Field

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.

Background

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. 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. While PD-1/PD-L1 blockade can reinvigorate some T cells, terminal exhaustion still limits the therapeutic efficacy of immune checkpoint blockade (ICB). Adoptive transfer of tumor-infiltrating lymphocytes (TILs) and genetically engineered T cells such as CAR T cells are changing the landscape of available treatments in hematological malignancies and solid tumors such as melanoma and lung cancer. Despite impressive clinical results, the efficacy of adoptively transferred T cells can likewise be compromised by an exhausted state. Thus, most patients with advanced cancers treated with immunotherapies still fail to achieve long-term responses.

The advent of single-cell technologies has re-shaped the understanding of the tumor-infiltrating immune compartment including exhausted T cells in human patients. Yet, these studies mainly provide a descriptive snapshot, while the mechanistic understanding and validation of potential targets for immunotherapy remain difficult. Most attempts to screen genes or molecules for their impact on T cell exhaustion are based on murine T cells that were exhausted either in vivo by chronic infection, ex vivo by repetitive stimulation with anti-CD3 antibodies, antigenexpressing tumor cells, or stimulation under hypoxia. Interestingly, one attempt to impose an exhaustion state in human T cells utilized repetitive stimulation ex vivo with peptide-loaded human dendritic cells, which resulted in a reduction in cytokine secretion, but not degranulation or killing capacity. Recently, ex vivo models have been developed using human tumor explants, which preserve many important features of the tumor microenvironment and mimic patient response. These patient-derived cultures, however, are short-lived with a scarce infiltration of tumor-specific T cells, which hampers extensive screening approaches. Likewise, the low abundance and unknown antigen specificity in TILs freshly obtained from patients hinder more comprehensive mechanistic studies.

To address these challenges, 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. Our findings suggest that 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.

Based on the above-mentioned state of the art, 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.

Summary of the Invention

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.

Terms and definitions

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference 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.”

As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

"And/or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods. The term Chimeric antigen receptor (CAR, also known as chimeric immunoreceptor, chimeric T cell receptor or artificial T cell receptor) in the context of the present specification relates to an artificial receptor protein that has been engineered to give T cells the ability to target a specific antigen. 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.

The term 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. In particular embodiments, the preparation substantially consists of CD3 positive T cells.

Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

Sequences

Sequences similar or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of the invention. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, 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.

In the context of the present specification, the terms 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. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).

One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1 .-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein 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.

Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).

General Molecular Biology: Nucleic Acid Sequences, Expression

The term 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. 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.

The term transgene in the context of the present specification relates to a gene or genetic material that has been transferred from one organism to another. In the present context, 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.

The term 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.

The terms gene expression or expression, or alternatively the term gene product, 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.

The term 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. The term 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. It further includes analogues of 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). Wherever reference is made herein to a hybridizing sequence, such hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.

The term phosphothioate as used herein is synonymous with the terms phosphorothioate and thiophosphate.

The terms 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.

In the context of the present specification, the term 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. In certain embodiments, 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. In certain embodiments, 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). In certain embodiments, the hybridizing sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and/or PNA nucleotides or mixtures thereof.

The term 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. Many sources exist for delivery of the actual physical oligonucleotide, which generally is synthesized by solid state synthesis.

The term 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. 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.

The term siRNA (small/short interfering RNA) 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). According to prevailing theory, the longer dsRNA is cleaved by an endo-ribonuclease (called Dicer) to form double stranded siRNA. In a nucleoprotein complex (called RISC), the double stranded siRNA is unwound to form single stranded siRNA. 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.

The term shRNA (small hairpin RNA) in the context of the present specification relates to an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi).

The term sgRNA (single guide RNA) in the context of the present specification relates to an RNA molecule capable of sequence-specific repression of gene expression via the CRISPR (clustered regularly interspaced short palindromic repeats) mechanism.

The term miRNA (microRNA) 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. Several tools for designing siRNAs, shRNAs, miRNAs, sgRNA and miRNAs are known to a practitioner skilled in the art. These include but are not limited to: siDirect, siRNA Wizard™ i- Score Designer (InvivoGen), PFRED, Silencei® Select siRNA (ThermoFischer Scientific), siMAX siRNA Design Tool (eurofins Genomics) and OriGene for the design of siRNA, Dharmacon™ Gene Knockdown, for designing siRNAs and shRNAs, Dharmacon™ 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.

The term 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. For vectors operating on the level of transcription and subsequent translation, 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. In certain embodiments, the viral genome is packaged into a capsid to become a viral vector, which is able to transduce the target cell.

As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) 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). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "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. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow.

Detailed Description of the Invention

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.

Another related aspect provides the 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.

In certain embodiments, the target cell is a T cell. As 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. In certain embodiments, 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. In certain embodiments, the nucleotide agent of the invention is administered as part of a T cell treatment.

In particular embodiments, 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.

In particular embodiments, the target cell is a recombinant immune effector cell. In particular embodiments, the target cell is a CD28-positive T cell, expressing a transgene encoding a transmembrane polypeptide. In particular embodiments, this transmembrane polypeptide is a T cell receptor polypeptide.

In certain more particular embodiments, this transmembrane polypeptide is a T cell receptor polypeptide capable of recognizing a specific cancer-related antigen in the context of an MHC molecule.

In particular embodiments, 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.

In more particular embodiment, the transmembrane polypeptide is a CAR targeting CD19.

In other more particular embodiment, 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.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting mesothelin.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting PD- 1.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting PD-

L1.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting Her-

2.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting CD22.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting EFGR.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting MUC1.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting a human leutocyte antigen (HLA) molecule.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting CD30.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting CD33.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting CD123.

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting epidermal growth factor receptor variant III (EGFRvlll).

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1).

In other more particular embodiment, the transmembrane polypeptide is a CAR targeting carcinoembryonic antigen (CEA). Nucleic acid agent format

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 ).

In certain embodiments, 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.

In certain embodiments, 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.

Other 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.

In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, the nucleic acid agent is an siRNA. In certain embodiments, 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 .

In certain embodiments, the nucleic acid agent is a miRNA.

In certain embodiments, the nucleic acid agent is shRNA. In certain embodiments, 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. In particular embodiments, 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.

In more particular embodiments, 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.

In more particular embodiments, 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.

In certain embodiments, 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.

In certain embodiments, the recombinant T cell expresses CD28.

In certain embodiments, the recombinant T cell expresses the intracellular domain of CD28.

In certain particular embodiments, 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.

In certain embodiments of the preparation of T cells according to the invention, 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.

In certain embodiments of the preparation of T cells according to the invention in any of its embodiments, the T cells are CAR T cells having a CAR using the intracellular domain of CD28.

In more particular embodiments of the preparation of T cells according to the invention in any of its embodiments, 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.

In certain embodiments of the preparation of T cells according to the invention, the T cells comprise a nucleic acid agent according to the first aspect of the invention. In particular embodiments thereof, the cells comprise an siRNA. In particular embodiments thereof, the cells comprise an shRNA.

In certain embodiments of the preparation of T cells according to the invention, 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.

In certain embodiments of the preparation of T cells according to the invention, 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.

Medical treatment

Similarly, within the scope of the present invention is 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.

Pharmaceutical Compositions, Administration/Dosage Forms and Salts

According to one aspect of the compound according to the invention, 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.

Similarly, a cell therapy product as described herein is provided for use in the prevention or treatment of an immune dysfunction. In particular embodiments, 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. Optionally, 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.

In certain embodiments, the T cell preparation is a preparation of adoptively transferred T cells. In particular embodiments, the condition is cancer.

Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

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. (b) Representative co-expression of PD-1 and TIM-3 measured by antibody staining and flow cytometry on day 12 after the first stimulation, (c) PD-1 expression measured by flow cytometry after antibody staining on day 12 after the first stimulation, n = 4-10 donors, (d) Degranulation measured by CD107a surface exposure during re-stimulation of all cells after 13 days of culture in the indicated conditions, n = 6-12 donors, (e-f) Representative flow cytometry plot and quantification of IFNy and TNFa production measured by intracellular cytokine staining, n = 7-14. (g) Specific lysis of T2-Luc+ tumor cells pulsed with peptide after 5 hours of co-incubation with T cells on day 13 post first stimulation measured by residual luminescence intensity after luciferin addition, n = 6-13 donors, (h) IFNy secretion in pg/ml shown on a log 10 transformed scale measured by ELISA after 4 days co-culture of T _e ff or T _ex with MDA-MB-231 cells loaded with 100nM NY-ESO-1- 9V peptide in presence of either 10 pg/ml human lgG4 isotype control or anti-PD-1 antibody (Nivolumab, hlgG4). Statistics is a paired 2-way-AN OVA with Holm-Sidak correction, n = 5 donors, (i) Heatmap of all differentially regulated genes between the four conditions showing row-scaled log-cpms and clustered using 6 k-means clusters (cluster numbers indicated on the left). Selected genes associated with exhaustion or T cell functionality are labeled on the right, n = 4 donors, (j) Gene set enrichment analysis comparing mRNA expression of the different conditions using camera (edgeR) for the Zheng, et al PanCancer T cell states. 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, (a) Schematic description of the targeted-pooled CRISPR-Cas9 KO screen, (b) Mean Iog2 fold changes for gRNA enrichment in the pooled CRISPR/Cas9 screen. Dots represent the mean enrichment of all 5 guides for each donor biological replicate. Values to the left in red indicate depletion = functional impairment upon KO, and values to the right in blue indicate enrichment = functional improvement upon KO. ZAP70, LAMP1 , and ZAP70 are significant (p < 0.01 ) in a 1-way ANOVA with Dunnett’s correction compared to intergenic controls. The highest-ranking genes did not reach significance (p > 0.05). (c) Representative flow cytometry plots of SNX9 vs. PD-1 and TIM-3 expression in tumor-infiltrating CD8 T cells of a nonsmall cell lung cancer (NSCLC) patient, (d) TIM-3 expression measured by flow cytometry in SNX9 positive vs. SNX9 negative in intratumoral PD-1 + CD8 T cells of NSCLC patients. Statistics are a two-tailed paired t-test. Mean and SD are shown, n = 10. (e) Representative flow cytometry plots and quantification of SNX9 expression measured by antibody staining in flow cytometry of human CD8 T cells of the ex vivo model with the different conditions. Mean and SD are shown, n = 11 . Statistics are a 1-way ANOVA with Holm-Sidak correction (f) Percentage of SNX9+ cells among CD8 T cells for each sample before treatment with ICI. Statistics are Mann-Whitney tests (non-normal distribution). The inventors used 1 log normalized counts as the threshold for SNX9 positivity. Samples with less than ten cells in either population were excluded, resulting in n = 10 non-responders and n = 9 responders, mean and SD are shown. * p<0.05, ** p <0.01 , *** p < 0.00 and **** p < 0.0001 .

Fig. 3 shows SNX9 KO improves effector functions of exhausted T cells and dampens the NFAT-NR4A1/3-TOX axis, (a) Schematic procedure to generate SNX9 KO T cells from healthy human CD8 T cells. 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 . (b) IFNy secretion by ELISA of T _e ff and T _ex in response to re-stimulation with peptide-loaded tumor cells. Dots represent individual healthy donor replicates. Statistics are paired t-tests. n = 10 (T _e tf) and 8 (T _ex ). (c) Top row: example multi-color images of T _ex that were co-incubated with peptide-pulsed T2 cells (CFSE labeled) for 30 min. Shown are spinning disk confocal images at 100x magnification, showing staining of SNX9 (magenta) together with either F-actin (Phalloidin), LAMP1 (CD107a) or Perforin all shown in green. Bottom row: example images of T _e ff electroporated with plasmids encoding CD28-EGFP or TCRz-EGFP fusion proteins, or stained with anti-CD45 all shown in green, together with anti- SNX9 in magenta (overlap generates white color). En face views (front view of the immune synapse) are shown below each image, (d) Translocation of NFATc2 to the nucleus in T _e ff upon stimulation with plate-bound 1 pg/ml anti-CD3 (low aCD3) or 1 pg/ml anti-CD3 + 2.5 pg/ml anti-CD28. Shown is the delta geometric mean between unstimulated and stimulated cells for the similarity metric between DAPI (nucleus) and NFATc2 (calculated in IDEAS). Statistics is a 2-way ANOVA with Holm-Sidak correction, n = 7 donors of n = 2 experiments, (e) Ca ²⁺ flux measured by Calbryte520-AM fluorescence in n = 8 donor replicates of 3 experiments induced by addition of CD2/3/28 beads. The signal was normalized to the first time point after beads addition and the area under the curve (AUC) was calculated for the first 40 min. Statistics is a paired-t-test (f) RT-qPCR quantifications of mRNAfor NR4A1 and NR4A3 in cells on day 6 of the T _ex culture. Ct values were normalized to the housekeeping control HPRT2. Statistics are two-sided paired t-tests. n = 7. (g) Antibody staining of TOX measured by flow cytometry in T _ex . The delta geometric mean to a fluorescence minus one (FMO, background) control is shown, n = 8 (h) Expression of CCR7 measured by flow cytometry antibody staining in T _ex . Statistics is a two-sided paired t-test. n = 8. (i) Geometric mean CD25 upregulation compared to unstimulated cells measured by flow cytometry for T _e ff stimulated 14h with T2 wt or T2 CD80 CD86 KO (T2 KO) cells loaded with 100 nM NY-ESO-9V peptide. Cells additionally received either 10 ug/ml human lgG1 isotype control or 10 ug/ml ipilimumab (anti-CTLA4 blocking antibody). Statistics is a paired 2-way ANOVA with Holm-Sidak correction, n = 6 donor replicates of n = 2 experiments, (j) Geometric mean fluorescence intensity of expanded T cells with or without SNX9 KO stimulated for 14h with the indicated dose of plate bound anti-CD3 (OKT3) and anti-CD28 (CD28.2) antibodies, n = 7 donors of n = 2 experiments. 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. T cell +/- SNX9 KO serum-starved for 1 h were incubated with soluble antibodies (low CD3/28: 1.25pg/ml of both OKT3 and CD28.2, or high CD3 = 2.5 pg/ml OKT3) and 10 pg/ml anti-mouse-Fc-antibodies on ice and then incubated at 37°C for 5min. Statistics are paired 2-way ANOVA with Holm-Sidak correction, n = 4 donors. * p<0.05, ** p <0.01 , *** p < 0.001 and **** p < 0.0001.

Fig. 4 shows Snx9 KO improves anti-tumor efficacy and reduces terminal exhaustion in vivo, (a) Schematic experimental setup of OTI isolation, OVA peptide stimulation, Cas9-RNP electroporation, and subsequent transfer to MC38-OVA bearing mice, (b) Mean and SEM of MC38-OVA tumor volumes in C57BL/6 mice. OTI Snx9 KO or control (intergenic) cells were transferred on day 13 post tumor injection. n= 6 mice per condition. All tumor volume curves are shown until the first mouse per group reached the humane endpoint. Statistics are individually performed 2-way ANOVAs followed by Bonferroni correction, (c) Percentage of PD-1 ^high Tim3+ OTI cells in MC38-OVA tumors in C57BL/6 mice on the indicated timepoints, n = 6 intergenic and n = 4 Snx9 KO, because one was excluded due to absence of OTI cells and one due to ulceration, (d) UMAP representation of single cell RNA sequencing data from intergenic and OTI Snx9 KO cells isolated from MC38-OVA tumors 13 days post transfer. OTI cells from five to six mice per condition were pooled before 10x library construction, (e) Dot plot for average expression of selected marker genes for different categories (color) while size indicates the percentage of cells with detected expression for each cluster, (f) Proportions of each cluster in the intergenic versus the Snx9 KO sample (n = 1 from 5-6 pooled mice per condition), (g) Dot plot of selected differentially expressed genes between intergenic and OTI Snx9 KO cells in each cluster. Size indicates the -Iog10 adjusted p-value and the color the mean Iog2 fold change, (h) quantification of endogenous CD8 T cells (left) and cDC1 s (CD11c+ MHCII+ F4/80- CD103+, right) in MC38-OVA tumors 3 days post transfer of OTI cells with and without Snx9 KO. Statistics are unpaired t-tests. n = 6 mice per group (i) Serum levels of IFNy and 11-10 for days 2,8 and 15 measured using the Murine Virus Response Legendplex system (Biolegend). The limit of detection (LOD) is indicated for IFNy. Statistics are unpaired 2-way ANOVA with Holm-Sidak correction, n = 6 mice per group, (j) Tumor volume of NSG (Nod-SCID-gamma) mice with subcutaneous MC38-OVA tumors with a transfer of OTI cells at day 12 post injection. n= 6 mice per condition except n = for untreated. Statistics are individually performed 2-way ANOVAs followed by Bonferroni correction * p<0.05, ** p <0.01 , *** p < 0.001. Fig. 5 shows deletion of SNX9 improves CAR T cell anti-tumor efficacy, (a) Schematic representation of the CAR T cell transfer experiments. 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. Statistics are 2-way ANOVAs followed by Bonferroni correction, (c) survival of these mice (until humane endpoint of 1500 m ³ tumor size). Statistics are log-rank Mantel-Cox tests followed by Bonferroni correction, (b and c): n = 8 for untreated, n = 7 for intergenic and SNX9 KO CAR T conditions, (d) Human cytokines measured by Legendplex (Biolegend) in the serum of Raji-bearing NSG mice treated with anti-19-28z-CAR T cells with and without SNX9 KO. Statistics are paired-2-way ANOVA with Holm-Sidak correction, n = 6 mice per condition, (e) Tumor volume in mm ³ of NSG mice treated 3 days post Raji tumor injection by i.v. transfer of 1 Mio human CD8+ CD28 KO anti-CD19-BBz CAR T cells with or without SNX9 KO. n = 6 for intergenic and SNX9 KO, n = 8 for untreated. Statistics are 2- way ANOVAs followed by Bonferroni correction. * p<0.05, ** p <0.01 , *** p < 0.001 .

