  • Tumor-specific CD8 T cells regularly enter a state of exhaustion due to chronic antigen stimulation within the tumor microenvironment. T cell exhaustion is characterized by impaired production of effector cytokines such as IFNy and high expression of inhibitory receptors such as PD-1 and TIM-3.
  • effector cytokines such as IFNy
  • inhibitory receptors such as PD-1 and TIM-3.
  • the pivotal role of tumor-specific CD8 T cells in anti-tumor immunity has fueled the development of therapeutic strategies that prevent or revert tumor-associated T cell exhaustion.
  • Antibodies targeting inhibitory receptors such as PD-1 and CTLA4 are now considered to be among the most critical advances in the field of oncology in the last decades, given their outstanding clinical success.
  • TILs tumor-infiltrating lymphocytes
  • CAR T cells genetically engineered T cells
  • the inventors developed a human ex vivo exhaustion model to generate tumor antigen-specific exhausted T cells that resemble patient-derived T cells on a phenotypic and transcriptional level.
  • the inventors performed a targeted pooled CRISPR-Cas9 screen with this model and discovered that a knockout of sorting nexin-9 (SNX9) improved T cell effector functions and memory differentiation, translating into enhanced anti-tumor efficacy of adoptively-transferred T cells including CAR T cells in vivo.
  • SNX9 amplifies TCR/CD28-mediated activation through PLCyl , Ca 2+ , and NFATc2 and this correlates with higher expression of NR4A1/3 and TOX.
  • the inventors thereby identify a therapeutic strategy to improve T cell-based immunotherapies by limiting excessive stimulatory signals and thereby alleviate T cell exhaustion.
  • the objective of the present invention is to provide means and methods to prevent or inhibit exhaustion in T cell products used in treatment of disease.
  • This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.
  • a first aspect of the invention relates to a nucleic acid agent capable of downregulating or inhibiting the expression or biological activity of SNX9 in a target cell, wherein the nucleic acid agent is selected from an antisense oligodeoxynucleotide, an siRNA, a miRNA and a shRNA.
  • a second aspect of the invention relates to a nucleic acid vector capable of expressing the nucleic acid agent in a target cell, particularly in a transgenic T cell.
  • Another aspect of the invention relates to a preparation of T cells with suppressed, inhibited or abrogated SNX9 expression.
  • any of the above aspects are provided for use in treatment of a condition characterized by or associated with exhaustion of T cell function, particularly cancer immunotherapy, more particularly in the context of cancer immunotherapy that benefits from the prevention of T cell exhaustion.
  • references to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”
  • Chimeric antigen receptor also known as chimeric immunoreceptor, chimeric T cell receptor or artificial T cell receptor
  • CAR Chimeric antigen receptor
  • the receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor.
  • Recombinant CAR structurally consist of an extracellular antigen binding domain, a transmembrane domain and an intracellular domain.
  • preparation of T cells in the context of the present specification relates to an ex-vivo generated plurality of T cells isolated from a patient.
  • the preparation may be isolated from blood, tumour tissue, lymph nodes or other sources of T cells commonly used.
  • the preparation of T cells may contain other blood cells, immune cells.
  • the preparation substantially consists of CD3 positive T cells.
  • sequences similar or homologous are also part of the invention.
  • sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.
  • substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to the complement of the strand.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position.
  • Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. AppL Math. 2:482 (1981 ), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci.
  • sequence identity values refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.
  • gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
  • ORF open reading frame
  • a polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.
  • transgene in the context of the present specification relates to a gene or genetic material that has been transferred from one organism to another.
  • the term may also refer to transfer of the natural or physiologically intact variant of a genetic sequence into tissue of a patient where it is missing. It may further refer to transfer of a natural encoded sequence the expression of which is driven by a promoter absent or silenced in the targeted tissue.
  • a recombinant in the context of the present specification relates to a nucleic acid, which is the product of one or several steps of cloning, restriction and/or ligation and which is different from the naturally occurring nucleic acid.
  • a recombinant virus particle comprises a recombinant nucleic acid.
  • gene expression or expression may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products.
  • the term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.
  • nucleotides in the context of the present specification relates to nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with RNA or DNA oligomers on the basis of base pairing.
  • nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymine), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine.
  • nucleic acids such as phosphothioates, 2’0-methylphosphothioates, peptide nucleic acids (PNA; N-(2- aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alphacarbon of the glycine) or locked nucleic acids (LNA; 2’0, 4’C methylene bridged RNA building blocks).
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.
  • phosphothioate as used herein is synonymous with the terms phosphorothioate and thiophosphate.
  • hybridizing sequences capable of forming a hybrid or hybridizing sequence in the context of the present specification relate to sequences that under the conditions existing within the cytosol of a mammalian cell, are able to bind selectively to their target sequence.
  • Such hybridizing sequences may be contiguously reverse-complimentary to the target sequence, or may comprise gaps, mismatches or additional non-matching nucleotides.
  • the minimal length for a sequence to be capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the energy of binding than A or T/U nucleotides, and on the backbone chemistry.
  • hybridizing sequence encompasses a polynucleotide sequence comprising or essentially consisting of RNA (ribonucleotides), DNA (deoxyribonucleotides), phosphothioate deoxyribonucleotides, 2’-O-methyl-modified phosphothioate ribonucleotides, LNA and/or PNA nucleotide analogues.
  • a hybridizing sequence according to the invention comprises 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
  • the hybridizing sequence is at least 80% identical, more preferred 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reverse complimentary sequence of the mRNA encoding SNX9 (GenBank code NM_016224.5).
  • the hybridizing sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and/or PNA nucleotides or mixtures thereof.
  • antisense oligonucleotide in the context of the present specification relates to an oligonucleotide having a sequence substantially complimentary to, and capable of hybridizing to, an RNA, here specifically the mRNA encoding SNX9. Antisense action on such RNA will lead to modulation, particular inhibition or suppression of the RNAs biological effect. For an mRNA, expression of the resulting gene product is inhibited or suppressed.
  • Antisense oligonucleotides can consist of DNA, RNA, nucleotide analogues and/or mixtures thereof. The skilled person is aware of a variety of commercial and non-commercial sources for computation of a theoretically optimal antisense sequence to a given target.
  • optimization can be performed both in terms of nucleobase sequence and in terms of backbone (ribo, deoxyribo, analogue) composition.
  • backbone ribo, deoxyribo, analogue
  • gapmer refers to a short DNA antisense oligonucleotide structure with RNA-like segments on both sides of the sequence, which are typically composed of locked nucleic acids (LNA), 2'-0Me, or 2'-F modified bases.
  • Gapmers often comprise nucleotides modified with phosphorothioate (PS) groups, particularly in their 5’ and 3’ terminal regions.
  • PS phosphorothioate
  • Gapmers are designed to hybridize to a target piece of RNA and silence the gene through the induction of RNase H cleavage. Binding of the gapmer to the target has a higher affinity due to the modified RNA flanking regions, as well as resistance to degradation by certain nucleases. Gapmers are being developed as therapeutics for a variety of cancers, viruses, and other chronic genetic disorders.
  • siRNA small/short interfering RNA
  • siRNA in the context of the present specification relates to an RNA molecule capable of interfering with the expression (in other words: inhibiting or preventing the expression) of a gene comprising a nucleic acid sequence complementary or hybridizing to the sequence of the siRNA in a process termed RNA interference.
  • the term siRNA is meant to encompass both single stranded siRNA and double stranded siRNA.
  • siRNA is usually characterized by a length of 17-24 nucleotides. Double stranded siRNA can be derived from longer double stranded RNA molecules (dsRNA).
  • RNA interference often works via binding of an siRNA molecule to the mRNA molecule having a complementary sequence, resulting in degradation of the mRNA. RNA interference is also possible by binding of an siRNA molecule to an intronic sequence of a pre-mRNA (an immature, non-spliced mRNA) within the nucleus of a cell, resulting in degradation of the pre-mRNA.
  • shRNA small hairpin RNA
  • RNAi RNA interference
  • sgRNA single guide RNA
  • CRISPR clustered regularly interspaced short palindromic repeats
  • miRNA in the context of the present specification relates to a small noncoding RNA molecule (containing about 22 nucleotides) that functions in RNA silencing and post-transcriptional regulation of gene expression.
  • siRNAs shRNAs, miRNAs, sgRNA and miRNAs are known to a practitioner skilled in the art.
  • siDirect siRNA WizardTM i- Score Designer (InvivoGen), PFRED, Silencei® Select siRNA (ThermoFischer Scientific), siMAX siRNA Design Tool (eurofins Genomics) and OriGene for the design of siRNA, DharmaconTM Gene Knockdown, for designing siRNAs and shRNAs, DharmaconTM Gene editing for the design of sgRNAs, WMD3 and AmiRNA Designer for the design of miRNAs, GenCRISPR gRNA Design Tool, SciTools® Web Tools and EnGen sgRNA Template (New England Biolabs) for sgRNA design.
  • nucleic acid expression vector in the context of the present specification relates to a plasmid, a viral genome or an RNA, which is used to transfect (in case of a plasmid or an RNA) or transduce (in case of a viral genome) a target cell with a certain gene of interest, or -in the case of an RNA construct being transfected- to translate the corresponding protein of interest from a transfected mRNA.
  • the gene of interest is under control of a promoter sequence and the promoter sequence is operational inside the target cell, thus, the gene of interest is transcribed either constitutively or in response to a stimulus or dependent on the cell’s status.
  • the viral genome is packaged into a capsid to become a viral vector, which is able to transduce the target cell.
  • treating or treatment of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof).
  • treating or treatment refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • treating or treatment refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
