Methods for producing car t cells

Abstract

The disclosure relates to methods for producing chimeric antigen receptor (CAR) cells, modified CAR cells and compositions thereof, and methods of using said CAR cells or compositions for cellular therapy.

Description

Methods for producing CAR T cells

[0001] This application claims priority from US provisional patent application 63 / 662789, the entire contents of which is incorporated herein by reference.Field of the invention

[0002] The present disclosure relates to methods for producing chimeric antigen receptor (CAR) cells, modified CAR cells and compositions thereof, and methods of using said CAR cells or compositions for cellular therapy. The present disclosure also relates to methods for identifying the expansion potential of a cell for use in producing CAR cells for cellular therapy.Background of the invention

[0003] Chimeric antigen receptor (CAR) cell therapy, such a CAR-T cell therapy, enables a patient or donor’s cells to be transformed into an adoptive immunotherapy for a range of diseases, such as cancer or autoimmune disease. Genetically modifying an immune cell to express a CAR re-directs the normal capacity of the cell towards killing of target cells (such as tumour cells) that express the antigen targeted by the CAR.Typically, cells collected by apheresis are modified to express a CAR, then the resultant cell product is provided to the patient by infusion.

[0004] A key limitation of CAR-based cellular therapy is the effective proliferation or expansion of cells in vitro and in vivo. In vitro expansion is required to manufacture the infusion product. Manufacturing cell therapy products can be laborious and expensive. In vivo expansion is required once the cell therapy is provided to the patient, to enable provision of an adequate in vivo dose of the therapeutic cell product to the patient. The in vivo CAR cell count in blood is a major determinant of clinical outcomes after CAR cell immunotherapy.

[0005] Previous pre-clinical and clinical trial data has highlighted the importance of differentiation state for the precursor or “origin” cells used to manufacture CAR cell therapies. For example, apheresis products that contain a greater proportion of less-differentiated subsets of T cells, naive (N) and central memory (CM) T cells, give rise to CAR-T infusion products (IP) associated with improved in vivo persistence and efficacy, but it is unclear precisely why or how.

[0006] Identifying molecular drivers that lead to cell infusion products with improved expansion, persistence and / or efficacy is crucial for improving cell infusion products and their manufacture. Selecting and culturing cells with preferred molecular characteristics, or genetically modifying cells to have said characteristics, are both means to enhance manufacture of the therapeutic product and the capabilities of the final therapeutic product.

[0007] However, identifying molecular drivers in cells and infusion products that are predictive of cell product quality and prognostic of desired therapeutic outcomes is challenging. Gene expression signatures, particularly for transcription factors associated with cell differentiation and cell proliferation, are dynamic and transient, and important genes can be expressed at low levels that are difficult to detect by transcriptomic techniques. During in vitro manufacture of cell therapies, gene expression signatures from immune cell activation can contribute statistical noise that obscures the identification of predictive factors.

[0008] There is a need for improved cells for use in chimeric antigen receptor cellular therapies, and methods for identifying and generating the same.

[0009] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0010] The present disclosure is based on the identification by the inventors of improved CAR cells, and methods of manufacturing CAR cell products, due to the characterisation of a key transcription factor, KLF7, involved in CAR cell expansion potential. Surprisingly, the provision of KLF7 improves expansion potential. The inventors further identified novel histone biomarker signatures for enabling the assessment of expansion potential. Advantageously, these histone biomarker signatures enable identification of expansion potential using methods that do not rely on transcriptomics.

[0011] In a first aspect of the disclosure, the disclosure provides a method for manufacturing a chimeric antigen receptor (CAR) cell product, the method comprising:- increasing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, in an immune cell comprising a chimeric antigen receptor (CAR immune cell) or a precursor thereof; and- then culturing the CAR immune cell or precursor thereof for a sufficient time and under suitable conditions to produce the CAR cell product.

[0012] In a second aspect of the disclosure, there is provided a method of producing a CAR immune cell or precursor thereof, the method comprising:- introducing a polynucleotide encoding a chimeric antigen receptor (CAR) into an immune cell or precursor thereof, wherein the polynucleotide enables the CAR to be expressed on the surface of the cell;- increasing the expression or amount of a KLF polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, in the cell; thereby producing the CAR immune cell or precursor thereof.

[0013] In a third aspect of the disclosure, the disclosure provides a CAR immune cell or a precursor thereof for manufacturing a chimeric antigen receptor (CAR) cell product, wherein the CAR immune cell or the precursor thereof is modified to increase the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same. The CAR immune cell or precursor thereof may be isolated. The CAR immune cell or precursor thereof may be in vitro, ex vivo or in vivo.

[0014] In any embodiment of any aspect of the disclosure, increasing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, comprises contacting a CAR immune cell or precursor thereof with an agent that increases expression of KLF7 polypeptide or functional variant or fragment thereof.

[0015] Preferably, the agent is selected from the group consisting of: a polynucleotide sequence (e.g. an mRNA or other polynucleotide encoding KLF7), a protein (e.g.purified and / or recombinant KLF7 protein), an aptamer and small molecule, ribosome, RNAi agent and peptide-nucleic acid (PNA) and analogues or variants thereof. In some embodiments, the agent is exogenous. The agent may comprise a histone-modification agent, such as a histone methylation or demethylation agent, such as histone demethylase KDM2B, or other epigenetic editors available to the art such as dCas9 fusion proteins. The present disclosure also contemplates the use of a transcriptional activation system (e.g., a gRNA for use in a gene activation system such as CRISPR / Cas9 or TALEN) for increasing the expression of the one or more transcription factors. In some embodiments, the amount of KLF7 may be indirectly increased by modifying the expression of genes upstream or downstream of KLF7.

[0016] In any embodiment of any aspect of the disclosure, increasing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, comprises introducing a KLF7 polypeptide or functional variant or fragment thereof or a polynucleotide encoding the same into the CAR immune cell or precursor cell thereof. Typically, the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, is increased by introducing at least one nucleic acid comprising a nucleotide sequence encoding a KLF7 polypeptide, or encoding a functional variant or fragment thereof, into the cell. In a preferred embodiment of the disclosure, the nucleic acid sequence encoding a KLF7 polypeptide or functional variant or fragment thereof is introduced into a cell by a plasmid.

[0017] In any embodiment of any aspect of the disclosure, (i) a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, and (ii) a chimeric antigen receptor polypeptide or a polynucleotide encoding the same may be introduced into the precursor cell, simultaneously or substantially at the same time. For example, the precursor may be provided an expression vector encoding both a KLF7 polypeptide and a chimeric antigen receptor polypeptide.

[0018] In other embodiments of any aspect of the disclosure, a genetic modification to introduce expression of a CAR into the immune cell or precursor thereof may be before or after the genetic modification to increase expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or polynucleotide encoding the same.

[0019] In any embodiment, the CAR immune cell or precursor thereof may be contacted, before or after increasing the expression of amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, with one or more agents known to favour immune cell proliferation and expansion (eg IL-2, IL-5, and / or IL-7 cytokines; FOXO1 ). In another embodiment, the CAR immune cell or precursor thereof may be genetically modified, before or after increasing the expression of amount of a KLF7 polypeptide or functional variant or fragment thereof, or polynucleotide encoding the same, to express or improve expression of one or more genes known to favour cell proliferation and expansion, particularly the expansion of T cells (eg / L-2, FOXO1).

[0020] The disclosure further provides for a CAR cell product obtainable or obtained by a method described herein.

[0021] The disclosure further provides for a population of cells comprising a plurality of the CAR immune cell or precursor thereof described herein or obtained by a method described herein; and provides for progeny of a cell described herein.

[0022] The disclosure further provides a pharmaceutical composition comprising a CAR cell immune, precursor, or a population of cells as described herein, or a CAR cell product obtained by a method described herein. In any embodiment the pharmaceutical composition may further comprise a pharmaceutically acceptable diluent, carrier, excipient, or combination thereof.

[0023] In another aspect of the disclosure, the disclosure provides a method for identifying the expansion potential of an immune cell comprising a chimeric antigen receptor (CAR immune cell) or a precursor thereof, or a cell population comprising a CAR immune cell or precursor thereof, for CAR cell therapy, the method comprising: assessing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, in the CAR immune cell, CAR immune cell precursor or cell population; wherein when the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, is increased compared to the expression or amount in a reference dataset, the CAR immune cell, CAR immune cellprecursor or cell population is identified as having high expansion potential for CAR cell therapy; and wherein when the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same is decreased compared to the expression or amount in a reference dataset, the CAR immune cell, CAR immune cell precursor or cell population is identified as having low expansion potential for CAR cell therapy.

[0024] In one embodiment of this aspect, the expansion potential of a precursor of a CAR immune cell is assessed prior to engineering the precursor to comprise a chimeric antigen receptor and / or prior to differentiating the precursor into a mature immune cell. In another embodiment of this aspect, the expansion potential of a CAR immune cell is assessed prior to in vitro expansion and / or further in vitro manufacturing steps to produce a CAR cell product. Thus, the disclosure provides means to screen starting material (eg apheresis product; sorted cells from apheresis product) for expansion potential before undertaking further laborious and / or expensive manufacturing steps. The disclosure also provides means to screen for starting material that is most likely to give rise to CAR cell product that expands well in vivo for improved therapeutic efficacy.