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. T _res t and T _tU mor conditions were performed as controls, n = 4 donors of 2 experimental replicates. 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 (e) Degranulation capacity measured by CD107a exposure upon re-stimulation with T2 tumor cells pulsed with peptide. Measurements were performed before and after seven days of resting, n = 4 (d-e) 2-way ANOVA with Holm-Sidak correction, (f) Specific killing of MDA-MB-231 tumor cells in a 4-day co-culture assay with Teff and Tex in presence of 100 nM NY-ESO- 9V peptide. The co-culture was conducted in presence of either 10 pg/ml Nivolumab (anti-PD-1 blocking antibody) or hlgG4 (isotype control). Statistics are a paired 2- way ANOVA with Holm-Sidak correction, n = 5 (g) Fold expansion of T _ex cells after the four rounds of stimulation compared to input on day 0. n = 5 (h) Log2 fold change of cell numbers of the different conditions after re-stimulation with T2 tumor cells and peptide compared to expansion in IL2 alone within six days. Measured by flow cytometry on day 19 (six days post stimulation) using precision counting beads. 1- way ANOVA with Holm-Sidak correction, n = 6 donors of n = 2 experiments, (i) Expression of PD-1 , co-production of IFNy and TNFa and degranulation capacity of NY-ESO-1 specific T cells stimulated with NA8 melanoma cells for the indicated conditions. For degranulation and cytokiens, the cells were re-stimulated for 5h with NY-ESO-9V loaded T2 cells in the presence of an anti-CD107a antibody and Monensin. 1-way ANOVA statistics with Holm-Sidak correction, n = 4 (j) Principal component analysis of individual replicates (n = 4 healthy donors) for the four investigated conditions. Axis indicate components and percentage of variance explained by these, (k) boxplots of the Iog2 cpm expression of selected genes among the four conditions for the n = 4 biological replicates. (I) ISMARA transcription factor activity scores. Shown are the average and SD activity of 4 donor replicates. Statistics are 1-way ANOVAs with Holm-Sidak correction, only significant differences are shown, (m) Column clustered heatmap showing row-scaled Iog2- cpm expression of the top 100 genes of the Zheng et al Tex-term gene set. Colors indicate the comparisons as in k. * p<0.05, ** p <0.01 , *** p < 0.001 , **** p < 0.0001 .

Fig. 7 shows (a) mRNA expression of SNX9, SERPINE1 and PHEX in Teff with the indicated genes knocked out by Cas9-crRNA-tracrRNA electroporation. Shown are -dtCt values to the HPRT1 house keeping control gene. Statistics are paired-t-tests. (b) Percentage of CD107a+ cells after 4h of restimulation of Tex with the indicated KOs normalized to the intergenic control of the same donor (to account for donor- to-donor variation). Statistics are 1 -sample t-tests against Ho = 1 . (c) Western blot of SNX9 protein levels in T _re st, T _e ff and T _ex conditions shown against ERK2 as a stable loading control for n = five different healthy donors. To the right, quantification of this blot using band densitometry in Fiji. Statistics are 1-way ANOVA with Holm-Sidak correction, (d) Fluorescence intensity (area) of SNX9 staining in T _e tf measured by flow cytometry. Shown is a control which was not stained with the primary antibody (“no primary), an isotype control (rabbit IgG) and the rabbit-anti-SNX9 antibody, (e) additional example plots showing PD-1 or TIM-3 versus SNX9 protein staining using flow cytometry of NSCLC infiltrating CD8 T cells. Shown as contour plots with 5% lines and including outliers, (f) Single-cell ATAC seq tracks displaying open chromatin regions from BCC TILs of Satpathy et al. are displayed using the Washll Epigenome Browser. The human SNX9 locus is shown. (RefSeq annotation of hg19) with the transcriptional start site found around the prominent OCR region to the left. An arrow indicates the OCR specific to exhaustion, Treg and Tfh specific at approx. +7 kbp of the TSS. (g) 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, (h) Mean log normalized counts and SD for CCR7, TCF7, TOX, and TOX2 in SNX9+ versus SNX9- CD8+ T cells. Expression of PDCD1 , HAVCR2, CCR7, TOX, TOX2, TCF7, NR4A1 , NR4A2 and NR4A3 in the scRNAseq data set by Sade-Feldman et al. Mean log counts of CD8 T cells for the two subsets (SNX9- and SNX9+, defined by> 1 log count) are shown per sample. Samples with less than ten cells in either population were excluded. Statistics are Mann-Whitney tests (non-normal distribution). Dots represent individual patient samples. Mean and SD are indicated. * p<0.05, ** p <0.01 , *** p < 0.001 , **** p < 0.0001 .

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. Statistics are paired t-tests. (d) Degranulation capacity for T _e ff (left) and T _ex (right) after re-stimulation with T2 + peptide shown as delta unstimulated geometric mean fluorescence intensity. Statistics are paired two-sided t-tests (e) Additional single-z slices of spinning disk confocal images for NY-ESO-1 specific cells co-incubated with NY-ESO-9V peptide- pulsed T2 cells for 30min. In the actin and LAMP1 images, tumor cells were stained with CFSE. All images show anti-SNX9 staining in magenta. 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. (f) Example images of the NFATc2 nuclear translocation readout using an Imagestream MK-IL Shown are an example of a cell that shows non-translocated and translocated NFATc2 signal towards the nucleus (in DAPI). (g) Calcium flux peak normalized to baseline. Shown in connected dot plots are the maximum peak intensities normalized to baseline and the raw baseline values for the Calbryte520- AM signal (before beads addition). Statistics are paired two-sided t-tests. (h) SNX9, NR4A2 and LDHA mRNA quantification by qPCR of T _ex at day 6 of culture. Shown are -dtCt values to the housekeeping control (HPRT1 ). Statistics are paired two- sided t-tests. n = 7 donors of n = 2 experiments, (i) Glucose dependence and FAO/AAO values from the SCENITH flow cytometry single cell metabolism assay. On the right, representative flow cytometry histogramsfor anti-puromycine-AF488 for the four conditions used to calculate the glucose dependence vs FAO/AAO are shown ⁸⁹ . Statistics are two-sided paired t-tests. (j) Histograms of flow cytometric determination of CD80/86 levels on T2 wt and T2 CD80 CD86 cell lines, (k) Geometric mean intensity of CD25 signal measured by flow cytometry for the unstimulated controls in the antibody titration assays, n = 6 healthy donors of n = 2 independent experiments. Statistics is a two-sided paired t-test. (I) Geometric mean intensities of CD28 and TCRP13.1 (variant of the NY-ESO-1 specific TOR) measured by flow cytometry on the surface of T _e ff- Statistics are paired two-sided t- tests. n = 10 healthy donors of n = 2 independent experiments, (m) Geometric mean area of fluorescence for anti-p-AKT-Ser473 shown as delta unstimulated controls measured by flow cytometry. T _e ff With or without SNX9 KO were stimulated for 30 min with plate-bound antibody (low CD3 + CD28 = 1.25pg/ml OKT3 + 2.5 pg/ml CD28.2, high CD3 = 5 pg/ml OKT3). Statistics is a paired-2 -way ANOVA with Holm- Sidak correction, n = 5 healthy donors. * p<0.05, ** p <0.01 , *** p < 0.001 , **** p < 0.0001.

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, (c) Tumor growth curve in mm ³ of MC38-OVA bearing tumors in C57BL/6 mice with a transfer of P14 (LCMV gp33 specific T cells) with and without a Snx9 KO. Statistic is a 2-way ANOVA. (d) Tumor weight of MC38-OVA tumors at timepoints for the flow cytometry analysis of intratumoral OTI cells. Statistics are 2-way ANOVA with Holm-Sidak correction, (e) Numbers of adoptively transferred OTI T cells found in the tumor (identified by CD45.1 , adjusted by Precision counting beads) and normalized per gram of tumor weight, n = 6 (for Snx9 KO at d 13 n = 5 due to ulceration), (f) Percentage of PD-1 ^high and Tim-3+ OTI cells at different timepoints post transfer. Statistics are 2-way ANOVA with Holm-Sidak correction, (g) Flow cytometry plots showing PD-1 versus TIM-3 for the samples on day 13 post OTI transfer shown in h. * p<0.05, ** p <0.01 , *** p < 0.001 , **** p < 0.0001.