  • a first aspect of the invention relates to a nucleic acid agent capable of downregulating or inhibiting the expression or biological activity of SNX9 in a target cell.
  • SNX9 is the abbreviation for a gene encoding the protein known as Sortin nexin-9, identified by Entrez: 51429 and Uniprot: Q9Y5X1. To the inventors’ knowledge, the data presented herein for the first time demonstrate the utility of inhibiting SNX9 expression to improve a medical condition.
  • a related aspect of the invention provides a nucleic acid agent capable of down regulating or inhibiting the expression or biological activity of SNX9 in a target cell, as set forth in detail below, or an expression vector, for medical use.
  • nucleic acid agent capable of downregulating or inhibiting the expression or biological activity of SNX9 for use in cancer, particularly in the context of adoptive T cell therapy.
  • One application is the use of the nucleic acid agents suppressing SNX9 in the context of chimeric antigen receptor (CAR) T cell treatments, particularly CAR T cells that make use of chimeric antigen receptors using the CD28 intracellular domain as a signalling I signal relay moiety.
  • CAR chimeric antigen receptor
  • the target cell is a T cell.
  • SNX9 is expressed and has a biological role in many if not all human cells, systemic administration at present seems to present a significant challenge.
  • the nucleic acid agent of the invention, or an expression vector encoding same is specifically provided to T cells isolated from a patient or prepared to be reinfused into a patient.
  • the nucleotide agent of the invention is administered as part of a T cell treatment.
  • the target cell is a CD28-positive T cell. While the inventors have observed CD28-independent effects of SNX9 knock-out at high CD3stimulation strengths, and without wanting to be bound by hypothesis, their current understanding indicates that exhausted CD28-signalling cells profit particularly from SNX9 inhibition. This applies both to physiological CD28 and to CAR constructs using the CD28 intracellular domain as a signal relay together with an extracellular sensing domain that is not CD28.
  • the target cell is a recombinant immune effector cell.
  • the target cell is a CD28-positive T cell, expressing a transgene encoding a transmembrane polypeptide.
  • this transmembrane polypeptide is a T cell receptor polypeptide.
  • this transmembrane polypeptide is a T cell receptor polypeptide capable of recognizing a specific cancer-related antigen in the context of an MHC molecule.
  • this transmembrane polypeptide is a chimeric antigen receptor (CAR) comprising an extracellular domain capable of specifically binding to (targeting) a target, and an intracellular domain.
  • CAR chimeric antigen receptor
  • the transmembrane polypeptide is a CAR targeting CD19.
  • the transmembrane polypeptide is a CAR targeting 13- cell maturation antigen (BCMA). In other more particular embodiment, the transmembrane polypeptide is a CAR targeting CD20.
  • the transmembrane polypeptide is a CAR targeting mesothelin.
  • the transmembrane polypeptide is a CAR targeting PD- 1.
  • the transmembrane polypeptide is a CAR targeting PD-
  • the transmembrane polypeptide is a CAR targeting Her-
  • the transmembrane polypeptide is a CAR targeting CD22.
  • the transmembrane polypeptide is a CAR targeting EFGR.
  • the transmembrane polypeptide is a CAR targeting MUC1.
  • the transmembrane polypeptide is a CAR targeting a human leutocyte antigen (HLA) molecule.
  • HLA human leutocyte antigen
  • the transmembrane polypeptide is a CAR targeting CD30.
  • the transmembrane polypeptide is a CAR targeting CD33.
  • the transmembrane polypeptide is a CAR targeting CD123.
  • the transmembrane polypeptide is a CAR targeting epidermal growth factor receptor variant III (EGFRvlll).
  • the transmembrane polypeptide is a CAR targeting Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1).
  • the transmembrane polypeptide is a CAR targeting carcinoembryonic antigen (CEA).
  • CEA carcinoembryonic antigen
  • the nucleic acid agent according to the invention is capable of hybridizing to an mRNA encoding SNX9.
  • An SNX9 mRNA sequence is accessible in GenBank at NM_016224.5. (SEQ ID NO 001 ).
  • the nucleic acid agent according to the invention is capable of hybridizing to an mRNA encoding SNX9 and comprises, particularly is, a sequence selected from SEQ ID NO 15 to SEQ ID NO 16316.
  • Current bioinformatic tools available to the skilled person identify these sequences as suitable candidates for antisense targeting of SEQ ID NO 001.
  • the nucleic acid agent is an antisense oligodeoxynucleotide.
  • Antisense technology has been developed for decades and a host of delivery technologies and chemistries to improve stability of the oligonucleotide exist.
  • a particularly useful modification encompassed by the present invention is the use of modified phosphodiester linkages, such as phosphothioates, which are more resistant to nuclease degradation than physiological phosphodiester-linked oligonucleotides.
  • modifications include locked nucleic acids and peptide nucleic acids, which increase stability of the double helical hybrid formed between the oligonucleotide and its target sequence.
  • the nucleic acid agent is an oligonucleotide having a sequence that hybridizes to SEQ ID NO 01 , and is selected from the sequences of SEQ ID NO 15 to SEQ ID NO 16316.
  • the nucleic acid agent is an oligonucleotide having a sequence that hybridizes to SEQ ID NO 01 , is selected from the sequences of SEQ ID NO 15 to SEQ ID NO 16316, and comprises on each of the 5'and 3' end of the oligonucleotide at least one, particularly two, more particularly three phosphothiate bonds.
  • the nucleic acid agent is an siRNA.
  • the nucleic acid agent is a siRNA described by a sequence selected from SEQ ID NO 15 to SEQ ID NO 16222. Current bioinformatic tools available to the skilled person identify these sequences as suitable candidates for siRNA targeting of SEQ ID NO 001 .
  • the nucleic acid agent is a miRNA.
  • the nucleic acid agent is shRNA.
  • the nucleic acid agent is an shRNA described by a sequence selected from SEQ ID NO 16223 to SEQ ID NO 16316. Current bioinformatic tools available to the skilled person identify these sequences as suitable candidates for shRNA targeting of SEQ ID NO 001 .
  • the invention further provides a polynucleotide vector encoding a nucleic acid agent capable of down regulating or inhibiting the expression or biological activity of SNX9 by interaction of the nucleic acid agent with an mRNA encoding SNX9.
  • the polynucleotide vector according to the invention encodes an RNA agent capable of hybridization to SNX9 that comprises or consists of a sequence selected from SEQ ID NO 15 to SEQ ID NO 16316.
  • the polynucleotide vector according to the invention encodes an siRNA described by a sequence selected from SEQ ID NO 15 to SEQ ID NO 16222.
  • the polynucleotide vector according to the invention encodes an shRNA described by a sequence selected from SEQ ID NO 16223 to SEQ ID NO 16316.
  • the polynucleotide vector as specified in the preceding paragraphs further encodes an immune receptor molecule selected from a chimeric antigen receptor polypeptide and a transgenic T cell receptor.
  • the invention further provides a recombinant immune cell, particularly a recombinant T cell, comprising the polynucleotide vector according to the previously described aspect of the invention.
  • the recombinant T cell expresses CD28.
  • the recombinant T cell expresses the intracellular domain of CD28.
  • the recombinant T cell is a CAR T cell and expresses the intracellular domain of CD28 comprised in a chimeric antigen receptor polypeptide.
  • the invention further encompasses a preparation of T cells, in which the expression of SNX9 is inhibited or abrogated.
  • a preparation of T cells according to the invention may be constituted of >70% of T cells in which the expression of SNX9 is lower than 50% of a control population of T cells, in which expression of SNX9 has not been targeted.
  • the T cells are characterized by a deletion of SNX9. In certain particular embodiments, >70% of the cells of the population are characterized by a deletion of SNX9.
  • the T cells are CAR T cells having a CAR using the intracellular domain of CD28.
  • the T cells are CAR T cells having a CAR using the intracellular domain of CD28 and their chimeric antigen receptor targets an antigen selected from the group consisting of CD19; BCMA; CD20; mesothelin; PD-1 ; PD-L1 ; Her-2; CD-22; EFGR; MUC1 ; an HLA molecule; CD-30; CD33; CD123, EFGRvlll; ROR1 ; CEA.
  • the T cells comprise a nucleic acid agent according to the first aspect of the invention.
  • the cells comprise an siRNA.
  • the cells comprise an shRNA.
  • the T cells comprise the polynucleotide vector according to the above-described aspect of the invention or any of the embodiments specified for the vector.
  • the T cells are CD28-positive. These may be for example T cell populations collected from the patient into whom they are to be retransferred after stimulation and I or transgene transfer.
  • a method or treating cancer in a patient in need thereof comprising administering to the patient a T cell product, particularly a preparation of T cells, according to the above description.
  • a nucleic acid agent or expression vector according to the invention is provided as a pharmaceutical composition.
  • This pharmaceutical composition is provided for use in preparing a T cell product or preparation of T cells for therapeutic administration to a patient in need thereof.
  • a cell therapy product as described herein is provided for use in the prevention or treatment of an immune dysfunction.
  • the immune dysfunction is T cell exhaustion of a preparation of adoptively transferred T cells, particularly in the treatment of cancer.
  • the invention further encompasses a kit comprising a composition comprising a nucleic acid agent or expression vector according to the invention; means for isolating a T cell preparation, particularly magnetic beads having T cell specific antibodies coated thereon, and optionally, buffers for the washing and recovery of the T cell preparation.
  • Certain embodiments of the cell preparations according to the invention relate to a dosage form for parenteral administration, such as subcutaneous, and particularly, intravenous injection forms.
  • a pharmaceutically acceptable carrier and/or excipient may be present.
  • the invention further encompasses, as an additional aspect, the use of a T cell preparation as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of a condition associated with immune dysfunction, in particular, an immune dysfunction characterized by T cell exhaustion.
  • the T cell preparation is a preparation of adoptively transferred T cells.
  • the condition is cancer.
  • Fig. 1 shows repetitive antigen-specific stimulation ex vivo of T cells results in exhaustion
  • (a) Scheme representing the principle of the exhaustion model T2 tumor cells are shown in brown with peptide-loaded MHC molecules on their surface. After lentiviral transduction with the NY-ESO-1 construct, cells were split into different conditions. Trest were only expanded in medium with IL2, and T tU mor were stimulated four times with unloaded T2 tumor cells (no TCR triggering). T e ff were stimulated once (3 days before the readout) and T ex four times with T2 tumor cells loaded with NY-ESO-1 - 9V peptide (every three days). Yellow dots represent cytokines such as IFNy.
  • Color represents the mean log fold change and the size the -Iog10 false discovery rate (FDR), (c-d; f-g) 1-way ANOVA statistics with Holm-Sidak correction for multiple comparison, Mean and SD are shown. Dots represent individual healthy donor biological replicates of >3 experiments. * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001 , **** p ⁇ 0.0001 .