[0025] In one embodiment of this aspect, the expansion potential of a cell population comprising a CAR immune cell or precursor thereof is identified. Thus, a cell population may be screened for likelihood to give rise to a final CAR product that expands well in vivo for improved therapeutic efficacy.

[0026] In one embodiment, assessing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same comprises assessing the relative proportion of cells within a cell population comprising a CAR immune cell or precursor thereof that demonstrate characteristics indicative of increased expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same.

[0027] For example, a cell population with high expansion potential may have an increased proportion or percentage of cells that:- have increased expression or amount of KLF7 polypeptide;- have increased expression or amount of one or more polynucleotides encoding KLF7; and / or- exhibit a histone marker signature associated with increased transcription of KLF7; compared to an appropriate control group (eg a cell population with known low expansion potential); or compared to another test group (eg another cell population being assessed for expansion potential).

[0028] Conversely, a cell population with low expansion potential may have a reduced proportion or percentage of cells that:- have increased expression or amount of KLF7 polypeptide;- have increased expression or amount of one or more polynucleotides encoding KLF7; and / or- exhibit a histone marker signature associated with increased transcription of KLF7; compared to an appropriate control group (eg a cell population with known high expansion potential) or compared to another test group (eg another cell population being assessed for expansion potential).

[0029] In one embodiment, assessing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, comprises assessing the expression or amount of KLF7 polypeptide or functional variant or fragment thereof present in a CAR immune cell or precursor thereof, or in a cell population, using any standard technique known to those in the art, such as western blotting, ELISA assay, Bradford or BCA assay, mass spectrometry, UV spectroscopy. Polypeptide analyses may also comprise assessing the expression or amount of one or more polypeptides encoding by genes downstream and / or upstream of KLF7, to indirectly assess the expression or amount of KLF7 polypeptide or functional variant or fragment thereof in a CAR immune cell or precursor thereof, or in a cell population.

[0030] In one embodiment, assessing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, comprises assessing the expression or amount of one or more polynucleotides encoding KLF7 polypeptide or functional variant or fragment thereof in a CAR immunecell or precursor thereof, or in a cell population. The expression or amount of one or more polynucleotides encoding KLF7 may be measured using any standard technique known to those in the art, such as reverse transcription polymerase chain reaction (RT- PCR), quantitative real-time polymerase chain reaction (qRT-PCR), microarrays, DNA or RNA sequencing (including next-gen and high-throughout), UV spectroscopy. Nucleotide analyses may also comprise assessing the expression or amount of one or more polynucleotides encoding genes downstream and / or upstream of KLF7, to indirectly assess the expression or amount of one or more polynucleotides encoding KLF7 in a CAR immune cell or precursor thereof, or in a cell population.

[0031] In one embodiment, assessing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or polynucleotide encoding the same, comprises assessing histone biomarker signatures for transcriptional repression or activation of KLF7, and / or one or more genes downstream of KLF7, and / or one or more genes upstream of KLF7.

[0032] Exemplary genes downstream of KLF7 include but are not limited to CCR7, TMEM45A, SLAMF7, UPC, CLIC3, ZFP57, and RHBDD2.

[0033] Exemplary genes upstream of KLF7 include but are not limited to EVI1 (encoding ecotropic viral integration site 1 ; also known as MECOM), CIZ (encoding cas interacting zinc finger protein; also known as NMP4, nuclear matrix transcription factor 4), HNF1 (encoding hepatocyte nuclear factor 1 ), DSP (encoding D site-binding protein) and HNF3 (encoding hepatocyte nuclear factor 1 , also known as FOXA).

[0034] In one embodiment, assessing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or polynucleotide encoding the same, comprises: assessing a histone biomarker signature of one or more genes selected from KLF7, CCR7, TMEM45A, SLAMF7, UPC, CLIC3, ZFP57, and RHBDD2 wherein when- one or more of KLF7, CCR7, and TMEM45A exhibit a transcriptionally active histone biomarker signature,- RHBDD2 exhibits a transcriptionally active histone biomarker signature, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 exhibit a transcriptionally repressive histone biomarker signature, the CAR immune cell, CAR immune cell precursor or cell population is identified as having high expansion potential for CAR cell therapy; and when- one or more of KLF7, CCR7, and TMEM45A exhibit a transcriptionally repressive histone biomarker signature,- RHBDD2 exhibits a transcriptionally repressive histone biomarker signature, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 exhibit a transcriptionally active histone biomarker signature, the CAR immune cell, CAR immune cell precursor or cell population is identified as having low expansion potential for CAR cell therapy.

[0035] In some embodiments, the method comprises assessing a histone biomarker signature of one or more genes selected from KLF7, CCR7, TMEM45A, SLAMF7, LIPC, CLIC3, ZFP57, and RHBDD2 in a CAR immune cell or CAR immune cell precursor, when- one or more of KLF7, CCR7, and TMEM45A is not H3K27me3 methylated,- RHBDD2 is H3K4me2 methylated, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 \s not H3K4me2 methylated, the CAR immune cell or CAR immune cell precursor is identified as having high expansion potential for CAR cell therapy; and when- one or more of KLF7, CCR7, and TMEM45A is H3K27me3 methylated,- RHBDD2 is not H3K4me2 methylated, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 \s H3K4me2 methylated, the CAR immune cell or CAR immune cell precursor is identified as having low expansion potential for CAR cell therapy.

[0036] In any embodiment of this aspect of the disclosure, the method comprises assessing a histone biomarker signature of one or more genes selected from KLF7, CCR7, TMEM45A, SLAMF7, UPC, CLIC3, ZFP57, and RHBDD2 in a cell population comprising a CAR immune cell or CAR immune cell precursor, wherein when a relatively high proportion of the cell population is characterised by:- one or more of KLF7, CCR7, and TMEM45A exhibiting a transcriptionally active histone biomarker signature,- RHBDD2 exhibiting a transcriptionally active histone biomarker signature, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 exhibiting a transcriptionally repressive histone biomarker signature, the cell population is identified as having high expansion potential for CAR cell therapy.

[0037] In some embodiments of this aspect of the disclosure, the method comprises assessing a histone biomarker signature of one or more genes selected from KLF7, CCR7, TMEM45A, SLAMF7, UPC, CLIC3, ZFP57, and RHBDD2 in a cell population comprising a CAR immune cell or CAR immune cell precursor, wherein when a relatively high proportion of the cell population is characterised by:- no H3K27me3 methylation of one or more of KLF7, CCR7, and TMEM45A,- H3K4me2 methylation of RHBDD2, and / or- no H3K4me2 methylation of one or more of SLAMF7, LIPC, CLIC3, ZFP57, the cell population is identified as having high expansion potential for CAR cell therapy; and when a relatively high proportion of the cell population is characterised by:- H3K27me3 methylation of one or more of KLF7, CCR7, and TMEM45A,- no H3K4me2 methylation of RHBDD2, and / or- H3K4me2 methylation of one or more of SLAMF7, LIPC, CLIC3, ZFP57, the cell population is identified as having low expansion potential for CAR cell therapy.

[0038] In a preferred embodiment of this aspect of the disclosure, the method comprises assessing a histone biomarker signature of KLF7, preferably wherein the histone biomarker signature comprises or consists of H3K27me3 methylation, wherein H3K27me3 methylation of KLF7 '\s associated with low expansion potential. The KLF7 histone biomarker signature may further comprise H3K4me2 methylation, wherein H3K4me2 methylation of KLF7 '\s associated with high expansion potential.

[0039] In another embodiment, the method comprises assessing a histone biomarker signature of KLF7 and at least one of CCR7, TMEM45A, SLAMF7, LIPC, CLIC3, ZFP57, and RHBDD2; optionally KLF7 and at least two, at least three, at least four, at least five, or at least six of: CCR7, TMEM45A, SLAMF7, LIPC, CLIC3, ZFP57, and RHBDD2.

[0040] In one embodiment, the method comprises assessing a histone biomarker signature of ZFP57, preferably wherein the histone biomarker signature comprises or consists of H3K4me2 methylation, wherein H3K4me2 methylation of ZFP57 is associated with low expansion potential.

[0041] In one embodiment, the method comprises assessing a histone biomarker signature of KLF7, preferably wherein the KLF7 histone biomarker signature comprises or consists of H3K27me3 methylation, wherein H3K27me3 methylation of KLF7'\s associated with low expansion potential; and a histone biomarker signature of ZFP57, preferably wherein the ZFP57 histone biomarker signature comprises or consists of H3K4me2 methylation, wherein H3K4me2 methylation of ZFP57 is associated with low expansion potential.

[0042] In one embodiment, the method comprises assessing a histone biomarker signature of KLF7, CCR7, TMEM45A, SLAMF7, UPC, CLIC3, ZFP57, and RHBDD2.

[0043] In another embodiment of this aspect, the method for identifying the expansion potential may further comprise assessing a histone marker of one or more genes set forth in Table 4; to identify an additional histone marker associated with improved expansion potential.