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. (e) Numbers of the indicated immune cell subsets in MC38-OVA tumors of C57BL/6 mice three days post OTI transfer with or without Snx9 KO. Numbers were corrected by Precision beads and normalized to the tumor weight, n = 6. Statistics are paired two-sided t-tests. (f) Serum cytokines in MC38-OVA bearing C57BL/6 mice at the indicated times post OTI transfer with or without Snx9 KO. n = 6 mice per condition, (g) Surival curve (humane endpoints) for NSG mice with MC38-OVA tumors with a transfer of OTI T cells 12 days post tumor injection. n= 6 mice per condition and statistics are Bonferroni-adjusted Mantel-Cox log-rank tests. * p<0.05, ** p <0.01 , *** p < 0.001 , **** p < 0.0001 .

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. Statistics are paired-2-way ANOV As with Holm-Sidak correction, n = 6 mice per condition, (d) Histograms for fluorescence intensity of CD28 on the left and SNX9 on the right for the indicated conditions measured by flow cytometry for anti-CD19-BBz CARs. A control without the primary anti-SNX9 is included. * p<0.05, ** p <0.01 , *** p < 0.001 , **** p < 0.0001 .

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.

Examples

Example 1: Ex vivo repetitive antigen-specific stimulation of T cells results in exhaustion

To develop an antigen-specific ex vivo model for human T cell 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 hypothesized that repetitive stimulation with antigen presented on tumor cells would lead to T cell exhaustion. Therefore, the inventors repetitively stimulated transduced antigen-specific T cells using HLA- A2 positive T2 tumor cells loaded with 1 pM NY-ESO-1 peptides for four cycles in three-day intervals (T _ex , Fig. 1 a). 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).

Both 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). However, only 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). At the same time, 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). Confirming the inventors’ results with repetitive T2 lymphoma line stimulation, 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). The inventors concluded that T _ex generated by repetitive antigen-specific stimulation ex vivo functionally resemble human TILs in their reduced capacity to degranulate, secrete cytokines, and proliferate ² . 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. Compared to T _res t and T _tU mor cells, 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. Along this line, 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). Additionally, the inventors used 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).

Next, the inventors performed gene set enrichment analyses for the comprehensive ‘PanCancer’ T cell gene signatures from Zheng et al. (Zheng, L. et al. Science (80-. ). 374, 2021 ). Ttumor cells enriched mainly for the Temra signature, while both Teff and T _ex showed high enrichment in the Tex-term signature (Fig. 1j). This is likely explained by the genes in the Texterm signature, which are associated with activation and cell cycle such as IL2RA (CD25), GZMB, TNFRSF9 and CCND2 (Fig. 6I). While 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

To discover genes regulating 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 inventors then sequenced the gRNAs of each sample (n = 5 biological donor replicates) and used the PinAPL- py platform for gRNA mapping and counting (Spahn, P. N. et al. Sc/. Rep. 7, 15854, 2017). For each donor replicate, the inventors calculated the mean Iog2 fold change of the n = 5 guides per gene in both cell fractions. The median of these values for n = 5 donor replicates was then used to rank the genes (Fig. 2b). As expected, the three controls with known essential functions in T cell functionality (LAT, LAMP1 , and ZAP70) showed the highest negative enrichment (p < 0.01 , 1-way ANOVA). The highest-ranking genes for positive enrichment of gRNAs in the functional fraction of cells included P2RY1 , SNX9, SERPINE1 , and PHEX (Fig. 2b, ns, p > 0.05). None of these genes had been associated with changes in T cell proliferation in the CRISPR screen by Shifrut and colleagues (P2RY1 KO had shown a trend towards lower proliferation, p = 0.057). Initial validation experiments were performed for these top ranked genes using crRNA-tracrRNA-Cas9 electroporation of the guides with the highest Iog2 fold change. Unlike for PHEX and P2RY1 , lower gene expression after electroporation was achieved for SNX9 and SERPINE1 (Fig. 7a). Repetitive stimulation of individual KO T cells confirmed higher CD107a degranulation in SNX9 KO T _ex (Fig. 7b).

Based on the individual gene KO data, the inventors decided to further investigate SNX9. First, 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). Furthermore, the inventors found that SNX9 is significantly co-expressed with TIM-3 among PD-1 + TILs from NSCLC patients (Fig. 2c-d, Fig. 7e). Mining a published single-cell ATACseq dataset of TILs from cancer patients, the inventors found an open chromatin region (OCR) 7 kbp downstream of the transcriptional start site of the SNX9 gene with increased accessibility in intermediate and late-exhausted compared to naive, memory, effector, and early exhausted CD8 T cells (Fig. 7f). This could suggest that epigenetic reprogramming associated with T cell exhaustion contributes to elevated SNX9 expression. Next, the inventors re-analyzed published single-cell RNA sequencing data of melanoma TILs. Within the CD8 tumor-infiltrating T cells cluster, 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. As TCF7+ CD8 TILs correlate with response to ICB ¹⁷ , the inventors investigated whether SNX9 expression would be associated with therapy resistance. In this cohort of melanoma patients, the percentage of SNX9+ cells among CD8 T cells before treatment correlated with poor response to ICB (Fig. 2f). These findings further strengthened the inventors’ hypothesis that SNX9 is involved in T cell exhaustion. Example 3: SNX9 KO improves effector functions of exhausted T cells and dampens the NFA T-NR4A 1/3-TOX axis

To mechanistically understand the contribution of SNX9 to the exhausted T cell state, the inventors knocked out SNX9 by Cas9-crRNA-tracrRNA electroporation and confirmed lower SNX9 protein levels by flow cytometry and Western blot (Fig. 3a, Fig. 8a-b). Cell expansion was unchanged by SNX9 KO, both for T _e ff and T _ex (Fig. 8d). As expected from the inventors’ CRISPR-Cas9 screen and the initial validation, SNX9 KO increased degranulation and IFNy secretion in T _ex , whereas reduced IFNy secretion was observed for SNX9 KO T _e ff (Fig. 3b, Fig. 8d). In agreement with reports on the role of SNX9 in CD28 signaling in cell lines, the inventors observed clustering of SNX9 at active immune synapses in primary human CD8 T cells with a qualitative co-localization of SNX9 with the central supramolecular activation cluster (cSMAC) components TCRz (CD3Q and CD28 (Fig. 3c, Fig. 8e). The inventors observed only marginal co-localization with the distal SMAC (dSMAC) component CD45, or with LFA1 and lytic granules. These experiments indicate that SNX9 may be implicated in regulating T cell activation at the immune synapse.

Next, the inventors asked whether SNX9 KO impacts signaling through the Ca ² 7NFAT pathways, as previously reported for cell lines ^{41 ,42} . Utilizing T _e ff cells, the inventors observed lower NFATc2 nuclear translocation and decreased Ca ²⁺ flux in SNX9 KO cells upon anti- CD3+anti-CD28 stimulation (Fig. 3d-e, Fig. 8f-g). 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. 3f-g, Fig. 8h). In addition to NFAT activation, CD28 activation is known to promote glycolysis and proliferation, which are, however, also linked to terminal differentiation. Therefore, the inventors further investigated how 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). Moreover, T _ex SNX9 KO cells maintained elevated expression of the central-memory associated receptor CCR7 (Fig. 3j). Overall, the inventors’ data suggests that 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 hypothesized that the observed reductions in Ca ² 7NFAT signaling, glycolysis, and terminal differentiation in SNX9 KO T cells might be explained by reduced CD28 signaling. Therefore, the inventors sought to investigate if SNX9 specifically affects CD28 signaling in the inventors’ experimental system with primary tumor-antigen specific T cells. For this purpose, the inventors generated T2 tumor cells with a double KO of both CD28 ligands CD80 and CD86 (termed “T2 KO”) (Fig. 8j). 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. As expected, stimulation with T2 KO resulted in lower 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. Identical results were obtained when a blocking CTLA4 antibody was added to the co-culture to avoid CTLA-4 inhibitory signaling through CD80/86. No effect of SNX9 KO was observed on CD25 expression in unstimulated T _e ff (Fig. 8k). SNX9 KO T _e ff cells showed decreased CD28 surface expression while expression of TCRp was not affected (Fig. 8I). In summary, the inventors provide evidence that a loss of SNX9 in primary tumor-antigen specific T cells reduces activation in the context of intact CD28-CD80/86 signaling. In the absence of the latter, T cell activation was independent of SNX9 expression.