  • Fig. 2 shows targeted CRISPR-Cas9 screen reveals SNX9 as a driver of T cell exhaustion
  • ZAP70, LAMP1 , and ZAP70 are significant (p ⁇ 0.01 ) in a 1-way ANOVA with Dunnett’s correction compared to intergenic controls.
  • Fig. 3 shows SNX9 KO improves effector functions of exhausted T cells and dampens the NFAT-NR4A1/3-TOX axis
  • Cells were electroporated with Cas9-crRNA- tracrRNA (Cas9-RNP) complexes after transduction with the NY-ESO-1 TCR.
  • TCR+ cells were then stimulated once with T2 tumor cells and NY-ESO-1 peptide (T e ff) or repetitively stimulated to generate T ex .
  • Statistics is a paired 2-way ANOVA with Holm-Sidak correction, (k) Geometric mean of phospho-PLCy1-Tyr783 fluorescence intensity measured by flow cytometry after subtraction of unstimulated background intensity.
  • Fig. 4 shows Snx9 KO improves anti-tumor efficacy and reduces terminal exhaustion in vivo
  • Size indicates the -Iog10 adjusted p-value and the color the mean Iog2 fold change
  • Healthy donor human CD8 T cells are stimulated ex vivo and lentivirally transduced with an anti-human- CD19(FMC63vH)-CD28-CD3zeta-T2A-copGFP CAR construct and electroporated with Cas9-crRNA-tracrRNA complexes to generate SNX9 KO cells and intergenic controls. These cells are then transferred to NSG mice with subcutaneous Raji tumors (CD19+). (b) Tumor volume in mm 3 of NSG mice treated 3 days post Raji tumor injection by i.v. transfer of 0.5 Mio human CD8 anti-CD19-28z CAR T cells with or without SNX9 KO.
  • Fig. 6 shows (a) representative scheme of the transduction and stimulation procedure underlying the ex vivo T cell exhaustion model. Days are indicated as circles with different treatments in different colors and shadings, (b) Expression of TIM-3 on cells stimulated in the indicated conditions 12 days after the first stimulation. 1 way ANOVA with Holm-Sidak correction, (c) Titration of NY-ESO-1 9V peptide dose at the four repetitive stimulations during the T ex culture showing the percentage of PD- 1 + TIM-3+ LAG-3+ cells (left), degranulation capacity measured by CD107a exposure over 4h (middle) and intracellular IFNy production (right) in response to peptide-loaded T2s.
  • Statistics are paired 1-way ANOVA with comparisons between T res t and all other conditions with Dunnett’s correction for multiple testing,
  • (d) Representative plot of PD-1 expression and geometric mean signal of PD-1 after 13 days of culture in the indicated conditions followed by 6 days of resting in fresh medium and IL-2.
  • N 6
  • An arrow indicates the OCR specific to exhaustion, Treg and Tfh specific at approx. +7 kbp of the TSS.
  • CD8 T cells extracted from the published human melanoma TIL scRNAseq data set by Sade-Feldman et al. are shown in a TSNE plot. Expression of SNX9, TCF7, and TOX are indicated by the size and color of the dots,
  • Fig. 8 shows (a) Example flow cytometry histogram and quantification of donor replicates of SNX9 expression in T ex with or without Cas9-RNP electroporation at the beginning of the procedure. Statistics is paired two-sided paired t-test. (b) Westernblot for protein expression of SNX9 and ERK2 as a loading control in Teff of four donors with or without SNX9 KO. The same antibody was used as for the flow cytometry experiments, (c) Fold cell expansion for T e ff (left) and T ex (right) from the first day of stimulation. Data are shown on a Iog2 scale. Donor replicates are shown as connected dots for intergenic and SNX9 KO conditions.
  • T e ff On the right T e ff are shown with electroporated CD28-EGFP or TCR-EGFP, or antibody-based staining of anti-CD45 and LFA1. “En face” images are 0.25 pm sections of the synapse region looking into the direction of the T cell from a 3D representation rendered in Imaris 9.
  • g Calcium flux peak normalized to baseline.
  • Fig. 9 shows (a) Example flow cytometry histogram of Snx9 staining in murine OTI cells for the indicated conditions and quantification thereof for Snx9 KO and intergenic for six experimental replicates. Statistics is a paired two-sided t-test. (b) Survival curve and of C57BL/6 mice with MC38-OVA tumors with adoptive transfer of OTI T cells with or without Snx9 KO at day 13 post tumor injection.
  • n 6 mice per condition and statistics are Bonferroni-adjusted Mantel-Cox log-rank tests,
  • Statistic is a 2-way ANOVA.
  • Fig. 10 shows (a - e) Data from the scRNAseq dataset of OTI cells 13 days post transfer in MC38-OVA tumors, (a) Heatmap showing row-scaled expression of the indicated marker genes defined by FindMarkers in Seurat for the clusters shown in columns. Yellow indicates high expression, magenta low expression. Cells were downsampled before plotting, (b) Average expression as color and percentage expression as size of the indicated transcription factors and nuclear genes with known involvement in T cell differentiation for each cluster, (c) Average expression as color and detection as size for the indicated gene sets for each cluster, (d) Differentially expressed genes between Snx9 KO and intergenic OTI cells for the indicated clusters: Tex-term, Tern and Tex-proflif.
  • Log2 fold change is indicated as x-axis, color and size of the dots, while the y-axis represents to -log 10 adjusted p- value.
  • Fig. 11 shows (a) Tumor volumes in mm 3 for subcutaneous Raji tumors in NSG mice either untreated or with 1.5 Mio anti-CD19-28z CARs with or without SNX9 KO. Statistics are individually performed 2-way ANOV As with Bonferroni correction, (b) Survival of these mice (humane endpoints) with Bonferroni-adjusted Mantel log-rank tests, (c) Legendplex-based measurement of the indicated human proteins in sera of Raji- bearing NSG mice with the indicated CAR treatments. The limit of detection (LOD) is indicated for IL2.
  • Fig. 12 shows graphical summary of the key findings.
  • Top panel shows the generation of the human ex vivo model for T cell exhaustion and the pooled targeted CRISPR- Cas9 screen.
  • the lower left panel shows the proposed mechanism how SNX9 amplifies TCR/CD28 signaling towards PLCyl , Ca 2+ , NFATc2, NR4A1/3, and TOX.
  • the lower right panel shows the effects of SNX9 KO in vivo.
  • Example 1 Ex vivo repetitive antigen-specific stimulation of T cells results in exhaustion
  • the inventors transduced human healthy donor peripheral CD8 T cells with a lentiviral construct encoding a TCR specific for the cancer-testis antigen NY-ESO-1.
  • the inventors compared T ex to T cells that only received medium and IL-2 (Trest) and to T cells that were co-cultured with T2 tumor cells without peptides (Ttumor).
  • the inventors also included a condition that is only stimulated once three days before the readout to mimic activated effector T cells (T e ff, Fig. 6a).
  • T e ff and T ex had increased expression of the inhibitory receptors PD-1 and TIM-3 compared to T res tand Ttumor controls (Fig. 1 b-c, Fig. 6b).
  • T ex showed a decreased capacity to degranulate (CD107a exposure upon restimulation), produce the inflammatory cytokines IFNy and TNFa, and kill tumor cells (Fig. 1 d-g).
  • the co-expression of PD-1 , TIM3, and LAG3 and the impairment in degranulation correlated with the peptide concentration used for the repetitive stimulation (Fig. 6c).
  • IFNy production was impaired even at the lowest concentration of NY-ESO-1 peptide.
  • T ex maintained increased PD-1 expression and impaired degranulation capacity after seven days of resting without further stimulation (Fig. 6d- e).
  • a therapeutic anti-PD1 antibody could improve the killing capacity and IFNy secretion of both T e ff and T ex in a co-culture assay with PD-L1 expressing tumor cells, however, T ex functionality remained impaired (Fig. 1 h, Fig. 6f). Loss of proliferative capacity has been described as another hallmark of exhaustion. T ex highly expanded during repetitive stimulation, however, their potential to proliferate in response to stimulation was reduced compared to controls (Fig. 6g-h).
  • T ex generated using a melanoma cell line for stimulation (NA8-Mel, HLA-A2+, loaded with NY-ESO-1 peptide) showed a similar exhausted phenotype (Fig. 6i).
  • Intratumoral T cells are characterized by a distinct transcriptional pattern compared to their functional counterparts. The inventors therefore investigated transcriptional changes among the different conditions by bulk mRNA sequencing.
  • T e ff showed a very distinct transcriptional profile (clusters 3, 5 and 6) which includes the upregulation of activation-associated genes such as NR4A1/2/3 and IFNG (Fig. 1 i, Fig. 6j-k). T ex share some of these activation-associated transcriptional changes to a lower extent (cluster 2), which includes the upregulation of co-stimulatory receptors TNFRSF9 (encoding 4-1 BB), and the inhibitory receptors LAG3 and CTLA4.
  • T ex showed pronounced upregulation of cluster 1 , which contains PDCD1 (PD-1 ), T0X2, and PTPN6 (SHP-1 ) - transcripts that are associated with T cell exhaustion and PD-1 signaling.
  • T res t displayed highest expression of clusters 4 and 6, which include the transcripts TNF and IL7R.
  • Ttumor and T ex showed higher expression of cluster 5, including the progenitor-associated transcripts TCF7 and SLAMF6, and NK-associated transcripts LILRB3, FCGR3A, and NKG7 (Fig. 1 i).
  • ISMARA ISMARA to estimate the activities of NFAT/NR4A transcription factors, which are associated with exhaustion.
  • the inventors observed higher motif activity for NFATc2/3 in T e ff and T ex compared to T res t, and elevated activity of NR4A1 in T ex compared to T eff (Fig. 6I).
  • T e ft express higher levels of these activation genes, other genes of the Tex-term signature, including PDCD1 , CD27, TOX, FUT8, LYST, and PDE7B, were more expressed in the T ex condition (Fig. 6m). Only T e ft showed high enrichment in the proliferation-associated NME1-T signature. At the same time, T ex enriched in the KIR+TXK+ NK-like signature, which could suggest that they undergo an NK-like transition as recently described for exhausted CAR T cells. Overall, the inventors’ data show that T ex resemble exhausted human TILs in terms of functionality and transcriptional changes.
  • Example 2 A targeted CRISPR-Cas9 screen reveals SNX9 as a driver of T cell exhaustion
  • the inventors utilized the ex vivo exhaustion model to perform a targeted pooled CRISPR-Cas9 screen. Briefly, among genes with upregulated expression in T ex , the inventors prioritized genes that were shared with published gene sets for human TILs and had little known functions in T cell exhaustion. To perform the CRISPR-Cas9 screen, the inventors simultaneously transduced primary human CD8 T cells with the NY-ESO-1 TCR construct and a pooled lentiviral library that encodes gRNAs and Cas9 (Fig. 2a).
  • Sorted co-expressing cells were repetitively stimulated, stimulated again for 4 hours and then sorted by flow cytometry into cells with maintained degranulation potential (CD107a+, “functional”) and cells with impaired degranulation (CD107a-, “exhausted”).
  • the three controls with known essential functions in T cell functionality showed the highest negative enrichment (p ⁇ 0.01 , 1-way ANOVA).
  • the inventors decided to further investigate SNX9.
  • the inventors confirmed that T e ff and T ex upregulated SNX9 on protein level both by flow cytometry and Western blot (Fig. 2e-f, Fig. 7c-d).
  • the inventors found that SNX9 is significantly co-expressed with TIM-3 among PD-1 + TILs from NSCLC patients (Fig. 2c-d, Fig. 7e).
  • SNX9 expression was negatively correlated with the expression of central-memory and progenitor markers, CCR7 and TCF7 (Fig. 7g-h). Consequently, SNX9+ T cells showed higher expression of inhibitory receptors PDCD1 and HAVCR2 (TIM3), and exhaustion-related transcriptional regulators TOX and TOX2.
  • TIM3 inhibitory receptors
  • TOX and TOX2 exhaustion-related transcriptional regulators
  • NFAT activation is important for T cell activation but has also been linked to the development of T cell exhaustion through downstream induction of NR4A1/2/3 and TOX/TOX2 expression 36,43,44 .