[0044] In another embodiment of this aspect, the method may further comprise assessing a histone marker of one or more transcription factors genes set forth in Table 2; to identify a CM-like phenotype likely to improve therapeutic potential.

[0045] In a further aspect there is provided a method of preventing or treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a CAR cell product manufactured using a method described herein, or comprising administering to the subject a CAR cell described herein.

[0046] In a further aspect there is provided a method of preventing or treating a disease, disorder, or condition in a subject in need thereof, the method comprising: assessing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, in an immune cell comprising a chimeric antigen receptor (CAR immune cell) or a precursor thereof; wherein when the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, is increased compared to the expression or amount in a reference dataset, the CAR immune cell, CAR immune cell precursor or cell population is identified as having high expansion potential for CAR cell therapy; and wherein when the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, is decreased compared to the expression or amount in a reference dataset, the CAR immune cell, CAR immune cell precursor or cell population is identified as having low expansion potential for CAR cell therapy, manufacturing a CAR cell product using a CAR immune cell, CAR immune cell precursor or cell population identified as having high expansion potential, treating the subject by administering the CAR cell product.

[0047] In one embodiment, the method of preventing or treating a disease, disorder, or condition in a subject in need thereof, comprises- assessing a histone biomarker signature of one or more genes selected from KLF7, CCR7, TMEM45A, SLAMF7, UPC, CLIC3, ZFP57, and RHBDD2 U a CAR immune cell, a CAR immune cell precursor or a cell population comprising a CAR immune cell or CAR immune cell precursor, wherein when- one or more of KLF7, CCR7, and TMEM45A exhibit a transcriptionally active histone biomarker signature,- RHBDD2 exhibits a transcriptionally active histone biomarker signature, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 exhibit a transcriptionally repressive histone biomarker signature, the CAR immune cell, CAR immune cell precursor or cell population is identified as having high expansion potential for CAR cell therapy; and when- one or more of KLF7, CCR7, and TMEM45A exhibit a transcriptionally repressive histone biomarker signature,- RHBDD2 exhibits a transcriptionally repressive histone biomarker signature, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 exhibit a transcriptionally active histone biomarker signature, the CAR immune cell, CAR immune cell precursor or cell population is identified as having low expansion potential for CAR cell therapy; manufacturing a CAR cell product using a CAR immune cell, CAR immune cell precursor or cell population identified as having high expansion potential, treating the subject by administering the CAR cell product.

[0048] In preferred embodiments, the disease, disorder, or condition is a cancer or an autoimmune disease, most preferably, the disease, disorder or condition is a cancer.

[0049] In any embodiment of any aspect herein, the method may further comprise obtaining or having obtained an apheresis product from the subject. In any embodiment, the method may comprise processing an apheresis product to obtain specific immune cells (eg CD3+ T cells), such a cell sorting using flow cytometry, or filtration.

[0050] In any embodiment of any aspect herein, the method may further comprise purifying or otherwise processing the CAR cell product so that it is ready for administrationinto the subject. For example, the CAR cell product may be adjusted for pH, adjusted for osmolarity, adjusted for cell dose (eg concentration per unit of infusion volume) depending on eg subject’s body weight, severity of disease, mixed with a buffer and / or a cryoprotectant (eg DMSO), stored for later use, and / or frozen.

[0051] The present disclosure also provides a kit for use in identifying the expansion potential of CAR immune cell, CAR immune cell precursor or cell population for CAR cell therapy, the kit comprising:- a means for assessing a histone biomarker signature as disclosed in any aspect herein; and / or- a means for determining the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same.

[0052] The disclosure provides for a kit when used in the methods described herein.

[0053] Preferably the kit also comprises instructions for use thereof according to a method herein.

[0054] Optionally the kit may comprise information pertaining a reference data set as described herein.

[0055] The present disclosure also provides for the use of agents in the manufacture of a kit as described herein.

[0056] In any embodiment of any aspect of the disclosure, the CAR cell product is a CAR-T cell product, or the CAR cell therapy is CAR-T cell therapy.

[0057] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0058] Further aspects of the present disclosure and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0059] Figure 1. A. Sorting strategy for isolation of naive (N), central memory (CM) and effector memory (EM) from human healthy donor (HD) CD8+ T cells following CD8+ T cell enrichment by negative selection from peripheral blood mononuclear cells (PBMCs). B-C. Expression of memory associated markers in post-sorted CD8+ T cells. Error bars represent standard deviation.

[0060] Figure 2. Epigenomic analyses highlight greater differences between CD8+ T cell subsets than transcriptomic approaches. A. Histogram (top row) of average log-normalized count of sequenced DNA bound to dimethylated lysine 4 of human histone 3 (H3K4me2, left column for each subset) or trimethylated lysine 27 of human histone 3 (H3K27me3, right column for each subset) within + 1 kb of the transcriptional start site (TSS). Tornado plots (second row) of CD8+ T cell subsets (N, left; CM, middle; EM, right) depict the relationship between transcriptome abundance and histone marks. Each line represents a genomic region within +1 kb of the TSS for an annotated gene. The genes in rows are ordered by transcript abundance by RNA-seq from high (top) to low (bottom). The heatmap is shaded by the intensity of H3K4me2 or H3K27me3 marks (histone marks) assessed by Cleavage Under Targets and Restriction Using Nuclease (CUT&RUN). H3K4me2 and H3K27me3 marks show an inverse relationship with transcript abundance. B-C. Numbers of differentially expressed (by RNA-seq) and differentially enriched (by CUT&RUN) genes (B) and transcription factors (C) when comparing CD8+ T cell subsets. Genes assigned to peaks + 1 kb of the transcriptional start site (TSS), no gene counted twice (adjusted P value < 0.05 and Log2 fold change (LogFC) > 1 ). D. Principal component analyses (PCA) and E. Uniform Manifold Approximation and Projection (UMAP) of RNA-seq (left) and H3K27me3 (middle) and H3K4me2 (right) histone marks show a distinction between CD8+ T cell subsets by histone marks that is less apparent by RNA-seq.

[0061] Figure 3. Diverse patterns of histone mark patterns and transcript abundance can be identified in CD8+ T cell subsets. A-B. Histogram of two- component Gaussian mixture model (GMM) to characterize distribution of H3K27me3- and H3K4me2-bound sequenced DNA reads from naive CD8+ T cells within 10kb of transcriptional start site (TSS). A bimodal distribution of read intensity is noted and modelled by GMM with negative reads on the left and positive reads on the right. The nadir of the distributions is represented by the dashed line, providing a positive andnegative cut-off used to integrate epigenomic and transcriptomics analyses in subsequent figures. Identical cut-offs were seen with CM and EM cells. C. Histogram (top row) of average log-normalized counts of genes that are deemed positive, negative and bivalent for H3K4me2- and H3K27me3-specific reads across subsets. Individual genes single- or bivalent for each subset are shown in histogram below, ordered and shaded by intensity of counts.

[0062] Figure 4. Histone mark analyses uncover differences in transcription factors in CAR-T manufactured from distinct starting cell subsets that are not detected by RNA-seq. A. PCA plots of RNA-seq, H3K27me3, or H3K4me2 of CAR-T manufactured from healthy donor (HD) CD8+ CM (CM-derived CAR-T) or EM (EM- derived CAR-T) T cells. B. Venn diagram showing numbers of differentially expressed (DEx by RNA-seq) or differentially enriched (DEn by H3K4me2 and H3K27me3) genes when comparing CM- and EM-derived CAR-T. C. Bar plots of scaled H3K4me2+1 signal in transcription factors (TFs) differentially enriched in CM- and EM-derived CAR- T. D. Bar plots of scaled H3K27me3+1 signal in TFs differentially enriched in CM- and EM-derived CAR-T. ****P<0.0001 , ***P<0.001 , **P<0.01 , *P<0.05 by Benjamini- Hochberg procedure.