The inventors sought to confirm these results in a reductionist antibody-based stimulation assay. To this end, 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). At an intermediate level of anti-CD3 (625 - 1250 ng/ml), the inventors observed increased CD25 expression when combined with anti-CD28 co-stimulation (Fig. 3j). In this intermediate anti-CD3 plus anti-CD28 stimulation condition, SNX9 KO T cells showed reduced CD25 upregulation, which is compatible with the inventors’ experiments above revealing CD28-dependent effects of SNX9. Unexpectedly, when increasing the level of anti-CD3 to a saturated range (2500 ng/ml), the inventors observed reduced CD25 upregulation on SNX9 KO T cells even in the absence of anti-CD28. In agreement, the inventors also observed lower PLCyl phosphorylation (upstream initiator of calcium/NFAT signaling) with SNX9 KO cells in response to low anti-CD3 + CD28 stimulation as well as high anti-CD3 stimulation alone (Fig. 3k). Contrastingly, the inventors did not observe an effect of SNX9 KO on the phosphorylation of AKT (downstream mediator of PI3K pathway) upon either stimulation (Fig. 8m). In summary, the inventors’ results suggest that SNX9 amplifies CD28- dependent upregulation of CD25 in the context of intermediate level TCR/CD3 signaling, while saturated TCR/CD3 stimulation provokes CD28-independent effects of SNX9 on CD25 and PLCyl .

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. To this aim, 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). No therapeutic effects of Snx9 KO were observed with P14 T cells, which recognize the LCMV gp33 peptide (Fig. 9c). These results demonstrate that Snx9 KO improves antigen-specific anti-tumor efficacy of adoptively transferred OTI T cells.

The inventors wanted to better understand how Snx9 KO improves anti-tumor efficacy in vivo, and, therefore, characterized OTI number and phenotype in MC38-OVA tumor-bearing mice. While the frequency of OTI cells in the tumor was unchanged (Fig. 9d-e), Snx9 KO OTI cells at day 13 post transfer co-expressed less PD-1 and Tim-3, which could indicate a less exhausted T cell state (Fig. 4c, Fig. 9f-g). To further investigate this, the inventors performed single-cell RNA sequencing analyses of intratumoral OTI cells 13 days post adoptive transfer. Our analysis pipeline in Seurat resulted in seven clusters ranging from naive-like cells to terminally exhausted populations, which the inventors termed based on differentially expressed genes, transcription factor expression, and gene set enrichment for published cell populations (Fig. 4d-e, Fig. 10a-c). Snx9 KO OTI cells were less frequently found in the Texterm cluster (terminally exhausted, high in Tox and Lag3), while they were enriched in Tern (effector-memory-like, high in Gzmb, Gzmc, Cxcr6, and Ly6c2) and the Tex-prolif clusters (proliferating exhausted T cells, high in Nme1 ).

The inventors next investigated the differentially expressed genes between Snx9 KO and intergenic OTI cells within each cluster and among all cells. First, the inventors observed that Snx9 itself and Il2ra (encoding CD25) were downregulated among multiple clusters, confirming the inventors’ ex vivo findings with human T cells (Fig. 4g, Fig. 10d). Further, 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. In two Tex clusters, the inventors found higher expression of Cxcr6, which is associated with effector functions and tumor infiltration. Unlike in the Tex-prolif cluster, the inventors observed reduced Tox expression in Snx9 KO OTI cells within the effector-memory-like Tern cluster. Additionally, Nr4a2 was reduced in the Tex-prolif cluster and among all cells, while Nr4a3 was changed only considering all cells. Nr4a1 and Ifng were not significantly changed, and 1110 was not detected. These results suggest that Snx9 KO OTI T cells express less IL2RA and Nr4a2/3 while they upregulate several genes associated with effector-memory differentiation and antitumor immunity.

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

As the inventors observed improved efficacy of Snx9 KO in murine adoptive transfer models, the inventors investigated whether SNX9 KO would also improve the efficacy of human CAR T cells in xenograft models. To test this, 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). Compared to the intergenic control, CART19-28z SNX9 KO cells improved long-term antitumor control and survival (Fig. 5b-c). Injecting three times the number of CARs did not improve the survival for CART19-28z intergenic, while CART19-28z SNX9 KO again led to higher survival (Fig. 11 a-b). Notably, the transfer of CART19-28z SNX9 KO was accompanied with increased serum levels of anti-tumor effector molecules IFNy, Perforin, and Granulysin, while the inventors detected a decrease in IL10 and IL6 (Fig. 5d, Fig. 11 a-b, human CCL5 could not be detected in the serum). To investigate the dependency on CD28 signaling in vivo, the inventors used a 4-1 BB domain-containing anti-CD19 CAR and additionally knocked out the endogenous CD28 (“CART19-BBz CD28 KO”, Fig. 11 d). The transfer of CART19-BBz CD28 KO intergenic induced delays in Raji tumor growth comparable to CART 19-28z intergenic (Fig. 5b, e), while the inventors did not observe an additional effect of SNX9 KO with CART19-BBz CD28 KO cells. Overall, this suggests that the effects of SNX9 KO in CAR T cells in vivo depend on the presence of CD28 signaling.

Example 6: Discussion

The inventors here developed a human ex vivo model for antigen-specific T cell exhaustion using primary CD8 T cells. This allowed the inventors to perform a targeted CRISPR-Cas9 screen for the identification of genes that regulate T cell exhaustion. The inventors thereby discovered that SNX9 promotes T cell activation through NFAT-NR4A1/3. Conversely, SNX9 KO T cells maintained effector functions and elevated expression of memory associated markers despite repetitive antigenic stimulation. The in vivo anti-tumor efficacy of adoptively transferred murine TCR transgenic T cells was improved by Snx9 KO, which correlated with increased effector-memory-like differentiation, enhanced chemokine expression, and elevated IFNy serum levels. Similarly, 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. Moreover, 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. Prospectively, 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.

Pooled 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. Recently, a genome-wide CRISPR/Cas9 screen in a murine polyclonal stimulation model to discover potential regulators of T cell exhaustion has been reported. Snx9 was not discovered in this screen and the inventors speculate that reasons for this include the lack of T cell - tumor cell interactions and their proliferation readout. Another exciting possibility for future studies utilizing the inventors’ model system might be the use of gain-of-fu notion CRISPR screens, as recently reported for murine CAR T cells.

Our targeted CRISPR-Cas9 screen revealed 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. They described interactions of SNX9 with the PI3K subunit p85 and PI(3,4,5)P3via its PX domain, while a more recent human CD28 interactome lists a direct interaction of SNX9 with the cytosolic part of CD28 (Skanland, S. S. & Tasken, K. J. Immunol. 203, 1055-1063, 2019). In agreement with its known functions linking different cytoskeletal proteins and membrane lipids, Ecker et al. showed that SNX9 contributes to positioning and stabilization of CD28 clusters at the immune synapse in the CD4 Jurkat T cell line (Ecker, M. et al. Elife 11 , 2022).

The inventors’ experiments utilizing a human antigen-specific model system (Fig. 3i) and the adoptive transfer of CAR T cells (Fig. 5a-c) indicate that the effects of SNX9 KO are largely CD28-dependent. However, with saturated levels of TCR/CD3 stimulation, SNX9 KO appears to lower T cell activation even in the absence of CD28 co-stimulation. A possible explanation for this observation is that the effects of SNX9 depend on synaptic PI(3,4,5)P3 accumulation levels induced upon strong TCR/CD28 stimulation. The latter may not be reached without CD28-costimulation at physiologically relevant TCR/CD3 stimulation strenghts. This is also supported by findings of Ecker et al. who observed lower but significant recruitment of SNX9 to the immune synapse already with low-dose anti-CD3 stimulation alone. Intriguingly, the inventors did not find evidence that 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.

The inventors discovered that while SNX9 KO reduces T cell activation, it also enhances antitumor immunity. At first, this is surprising given that current approaches to alleviate T cell exhaustion mainly aim to increase co-stimulation or decrease co-inhibition. For example, 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. On the other hand, there is mounting evidence that lack of co-inhibition during antigenic stimulation can lead to T cell exhaustion. For example, the genetic absence of PD-1 increases T cell exhaustion in mice, presumably due to overactivation. These findings imply that an optimal balance of co-stimulation is necessary to achieve and maintain functional, tumor-specific effector T cells. SNX9 KO appears to induce a small but significant decrease in NFAT signaling and is therefore an attractive way to finetune T cell activation.

Mechanistically, the inventors’ ex vivo results show that SNX9 KO T _ex have a lower expression of NR4A1, NR4A3, and TOX, which have been implicated in T cell exhaustion. In vivo, the inventors also found that Snx9 KO OTI cells express lower levels of Nr4a2 and Nr4a3, while effects on Tox expression were less consistent. The inventors discovered that 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. Along this line, 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. In summary, SNX9 KO alleviates exhaustion, increases chemoattraction, and prolongs IFNy secretion of tumor-specific T cells.