  • the inventors identified that the expression of NR4A1 , NR4A3, and TOX was reduced in SNX9 KO T ex (Fig.
  • SNX9 KO affects T cell metabolism and differentiation.
  • the inventors found that SNX9 KO decreased the expression of lactate dehydrogenase A (LDHA), a critical enzyme in the glycolytic pathway (Fig. 8h), and that SNX9 KO T ex had a lower glucose dependence with a switch towards fatty acid or amino acid oxidation (FAO/AAO), reminiscent of memory T cells (Fig. 8i).
  • LDHA lactate dehydrogenase A
  • FEO/AAO fatty acid or amino acid oxidation
  • Fig. 8i T ex SNX9 KO cells maintained elevated expression of the central-memory associated receptor CCR7 (Fig. 3j).
  • SNX9 KO decreases signaling through NFATc2-NR4A1/3-TOX and induces metabolic changes, which may both contribute to decreased exhaustion and increased central-memorylike differentiation.
  • the inventors then stimulated both intergenic and SNX9 KO Teff, either with NY-ESO-1 peptide-loaded T2 wildtype (“T2 wt”), or T2 KO cells and quantified CD25 upregulation as a marker of NFAT signaling.
  • T2 wt NY-ESO-1 peptide-loaded T2 wildtype
  • T2 KO T2 KO cells
  • CD25 upregulation compared to stimulation with T2 wt cells (Fig. 3i, Fig. 8k).
  • the KO of SNX9 in T e ff resulted in lower upregulation of CD25 after stimulation with T2 wt cells, while no significant change was observed with T2 KO cells.
  • the inventors sought to confirm these results in a reductionist antibody-based stimulation assay.
  • the inventors stimulated intergenic and SNX9 KO T cells with plate-bound anti-CD3 antibody (OKT3 clone) alone or in combination with stimulatory plate-bound anti- CD28 antibody (CD28.2 clone).
  • plate-bound anti-CD3 antibody OKT3 clone
  • CD28.2 clone stimulatory plate-bound anti- CD28 antibody
  • Fig. 3j the inventors observed increased CD25 expression when combined with anti-CD28 co-stimulation
  • SNX9 KO T cells showed reduced CD25 upregulation, which is compatible with the inventors’ experiments above revealing CD28-dependent effects of SNX9.
  • Example 4 Snx9 KO improves anti-tumor efficacy and reduces terminal exhaustion in vivo
  • the inventors then asked whether the reduced initial activation coupled to a later reduction in exhaustion observed with an SNX9 KO in the ex vivo model would also translate into improved in vivo efficacy.
  • the inventors knocked out Snx9 in pre-stimulated OTI splenocytes (murine OVA-specific CD8 T cells) by Cas9-crRNA-tracrRNA electroporation, which significantly reduced Snx9 protein expression (Fig. 4a, Fig. 9a).
  • the adoptive transfer of 1.5 Mio Snx9 KO OT cells to MC38-OVA tumor-bearing mice reduced tumor growth and improved survival (Fig. 4b, Fig. 9b).
  • the inventors next investigated the differentially expressed genes between Snx9 KO and intergenic OTI cells within each cluster and among all cells.
  • Snx9 itself and Il2ra encoding CD25
  • the inventors found that Snx9 KO OTI cells expressed higher levels of the chemokines Ccl3, Ccl4, and Ccl5 in the Tex-term cluster (and for Ccl5 also the Tex-prolif cluster), while Xcl1 was reduced.
  • Cxcr6 which is associated with effector functions and tumor infiltration.
  • Ccl3, Ccl4, and Ccl5 are well known chemokines that attract other immune cells into tumors including dendritic cells, monocytes, and T cells. Therefore, the inventors investigated the number of endogenous immune cells within MC38-OVA tumors 3 days post OTI transfer. In agreement with a higher chemokine expression, the inventors observed more endogenous CD8 T cells and cDC1 s (CD11 c+ MHCII+ F4/80- CD103+) in tumors after transfer of Snx9 KO OTI cells (Fig. 4h, Fig. 10e). These changes in the intratumoral immune composition were accompanied with higher serum levels of IFNy and lower levels of the immunosuppressive cytokine 11-10 (Fig. 4i).
  • the inventors detected no change in Ccl5, Cxcll O, and II-6 in the serum (Fig. 10f). Notably, the inventors observed a similar improvement in anti-tumor efficacy by Snx9 KO when OTI cells were transferred to (immunodeficient) NSG mice with MC38-OVA tumors (Fig. 4j, Fig. 10g). This suggests that while Snx9 KO OTI cells promote the recruitment of other immune cells, Snx9 KO OTI cells can directly mediate anti-tumor effects in NSG mice independent of an intact endogenous immune system.
  • Example 5 Deletion of SNX9 improves CAR T cell anti-tumor efficacy
  • the inventors investigated whether SNX9 KO would also improve the efficacy of human CAR T cells in xenograft models.
  • the inventors generated human anti-CD19 CAR T cells harboring a CD28-CD3 ⁇ co-stimulation domain with or without SNX9 KO and transferred them to NSG mice with subcutaneous human CD19+ Raji tumors (CART19-28z, Fig. 5a).
  • CART19-28z SNX9 KO cells improved long-term antitumor control and survival (Fig. 5b-c).
  • CART19-BBz CD28 KO 4-1 BB domain-containing anti-CD19 CAR and additionally knocked out the endogenous CD28
  • CART19-BBz CD28 KO 4-1 BB domain-containing anti-CD19 CAR and additionally knocked out the endogenous CD28
  • the inventors did not observe an additional effect of SNX9 KO with CART19-BBz CD28 KO cells.
  • SNX9 KO improved human CART19-28z efficacy in vivo which was accompanied by elevated IFNy, perforin, and granulysin levels in the serum, whereas IL10 and IL6 were reduced.
  • SNX9 expression in CD8 T cells correlated with resistance to immunotherapy in melanoma patients (graphically summarized in Fig. 12).
  • the ex vivo model developed here enables the generation of millions of cancer-associated exhausted T cells from peripheral human blood.
  • the cells generated with this approach acquire features of T cells in human tumors, such as co-expression of inhibitory receptors, reduced effector functions, and impaired proliferation, a phenotype recently also observed with continuously stimulated CAR T cells.
  • the simplicity and versatile nature of the current ex vivo exhaustion model enables immediate testing of novel compounds or drug targets in a fully human system.
  • the model can be refined to investigate mechanisms beyond persistent antigenic stimulation by including metabolic restriction, low oxygen availability, suppressive cytokines, or immunosuppressive cells which would allow the replication of important aspects of the tumor microenvironment.
  • CRISPR-Cas9 screens have been previously used in cancer cells to discover important genes for immunotherapy resistance. Additionally, in vivo CRISPR-Cas9 KO screens in murine T cells have been published to improve anti-tumor efficacy. However, due to low T cell infiltration in the tumor, in vivo screens often suffer from guide underrepresentation limiting the discovery of new targets. Notably, several studies performed genome-wide CRISPR-Cas9 screens in human T cells using an improved electroporation-based protocol. Their protocols provide a perspective to perform larger screens utilizing the inventors’ human exhaustion model.
  • SNX9 as a potential mediator of T cell exhaustion, which the inventors confirmed ex vivo and in vivo. While SNX9 has been identified in expression analyses of tumor-infiltrating T cells before, its role in T cell exhaustion has remained unexplored. SNX9 was reported to enhance CD28 signaling, which is generally considered to be beneficial for CD8 T cells. Badour and colleagues described that SNX9 amplifies CD28 signaling by increasing its internalization.
  • SNX9 modulates PI3K/AKT signaling directly, in contrasts to its effects on PLCy1/Ca 2 7NFAT signaling.
  • SNX9 is known to bind PI(4,5)P2, PI(3,4,5)P3, N- WASP, and trigger Arp2/3-dependent polymerization of actin filaments. This raises the interesting possibility that SNX9 functions downstream of TCR/CD28-evoked PI3K activity by enhancing the assembly of ITK, TEK, and WASP-dependent actin regulatory processes, which are known to promote PLCyl signaling. More research is required to elucidate in detail how SNX9 promotes TCR/CD28 downstream signaling.
  • SNX9 KO reduces T cell activation, it also enhances antitumor immunity.
  • current approaches to alleviate T cell exhaustion mainly aim to increase co-stimulation or decrease co-inhibition.
  • PD- 1 blockade is thought to primarily rescue impaired CD28 co-signaling and CD28 is required for the efficacy of anti-PD1 antibodies in vivo.
  • lack of co-inhibition during antigenic stimulation can lead to T cell exhaustion.
  • the genetic absence of PD-1 increases T cell exhaustion in mice, presumably due to overactivation.
  • SNX9 KO T ex have a lower expression of NR4A1, NR4A3, and TOX, which have been implicated in T cell exhaustion.
  • Snx9 KO OTI cells express lower levels of Nr4a2 and Nr4a3, while effects on Tox expression were less consistent.
  • Snx9 KO OTI cells expressed more effector-memory associated proteins such as Cxcr6, Ccl5, Ccl4, and Cc/3 and induced higher recruitment of CD8 T cells and cDC1 s.
  • Cxcr6 was recently described to promote the maintenance of cytotoxic CD8 T cells in tumors through interactions with Ccr7+ dendritic cells, a potential factor how Snx9 KO OTI cells may enhance the endogenous antitumor immune response.
  • adoptive transfer of CART 19-28z SNX9 KO provoked higher serum levels of cytotoxic granule proteins and IFNy, while the immunosuppressive cytokine IL10 was markedly reduced.
  • IL10 secretion is known to correlate with TCR signaling strength, inhibit CD28-signaling, and increase the activation threshold of T cells in a negativefeedback loop, which might be dampened by SNX9 KO.
  • SNX9 KO alleviates exhaustion, increases chemoattraction, and prolongs IFNy secretion of tumor-specific T cells.
  • the inventors findings suggest that SNX9 fine tunes T cell activation and is a potential target to improve the efficacy of cellular immunotherapies in cancer patients.
  • human genes and transcripts are written in all capital letters and italic (e.g. SNX9).
  • Human proteins are indicated in all capital letters (e.g. SNX9).
  • Murine genes and transcripts are written in italic with the first letter in capital letters (e.g. Snx9).
  • Murine proteins are written with the first letter in capital letters (e.g. Snx9).
  • Official gene symbols and protein names are used when applicable according to the Uniprot Consortium (uniprot.org). CD nomenclature is used for CD107a (official protein name LAMP1 ).
  • the NY-ESO-9V peptide used in this manuscript has a 4500-fold relative competitor activity towards HLA-A2010 and 200-fold higher antigenic activity (half-maximal dose required for killing of T2 tumor cells) compared to the NY-ESO-9C endogenous peptide.
  • RPMI1640 was supplemented as described above, but FCS was replaced with 8% heat-inactivated AB+ male donor serum and 50 pM normocin (Invivogen) was added.
  • Recombinant human IL-2 (Peprotech or Proleukin) was always added freshly at the indicated doses.
  • DMEM heat-inactivated (56°C 30min) fetal bovine serum (FBS, PAN Biotech), 100 ng/ml penicillin/streptomycin (Sigma), 2 mM L-Glutamine (Sigma), 1 mM Sodium Pyruvate (Sigma) and 1 % MEM non- essential amino acids (Sigma) and 10mM HEPES (Sigma).
  • T2 cells (ACC598, RRID:CVCL_2211 ) and Jurkat (ACC282, RRID:CVCL_0065) were purchased from DSMZ, Leibnitz Institute.
  • HEK293T cells (ATCC CRL-3216, RRID:CVCL_0063) and Raji (ATCC CCL-86, RRID:CVCL_0511 ) were purchased from ATCC.
  • the melanoma cell line NA8-Mel (RRID:CVCL_S599) was kindly provided by Dr. Romero (University of Lausanne) and cultured in RPMI-1640 supplemented as described above.
  • Murine MC38-OVA colon cancer cells (provided by Mark Smyth, Peter MacCallum Cancer Centre, Melbourne, Australia) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% heat-inactivated FBS (Gibco), sodium pyruvate (Sigma), penicillin/streptomycin (Gibco), and minimal essential medium nonessential amino acids (Sigma). All cells were confirmed to be negative for mycoplasma by PCR as described (Choppa, P. C. et al., Mol. Cell.