[0063] Figure 5. Epigenomic marks in LBCL PT CM-derived CAR-T associated with in vivo accumulation of CAR-T after adoptive transfer. Epigenomic marks in LBCL PT CM-derived CAR-T associate with in vivo accumulation of CAR-T after adoptive transfer. A. PCA plots of RNA-seq, H3K27me3 or H3K4me2 histone marks of CAR-T manufactured from CM-enriched CD8+ T cells from LBCL PTs who achieved CR after CD19-targeted CAR-T immunotherapy in a phase 1 / 2 clinical trial (NCT01865617). Red points indicate data from PTs in whom infused CAR-T resulted in peak CAR-T counts below the median (Low_Expander) and blue points indicate data from PTs in whom infused CAR-T resulted in peak CAR-T counts above the median (High_Expander; Fig. S18). B. Number of differentially expressed (by limma, LogFC >1 , adjusted P<0.1 ) or enriched (by H3K4me2 and H3K27me3 histone marks, LogFC >1 , adjusted P<0.1 ) genes when comparing low expanding and high expanding CAR-T infused products. C. Volcano plot of -log(adjusted P value) in transcript (left panel) or H3K4me2 (middle panel) or H3K27me3 (right panel) signal vs logFC. Red points indicate genes enriched in low expanding CAR-T and blue points indicate genes enriched in high expanding CAR-T. D. Bar plot of scaled H3K27me3+1 signal ofproliferation-associated transcription factor (KLF7) DEn by H3K4me2 histone marks analysis when comparing low expanding and high expanding CAR-T. E-F. Bar plots of scaled H3K27me3+1 signal (E) and H3K4me2 signal (F) of targets of KLF7 DEn by histone mark analysis when comparing low expanding and high expanding CAR-T. All genes have an empirically demonstrated association (or inverse association) with cell proliferation concordant with the respective histone mark. ****P<0.0001 , ***P<0.001 , **P<0.01 , *P<0.05, #P<0.1 by Benjamini-Hochberg procedure. G. Fold change in accumulation of KLF7_P2A_GFP- and control P2A_GFP-lentiviral transduced T cells at increasing viral volumes. n=5 donors; ****P<0.0001 by two-way ANOVA with post-hoc Tukey correction. H. Change in geometric mean CellTrace Violet (CTV) as an inverse measure of cell proliferation of CD19-directed CAR-T with and without KLF7 cotransduction. n=2 donors in technical duplicate, **P<0.01 by two-way ANOVA with post- hoc Tukey correction. I. Frequency of CD45RA- CCR7+ phenotype cells in CD19- directed CAR-T with and without KLF7 co-transduction. n=2 donors in technical duplicate, *P<0.05 by t test. J. Promoter region of / L2 in the human genome and predicted upstream KLF7 binding site. K. Intracellular IL-2 expression in CD19-directed CAR-T with and without KLF7 co-transduction. n=2 donors in technical duplicate, **P<0.01 by t test.

[0064] Figure 6. Comparison of histone marks from CM-derived CAR-T cells from healthy donors and CM-enriched CAR-T cells from patients reveal exhaustion- associated transcription factors not seen by transcriptomic approaches. A. PCA plots of RNA-seq, H3K27me3 or H3K4me2 marks of CART cells manufactured from healthy donor (HD) CM-derived and patient (PT) CM-derived CD8+ T cells. B. Number of differentially expressed (by RNA-seq) or enriched (by H3K4me2 and H3K27me3) genes when HD CM-derived CART are compared to PT CM-derived CART cells. C. Volcano plot of -log(adjusted P value) in transcript (left panel) or H3K4me2 (middle panel) or H3K27me3 (right panel) signal vs log(fold-change, FC). DEx / DEn genes are denoted purple for HD CM-derived CART cells, or peach for PT CM-enriched CART cells. Named genes are transcription factors (TFs) that are DEx / DEn in each analysis. D. Bar plot of transcript level of exhaustion-resistance-associated TF (TCF7) from Good et. al. 2021 differentially expressed in PT CM-enriched and HD CM-derived CAR-T. E. Bar plots of scaled H3K27me3+1 signal of exhaustion-resistance-associated and exhaustion-associated TFs differentially enriched in PT CM-enriched and HD CM- derived CAR-T. F. Bar plots of scaled H3K4me2+1 signal of exhaustion-resistance-associated and exhaustion-associated TFs differentially enriched in PT CM-enriched and HD CM-derived CAR-T. ****P<0.0001 , ***P<0.001 , **P<0.01 , *P<0.05 by Benjamini-Hochberg procedure.Detailed description of the embodiments

[0065] Reference will now be made in detail to certain embodiments of the disclosure. While the disclosure will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the disclosure to those embodiments. On the contrary, the disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present disclosure as defined by the claims.

[0066] One skilled in the art will recognise many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described.

[0067] It will be understood that the disclosure disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the disclosure.

[0068] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0069] Table 1: Sequence information

[0070] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0071] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook etal. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1 -4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0072] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0073] As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.CARs and CAR cell therapy

[0074] As used herein, the term “chimeric antigen receptor” or CAR, refers to an artificially constructed protein for expression on the surface of an immune cell, the protein comprising an extracellular domain (extracellular part) comprising an antigen binding domain (also called an antigen recognition domain), a transmembrane domain and an intracellular signaling domain. The extracellular domain may be linked to the transmembrane domain by a linker. The extracellular domain may also comprise a signal peptide.

[0075] The terms "binds to", “specifically binds to” or "specific for" with respect to the antigen-binding domain of a CAR, refers to a domain that recognises and binds to a specific antigen and does not substantially recognise or bind to other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species also may bind to that antigen from another species. This cross-species reactivity is typical of many antibodies and therefore not contrary to the definition that the antigen-binding domain is specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.) or homologous variants of this antigen from the same gene family. This cross reactivity is typical of many antibodies and therefore not contrary to the definition that the antigen-binding domain is specific.

[0076] Exemplary CARs include but are not limited to CD-19 targeting CARs (eg Gauthier, J. et al. Blood 2020;135(19): 1650-1660), or CARs directed to CD171 , EGFR, MSLN, CD123, Lewis Y, FAP or CD131 . It will be appreciated that the method is not limited to the type of antigen-binding domain of the CAR, or the type of tumour antigen expressed by a cancer.

[0077] The skilled person will be familiar with in vitro and in vivo methods in the art for introducing one or more CARs into an immune cell or precursor thereof to generate a CAR immune cell or precursor thereof.

[0078] To produce a histocompatible CAR cell product, autologous immune cells may be obtained from a subject via apheresis. For example, apheresis using standard techniques may be used to obtain CD3+ T lymphocytes to manufacture CAR T cells.

[0079] Alternatively, CAR-T cells may be manufactured using in vivo techniques where gene delivery systems are used to introduce a CAR into a target immune cell or cells, without the immune cell or cells needing to be collected from the subject and modified in vitro. For example, a lentiviral vector may be used to deliver a CAR gene to a target immune cell in vivo.

[0080] The term "autologous" as used herein refers to any material derived from the same subject to whom it is later re-introduced.

[0081] The term "allogeneic" as used herein refers to any material derived from a different subject of the same species as the subject to whom the material is re-introduced.

[0082] As referred to herein, “cell product”, “CAR cell product”, “therapeutic product”, or “infusion product” are used interchangeably to define the cell therapy product that is to be provided to the patient.

[0083] The skilled person will appreciate the steps required to manufacture a CAR cell product once a desired CAR immune cell or population thereof is obtained. Typically, manufacturing a CAR cell product involves:- expanding the CAR cell in vitro under suitable conditions and for sufficient time to achieve the desired number of CAR cells;- removing unwanted cells and materials (eg cells not comprising the CAR; dead cells; residual cytokines, vectors and serum proteins);- formulating the CAR cells for infusion (eg suspending the CAR cells in an infusion solution);- conducting potency tests in vitro to verify that the cells in the CAR cell product are viable and able to proliferate (including if the product has been thawed);- conducting safety tests to ensure no harmful materials remain in the infusion product.

[0084] Formulation or formulating the CAR cells for infusion may comprise: adjusting for CAR cell dose (calibrating concentration of cells per volume), pH (to match physiological conditions), osmolarity (to match physiological conditions); and / or adding cryoprotectants (to protect the product if it is to undergo freezing before use).

[0085] As referred to herein, a “CAR immune cell” is used to define an immune cell comprising at least one chimeric antigen receptor (CAR).

[0086] As referred to herein, the term "immune cell" refers to a cell that may be part of the immune system, either the adaptive (i.e. cellular or humoral) or innate immune system, and executes a particular effector function such as alpha-beta T cells, NK cells, NKT cells, B cells, B regulatory (Breg) cells, T regulatory (Treg) cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes or macrophages, or any hematopoietic progenitor cells such as pluripotent stem cells and early progenitor subsets that may mature or differentiate into somatic cells.The cells may be naturally occurring (eg isolated directly from apheresis product) or generated by culturing and differentiating cells with exposure to certain cytokines and / or growth factors. The immune cell may be an artificial cell subset including induced pluripotent stem cells and cells maturated therefrom.

[0087] The cells may be isolated from a histocompatible donor, or from the subject.

[0088] "Effector function" means a specialised function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper cell activity including the secretion of cytokines. Preferred immune cells are cells with cytotoxic effector function such as alphabeta T cells (that may be CD8+ and / or CD4+), NK cells, NKT cells, ILC, CIK cells, LAK cells or gamma-delta T cells.

[0089] In more preferred embodiments, the immune cell is a T cell. T cells may be selected from the group consisting of tumour infiltrating lymphocytes, peripheral blood lymphocyte, yb T cells. A cell population or CAR cell product described herein may comprise more than one type of T cells, comprising any one or more types described herein. For example, the population of cell or CAR cell product may include naive, activated, central memory, and / or effector memory T cells.

[0090] In other embodiments, the immune cell is an NK cell, preferably an NKT cell.