Despite its upregulation in exhausted TILs, SNX9 is rather ubiquitously expressed in humans. Thus, a knockout of SNX9 ex vivo, e.g. in TILs and genetically engineered T cell products appears to be an ideal strategy for clinical use. As a proof-of-concept, the inventors showed that Snx9 KO improved the anti-tumor efficacy of adoptively transferred TCR-transgenic T cells and human CAR T cells. The inclusion of costimulatory domains, e.g. derived from CD28 or 4- 1 BB, is required for optimal CAR T cell persistence, cytokine production, and tumor rejection in vivo. There is evidence that addition of a CD28 domain drives effector memory differentiation with metabolic reprogramming towards aerobic glycolysis. This is in line with the inventors’ findings for SNX9 KO T cells, which exhibited less CD28/TCR signaling, lower glucose dependence and increased expression of CCR7. Several studies have demonstrated that CAR T cells with a CD28 costimulatory domain release higher quantities of cytokines that may result in adverse effects. Notably, in the NSG mice treated with SNX9 KO CART19-28z, the inventors found lower serum levels of IL6, one of the main cytokines involved in CAR T-mediated cytokine release syndrome. Therefore, it is intriguing to speculate that lowering TCR/CD28 signaling, e.g., by SNX9 KO, may also reduce the toxicities associated with CAR T cells.

In summary, 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.

Example 7: Material and Methods

CRISPR-Cas9 Guide Sequences

Table 1 :

Nomenclature

According to the HUGO convention, 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 ).

Peptides

Peptides were purchased in >95% purity from EZ Biolabs. Lyophilized peptides were resuspended at 10mM in sterile dimethyl sulfoxide (DMSO) and stored at -20°C until use. The endogenous NY-ESO-1 peptide SLLMWIQC (SEQ ID NO: 16320) was shown to elicit half- maximal response by LAU155 TCR transduced T cells at an EC50 of 12 nM (Romero, P. etal. Clin. Cancer Res. 7, 766-772, 2001). 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.

Cell Culture Media

For the culture of T2, Jurkat and NA8-Mel cells, RPMI1640 (Sigma) was supplemented with 10% heat-inactivated (56°C 30min) fetal calf serum (PAN Biotech), 100 ng/ml penicillin/streptomycin (Sigma), 2 mM L-Glutamine (Sigma), 1 mM Sodium Pyruvate (Sigma), 1 % MEM non-essential amino acids (Sigma), 50 nmol/l beta-mercaptoethanol (Thermo Fisher) and 10mM HEPES (Sigma). In later experiments T2, Jurkats, Raji and NA8- Mel cells were cultured in the media described above but replacing the 10% FCS with 2% FCS and 10% of Panexin Basic FCS Replacement (PAN Biotech) which results in identical growth kinetics.

For the culture of human T cells, 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.

For the culture of HEK293T cells, DMEM (Sigma) was supplemented with 5% 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).

Cell Lines

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. T2, Jurkat, Raji and NA8-Mel cells were cultured in supplemented RPMI as described in Section Cell Culture Media 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. Probes 12, 301-308, 1998) after every freeze-thaw cycle and then passaged every 2-3 days for a maximum of 15 passages. For adherent cell lines, TrypLE Express (recombinant Trypsin replacement, Thermo Fisher) or 0.05% Trypsin-EDTA (Thermo Fischer) was used.

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).

Tumor models

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.

Cellular tumor assessment

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. 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. Cells were then incubated with fluorophore-conjugated antibodies directed against cell surface antigens in Brilliant Stain buffer (BD) for 20 min at 4°C, washed, and fixed and permeabilized using Foxp3/transcription factor staining buffer set (eBioscience) prior to incubation with antibodies directed against intracellular antigens in FACS buffer. Cell populations were analyzed on a Cytek Aurora. The following gating strategies were used: 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 : live singlet CD19- Ly6G+ CD11 b+; Macrophages: live singlet CD19- Ly6G- CD45.2+ CD45.1- CD11 b+ F4/80+ Ly6Ci _0W ; M2 macrophages: live singlet CD19- Ly6G- CD45.2+ CD45.1- CD11 b+ F4/80+ Ly6Ci _0W CD206+; Monocytes: live singlet CD19- Ly6G- CD45.2+ CD45.1- CD11 b+ F4/80i _ow Ly6Chigh;

Isolation of Primary Immune Cells

Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Histopaque-1077 (Sigma) from buffy coats obtained from healthy blood donors (HD) (Blood Bank, University Hospital Basel). Briefly, 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.

Fresh tumor tissues were collected from patients with NSCLC undergoing surgery at the University Hospital Basel, Switzerland. The study was approved by the local ethical review board (Ethikkommission Nordwestschweiz, EK321/10), and all patients consented in writing to the analysis of their tumor samples. Tumor lesions were mechanically dissociated and digested using accutase (PAA), collagenase IV (Worthington), type V hyaluronidase from bovine testes (Sigma), and DNAse type IV (Sigma), directly after excision. Single-cell suspensions were prepared and frozen as above. For the SNX9 staining in NSCLC patient TILs, the median age at resection was 70.6 years, average 69.9 years (min-max: 54.6-83 years). 6 patients were male and 5 female.

T Cell Receptor Construct

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). This TCR has a KD = 21.4 pM for its endogenous NY-ESO-1 SLLMWITQC (SEQ ID NO: 16319) peptide.

Generation of Lentivirus

To generate lentivirus, 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. After two days, supernatants were collected from cells (exchange medium on plates) and filtered through a 0.45 pm PES filter. Supernatants were stored for 1 day at 4°C until the second batch of supernatant was collected 24 h later. The supernatant containing lentiviral particles was concentrated by ultra-centrifugation at 40’000 x g for 2 h at 4°C, resuspended in 0.1 % BSA in PBS, and frozen to -80°C. To ease production of antigen specific T cells, 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).

Transduction of Human T Cells

To generate NY-ESO-1 TCR specific T cells, human healthy donor PBMCs were thawed and washed in PBS. 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. These beads were washed in medium and added to the CD8 T cells at a 1 :1 ratio together with 150 U/ml IL-2. 24 h later, NY-ESO-1 TCR lentiviral particles produced as described above were added at a multiplicity of infection (MOI) of 2. Cells were then expanded every 2 days with fresh medium and replenishing 50 U/ml IL-2 for 5 days. The percentage of transduced cells was then calculated by staining for TCR Vbeta13.1 + T cells in comparison to non-transduced cells. 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).

Repetitive Stimulation

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. For T _res t, 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.

Immunofluorescence Staining for Flow Cytometry

At the indicated time points, 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. For surface staining, cells were washed and resuspended in FACS buffer (PBS supplemented with 2 mM EDTA, 0.1 % Na-Azide, 2% FCS), and stained with the appropriate antibodies for 30 min at 4°C. All antibodies used in this study are listed above. For intracellular (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. For staining of nuclear proteins, 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). For the staining for 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). Data were collected using the BD FACS Diva Software version 7 (for Fotessa), Beckmann Culture CytExpert, or SpectraFlow (for Aurora) and further analyzed with FlowJo v10.1.6 (Tree Star Inc.) and GraphPad Prism v8 (GraphPad Software Inc.). All results unless indicated show integrated fluorescence area on a biexponential scale.

Flow Cytometry-based Cell Sorting

For cell sorting, cells were kept on ice, washed in PBS, and stained with appropriate antibodies for 30 min at 4°C in PBS + 2% FCS and 2 mM EDTA (without Azide). Antibodies targeting CD14, CD11 b, CD4 and CD19 were used to gate out potentially contaminating other immune cell populations. Following incubation, cells were washed, resuspended in the same buffer and filtered through a 70 pm mesh. Sorting of cells was performed using a FACSAria III or FACS SorpAria (BD), and the purity of sorted populations was routinely tested to be >98%. Degranulation and Cytokine Production Assay

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. For this purpose, T2 cells were incubated at a density of 1 mio/ml with the respective peptides diluted in full RPMI supplemented medium. Afterward, 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). Following incubation, the cells were stained for dead cells, surface antibodies, fixed using IC fixation buffer (eBioscience), and then stained for the accumulation of IFNy and TNFa within cells (in 1x Permeabilization Buffer, eBioscience). Samples were analyzed on a Fortessa LSR (BD) or Cytoflex (Beckmann Coulter).