  • TrypLE Express replacement, Thermo Fisher
  • Trypsin-EDTA Thermo Fischer
  • mice Wildtype (CD57BL/6NRj), OT-I (C57BL/6-Tg(TcraTcrb)110OMjb/J, RRID:IMSR_JAX:003831 ), and NSG (NOD.Cg-Prkdc ⁇ scid>ll2rg ⁇ tm1 Wjl>SzJ, RRID:IMSR_JAX:005557) mice were bred in-house at the University Hospital Basel, Switzerland. Animals were housed under specific pathogen-free conditions. Sex-matched littermates at 8-12 weeks of age at the start of the experiments were used. All animal experiments were performed in accordance with Swiss federal regulations and licenses were approved by the cantonal veterinary office of Basel-Stadt (CH).
  • CH Basel-Stadt
  • C57BL/6NRj and NSG mice were injected subcutaneously into the right flank with 1 Mio (CD57BL/6NRj) or 0.25 Mio (NSG) syngeneic murine MC38-OVA colon cancer cells suspended in phenol red-free DMEM (without additives) or 0.5 Mio human Raji lymphoma cells suspended in Corning® Matrigel® Matrix High Concentration Phenol-Red-Free diluted 1 :1 in phenol red-free DMEM without additives. Cell lines were tested for mycoplasma contamination before injection by PCR as above. Tumor volume was calculated according to the formula: D/2 x d x d, with D and d being the longest and shortest tumor diameter in mm, respectively.
  • Tumor tissue was isolated from mice, weighed, and minced using razor blades. Tissue was then digested using accutase (PAA), collagenase IV (Worthington), hyaluronidase (Sigma), and DNAse type IV (Sigma) for 60 min at 37 °C with constant shaking. Cell suspensions were filtered using a cell strainer (70 pm). Precision Counting beads (Biolegend) were added before staining to quantify the number of cells per gram tumor.
  • PAA accutase
  • collagenase IV Worthington
  • hyaluronidase Sigma
  • DNAse type IV Sigma
  • Fc-Block Single cell suspensions were blocked with rat anti-mouse Fcylll/ll receptor (CD16/CD32) blocking antibodies (“Fc-Block”) and stained with live/dead cell-exclusion dye (Zombie UV dye; Biolegend) for 20 min at 4°C then washed with FACS buffer (PBS supplemented with 2 mM EDTA, 0.1 % Na-Azide, 2% FCS) by centrifugation at 500 g for 3 min.
  • FACS buffer PBS supplemented with 2 mM EDTA, 0.1 % Na-Azide, 2% FCS
  • OTI T cells live singlet CD19- Ly6G- CD45.2- CD45.1 + CD8+; Endogenous T cells: live singlet CD19- Ly6G- CD45.2+ CD45.1- F4/80- CD11c- CD8+ or CD4+; NK cells: live singlet CD19- Ly6G- CD45.2+ CD45.1- F4/80- CD11c- CD8- CD4- CD3- MHCII- NKp46+; cDC1 : live singlet CD19- Ly6G- CD45.2+ CD45.1- CD11 c+ F4/80- MHCII+ CD3- Ly6C- CD103+ CD11 b- ; cDC2: live singlet CD19- Ly6G- CD45.2+ CD45.1- CD11c+ F4/80- MHCII+ CD3- Ly6C- CD103- CD11 b+; B cells: live singlet CD19+; Neutrophils: cDC1
  • PBMCs Human peripheral blood mononuclear cells
  • HD healthy blood donors
  • buffy coat was diluted in PBS and layered on top of Histopaque-1077 and spun in SepMate (Stem Cell) according to manufacturer’s instructions.
  • Red blood cells were lysed using Red Blood Cell Lysis Kit (eBiosciences), washed and frozen in 10% DMSO 90% FCS in a Styrofoam container to - 80°C. For long term storage (> 1 week), cells were transferred to liquid nitrogen.
  • the lentiviral construct encoding for the codon-optimized pRRL 131 (WT) T2A 1xATG Cys LAU155 NY-ESO-1 T cell receptor consists of alpha and beta chains under an hPGK promotor separated by a T2A sequence and was kindly provided by Dr. Michael Hebeisen and Dr. Natalie Rufer at the University of Lausanne (Schmid, D. A. et al. J. Immunol. 184, 4936-4946, 2010; Hebeisen, M. et al. Front. Immunol. 4, 1-10, 2013).
  • HEK293T cells 2.5 million low passage HEK293T cells were cultured in DMEM medium and seeded into a 15cm tissue-culture treated dish. Three days later, 2 nd generation LTR- containing donor plasmid, packaging plasmid pCMV-delta8.9 and the envelope plasmid VSV- G were mixed at a ratio of 4:2:1 ratio in unsupplemented Opti-MEM (ThermoFisher) and sterile filtered. This solution was then mixed with polyethyleneimine 25 kDa (Polysciences Inc.), also diluted in Opti-MEM at a DNA:PEI ratio of 1 :3. 28 pg of DNA was transfected per 15cm dish.
  • Opti-MEM unsupplemented Opti-MEM
  • polyethyleneimine 25 kDa Polysciences Inc.
  • virus production for the NY-ESO-1 TCR vector was later outsourced to Vectorbuilder Inc (USA), who provided stocks of > 1 *10 A 9 TU/ml lentivirus produced by PEG precipitation (measured by p24 ELISA).
  • CD8 T cells were then isolated using the CD8 microbeads kit (Miltenyi, positive selection) according to the manufacturer’s instructions on an AutoMACS. Isolated cells were washed and resuspended in suppl. RPMI with 8% human serum as described above and plated at 1.5 Mio/ml. T cell activation and expansion kit (Miltenyi) anti-CD3+anti-CD28 stimulatory magnetic beads were coated overnight at 4°C on a rotator shaker according to the manufacturer’s instructions.
  • TCR Vbeta 13.1 + cells were sorted by staining of TCR- Vbeta13.1 antibody by flow cytometry (H131 clone PE-Cy7, 1 :25, SorpAria) or using purified antibody (H131 clone purified, 1 :50 dilution) followed by magnetic column purification (antimouse IgG Microbeads, Milentyi).
  • NY-ESO-1 TCR specific T cells were plated at 0.125 mio/ml specific cells.
  • T2 tumor cells were irradiated with 3000 rad gamma rays using a GammaCell irradiator.
  • Irradiated cells were mixed with the indicated dose (1 pM) of NY-ESO-1 9V peptide and added to the T cells at an effector to target ratio of 1 :3 in the presence of 50 U/ml IL-2. This procedure was repeated (with IL-2 stimulation at each stimulation) according to the scheme in Fig. 6A. At each stimulation, 75% of the medium was replaced.
  • the cells were expanded 1 :2 on the day of the second stimulation with tumor cells and peptide.
  • T tU mor were treated identically, but without the addition of NY- ESO-1 9V peptide.
  • the inventors only exchanged 75% of the medium every three days replenishing IL2.
  • Acute stimulation controls were only cultured as T res t with IL2 on days 0, 3, 6, and with tumor cells + peptide + IL2 on day 9 after plating. After 12 days (thus 3 days after last stimulation), cells were stained and analyzed by flow cytometry. Functional assays were performed the same or the next day normalized to cell numbers.
  • T cells were stained with the following protocol.
  • Cells are washed in PBS, resuspended in PBS, and blocked with 1 :100 human Fc-receptor-inhibitor (eBioscience) in PBS and stained with Fixable Viability Dyes (Biolegend or eBioscience) 1 :200 for 20min on ice.
  • FACS buffer PBS supplemented with 2 mM EDTA, 0.1 % Na-Azide, 2% FCS
  • All antibodies used in this study are listed above.
  • cytoplasmic staining including SNX9 and cytokines
  • cells were fixed and permeabilized using IC Fixation Buffer (eBioscience) for 20min at room temperature. Intracellular antibodies were then stained in 1 x Permeabilization buffer (eBioscience) for 30min at 4°C. For secondary staining, this procedure was repeated, including washing steps.
  • the Fixation/Permeabilization kit eBioscience was used for 30min at room temperature followed by two wash cycles in 1x permeabilization buffer and antibody staining in 1x permeabilization buffer for 45 min at room temperature.
  • the inventors added 10’000 Precision counting beads (Biolegend) to each sample before the first washing step to adjust cell counts after acquisition based on the bead count (population high in SSC and positive in any channel ⁇ 640 lasers).
  • phospho-AKT-Ser473 cells were fixed with IC fix for20min at room temperature and then permeabilized in a custom 0.1 % Triton- Xi 00 + 1 % BSA in PBS buffer for 5min. Then cells were stained in FACS buffer with antibodies as described above. After staining, cells were analyzed on a BD LSR Fortessa Cell analyzer (BD Bioscience), Cytoflex S (Beckmann) flow cytometer or an Aurora Spectra Analyzer (CyTek).
  • the inventors performed a co-culture of peptide-loaded T2 cells with T cells in the presence of CD107a antibodies to assess the degranulation of cytokine production of T cells.
  • T2 cells were incubated at a density of 1 mio/ml with the respective peptides diluted in full RPMI supplemented medium.
  • 10’000 NY-ESO-1 specific T cells (measured by TCR Vpi3.1 staining as above) were co-cultured with 10’000 of these peptide-loaded T2 cells (1 :1 E:T Ratio) for 5h in the presence of 20 ng/ml anti-CD107a-PE antibody and 1x Monensin (Biolegend).
  • T2 cells expressing Luciferase and tdTomato were generated by the transduction of a lentivirus made with the pFU-Luc2 -tdTomato construct and sorting for tdTomato expression.
  • T2-Luc2 cells were washed and resuspended at 1 mio/ml with 1 pM NY-ESO-9V peptides and incubated at 37°C for 30 min. T2-Luc cells were then washed and plated at 20’000 cells per well of a V-bottom 96-well white plate.
  • NY-ESO-1 specific T cells were then added to these cells and incubated for the indicated time. Afterward, 0.15mg/ml D-Luciferase (Perkin Elmer) was added to each well and immediately analyzed on a BioTek H1 Spectro/luminometer, acquiring for 1 sec I well. Averages of three successive reads were used. Controls without T cells (maximal signal) and one with 0.1 % Triton-X100 (minimal signal) were used to calculate % specific lysis:
  • This breast cancer cell line naturally expresses high levels of PD-L1 and is HLA-A2+, which allows for loading of NY-ESO-1 peptides (Muller, P. et al. Cancer Immunol. Res. 2, 741-755, 2014).
  • T cells from four donors were isolated by CD8 MACS isolation. Cells were stimulated and transduced as described above for the ex vivo exhaustion model. 12 days after the first stimulation with tumor cells and peptide these Trested, T tU mor, T ex were sorted for TCR VP13.1 + (NY-ESO-1 TCR) CD8 CD56- CD4- DAPI- cells. Minimally 415’000 (most samples 600’000) T cells were sorted into cold FACS buffer, then centrifuged at 500g for 8min, and washed by centrifugation with cold PBS. Cells were then resuspended in 750 pl Trireagent (Sigma).
  • RNA Total RNA were purified using the kit Direct-Zol RNA Miniprep (ZymoResearch, Cat# R2050). The purified RNA was quality-checked on the Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA) using the RNA 6000 Pico Chip (Agilent, Cat# 5067-1513). RNA was quantified by Fluorometry using the QuantiFluor RNA System (Cat# E3310, Promega, Madison, Wl, USA). Library preparation was performed, starting from 200ng total RNA, using the T ruSeq Stranded mRNA Library Kit (Cat# 20020595, Illumina, San Diego, CA, USA) and IDT TruSeq RNA UD Indexes.
  • Bioinformatics 30, 923-930, 2014) using gene annotation from Ensembl release 105, and options “-0 -M -read2pos 5 —primary -s 2 -p -B” to count the number of reads (5’ ends) overlapping with the exons of each gene assuming an exon union model.
  • transcript count data was analyzed in R 4.2.1 using edgeR_3.38.1 .
  • Transcripts coding for protein coding genes were normalized for library size.
  • a donor-paired design matrix an edgeR Robot, M. D. et al., Bioinformatics 26, 139-140, 2009
  • functions glmQLFit, glmQLFTest, and topTags were used to investigate all significantly dysregulated genes among any conditions (ANOVA-like) or between each combination of conditions.
  • a Benjamini- Hochberg adjusted p-value cutoff of ⁇ 0.01 and Iog2-fold-change cutoff of 0.75.
  • the inventors ranked all significantly upregulated genes in T ex vs T res t condition according to their overlap with published patient TIL datasets available at the time.
  • the inventors focused the inventors’ analysis on genes with more than two counts per million reads after repetitive stimulation (T ex ) and at least two-fold upregulation compared to T res t.
  • the inventors further selected genes based on literature review and excluded genes that were already highly studied in T cell exhaustion (for example, HAVCR2 and CTLA4).