[0091] A “precursor” or “progenitor” cell are terms used interchangeably herein to refer to a cell capable of differentiating into a CAR immune cell (e.g. a NK cell or a T cell that will express the CAR). A cell that is capable of differentiating into an immune cell includes a non-totipotent stem cell (eg embryonic stem cell, adult stem cell, fetal stem cell, preferably an embryonic stem cell (ESC)), multipotent stem cell (eg haematopoietic stem cell (HSC)), haematopoietic progenitor cell (eg haematopoietic progenitor cell (HPC), common lymphoid progenitor cell (CLP), or common myeloid progenitor cell (CMP)), induced pluripotent stem cell (iPSC), lymphoblast, prolymphocyte, thymocyte. A precursor or progenitor may be differentiated to a more differentiated or mature immune cell using any standard technique known to those in the art.

[0092] As referred to herein, a “CAR-T” or “CART” cell are used interchangeably to define a T cell comprising a CAR.

[0093] "in vivo persistence” as used herein refers to how long a CAR immune cell may be detected in vivo following administration the subject, or the in vivo generation of the CAR immune cell. Persistence may be influenced by the rate of in vivo expansion / proliferation / accumulation (with more expansion favouring increased persistence), and the rate of contraction (cell death and decline in CAR immune cell count; with less cell death and / or exhaustion favouring increased persistence).

[0094] “Therapeutic potential” as used herein refers to the potential for a cell to provide a desired therapeutic effect. This may include characteristics that enable in vivo persistence, proliferation / expansion, potency; to ensure that the CAR immune provides a sufficient in vivo dose to achieve its therapeutic outcome.Expansion potential

[0095] “Expansion potential” as used herein refers to the potential for a cell to successfully expand / proliferate in vitro, ex vivo and / or in vivo. Expansion potential may be measured in vitro or ex vivo by:- ability of cells to proliferate I increase in number- autocrine production of proliferative cytokines eg IL-2- cytokine polyfunctionality ie identification of how many different cytokines (eg IL-2, interferon-gamma, tumour necrosis factor alpha) an immune cell can produce,- proportion of less-differentiated cell types after being cultured (eg proportion of cells exhibiting a naive or central-memory (CM) phenotype; percentage of CD62L+ and / or CD45RA+ cells)- proportion of more-differentiated and / or exhausted cell types after being cultured (eg proportion of cells exhibiting an effector-memory (EM) phenotype; exhausted cell types may be identified by reduction in ability to produce IFN-gamma and / or tumour necrosis factor (TNF)).

[0096] “High expansion potential” may be identified in vitro or ex vivo by- increased proliferation I cell count- increased autocrine production of proliferative cytokines eg IL-2;- increased cytokine polyfunctionality;- increased proportion of less-differentiated cell types after being cultured eg increased percentage of cells exhibiting a naive or central-memory (CM) phenotype; increased percentage of CD62L+ and / or CD45RA+ T cells; and / or- reduced proportion of more-differentiated and / or exhausted cell types after being cultured eg decreased percentage of cells exhibiting an effector-memory (EM) phenotype; decreased percentage of CD62L- CD45RA- cells; compared to an appropriate control group.

[0097] “An appropriate control group” or “reference dataset” are herein used interchangeably to describe a control / reference data obtained wherein the CAR immune cell or precursor is not modified as described herein, or the CAR cell product has been manufactured using standard methodology (not a method described herein).

[0098] Expansion potential may be determined or corroborated in vivo by administering a test subject, for example a mouse, with a CAR cell product manufactured using the methods, CAR immune cell or precursor thereof of the disclosure, and comparing the in vivo proliferation of the CAR cells with proliferation of CAR cells in a control subject that has been administering a CAR cell product produced by standard methods.

[0099] “High expansion potential” may be identified in vivo by:- increased proliferation I cell count, including accumulation in a specific tissue or organ;- increased production of proliferative cytokines eg IL-2;- increased cytokine polyfunctionality;- increased proportion of less-differentiated cell types eg increased percentage of cells exhibiting a naive or central-memory (CM) phenotype; increased percentage of CD62L+ and / or CD45RA+ T cells, including accumulation in a specific tissue or organ; and / or- reduced proportion of more-differentiated and / or exhausted cell types eg decreased percentage of cells exhibiting an effector-memory (EM) phenotype; decreased percentage of CD62L- CD45RA- cells;compared to an appropriate control subject / group of control subjects.

[0100] The skilled person will appreciate suitable methods known in the art to determine cell proliferation, cytokine production, and proportion of different cell types within a cell population. The Examples provided herein further demonstrate suitable methods to identify expansion potential.

[0101] “Increased proliferation” may be at least about a 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 3-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, or 60-fold or higher increase in cell count compared to a reference dataset.

[0102] “Increased production of proliferative cytokines” preferably includes increased production of IL-2. Increased IL-2 production may be at least about a 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% increase in the percentage of IL-2+ cells compared to a reference dataset, preferably at least about 10 to about 15% higher.

[0103] “Increased proportion of less-differentiated cell types after being cultured” may comprise at least about a 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 355, 36%, 37%, 38%, 39%, 40% increase in the percentage of less-differentiated cell types, eg CD45RA CCR7+ or CD45RA CD62L+ cells, compared to a reference dataset, preferably at least about 10% to about 20% higher.“Reduced proportion of more-differentiated and / or exhausted cell types after being cultured” may comprise at least about a 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 355, 36%, 37%, 38%, 39%, 40% decrease in the percentage of more-differentiated cell types, eg CD45RA-CD62L- cells, compared to a reference dataset, preferably at least about 10% to about 20% lower.

[0104] “Relatively high proportion” of the cell population may comprise at least about a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cell population.

[0105] “Relatively low proportion” of the cell population may comprise between about 0% to at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 49% of the cell population.

[0106] Proportion of cells may also be assessed by fold-change (FC) or log foldchange (logFC), for example as described herein in the Examples. For example, increased proportion of cells exhibiting a histone biomarker signature may be characterised by at least about a 1 -logFC, 1 .5-logFC, 2-logFC, 2.5logFC, 3-logFC, 3.5- logFC, 4-logFC, 4.5-logFC, 5-logF, 5.5-logFC, 6-logFc, 6.5-logFC, 7-logFC, 7.5-logFC, 8-logFC, 8.5-logFc, 9-logFC, 9.5-logFC or about 10-logFC compared to a reference dataset.Transcription factors

[0107] As used herein, KLF7 refers to the transcription factor Kruppel-like factor 7 encoded by the gene KLF7 (Chromosome 2: 207,079,115-207,166,018; Ensembl gene reference: ENSG000001 18263.16). KLF7 is also known as UKLF or Kruppel-like factor 7 (ubiquitous). KLF7 (UniProt reference: 075840) has isoform variants due to alternative splicing (eg Ensembl transcript reference: ENST00000703734.1 ). Exemplary polypeptide sequences for KLF7 are provided in SEQ ID NO:1 and SEQ ID NO:2. Exemplary polynucleotide sequences encoding KLF7 are provided in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

[0108] “A KLF7 polypeptide or functional variant or fragment thereof” encompasses the full-length KLF7 protein, or a variant or a fragment which achieves the desired functionality as described herein ie provision of the functional variant or fragment to a CAR immune cell or precursor thereof improves expansion potential.

[0109] “A KLF7 polypeptide or functional variant or fragment thereof” may comprise a polypeptide or fragment thereof that is at least 70%, 75%, 80%, 85%, 90%, 91 %, 2%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to the sequence set forth in SEQ ID NO:1 .

[0110] “A KLF7 polypeptide or functional variant or fragment thereof’ may comprise a polypeptide or fragment thereof that is at least 70%, 75%, 80%, 85%, 90%, 91 %, 2%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to the sequence set forth in SEQ ID NO: 2.

[0111] “ A KLF7 polypeptide or functional variant or fragment thereof” may be encoded by a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91 %, 2%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in nucleotide sequence to the sequence set forth in SEQ ID NO:3.

[0112] “A KLF7 polypeptide or functional variant or fragment thereof” may be encoded by a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91 %, 2%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in nucleotide sequence to the sequence set forth in SEQ ID NO:4.

[0113] “A KLF7 polypeptide or functional variant or fragment thereof” may be encoded by a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91 %, 2%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in nucleotide sequence to the sequence set forth in SEQ ID NO:5.

[0114] “Increasing expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same” or “increased expression or amount of KLF7” may comprise increased or increasing the expression or amount of KLF7 polypeptide or functional variant or fragment thereof by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0115] “Increasing expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same” may comprise or “increased expression or amount of KLF7” may comprise increased or increasing the expression or increasing the amount of one or more polynucleotides encoding a KLF7 polypeptide or a functional variant or fragment thereof by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98, 99% or 100%.

[0116] As used herein, CCR7 refers to C-C chemokine receptor type 7 protein encoded by the gene CCR7. CCR7 is also known as BLR2, CC-CKR-7, CCR-7, CD197, CDw197, CMKBR7, EBI1 , or C-C motif chemokine receptor 7.

[0117] As used herein, TMEM45A refers to transmembrane protein 45 encoded by the gene TMEM45A. TMEM45 is also known as DNA polymerase-transactivated protein 4 (DNAPTP4) or dermal papilla-derived protein 7 (DERP7).

[0118] As used herein, SLAMF7 refers to SLAM family member 7 protein encoded by the gene SLAMF7. SLAMF7 is also known as SLAF7, CD2 subset 1 , CD2-like receptoractivating cytotoxic cells (CRACC), Membrane protein FOAP-12, Novel Ly9, CD319, Protein 19A.