Killing Assay

Killing capacity was measured using a luminescence-based cell system. T2 cells expressing Luciferase and tdTomato (abbreviated “T2-Luc”) 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. 20’000 (or else according to the E:T ratio) 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:

Proliferation and Stability Assay

To measure proliferation and stability after repetitive stimulation, cells on day 13 after the first stimulation, were washed and plated at 10’000 specific cells per 96-well with 50 U/ml IL-2. Cells were then either left to rest or stimulated again with a 1 :1 ratio of irradiated NY-ESO-1 9V loaded T2 tumor cells as above. The medium was exchanged three days later, and cells were stained and analyzed after another 3 days (total 6 days). The next day (7 days total), the degranulation capacity and production of cytokines of the cells was measured as above. PD-1 blockade in T _ex Model

The inventors assessed the responsiveness of Teff and Tex (thus both 3 days post last stimulation), which both upregulate PD-1 , to PD-1 blockade in a co-culture assay with the MDA-MB-231 cell line. 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). The inventors seeded 5000 MDA-MB-231 cells per flat bottom 96-well the day before the assay in 100 pl IMDM complete medium (same supplements as for other media, 10% FCS). The next day, 10 nM NY-ESO-1 9V peptide and 10 U/ml recombinant human IL-2 in 100 pl human serum RPMI medium (see above) was added to these wells. Next, T _e ff or T _ex were added either at an effector to target E:T ratio of 1 :1 for the killing readout or 4:1 for the IFNy ELISA readout (to stay in detectable range for both assays). Cells were then co-cultured for 4 days. The supernatant of the 4:1 E:T ratio co-culture was used to measure IFNy by ELISA (BD). Tumor cell killing was evaluated using an MTT assay. Briefly, wells were washed carefully with PBS, then incubated with fully supplemented RPMI containing 0.5 mg/ml MTT ((3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide, Sigma) for 2 h. Then solution was then aspirated carefully and the deposited color solubilized using 90 pl of DMSO (Sigma). Absorption at 550 nm was then measured using a BioTek H1 plate reader. Specific lysis was calculated as above (killing assay).

Transcriptomics Analysis

Human Healthy donors CD8 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). 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. Libraries were quality-checked on the Fragment Analyzer (Advanced Analytical, Ames, IA, USA) using the Standard Sensitivity NGS Fragment Analysis Kit (Cat# DNF-473, Advanced Analytical). Samples were pooled to equal molarity. The pool was quantified by Fluorometry using the QuantiFluor ONE dsDNA System (Cat# E4871 , Promega, Madison, Wl, USA). Sequencing was performed on the Illumina Novaseq 6000 platform to produce paired-end 51 nt reads. Read quality was assessed with the FastQC tool (version 0.11.5). Reads were mapped to the human genome (hg38) with STAR (version 2.7.9a) (Dobin, A. et al. Bioinformatics 29, 15-21 , 2013)) with default parameters, except filtering out multimapping reads with more than 10 alignment locations (outFilterMultimapNmax=10) and filtering reads without evidence in the spliced junction table (outFilterType- 'BySJout”). Gene expression was quantified with featureCounts from the Subread package (v2.0.1 ) (Liao, Y., Smyth, G. K. & Shi, W. 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.

Briefly, 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 (Robinson, 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. Heatmaps and k-means clusters (n = 6) were created using ComplexHeatmap_2.12.0 from row-scaled counts per million (cpms). Gene sets from the comprehensive single-cell PanCancer T cell Atlas by Zheng et al. ibid were retrieved. The top 100 genes per gene set (ranked based on their reported effect size) were extracted to better account for the differences in gene set sizes. Gene set enrichment was calculated using the camera function in edgeR_3.38.1 and plotted using ggplot2_3.3.6.

Selection of genes for the targeted CRISPR/Cas9 screen

To prioritize genes, 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. Of this list of genes, 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). In addition to the 29 selected genes, 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.

Cloning gRNA CRISPR Screen Library

5 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.

CRISPR Screen and Analysis

Freshly isolated healthy donor CD8 T cells were stimulated as above. In addition, cells were co-transduced with LentiCRISPRv2-mCherry virus at an MOI of 0.5. For this purpose, 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. Plates were then washed with 0.1 % BSA in PBS, and CD8 T cells were added on top. Then the NY-ESO-1 TCR virus was added, and cells were incubated for 2 days. Fresh medium and 50 U/ml IL-2 was added every 2 days, and after 6 days, cells were sorted for live CD8+ TCR+ mCherry+ cells (co-transduction efficacy for the two donors was 0.57% and 0.99% respectively). Cells were then repetitively stimulated with NY-ESO-9V peptide, as described above. After four rounds of stimulation, cells were re-stimulated with NY-ESO-9V loaded T2 cells in the presence of an anti-CD107a-APC-H7 antibody. 4h later, cells were stained and sorted based on live CD8+ VP13.1 + CD19- CD4- CD56- CD107a+Z- fractions into 200ul PBS. 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).

Cas9-RNP-mediated KO

To knock out genes using Cas9-crRNA-tracrRNA ribonuclear protein complexes, the Lonza Nucleofector 4D system was used. Both gene-specific Alt-R-crRNA and Alt-R-tracrRNA were mixed at 200 pM in nuclease-free duplex buffer (IDT) and heated to 95°C for 5min, then cooled to 25°C at -0.1 °C per minute. 1.5 pl of these annealed crRNA-tracrRNA complexes was then incubated with 1.5 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). 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. Immediately after electroporation, 80 pl prewarmed medium was added, and incubated for 20 min at 37°C. Cells were then transferred to 24 well plates at 1 mio/ml in fresh medium with 50 U/ml IL-2, 1 :4 bead to cell ratio of anti-CD3+anti-CD28 stimulatory beads and if indicated additional NY-ESO-1 TCR virus at 1 MOI. Cells were then expanded 1 :2 every two days with fresh medium and 50 U/ml IL-2. On day 6 post electroporation, cells were sorted for VP13.1 + cells using a mouse anti-Vpi3.1 antibody (Biolegend, H131 clone) and anti-mouse IgG microbeads (Miltenyi). Cells were rested overnight in fresh medium and 50 U/ml IL-2 and then stimulated either 1x with T2 + peptide and analyzed on day 3 (T _e ff) or repetitively for 4x T2 + peptide as described above (Tex).

Immunofluorescence Staining for Imaging

To perform immunofluorescence images of T cell - tumor cell conjugates, T2 tumor cells were loaded with 1 pM NY-ESO-1 (9V) peptide for 30min at 37°C. When indicated 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. Then T2 tumor cells were washed 2x in serum-free prewarmed RPMI and resuspended at 1 Mio/ml in the same medium. Repetitively stimulated NY-ESO-1 TCR transduced T cells produced as above, were washed 2x in serum- free prewarmed RPMI and resuspended at 1 Mio/ml. Both cells were then mixed at a 1 :1 ratio and incubated for 5min at 37°C. 50 pl of this cell mixture was then plated per microscopy slide well (25'000 T cells and tumor cells respectively). Cells were then incubated on the slide for 30min and then fixed with -20°C 100% Methanol for 5min. For LAMP1 and phalloidin stainings, cells were instead fixed for 20min at room temperature in 4% PFA (Electron Microscopy Services, diluted with PBS), extracted with 0.1 % Triton-X100 in PBS for 5min and quenched with 50mM Glycine in PBS for 20min. For the images of CD28 and TCRz, 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.

Both fixation procedures were then followed by blocking in 1 % 0.2 pm filtered bovine serum albumin (Sigma) in PBS (blocking buffer) for 15 min. Primary antibodies were then incubated in the same blocking buffer for 1 h at room temperature or overnight at 4°C. Samples were then washed 4x times with blocking buffer, incubated for 2x 5 min in blocking buffer, and then incubated for 1 h with secondary antibody in blocking buffer at room temperature. For the amplification of EGFP’s signal, it was counterstained with chicken anti-GFP followed by anti- chicken-AF488 antibody. If indicated, samples were washed again as above and incubated with 1 :500 dilution of DAPI in blocking buffer for 5 min. Samples were washed again and mounted with Vectashield Vibrance mounting medium and Nr. 1.5 coverslips and sealed with clear nail polish. Samples were stored at 4°C until acquisition.

Imaging

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. For the actin, LAMP1 , and perforin stainings, raw nd2 format image stacks of 110x110x1 OOnm were deconvoluted using Huygens using a theoretical point spread function, classical maximum likelihood estimation using 100 iterations and a quality stop criterion of 0.01. Images were visualized using Imaris 9 (Bitplane) and OMERO (www.openmicroscopy.org, managed by Biozentrum Basel) was used to generate figures. Single slices or sections are shown with linear display adjustments (brightness). Immunoblotting (Western blot)

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. Denaturized proteins and Precision Plus Protein Dual Color Standards (BioRad #1610374) were separated by SDS-PAGE and later transferred to a PVDF membrane (Immobilon-P, Sigma #IPVH85R) at 100 V for 60 min in tris/glycine buffer (BioRad #1610771 ) supplemented with 20% methanol. The membrane was blocked for 1 h at room temperature with 5% BSA in TBS-T (TBS with 0.05% Tween 20). Membranes were incubated with primary antibodies overnight at 4°C followed by an incubation IRDye secondary antibodies (LI-COR #925-68070 and #925-32211 ) for 1 h at room temperature. OTI KO generation

For the generation of murine antigen-specific Snx9 KO T cells, 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. Cells were then resuspended at 1 .5 Mio/ml in murine T cell medium (supplemented RPMI medium as above for human T cells, but with 10% heat inactivated FCS instead of human AB+ serum). Cells were then stimulated by addition of 100 ng/ml SIINFEKL (Invivogen, OVA257-264) and 100 U/ml recombinant human IL2 (cross-reactive with murine IL-2 receptor; Proleukin, Clinigen Healthcare) and incubated at 37°C. After two days of stimulation, 900 pmol Alt-R-crRNA (sgSNX9_9 and sgSNX9_IDT_AF; IDT) and 900 pmol Alt-R-tracrRNA (IDT) were annealed by heating to 95°C for 5min, then cooled to 25°C at -0.1 °C per minute. 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). 2 days after stimulation, 10mio of the splenocytes and lymphocyte mixture was collected per condition, spun at 90g for 10min and then resuspended in 10Oul P4 nucleofection buffer, the Cas9-RNPs and electroporated with the CM137 program. Immediately 900ul medium was added and rested for 20min at 37°C. Then cells were transferred to wells at 1 .5 Mio/ml with 100 U/ml IL-2. Cells were expanded to reach 1.5 Mio/ml with fresh 100 U/ml IL-2 daily for 4 days. Cells were then washed in PBS and 1.5 Mio or 2.7 Mio were transferred per mouse as indicated. Calcium Flux

For Ca ²⁺ flux measurements, 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. On the day of the assay, 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.