  • three genes known to be essential for T cell functionality ZAP70, LAT, LAMP1 ) (Shifrut et al. 2018) were used as positive and negative controls for the CRISPR-Cas9 screen.
  • gRNA sequences for each of the selected genes and 20 intergenic controls were extracted from a published highly optimized gRNA library (Wang et al. 2017) and ordered as a DNA oligo pool with the required overlaps for assembly from Twist Biosciences according to Wang et al. (Wang, T., Lander, E. S. & Sabatini, D. M. Cold Spring Harb. Protoc. 2016, 283-288, 2016).
  • gRNA DNA oligonucleotides were amplified by high fidelity PCR, purified over a 2% agarose Tris-acetate EDTA gel, and extracted using Machery Nagel PCR cleanup kit.
  • LentiCRISPRv2- mCherry (Addgene 99154) was digested by BsmBI and cut plasmid extracted from a 1 % TAE agarose gel. This fragment was fused with the PCR amplified gRNAs at a 1 :30 molar ratio by Hifi-DNA Assembly for 1 h at 50°C (New England Biolabs, failed using Gibson Assembly standard kit). The product was amplified by the transformation of Stbl3 E. coll with over 1000 colonies per guide, cultured in LB with 100 pg/ml ampicillin (Sigma), and extracted using Midi Prep (Machery Nagel).
  • the plasmid library was barcoded by PCR and sequenced at the D- BSSE Genomics Facility on an Illumina MiSeq 50 cycle v2 run, which proved successful cloning and representation of all guides (Gini index 0.188).
  • 2nd generation lentivirus was then prepared on a larger scale (>1000 cells / guide) from the library and the NY-ESO-1 TCR, which were then concentrated by ultracentrifugation and titrated on Jurkat cells.
  • Freshly isolated healthy donor CD8 T cells were stimulated as above.
  • cells were co-transduced with LentiCRISPRv2-mCherry virus at an MOI of 0.5.
  • nontreated 6-well polystyrene plates were coated with 2 pg/cm 2 Retronectin (Takara) overnight in PBS at 4°C and then blocked using 2% BSA in PBS for 20 min at room temperature.
  • the LentiCRISPRv2-mCherry virus was diluted according to the desired MOI in PBS with 0.1 % BSA and added to the plates in 2ml per well. Plates were then centrifuged 90min at 2000 g at 32°C.
  • Genomic DNA from these samples was extracted using the Qiagen Blood DNA mini kit, including a Proteinase K digestion step. DNA was eluted in 5mM Tris-HCI (no EDTA), and a barcoded PCR amplification of gRNA sequences was performed as described (Wang et al., ibid) with PCR cycles: 95°C 2’; 35x: 98°C 10”, 60°C 15”, 72°C 30”; 72°C 5’.
  • gRNA PCR product was isolated from a 2% agarose in Tris-Acetate-EDTA gel and eluted using Machery Nagel PCR cleanup kit and dried for 10 min at 56°C. DNA was eluted in 25 pl 5 mM Tris-HCI. This DNA library was loaded onto a 50 cycle MiSeq v2 Illumina Run on an Illumina MiSeq. gRNA sequencing reads retrieved from this sequencing were demultiplexed and uploaded to PinAPL-py.ucsd.edu (Spahn et al., ibid).
  • PinAPL-py was then used to align, control for quality, and count guides with the 5' adapter ATTTTAACTTGCTATTTCTAGCTCTAAAAC (SEQ ID NO: 16317). Sequencing results are deposited under GSE190246. R Studio (R version 4.2.1 ) was then used to calculate the average Iog2 fold change per gene and donor (average over the 5 guides per gene) and plot these values. Genes were ranked based on median Iog2 fold change (of donor replicates).
  • CD8 T cells were stimulated for 1 day using anti-CD3+anti-CD28 stimulatory beads (Miltenyi) with 150 U/ml of IL-2 in 8% human serum containing supplemented RPMI as described above. The next day, beads were removed by magnetic separation. Cells were spun down at 500g 3min and resuspended in supplemented electroporation P3 buffer (1 :4.5 supplement to buffer ratio) at 1 Mio I 20 pl according to manufacturer’s instruction. 20 pl of this solution was then mixed with the 3 pl of Cas9-RNP and 0.75 pl of 200 pM Electroporation enhancer. This mixture was then electroporated using the EH115 setting in an X-unit Lonza Nucleofector 4D.
  • T2 + peptide 1x with T2 + peptide and analyzed on day 3 (T e ff) or repetitively for 4x T2 + peptide as described above (Tex).
  • T2 tumor cells were loaded with 1 pM NY-ESO-1 (9V) peptide for 30min at 37°C.
  • T2 cells were before also stained with 1 pM Cell Trace Violet or CFSE for 15 min in PBS at 37°C, then washed in complete medium 2x before co-culture.
  • T2 tumor cells were washed 2x in serum-free prewarmed RPMI and resuspended at 1 Mio/ml in the same medium.
  • Teff cells (3 days post stimulation with T2-peptide) were electroporated with 800ng of p-human-TCRzeta-EGFP or p-human-CD28-EGFP plasmids using the EH115 P3 protocol as described above. The cells were then rested in 50 U/ml IL2 in human serum medium for 24h, before the co-culture with T2 tumor cells as described above.
  • Images from immunofluorescence images were recorded on a Nikon Ti with a Yokogawa CSU- W1 spinning disk module on a Photometries 95B (22mm back-illuminated sCMOS) camera.
  • a Nikon CFI Apo TIRF NA 1.49 100x objective together with a 1.5x additional magnification unit was used with 1.515 NA oil mounted samples.
  • Diode-pumped solid-state lasers at 405, 488, 561 , and 647nm were used together with filters for DAPI (ET460/50nm), AF488 (ET525/50nm), AF568 (ET630/75nm) and AF647 (ET700/75nm) with a Quad BS Dichroic mirror.
  • T cells were collected and washed 2x in ice-cold PBS and then lysed in 8 M urea (in H2O, Cell Signaling #7900) supplemented with 0.5% Triton-X100 (Merck #1.08643.1000), 1 x complete mini protease inhibitor cocktail (Roche #11836153001 ). DNA was sheared by sonication, then the DNA was pelleted before samples were complemented with 5x Laemmli buffer (2% SDS, 5% 2p-mercapto-ethanol, 10% glycerol, 0.002% bromophenol blue in 62.5 mM Tris-HCI) and boiled at 95°C for 5 min.
  • Laemmli buffer 2% SDS, 5% 2p-mercapto-ethanol, 10% glycerol, 0.002% bromophenol blue in 62.5 mM Tris-HCI
  • the inventors harvested spleen and axial, cervical and inguinal lymph nodes from “OTI” mice (C57BL/6- Tg(TcraTcrb)1100Mjb/J, RRID:IMSR_JAX:003831 ), in which all CD8 T cells recognize ovalbumin (OVA257-264, H-2Kb). Spleens and lymph nodes were strained over a 70 pm strainer and washed with PBS, incubated in red blood cell lysis buffer for 1 min and wash with PBS again.
  • 900 pmol Alt-R-crRNA sgSNX9_9 and sgSNX9_IDT_AF; IDT
  • 900 pmol Alt-R-tracrRNA IDT
  • 1 .5 pl of these aligned crRNA-tracrRNA complexes was then incubated with 15 pl of 40 pM Cas9-NLS protein (Berkeley, QB3) for 30min at room temperature in the dark and used immediately (referred to as Cas9-RNP).
  • T e ff were used (1x T2-peptide stimulation of the repetitive stimulation procedure described above, then used on day 3 post stimulation).
  • Black well, clear bottom pCIear 96-well polystyrene plates were coated with 0.01 % Poly-L-Lysine for 1 h at room temperature, then washed 3x in distilled water and dried for >2h.
  • Stimulatory beads were prepared from the Human T cell Activation and Expansion Kit (Milteniy) by coating anti- CD2/3/28 antibodies each at 10 pg/ml in MACS buffer (0.5% FCS, 2mM EDTA in PBS) overnight onto the beads in a rotator at 4°C.
  • anti-CD2 was included to increase binding strength of cells to the beads.
  • cells were loaded for 45min at 37°C in a solution consisting of phenol free RPMI (base medium, supplemented with 1 % PenStrep, 1 mM Pyruvate, 1 % NEAA, 10mM HEPES) with 0.04% PluronicF127 (Thermo Fisher) and 2 pM Calbryte520-AM (AAT Bioquest). Cells were then washed 2x in base medium with 2% FCS (termed assay medium) and plated at 250’000-500’000 cells per well in assay medium onto the Poly-L-Lysine coated plate and incubated for 30 min at 37°C for cell attachment.
  • Beads were washed 2x in assay medium and rapidly added to each well at 1.5 Mio beads per well (approx. 4:1 ratio). The plate was then recorded immediately in a BioTech H1 fluorescence reader at 490 nm excitation and 525 nm absorbance for 30 min. The signal for each timepoint was normalized to the first measurement (seconds after the bead addition) to adjust for baseline differences. Normalizing to a read acquired before bead addition proved to be less accurate, because of differences in changes of fluorescence by addition of beads. The normalized signal was the visualized in Graphpad Prism and the area under the curve (AUC) and maximum increase in intensity calculated for the first 30 min after beads addition.
  • AUC area under the curve
  • non-treated flat bottom 96-well plates were coated with 1 pg/ml anti-CD3 (OKT3) or 1 pg/ml anti-CD3 plus 2.5 pg/ml anti-CD28 (CD28.2) in PBS overnight at 4°C.
  • Wells were then washed with PBS.
  • T e ff were collected, counted and resuspended in fresh human T cell medium. 200’000 cells were then seeded per well and the plate was centrifuged for 1 min at 500g. Cells were then incubated for 3h at 37°C in the incubator.
  • the cells were stained in FACS buffer sequentially for 1 :800 NFATc2 (D43B1 clone, CST) and then with 1 :500 anti-rabbit IgG (highly cross absorbed, Thermo Fisher) and DAPI 5 pg/ml , each step for 30 min at RT.
  • Cells were then washed twice in FACS buffer and then acquired on an Imagestream MKII imagestream instrument using automated plate handling. Between 1000-5000 single (aspect ratio M01 >0.5) CD8+ cells were recorded. Cells were then gated for focused, DAPI+ CD8+ NFATc2+ cells.
  • the nuclear localization wizard was used to quantify the Similarity_Morphology metric between DAPI and NFATc2 for each cell. The geometric mean of the distribution of this similarity metric was used to compare the different conditions.
  • T e ff cells +/- SNX9 KO thus stimulated three days before with tumor cells, peptide, and IL2.
  • 96-well flat bottom plate wells were coated with either 1.25 pg/ml anti-CD3 (OKT3) and 2.5 pg/ml anti- CD28 (28.2 clone) antibodies (low CD3 + CD28 condition), or with 5 pg/ml anti-CD3 (OKT3) alone overnight at 4°C in PBS.
  • Wells were washed with PBS and replaced with 100 pl human Serum medium as described above.
  • T cells were washed in medium and then added to the plates, spun for 10 seconds at 500g and then incubated for 30 min. Then cells were rigorously resuspended using ice cold FACS buffer (PBS with 2% FCS, 5mM EDTA, and 0.1 % Sodium azide) and spun at 4°C. Cells were then fixed for 10 min using IC fix at RT. Then cells were permeabilized using self-made Perm-Wash buffer (PBS with 0.1 % Triton-X100 and 0.1 % BSA) for 10 min at RT.
  • PBS Perm-Wash buffer
  • Cells were then washed 2x and stained in FACS buffer with the indicated antibodies (AKT-phospho-Ser473, clone 98H9L8, Thermo Fisher) and counterstained using PE-labeled anti-rabbit IgG polyclonal highly cross absorbed antibody (Thermo Fisher). Cells were measured using a BD Fortessa.
  • the inventors had to change the assay procedure due to difficulties to detach T cells from the plate after stimulation and the very dynamic signaling through PLCyl . Therefore, the inventors rested T cells +/- SNX9 KO at 6 days post electroporation, in fresh human serum medium and 10 U/ml IL2 overnight. Then the inventors stained the cells at RT for 5 min with fixable viability dye eF450 before the assay. Afterwards the inventors washed and incubated them in serum-free cytokine-free medium (RPMI with other supplements as above but without serum, but with 0.1 % BSA to reduce attachment) for 1 h at 37°C.
  • serum-free cytokine-free medium RPMI with other supplements as above but without serum, but with 0.1 % BSA to reduce attachment
  • cells were stained on ice with low anti-CD3 + anti-CD28 (1 .25 pg/ml OKT3 + 1.25 pg/ml CD28.2) or high anti-CD3 (2.5 pg/ml OKT3) in medium for 15min. Then cells were washed and stained on ice with 10 pg/ml anti-mouse IgG (Jackson, to crosslink antibodies for activation) for 15min and then washed Afterwards, cells were resuspended in 37°C warm medium and immediately added to a pre-warmed metal plate in an 37°C incubator (activation). After 5min, cells were immediately fixed by the addition of an equal volume of IC fix and incubate for 20 min at RT.