[0119] As used herein LIPC refers to hepatic triacylglycerol lipase encoded by the gene LIPC. LIPC is also known as hepatic lipase (HL), lipase member C, Lysophospholipase (EC:3.1 .1 .5), Phospholipase A1 (EC:3.1 .1 .32).

[0120] As used herein CLIC3 refers to chloride intracellular channel protein 3 encoded by the gene CLIC3.

[0121] As used herein ZFP57 refers to Zinc finger protein 57 homolog protein encoded by the gene ZFP57. ZFP57 is also known as Zfp-57, zinc finger protein 698.

[0122] As used herein RHBDD2 refers to Rhomboid domain-containing protein 2 encoded by the gene RHBDD2. RHBDD2 is also known as RHBD2, RHBDL7.Histone markers

[0123] “Histone marker” or “histone mark” as used interchangeably herein, refers to a modification of histone protein in chromatin that indicates the accessibility of the chromatin for transcription and expression of one or more genes. Histone marks may be intergenic or intragenic (eg located on promoter, enhancer sequences). Histone marks may be present on the core / nucleosomal histone proteins: H2A, H2B, H3, H4. Histone modifications include methylation (me), acetylation (ac), ubiquitylation, and phosphorylation.

[0124] Transcriptionally repressive histone marks are marks are associated with reduced gene expression, due to increased chromatin condensation (formation of heterochromatin). Exemplary repressive histone marks include H3K27me3, H3K9me1 , H3K9me2, H3K9me3 (where K# represents the lysine residue modified in the histone (H#) protein, as per standard nomenclature). Due to the sensitivity limitations of methods used to detect gene transcription, a “transcriptionally repressive” state due to increased chromatin condensation may or may not result the absence of detectable transcript, or a decrease in detectable transcript.

[0125] Transcriptionally active histone marks are modifications are associated with increased gene expression, due to reduced chromatin condensation (decreased heterochromatin in favour of euchromatin). Exemplary active histone marks include H3K4me2, H3K4me1 , H3K4me3, H3K36me3, H3K79me2, H3K9Ac, H3K27Ac, H4K16Ac. Due to the sensitivity limitations of methods used to detect gene transcription, a “transcriptionally active” state due to reduced chromatin condensation may or may not result in the presence of detectable transcript, or an increase in detectable transcript.

[0126] “Histone biomarker” as used herein refers to a histone marker of a gene that is a biomarker indicative of expansion potential. Presence or absence of a histone biomarker may, for example, be indicated by the methylation status (methylated or nonmethylated) of a histone protein at a particular gene locus.

[0127] “Histone biomarker signature” as used herein refers to a histone biomarker or combination of histone biomarkers indicative of expansion potential.

[0128] A “transcriptionally active histone biomarker signature” is defined herein as a histone biomarker signature where transcriptionally active histone biomarker / s is / are favored at a gene locus, such that transcription of the gene is or is likely to be increased or enhanced.

[0129] A “transcriptionally repressive histone biomarker signature” is defined herein as a histone biomarker signature where transcriptionally repressive histone biomarker / s is / are favored at a gene locus, such that transcription of the gene is or is likely to be reduced or inhibited.

[0130] “Assessing a histone biomarker signature” as used herein refers to determining the presence or absence of a histone biomarker signature within a cell or cell population of interest.

[0131] In some embodiments, assessing a histone biomarker signature may comprise or consist of assessing a single histone biomarker (eg H3K27me3) at a single gene locus (eg KLF7). In other embodiments, assessing a histone biomarker signature may comprise or consist of assessing multiple histone biomarkers (eg H3K4me2 and H3K27me3) at a single locus (eg KLF7). In other embodiments, assessing a histone biomarker signature may comprise or consist of assessing multiple histone biomarkers (eg H3K4me2, H3K27me3) at multiple loci (eg KLF7, ZFP57).

[0132] “Assessing a histone biomarker signature” may further refer to assessing the relative proportion of cells within a cell population that exhibit a preferred histone biomarker at a particular gene locus; for example the proportion of cells that exhibit transcriptionally active mark / s compared to transcriptionally repressive mark / s. For example, a transcriptionally active histone signature of KLF7 may comprise increased proportion of cells demonstrating H3K4me2 (a transcriptionally active histone marker) at the KLF7 locus and reduced proportion of cells demonstrating H3K27me3 (a transcriptionally repressive histone marker) at the KLF7 locus.

[0133] Preferably, assessing a histone biomarker signature to identify expansion potential comprises assessing one or more of the histone biomarkers set forth in Table 5.

[0134] In some embodiments, methods comprising assessing a histone biomarker signature to identify expansion potential may further comprise assessing one or more of the histone biomarkers set forth in Table 4.

[0135] In some embodiments, methods comprising assessing a histone biomarker signature to identify expansion potential may further comprise assessing a histone marker phenotypic of CM-derived CART cells, because CM-derived cells are associated with less differentiated cell status and improved in vivo proliferation. For example, assessing a histone marker of a CM-like CAR cell may comprise assessing histone markers of one of more of: ETV6, ZNF407, DPF3, SCML4, OLIG3, KLF7, LEF1, TRPS1 and ZEB2; preferably one or more of the histone markers set forth in Table 2.

[0136] The skilled person is familiar with standard methods for identifying histone markers, and thus identifying histone biomarker signatures as described herein. For example, immunoblotting (eg western blot for certain histone marks), chromatin immunoprecipitation (ChIP) assays, ChlP-sequencing (ChlP-seq), or CUT&RUN (eg as described herein).

[0137] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell.Engineered cells

[0138] The terms "engineered cell", “modified cell” and "genetically modified cell" as used herein can be used interchangeably. The terms mean containing and / or expressinga foreign gene or nucleic acid sequence that in turn modifies the genotype or phenotype of the cell or its progeny. Especially, the terms refer to the fact that cells, preferentially immune cells, can be manipulated by recombinant methods well known in the art to express stably or transiently RNA, peptides or proteins that are not expressed in these cells in the natural state.

[0139] For example, nucleic acid sequences may be delivered into cells using an adenoviral, adeno-associated viral (AAV)-based, retroviral or lentiviral vector or any other pseudotyped variations thereof or any other gene delivery mechanism such as electroporation, nano-injection, or use of non-viral vectors vectors (eg nanoparticles, lipid nanoparticles, exosomes, extracellular vesicles, microvesicles, liposomes, polyplexes). Delivery may comprise gene editing such as CRISPR / Cas9, transposons (e.g. sleeping- beauty) or variations thereof. The gene delivery may be in the form of mRNA (transient) or DNA (transient or permanent). The skilled person will be familiar with appropriate delivery systems available in the art.

[0140] For example, immune cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface.

[0141] In another example, immune cells or precursors thereof are engineered to express a CAR and engineered to overexpress the transcription factor KFL7. For example, nucleic acid sequences encoding a KLF7 polypeptide or functional variant or fragment thereof may be transduced or delivered into cells using delivery methods such as those described herein.

[0142] In another embodiment, the cell may be genetically modified to indirectly result in increased KLF7 expression, by modifying genes or polypeptides upstream or downstream of KLF7 expression to increase the levels of KLF7 expressed in the cell. The skilled person will appreciate suitable methods to identify KLF7 overexpression by the cell; such as measuring protein levels by western blot, measuring a reporter linked to the KLF7 (eg detecting a fluorescent marker), measuring mRNA levels.

[0143] The cell may be an "engineered cell", "genetically modified cell", “immune cell” or “immune effector cell” as described herein.

[0144] As referred to herein, a "genetically modified cell" includes any cell comprising a non-naturally occurring and / or introduced nucleic acid molecule or nucleic acidconstruct encompassed by the present disclosure. The introduced nucleic acid molecule or nucleic acid construct may be maintained in the cell as a discreet DNA molecule, or it may be integrated into the genomic DNA of the cell.

[0145] Genomic DNA of a cell should be understood in its broadest context to include any and all endogenous DNA that makes up the genetic complement of a cell. As such, the genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA and the like. As such, the term "genomically integrated" contemplates chromosomal integration, mitochondrial DNA integration, and the like. The "genomically integrated form" of the construct may be all or part of the construct.

[0146] In some embodiments of the disclosure, the genetically modified cell is a genetically modified cell autologous to the target cell from the subject.Pharmaceutical compositions

[0147] The cell or CAR cell product of the present disclosure may be administered to a subject perse, or in a pharmaceutical composition.

[0148] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0149] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. The composition may further include a cytokine for enhancing cell killing, such as IL-2 or interferon-gamma (IFNy).

[0150] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatine, vegetable oils and polyethylene glycols.

[0151] Pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0152] Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0153] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

[0154] Typically, the pharmaceutical composition is administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

[0155] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0156] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0157] Pharmaceutical compositions suitable for use in context of the present disclosure include compositions wherein the active ingredients are contained in anamount effective to achieve the intended purpose. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0158] Compositions of the present disclosure may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser advice may be a syringe. The syringe may be prepacked with the cells. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labelling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the disclosure formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition, as if further detailed above.Methods of treatment and administration

[0159] As discussed further in this document, the present disclosure finds application in the prevention or treatment of a variety of conditions, although preferably in the prevention or treatment of a cancer or autoimmune disease.