NFAT nuclear translocation assay

To measure NFAT activation, 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. Then cells were resuspended, the solution was transferred, the wells washed with FACS buffer and both fractions combined in one V-bottom well per condition. The cells were centrifuged and resuspended in cold FACS buffer with 1 :50 dilution of anti-CD8 (SK1 , Biolegend) in FACS buffer and incubated for 10min on ice. Cells were then washed twice in FACS buffer and then fixed with 4% PFA in PBS and incubated for 20 min at RT. Cells were then washed with permeabilization wash buffer (PBS with 0.1 % TritonX100 (Sigma), 1 % BSA and 0.01 % Sodium Azide) 2 times. Then 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.

Signaling Flow Cytometry

To determine the phosphorylation of AKT upon stimulation, the inventors used 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. 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.

To determine phospho-PLCyl , 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. Then 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. Then 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.

Quantitative real-time PCR

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 ViiA™ 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.

SCENITH

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. Then the drug solutions with 2-deoxyglucose (DG), oligomycin (O), the combination (DGO), or DMSO only (control) were prepared in the same medium and equilibrated in the incubator for 10 min. Then the drugs were added to the cells, mixed rapidly and incubated again. For the DGO condition, DG was added first for 10min and then O was added for the last 5min, according to the manufacturer’s instruction. After the 15min total incubation time, 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. Cells were then fixed with the Foxp3 Transcription Factor Fixation Permeabilization Kit (eBioscience) for 20 min at room temperature (RT). Cells were then washed (500g 3min centrifugation) in 1x Permeabilization buffer (eBioscience) and incubated in blocking solution (1x Permeabilization buffer with 10% final concentration of FCS) for 10 min at RT. Then 1 :250 diluted anti- Puromycine-AF488 antibody (Gift from R. Arguello) was added on top in blocking buffer and incubated for 1 h at 4°C. Cells were then washed (1000g 5min centrifugation) 2x with FACS buffer (described above, 2% FCS, 5mM EDTA in PBS) and then kept in FACS buffer until acquisition on a Beckman Coulter Cytoflex. The following formula was used to calculate the glucose and FAO/AAO dependence. DG inhibits glucose usage, thereby remaining ATP production measured by Puromycin incorporation is dependent on FAP/AAO. Background signal is obtained by inhibiting glucose usage (DG) and mitochondrial respiration (O, oligomycin). control — DG qlucose dependence = 1 — FAO or AAO = - - - control - DGO scRNASeq Sade-Feldman et al. reanalysis

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. Cells with a high fraction mitochondrial reads (>3 standard deviations from the mean; dying cells) and very many detected genes (> 4 standard deviations from the mean; doublets) were excluded. PCA and UMAP dimension reductions were calculated as above. 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. scRNASeq library preparation of intratumoral OTI cells

OTI T cells with or without Snx9 KO were generated as described in “OTI KO generation” and confirmed using flow cytometry staining. As above 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). The mixture was then incubated for 15min on a rotating shaker (200 rpm) at 37°C. Then the mixture was again dissociated using the m_impTumor_03 program on the gentle MACS dissociator. Cells were then strained through a 100 pm MACS Smartstrainer (Miltenyi) and washed using PBS with 2% FCS. Cells were stained for CD45 (30-F11 clone in V450, BD 560501 ) and CD19 (1 D3 clone in FITC, BD 553785) and analyzed for lymph node contamination. Samples with more than 5% CD19+ cells among CD45+ live cells were excluded due to lymph node contamination (3 samples: 2 intergenic and 1 Snx9 KO). The remaining samples were pooled into 2 pools (2- 3 mice) per condition. These pools were sorted for DAPI- CD3e+ F4/80- Ly6G- CD19- CD8a+ CD45.1 + CD45.2- single cells using an Arialll sorter (intergenic A: 6787 cells, intergenic B: 1975 cells; Snx9 KO A: 6163 cells, Snx9 KO B: 2295). Sorted single cells were then loaded onto a 10x Genomics Chromium NEXT GEM chip G. Libraires were prepared following the 10x Genomics protocol for 3' gene expression profiling (CG000315, Rev C) and cDNA or library quality was assessed using a 4200 TapeStation System (Agilent). 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. The options “—outFilterType=BySJout -outFilterMultimapNmax=10 -outSAMmultNmax=1 - outFilterScoreMin=30 -soloCBmatch WLtype= 1 MM_multi_Nbase_pseudocounts soloUMIIen=12 -soloUMIfiltering=MultiGeneUMI_CR -soloUMIdedup=1MM_CR soloCellFilter=None” were used for STARsolo. For each sample, empty droplets were detected and removed using the emptyDrops() function from the Bioconductor DropletUtils package (version 1 .14.0; using 5000 iterations, the option test.ambient=TRUE, a lower threshold of 100 UMIs and an FDR threshold of 0.1 %) (Lun, A. T. L. et al. Genome Biol. 20, 63, 2019).

Analysis of intratumoral OTI scRNAseq

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. For the remaining cells, a 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. et al. Nat. Immunol. 20, 326-336, 2019). ggplot2_3.3.6, ggrepel_0.9.1 , tidyr_1 .2.0 and Seurat_4.1.1 were used to create visualizations. FindMarkers in Seurat was used to calculate differentially expressed genes for each cluster and for all cells in total between Snx9 KO and intergenic.

Generation of T2 CD80 CD86 KO and co-culture assays

Low passage T2 cells (source DSMZ) 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 crRNAwas 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. For the co-culture assays with T2 wt and T2 CD80 CD86 KO cells, T _e ff cells (day 3 post first stimulation with T2 wt + NY-ESO-1 9V peptide) with or without SNX9 were washed and reseeded at 1 mio cells per ml in 10 U/ml IL2 in human serum medium as above. After 24h of resting at 37°C, the 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. Additionally, either 10 pg/ml human lgG1 (Ultra-LEAF, Biolegend 403502) or Ipilimumab (clinical grade, Yervoy, BMS) was added to the culture. After 14h of incubation at 37°C, the co-culture was stained for CD25 in addition to CD8 and Zombie NIR. Fixed stainings (IC fix, eBiosciences) were acquired on a Cytoflex (Beckmann Coulter).

Cell-free anti-CD3 anti-CD28 stimulation assays

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. These rested cells, were then restimulated on nontreated flat bottom 96-well plates coated for 2h at 37°C with the indicated concentrations of Ultra-LEAF purified anti-CD3 (OKT3, Biolegend Cat Nr. 317347) and/or anti-CD28 (CD28.2, Biolegend Cat Nr. 302943). Cells were then stained for CD25, CD8 and Zombie NIR, fixed and acquired on a Cytoflex (Beckmann Coulter).

CAR T cell production and adoptive transfer

To generate human CD8+ CAR T cells, the inventors isolated 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). Cells were stimulated on the same day with 1 :1 ratio of CD3/CD28 beads (Human T cell Activation and Expansion kit, Miltenyi) and 150 U/ml rh-IL-2 (Proleukin). The next day, cells were collected into a falcon tube and 4 pg/ml Polybrene (Sigma) was added together with 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). For the anti-CD19- CART19-BBz 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).

Serum protein analyses

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.

For the serum analysis after OTI transfer, 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.

For the CAR T cell experiments, 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). Of note, although 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. Statistics and Data Visualization

The statistical analysis and graph preparation were performed using the software package Prism version 8.0a (GraphPad Software, La Jolla, CA). Functional data are representative of at least two experiments each with multiple human donors. Data is displayed as scatter dot plots where applicable and single points represent different healthy donor replicates. Data were considered statistically significant with p values < 0.05. Normality tests were used to choose parametric or non-parametric tests. Data are shown as mean ± standard deviation with symbols representing individual patients or donors where applicable. All t-tests were performed as two-sided tests.

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