  • low anti-CD3 + anti-CD28 (1 .25 pg/ml OKT3 + 1.25 pg/ml CD28.2
  • high anti-CD3 2.5 pg/ml OKT3
  • cells were spun and resuspended in 20 pl FACS buffer. Then cells were permeabilized by the addition of 180 pl Methanol at -20°C and incubated at -20°C for 15 min. Cells were then 2x washed by centrifugation (from now on 1000g 3min) in FACS buffer, stained for antibodies (CD8 SK1 FITC and anti-PLCy1-Tyr783 CST) in FASC buffer for 30 min and then counterstained with anti-rabbit antibodies as above. Cells were measured on a Beckmann Coulter Cytoflex.
  • T ex were purified by human CD8 microbeads (Miltenyi) according to manufacturer’s protocol. Cells were washed 1x by centrifugation at 500g 3min in cold PBS. RNeasy Plus Mini Kit (Qiagen; # 74136) was used to isolate total RNA from approx. 1 Mio T cells according to the manufacturer’s protocol. Isolated RNA was reverse transcribed using the iScript cDNA synthesis kit (BioRad; #170-8891 ).
  • Quantitative real-time PCR was performed according to the manufacturer’s protocol using the PrimeTime Gene Expression Master Mix (IDT; #1055771 ), the equivalent of 12.5 ng cDNA, and PrimeTime qPCR assay probes (IDT) in a volume of 10 pl.
  • a ViiATM 7 Real-Time PCR System (Applied Biosystems) was used for the fluorescence readout.
  • the following assay probes were used: SNX9 (Hs. PT.58.21424684), TOX (Hs. PT.58.28002606), TOX2 (Hs.PT.58.39787291 ), NR4A1 (Hs.PT.58.39997829), NR4A2 (Hs.
  • PT.58.704850 NR4A3 (Hs.PT.58.14945655), LDHA (Hs.PT.40245343) and the house keeping gene HPRT1 (Hs.PT.58.v.45621572).
  • Ct values of the house keeping gene were subtracted from the other transcripts Ct values, yielding a delta Ct value. This value equals the Iog2 fold change in transcript abundance shown in the figures.
  • the SCENITH flow-based single cell metabolic analysis was performed according to and with reagents provided by Arguello et al (Arguello, R. J. et al. Cell Metab. 32, 1063-1075. e7, 2020).
  • T ex (3 days after the fourth stimulation with T2 tumor cells and peptide) were collected, counted and washed in fresh T cell media (as described above 8% human serum) and plated at 150’000 cells per 96 V-bottom plate well. Cells were equilibrated in the incubator at 37°C and 5% CO2 for 30 min.
  • DG 2-deoxyglucose
  • O oligomycin
  • DGO combination
  • DMSO only control
  • the drugs were added to the cells, mixed rapidly and incubated again.
  • DG was added first for 10min and then O was added for the last 5min, according to the manufacturer’s instruction.
  • the cells were washed 1x in PBS at 4°C and then stained in PBS for Zombie NIR Viability Dye (Biolegend) and anti-CD8-BV605 (SK1 clone, Biolegend) for 20 min on ice.
  • the inventors obtained the scRNAseq dataset described in Sade-Feldmann et al. under GEO accession number GSE120575 and analyzed in R 4.0.2, SingleCellExperiment_1 .10.1 , ggplot2_3.3.5 and scater_1.16.2. As described in their original publication, genes were filtered for protein coding genes and min expression of >4.5 logcounts in at least 10 cells. Cells were filtered to have >2.5 mean logcounts of their listed housekeeping genes and >0 logcounts for PTPRC (CD45) to exclude non-immune cells.
  • CD8 T cells were defined as having at least 1 read of CD3E and CD8A or CD8B', and having no detected read for NCR1, NCAM1, CD4 (excluding NKs and CD4 T cells).
  • a tSNE dimensionality reduction (based on PCA components and perplexity of 30) was calculated for these CD8 T cells.
  • tSNE plots with selected transcripts highlighted as dot size and color were generated using ggplot2.
  • SNX9 positive CD8+ T cells were additionally defined as having > 1 logcount for SNX9. The percentage of SNX9+ cells among all CD8+ T cells before immunotherapy was exported and visualized in Graphpad Prism according to treatment response.
  • OTI T cells with or without Snx9 KO were generated as described in “OTI KO generation” and confirmed using flow cytometry staining.
  • CD57BL/6 mice were injected subcutaneously with 1 Mio MC38-OVA and 1 .5 Mio OTI T cells were transferred intravenously 12 days post tumor injection. After 12 days, the tumors (7 per condition) were removed separated from fibrous, necrotic and lymphoid tissue and cut into pieces of 1 mm 3 . These pieces were then digested in the digestion mix as described above and dissociated using a gentle MACS dissociator (Miltenyi, using a C-tube, program m_impTumor_02).
  • Sequencing was performed on Illumina Novaseq 6000 platform to produce paired-end 101 nt R2 reads. Read quality was assessed with the FastQC tool (version 0.11.5). Sequencing files were processed with STARsolo (STAR version 2.7.9a) (Kaminow, B., Yunusov, D. & Dobin, A. bioRxiv 2021 .05.05.442755, 2021 ) to perform sample and cell demultiplexing, and alignment of reads to the mouse genome (mm10) and UMI counting on gene models from Ensembl 102.
  • STARsolo STAR version 2.7.9a
  • Transcript counts per cell were used for downstream analysis in R version 4.2.1 (within R Studio for Mac 2022.07.01 ) using Seurat_4.1 .1 (Hao, Y. etal. Cell 184, 3573-3587.e29, 2021 ). Briefly, Seurat was used to Iog10 normalize counts and the number of unique features, RNA counts, and the percentage of mitochondrial transcripts were calculated. biomaRt_2.52.0 was used to find the murine orthologues for the human cell cycle genes provided in Seurat. Cells with fewer then 2000 unique RNAs, less than 5000 transcripts, or over 15 percentage of mitochondrial transcripts were discarded.
  • FeatureScore for cell cycle genes (S phase and G2M phase), histone genes and interferon-stimulated genes together with the number of unique features per cell were used to scale the data (ScaleData).
  • the ‘future’ Bioconductor package was used for parallelization. UMAPs were generated from the first 20 principal components and clusters defined with a resolution of 0.5. Cells from the two different pools per condition were merged. Signatures were retrieved from original publications: Andreatta (Andreatta, M. et al. Nat. Commun. 12, 1-19, 2021 ), Schietinger (Schietinger, A. et al. Immunity 45, 389-401 , 2016), and Miller (Miller, B. C.
  • Low passage T2 cells were electroporated with crRNA-tracrRNA-Cas9 complexes targeting human CD80 and CD86 (see sequences above) using the CA148 program in SE buffer at 0.4 Mio cells / 20 pl in a 16-well strip cuvette.
  • Alt-R crRNA was obtained from IDT, Cas9-NLS protein from Q3 Macrolab (Berkeley). Cells were then expanded for 7 days before sorting for CD80- CD86- live cells using an Arialll sorter (Beckmann). Cells were then expanded for 7 days before resorting for highly pure CD80- CD86- cells.
  • T e ff cells day 3 post first stimulation with T2 wt + NY-ESO-1 9V peptide
  • SNX9 SNX9
  • T2 wt or T2 KO cells were co-incubated with T2 wt or T2 KO cells at an E:T ratio of 1 :2 in presence of 100 nM NY- ESO-1 9V peptide.
  • Fresh SNX9 KO and control CD8+ human T cells were generated by electroporation as described above but without TCR transduction. SNX9 KO efficacy was confirmed by flow cytometry staining 4 days post electroporation. Cells were expanded 1 :2 every 2 days in 150 U/ml IL-2 containing human serum RPMI medium as above. 6 days post electroporation, the cells were removed from the magnetic stimulatory beads, washed and reseeded at 1 mio/ml cells with 10 U/ml IL2 and rested for 24h.
  • CD8+ T cells from heathy donor PBMCs using the CD8 human microbead MACS kit according to manufacturer’s instruction.
  • Cells were then cultured in RPMI-1640 (Sigma) with 10% heat inactivated human male AB+ serum with 1 mM Sodium Pyruvate (Sigma), 2mM Glutamine (contained in RPMI formulation), 10mM HEPES (Gibco), 5mM beta-mercaptoethanol (Gibco), 1 % PenicilinStreptomycin (Sigma).
  • CD3/CD28 beads Human T cell Activation and Expansion kit, Miltenyi
  • rh-IL-2 Proleukin-2
  • VSV- g pseudotyped lentivirus encoding an anti-human-CD19-FMC63vH chimeric antigen receptor with a CD28 transmembrane domain and a CD28 and CD3 ⁇ signaling domain with a c-terminal T2A self-cleaving copGFP protein (anti-CD19-CD28z-T2A-copGFP).
  • the same procedure was performed with a pLV-EFS-FMC63-BBz-P2A-mCherry CAR construct encoding the same single chain variable fragment targeting human CD19 but coupled to a CD8 transmembrane domain and CD3zetta and 4-1 BB signaling domains.
  • the cell - lentiviral mixture was centrifuged for 90 min at 1000 g (spinfection) and the resuspended and plated for 24h at 37°C. Then cells were expanded 1 :2 every 2 days for 2 iterations with fresh medium and 50 U/ml rh-IL-2.
  • Cells were then stained for CD8 (CD8-APC SK1 clone, Biolegend) and DAPI (Sigma) and analyzed for GFP+ cells (mCherry+ for BBz-CARs) using a Cytoflex flow cytometer. Cells were then counted, washed by centrifugation at 500g 3min in PBS, adjusted in volume for equal numbers of CARs (based on GFP/mCherry positivity), and transferred in PBS intravenously to NSG mice subcutaneously injected 3 days before with 0.5 Mio Raji (ATCC) in 8-12 mg/ml Matrigel (Corning, standard formulation).
  • CD8 CD8-APC SK1 clone, Biolegend
  • DAPI Sigma
  • Serum was collected from mice from the tail once a week into Monovette 200 Z-Gel (Sarstedt, Cat Nr. 20.1291 ) tubes, centrifuged as instructed and frozen to -80°C.
  • the Legendplex MurineVirusResponse 13-plex, Biolegend Cat. Nr. 740621 ) was used according to the manufacturer’s instructions and measured on a Fortessa (BD).
  • the Biolegend Legendplex analysis online tool was used to calculate concentrations according to internal standards. TNFa and IFNp were not detected but can be found in the source data.
  • the Legendplex Human CD8/NK Panel (13-plex, Biolegend Cat Nr. 741065) was used according to the manufacturer’s instruction and measured on a Fortessa (BD). Analytes that were not detected in fraction of samples were not displayed in figures, but can be found in source data (IL4, IL17, TNFa, sFAS, Granzyme A, Granzyme B). sFasL was not displayed as it only correlated with tumor size (also in source data).
  • IL4, IL17, TNFa, sFAS, Granzyme A, Granzyme B sFasL was not displayed as it only correlated with tumor size (also in source data).
  • the inventors used a Legendplex kit for the detection of human proteins (which binds proteins using two anti-human antibodies per analyte), the inventors cannot exclude that this kit also cross-recognizes the murine orthologues.
  • PinAPL-Py A comprehensive web-application for the analysis of CRISPR/Cas9 screens. Sci. Rep. 7, 15854 (2017).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Un premier aspect de l'invention concerne un agent d'acide nucléique capable de réduire ou d'inhiber l'expression ou l'activité biologique de SNX9 dans une cellule cible, l'agent d'acide nucléique étant choisi parmi un oligodéoxynucléotide antisens, un ARNsi, un miARN et un ARNsh. Un deuxième aspect de l'invention concerne un vecteur d'acide nucléique pouvant exprimer l'agent d'acide nucléique dans un lymphocyte T cible, plus particulièrement dans un lymphocyte T transgénique. Un autre aspect de l'invention concerne une préparation de lymphocytes T dont l'expression de SNX9 est supprimée, inhibée ou abrogée. L'un quelconque des aspects ci-dessus est prévu pour être utilisé dans le traitement d'une affection caractérisée par ou associée à l'épuisement de la fonction des lymphocytes T, en particulier l'immunothérapie contre le cancer, plus particulièrement dans le contexte de l'immunothérapie contre le cancer qui bénéficie de la prévention de l'épuisement des lymphocytes T.
PCT/EP2022/077391 2022-09-30 2022-09-30 Cibler snx9 sauve les lymphocytes t recombinés dans le cadre d'une thérapie adoptive Ceased WO2024068010A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/EP2022/077391 WO2024068010A1 (fr) 2022-09-30 2022-09-30 Cibler snx9 sauve les lymphocytes t recombinés dans le cadre d'une thérapie adoptive