[0160] As used herein, the term "subject" refers to a mammal capable of suffering from a disease, disorder or condition. Particular subjects of interest are human beings, and scientifically relevant species such as mice, rats, ferrets, guinea pigs, hamsters, nonhuman primates, dogs, pigs and sheep, or economically relevant animals such as horses, dogs, cats and cattle. In a preferred embodiment of the disclosure, the subject is a human.

[0161] The subject may be a subject suffering from a disorder such as cancer or an autoimmune disease (a patient), but the subject also may be a healthy subject. As used herein, the terms “subject”, “individual” and “patient” may be used interchangeably.

[0162] The term "treat" (treatment of) a disease, disorder or condition as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease, disorder or condition experienced by a subject.

[0163] The terms "treat", "treating" or "treatment" of a cancer as used herein are to be understood to include within their scope one or more of the following outcomes: (i) inhibiting to some extent the growth of a primary tumour in a subject, including, slowing down and complete growth arrest, and including reducing the growth of the primary tumour after resection; (ii) inhibiting to some extent the growth and formation of one or more secondary tumours in a subject; (iii) reducing the number of tumour cells in a subject; (iv) reducing the size of a tumour in the subject; (v) inhibiting (i.e. reduction, slowing down or complete stopping) of tumour cell infiltration into peripheral organs; (vi) inhibiting (i.e. reduction, slowing down or complete stopping) of metastasis; (vii) improving the life expectancy of a subject as compared to the untreated state; (viii) improving the quality of life of a subject as compared to the untreated state; (ix) alleviating, abating or ameliorating at least one symptom of cancer in a subject; (x) causing regression or remission of cancer in a subject; (xi) relieving a condition in a subject that is caused by cancer; and (xii) stopping symptoms in a subject that are associated with cancer.

[0164] The terms "treat", "treating" or "treatment" of an autoimmune disease as used herein are to be understood to include within their scope one or more of the following outcomes: (i) inhibiting to some extent progression of disease in a subject, including, slowing down and complete arrest, and including reducing spread of disease through tissue or to other tissues; (ii) inhibiting to some extent the aberrant release of cytokines and / or autoantibodies associated with the disease; (iii) reducing the number of auto- reactive cells in a subject; (iv) reducing the size of a tumour in the subject; (v) inhibiting (i.e. reduction, slowing down or complete stopping) of infiltration of auto-reactive cells into peripheral organs; (vi) improving the life expectancy of a subject as compared to the untreated state; (vii) improving the quality of life of a subject as compared to the untreated state; (viii) alleviating, abating or ameliorating at least one symptom of the autoimmune disease in a subject; (ix) causing regression or remission of the autoimmune disease in a subject; (x) relieving a condition in a subject that is caused by the autoimmune disease; and (xi) stopping symptoms in a subject that are associated with the autoimmune disease.

[0165] The terms “prevent” and “prevention” as used herein, are intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a diseaseor disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in an individual that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). Biological and physiological parameters for identifying such patients are well known by physicians.

[0166] For example, prevention of a cancer may be characterized by inhibiting the formation of a primary tumour in a subject, inhibiting the formation of one or more secondary tumours in a subject, or reducing or eliminating the recurrence of cancer in a subject in remission. For example, prevention of an autoimmune disease or cancer, may be characterized by preventing an aberrant immune response as indicated by an absence of an increased release of particular cytokines or auto-antibodies.

[0167] The term “inhibiting” as used herein is taken to mean a decrease or reduction in the growth of a cancer, cancerous cell or tumour when compared to the growth in a control, such as an untreated cell or subject. In some embodiments, growth may be decreased or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to an untreated control.

[0168] Inhibition of the growth of a cancer, tumour or cancerous cell may be assessed by a range of methods known in the art. For example, for a cancerous cell in vitro, the growth of the cell may be determined by a suitable proliferation assay, or by method which assess the extent of incorporation of tritiated thymidine into cellular DNA over a given period of time. For a tumour or cancerous cell present in vivo, the growth of the tumour or cell may be determined for example by a suitable imaging method known in the art.

[0169] Subjects requiring treatment include those already having a benign, pre- cancerous, or non-metastatic tumour as well as those in which the occurrence or recurrence of cancer is to be prevented. Subjects may have metastatic cells, including metastatic cells present in the ascites fluid and / or lymph node.

[0170] The subject who has received the treatment for cancer may be in partial or complete remission.

[0171] A "therapeutically effective amount" of an agent, e.g. a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effectiveamount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.

[0172] The term "administered" means administration of a therapeutically effective dose of the aforementioned CAR cell product or pharmaceutical composition to a subject. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localised delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art

[0173] The objective or outcome of treatment may be to reduce the number of cancer cells; reduce the primary tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; and / or relieve to some extent one or more of the symptoms associated with the disorder.

[0174] Efficacy of treatment can be measured by assessing the duration of survival, time to disease progression, the response rates (RR), duration of response, and / or quality of life.

[0175] The term “cancer” will be understood to include benign, pre-cancerous, pre- neoplastic, non-metastatic tumours, or metastatic tumours. The cancer may be a solid or a “liquid” tumour. In other words, the cancer may be growth in a tissue (carcinoma, sarcoma, adenomas etc) or it may be a cancer present in bodily fluid such as in blood or bone marrow (e.g., lymphomas and leukaemias). The cancer may be a secondary cancer or metastases.

[0176] Examples of particular cancers include but are not limited to: adenocarcinoma, adenoma, adenofibroma, adenolymphoma, adontoma, AIDS related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma sclerosing, angiomatosis, apudoma, anal cancer, angiosarcoma, aplasticanaemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS tumours, breast cancer (preferably triple negative breast cancer), branchioma, CNS tumours, carcinoid tumours, cervical cancer, childhood brain tumours, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma, carcinoma (e.g. Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumour, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), carcinosarcoma, cervical dysplasia, cystosarcoma phyllodies, cementoma, chordoma, choristoma, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangioma, cholangiocarincoma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma- protuberans, desmoplastic-small-round-cell-tumour, ductal carcinoma, dysgerminoam, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconi anaemia, fibroma, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumour, genitourinary cancers, germ cell tumours, gestationaltrophoblastic-disease, glioma, gynaecological cancers, giant cell tumours, ganglioneuroma, glioma, glomangioma, granulosa cell tumour, gynandroblastoma, haematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, hemangiosarcoma, histiocytic disorders, histiocytosis malignant, histiocytoma, hepatoma, hidradenoma, hondrosarcoma, immunoproliferative small, opoma, ontraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, langerhan's cell-histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, li-fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leigomyosarcoma, leukemia (e.g. B-cell, mixed cell, null-cell, T-cell, T-cell chronic, HTLV-llassociated, lymphangiosarcoma, lymphocytic acute, lymphocytic chronic, mast-cell and myeloid), leukosarcoma, leydig cell tumour, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphagioma, lymphagiomyoma, lymphangiosarcoma, male breast cancer, malignant-rhabdoid-tumour-of-kidney, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma, myeloproliferative disorders, malignant carcinoid syndrome carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymoma, mesonephroma, mesothelioma, myoblastoma, myoma, myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, nonmelanoma skin cancer, non-small-cell-lung-cancer (NSCLC), neurilemmoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, neoplasms (e.g. bone, breast, digestive system, colorectal, liver), ocular cancers, oesophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral- neuroectodermal-tumours, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian carcinoma, papilloma, paraganglioma, paraganglioma nonchromaffin, pinealoma, plasmacytoma, protooncogene, rare-cancers-and-associated- disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc), small intestine cancer, soft tissue sarcoma, spinal cord tumours, squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma, sarcoma (e.g. Ewing's experimental, Kaposi's and mast-cell sarcomas), Sertoli cell tumour, synovioma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-(bladder), transitional-cell-cancer-(renal-pelvis- / -ureter), trophoblastic cancer, teratoma, theca cell tumour, thymoma, trophoblastic tumour, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom' s-macroglobulinemia and Wilms' tumour. Preferred particular examples include lymphoma, leukemia, pleural or peritoneal mesothelioma, gastric cancer, endometrial cancers, colorectal cancer, non-small cell lung adenocarcinoma, cholangiocarcinoma, ovarian carcinoma, esophageal cancer, pancreatic ductal adenocarcinoma, triple negative breast cancer and any other mesothelin-positive cancers.Compositions and administration

[0177] The delivery or administration of the cell or cell product according to the disclosure may be delivery or administration of the cell or cell product alone, or delivery or administration of the cell formulated into a suitable pharmaceutical composition. Accordingly, the present disclosure provides a pharmaceutical composition including a CAR immune cell or CAR cell product of the disclosure, and a pharmaceutically acceptable carrier.