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2022/077391 WO2024068010A1 (fr) 2022-09-30 2022-09-30 Cibler snx9 sauve les lymphocytes t recombinés dans le cadre d'une thérapie adoptive

Publications (1)

Publication Number Publication Date
WO2024068010A1 true WO2024068010A1 (fr) 2024-04-04

Family

ID=84329653

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2022/077391 Ceased WO2024068010A1 (fr) 2022-09-30 2022-09-30 Cibler snx9 sauve les lymphocytes t recombinés dans le cadre d'une thérapie adoptive

Country Status (1)

Country Link
WO (1) WO2024068010A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006094673A2 (fr) * 2005-03-08 2006-09-14 Ludwig-Maximilians-Universität München Nexines de tri dans une intervention medicale de troubles neurologiques et/ou metaboliques
US10144929B2 (en) * 2016-02-16 2018-12-04 Mayo Foundation For Medical Education And Research Polypeptide inhibitors of Smad3 polypeptide activities
WO2019084495A1 (fr) * 2017-10-27 2019-05-02 The Trustees Of The University Of Pennsylvania Identification de cibles épigénétiques et transcriptionnelles pour prévenir et inverser l'épuisement des lymphocytes t
US20200103416A1 (en) * 2013-10-11 2020-04-02 Yissum Research Development Company Of The Hebrew University Of Jerusalem SNX9 as a novel biomarker for chronic inflammation and associated immunosuppresion and a new regulator of T cell receptor expression and function

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006094673A2 (fr) * 2005-03-08 2006-09-14 Ludwig-Maximilians-Universität München Nexines de tri dans une intervention medicale de troubles neurologiques et/ou metaboliques
US20200103416A1 (en) * 2013-10-11 2020-04-02 Yissum Research Development Company Of The Hebrew University Of Jerusalem SNX9 as a novel biomarker for chronic inflammation and associated immunosuppresion and a new regulator of T cell receptor expression and function
US10144929B2 (en) * 2016-02-16 2018-12-04 Mayo Foundation For Medical Education And Research Polypeptide inhibitors of Smad3 polypeptide activities
WO2019084495A1 (fr) * 2017-10-27 2019-05-02 The Trustees Of The University Of Pennsylvania Identification de cibles épigénétiques et transcriptionnelles pour prévenir et inverser l'épuisement des lymphocytes t

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
ALSDORF W ET AL: "BNT211: A PHASE I TRIAL EVALUATING SAFETY AND EFFICACY OF CLDN6 CAR-T CELLS AND CARVACMEDIATED IN VIVO EXPANSION IN PATIENTS WITH CLDN6-POSITIVE ADVANCED SOLID TUMORS", HUMAN GENE THERAPY, vol. 33, no. 7-8, 1 April 2022 (2022-04-01), GB, pages A2, XP093033028, ISSN: 1043-0342, Retrieved from the Internet <URL:https://www.liebertpub.com/doi/abs/10.1089/hum.2022.29200.abstracts> DOI: 10.1089/hum.2022.29200.abstracts *
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ANDREATTA, M. ET AL.: "Interpretation of T cell states from single-cell transcriptomics data using reference atlases", NAT. COMMUN., vol. 12, 2021, pages 1 - 19
ARGUELLO, R. J. ET AL.: "SCENITH: A Flow Cytometry-Based Method to Functionally Profile Energy Metabolism with Single-Cell Resolution", CELL METAB., vol. 32, 2020, pages 1063 - 1075
AUSUBEL ET AL.: "Short Protocols in Molecular Biology", 2002, JOHN WILEY & SONS, INC.
CHOPPA, P. C.VOJDANI, A.TAGLE, C.ANDRIN, R.MAGTOTO, L.: "Multiplex PCR for the detection of Mycoplasma fermentans, M. hominis and M. penetrans in cell cultures and blood samples of patients with chronic fatigue syndrome", MOL. CELL. PROBES, vol. 12, 1998, pages 301 - 308
DOBIN, A. ET AL.: "STAR: Ultrafast universal RNA-seq aligner.", BIOINFORMATICS, vol. 29, 2013, pages 15 - 21, XP055500895, DOI: 10.1093/bioinformatics/bts635
ECKER MANUELA ET AL: "SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling", DEPARTMENT OF BIOLOGY, UNIVERSITY OF KONSTANZ, vol. 11, 12 January 2022 (2022-01-12), XP093041102, Retrieved from the Internet <URL:https://cdn.elifesciences.org/articles/67550/elife-67550-v2.xml> DOI: 10.7554/eLife.67550 *
ECKER, M. ET AL.: "SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling", ELIFE, vol. 11, 2022
HAO, Y. ET AL., CELL, vol. 184, 2021, pages 3573 - 3587
HAO, Y. ET AL.: "Integrated analysis of multimodal single-cell data", CELL, vol. 184, pages 3573 - 3587
HEBEISEN, M. ET AL.: "Molecular insights for optimizing T cell receptor specificity against cancer", FRONT. IMMUNOL., vol. 4, 2013, pages 1 - 10
KAMINOW, B.YUNUSOV, D.DOBIN, A.: "STARsolo: accurate, fast and versatile mapping/quantification of single-cell and single-nucleus RNA-seq data", BIORXIV 2021.05.05.442755, 2021
LIAO, Y.SMYTH, G. K.SHI, W.: "FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features", BIOINFORMATICS, vol. 30, 2014, pages 923 - 930, XP055693027, DOI: 10.1093/bioinformatics/btt656
LUN, A. T. L. ET AL.: "EmptyDrops: Distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data", GENOME BIOL., vol. 20, 2019, pages 63
MILLER, B. C. ET AL.: "Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade", NAT. IMMUNOL., vol. 20, 2019, pages 326 - 336, XP036898535, DOI: 10.1038/s41590-019-0312-6
MULLER, P. ET AL., CANCER IMMUNOL. RES., vol. 2, 2014, pages 741 - 755
MULLER, P. ET AL.: "Microtubule-depolymerizing agents used in antibody-drug conjugates induce antitumor immunity by stimulation of dendritic cells", CANCER IMMUNOL. RES., vol. 2, pages 741 - 755, XP055334020, DOI: 10.1158/2326-6066.CIR-13-0198
ROBINSON, M. D.MCCARTHY, D. J.SMYTH, G. K.: "edgeR: A Bioconductor package for differential expression analysis of digital gene expression data", BIOINFORMATICS, vol. 26, 2009, pages 139 - 140, XP055750957, DOI: 10.1093/bioinformatics/btp616
ROMERO, P. ET AL.: "CD8+ T-cell response to NY-ESO-1: Relative antigenicity and in vitro immunogenicity of natural and analogue sequences", CLIN. CANCER RES., vol. 7, 2001, pages 766 - 772
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
SCHIETINGER, A. ET AL.: "Tumor-Specific T Cell Dysfunction Is a Dynamic Antigen-Driven Differentiation Program Initiated Early during Tumorigenesis", IMMUNITY, vol. 45, 2016, pages 389 - 401, XP029687762, DOI: 10.1016/j.immuni.2016.07.011
SCHMID, D. A. ET AL.: "Evidence for a TCR Affinity Threshold Delimiting Maximal CD8 T Cell Function", J. IMMUNOL., vol. 184, 2010, pages 4936 - 4946, XP055653362, DOI: 10.4049/jimmunol.1000173
SKANLAND, S. S.TASKEN, K.: "Carboxyl-Terminal Src Kinase Binds CD28 upon Activation and Mutes Downstream Signaling", J. IMMUNOL., vol. 203, 2019, pages 1055 - 1063
SPAHN, P. N. ET AL.: "PinAPL-Py: A comprehensive web-application for the analysis of CRISPR/Cas9 screens", SCI. REP., vol. 7, 2017, pages 15854
WANG, T.LANDER, E. S.SABATINI, D. M.: "Single guide RNA library design and construction", COLD SPRING HARB. PROTOC., 2016, pages 283 - 288
ZHENG, L. ET AL.: "Pan-cancer single-cell landscape of tumor-infiltrating T cells", SCIENCE, no. 80, 2021, pages 374

Similar Documents

Publication Publication Date Title
Trefny et al. Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy
Mandula et al. Ablation of the endoplasmic reticulum stress kinase PERK induces paraptosis and type I interferon to promote anti-tumor T cell responses
Johnson et al. The immunostimulatory RNA RN7SL1 enables CAR-T cells to enhance autonomous and endogenous immune function
Chen et al. Human Brat ortholog TRIM3 is a tumor suppressor that regulates asymmetric cell division in glioblastoma
US20220135662A1 (en) Novel use of regulatory t cell-specific surface protein lrig-1
Leoncini et al. MicroRNA fingerprints in juvenile myelomonocytic leukemia (JMML) identified miR-150-5p as a tumor suppressor and potential target for treatment
Giladi et al. RTVP-1 promotes mesenchymal transformation of glioma via a STAT-3/IL-6-dependent positive feedback loop
Chen et al. CDK2 inhibition enhances antitumor immunity by increasing IFN response to endogenous retroviruses
CN106574241A (zh) 癌症免疫疗法组合物和方法
CN110709507A (zh) 自然杀伤细胞
CN111166867B (zh) Pd-1泛素化激动剂的功能与用途
JP7225107B2 (ja) ナチュラルキラー細胞
EP3733212B1 (fr) Composition pharmaceutique permettant de prévenir ou de traiter la métastase du cancer vers le poumon, contenant un petit arn interférent de chi3l1 à titre de principe actif
EP3789485A1 (fr) Thérapies cellulaires pour le cancer
WO2020128006A1 (fr) Miarn destiné à être utilisé en thérapie
EP2816107B1 (fr) Procédé d&#39;expansion de cellules souches hématopoïétiques
Khakpoor-Koosheh et al. MicroRNA-124 enhances T cells functions by manipulating the lactic acid metabolism of tumor cells
KR20230004456A (ko) 면역 요법에 대한 종양 세포의 감작화를 위한 방법 및 조성물
CN108697762A (zh) 治疗癌症的组合物和方法
WO2024068010A1 (fr) Cibler snx9 sauve les lymphocytes t recombinés dans le cadre d&#39;une thérapie adoptive
Li et al. Combination epidermal growth factor receptor variant III peptide-pulsed dendritic cell vaccine with miR-326 results in enhanced killing on EGFRvIII-positive cells
KR102352127B1 (ko) Mitf 억제제를 유효성분으로 함유하는 골수-유래 억제세포 저해용 조성물
KR20230084416A (ko) Wnk3 억제제를 유효성분으로 포함하는 면역관문 억제용 조성물
US20150283164A1 (en) Treatment of Myelodysplastic Syndrome by Inhibition of NR2F2
Gordino Regulation of human γδ T cell type 1 functional differentiation by microRNAs

Legal Events

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

Ref document number: 22800610

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22800610

Country of ref document: EP

Kind code of ref document: A1