[0178] Methods are known in the art for providing CAR-containing cells for immunotherapy (see for example Kershaw, MH. et al. Clin Cancer Res. 2006;12(20): 6106-15; Parker LL. et al. Hum Gene Ther 2000;11 : 2337-87). Furthermore, protocols and methods are known in the art for the preparation, expansion and assessment of mammalian CAR-expressing cells (see for example Cheadle, EJ. et al. Antibody Engineering: Methods and Protocols, Second Edition, Methods in Molecular Biology, vol. 907: 645-66) and include those aforementioned and summarised in the Examples below.

[0179] Administration of the pharmaceutical composition or CAR product may also be via parenteral means which include intravenous, intraventricular, intraperitoneal, intramuscular, intrapleurally or intracranial injection, or local injections to the site of the disease eg a tumour or cancerous mass.ExamplesExample 1 - Materials and methodsStudy design

[0180] Sample size was determined by known variation in CUT&RUN and RNA-seq of healthy donor T cells. All data was included except biological replicates that failed QC assessments. All studies were approved by the Institutional Review Board of the Fred Hutchinson Cancer Center.Flow cytometry and sorting

[0181] Cryopreserved healthy human donor peripheral blood mononuclear cells were thawed, negatively selected using the CD8+ T cell isolation kit, human (Miltenyi Biotec) and sorted into CD8+ T cell subsets using antibodies against CD3, Va7.2, CD161 ,CD62L, CD45RA, CD45RO, CD8, CD4, CD95, and / or CD27 with gating as outlined in Fig. 1 A on a BD FACSAria. Flow cytometry was performed using antibodies against CD3, Va7.2, CD161 , CD62L, CD45RA, CD45RO, CD8, CD4, CD95, and / or CD27 according to manufacturers’ recommendations on a BD FACSymphony and analyzed on FlowJo. CAR-T cells were similarly thawed and sorted by truncated EGFRt.CAR-T manufacturing and retroviral transduction

[0182] Patient (PT) CM-derived CAR-T were manufactured from CM-enriched CD8+ T cells isolated from PTs participating in a phase 1 / 2 clinical trial, as described in Gauthier, J. et al. Blood 2020;135(19): 1650-1660 (incorporated herein by reference in its entirety). Healthy donor (HD) CM- and EM-derived CAR-T were manufactured from CM or EM CD8+ T cells isolated from apheresis products, as described above, then stimulated and transduced as described in Gauthier, J. et al. Blood 2020;135(19): 1650- 1660. KFL7 open reading frame from sequence NM_001270942 was cloned into an epHIV7 retroviral plasmid containing P2A and GFP, and virus was grown and transduced into T cells as described in Gauthier, J. et al. Blood 2020;135(19): 1650- 1660.CUT&RUN Assay

[0183] CUT&RUN was performed as described in Janssens, DH. et al. Nat Genet 2021 ;53(11 ): 1586-1596. Briefly, live sorted cells were incubated with concavalin A beads, then permeabilized and incubated overnight at 4C with anti-H3H3K27me3me3, anti-H3H3K4me2me2 or IgG isotype control with rabbit-anti-mouse as an adapter. Samples were then processed on a Beckman Biomek FX liquid-handling robot. pA- MNAse was added to samples, DNA cleaved and released, and chromatin targets purified, followed by adapter ligation, Ampure cleanup and PCR amplification of libraries. Quality check (QC) of amplified DNA was assessed using an Agilent 4200 TapeStation and quantified on a QuBit Fluoremeter (ThermoFisher). Samples were then pooled and paired-end sequenced on an Illumina HiSeq 2500.CAR-T counts

[0184] CAR-T cell peak expansion was quantified as the highest percentage of CD8+ EGFRt+ cells falling within the CD45+ vs SSC lymphocyte gate multiplied by the absolute lymphocyte count. In vitro CAR-T expansion was quantitated with acridineorange / propidium iodide dye on a Cellaca PLX (Revvity) as per manufacturer’s instructions.CUT&RUN and RNA-seq data processing and analysis

[0185] Packages and / or programs including FastQC, Bowtie2, SEACR, RSEM, STAR, GenomicRanges, Limma, DeepTools, mixtools, GSVA, fgsea, ggVennDiagram, GeneOverlap, tidyveRse, ggPlot2, ggrepel, viridis, RColorBrewer, clipr, readxl, Metascape.org, and / or Graphpad Prism were used for analysis of differential detection, gene ontology, geneset enrichment, and / or statistical analysis.

[0186] CUT&RUN data processing and analysis generally follows the pipeline published in detail on protocols.io (https: / / www.protocols.io / view / cut-amp-tag-data- processing-and-analysis-tutorial-e6nvw93x7gmk / v1 ). Peaks were called using SEACR epigenomic enriched peaks were defined simultaneously following two criteria: peaks that were among the top 10% enriched peak regions and peaks identified above background noise (profiled by IgG isotype control). To generate the heatmap and summary plot of histone modifications at each TSS region (from 1 kb downstream to 1 kb upstream), the countMatrix and plotHeatmap functions from deepTools version 3.3.1 were used to sum the total signals.

[0187] RNA-seq raw data that passed the FASTQC quality check were processed by RSEM using STAR as the aligner. The expected count was used as the gene expression raw count. Log-transformed count per million (logCPM) was used to avoid sequencing depth biases unless otherwise specified. UCSC genome browser is employed to visualize the epigenomic landscape around the genes or regions of interest. When integrating RNA-seq and CUT&RUN gene lists, a single differentially expressed (DEx) or differentially enriched (DEn) gene was counted only once by selecting the transcript or peak with the highest logFC.

[0188] Data processing and analysis was conducted in R, Python and Linux. The Limma R package was used for differential detection analyses for both CUT&RUN and RNA-seq data, HD or PTs were considered as random effect variables to remove donor effect. Benjamini-Hochberg procedure was used to control for false discovery rate. Principal Component Analyses (PCA) and Uniform Manifold Approximation and Projections (UMAP) provided visualizations of high-dimensional data in two-dimensionalspace. Two-component GMM was used to characterize the distribution of reads mapping in a 2kb window surrounding the TSS for each gene and was fitted using the normalmixEM function from mixtools library in R. Distinct thresholds at 4.5 for H3K4me2 and 2.5 for H3K27me3 were inferred from the intersection point of the GMM distribution and separated genes into positive and negative groups per histone marker. All statistical tests were two-sided unless otherwise specified.Example 2 - CUT&RUN analyses of histone marks distinguishes distinct human CD8+ T cell subsets

[0189] The inventors set out to compare transcriptomic and epigenomic differences in T cell precursors and CAR-T products that are prognostic of therapeutic expansion potential.

[0190] CD8+ T cells were sorted from healthy donors (HD) into CD62L+ CD45RAhiCD45RO- (naive immunophenotype; N), CD62L+ CD45RA- CD45RO+ (central memory immunophenotype; CM), and CD62L- CD45RA- (effector memory immunophenotype; EM) subsets (Fig. 1 A). The sorted N, CM, and EM subsets were of high purity and demonstrated expression of canonical immunophenotypic markers of N, CM, and EM cells (Fig. 1 B-C).

[0191] To investigate the relationship of transcriptomic output and histone marks in CD8+ T cell subsets, the inventors performed RNA-seq analyses and CUT&RUN analyses of transcriptionally repressive H3K27me3 and permissive H3K4me2 histone marks were performed on each subset. Quality check (QC) metrics were satisfied for RNA-seq samples and data (6 HD, 3 T cell subsets / HD) and CUT&RUN data (4 HD, 3 T cell subsets / HD), with consistent findings across donors. The high quality of RNA-seq data was confirmed by the observed correlation between genes differentially expressed in CM vs EM cells with recently published human CD8+ T cell subset data (P=4.85x10‘13, odds ratio = 31 .54 by Fisher’s exact test).

[0192] H3K27me3 marks were mostly located within gene bodies and in intergenic regions, whereas a greater proportion of H3K4me2 marks were associated with promoter regions, confirming the specificity of the CUT&RUN profiles.

[0193] A genome-wide peak calling strategy was implemented to identify regions enriched for H3K27me3 and H3K4me2 marks in an unbiased manner, which organizedthe T cell subsets into distinct clusters. Genomic tracks coding for TFs that have been typically associated with N and CM subsets were marked by low H3K27me3 in N and CM subsets and low H3K4me2 in EM subsets, and vice versa for TFs that associated with effector differentiation.

[0194] To allow direct comparison of epigenomic and transcriptomic data the inventors focused on gene units, by assigning H3K27me3 and H3K4me2 marks within + 1 kb of the transcriptional start site (TSS) to an equivalent transcript.

[0195] When comparing subsets, analysis of H3K4me2 and H3K27me3 profiles identified more differentially enriched (DEn) genes than differentially expressed (DEx) genes identified by RNA-seq (Fig. 2B). A larger number of DEn TFs between subsets were identified than by DEx TFs by RNA-seq (Fig. 2C). Analyses of histone marks on genes +1 kb of the TSS by Principal Component Analyses (PCA, Fig. 2D) and Uniform Manifold Approximation and Projection (UMAP, Fig. 2E) more clearly distinguished T cell subsets than the same analysis performed on the RNA-seq data, and this difference was particularly prevalent in the first projection (PCA1 and UMAP1 ).

[0196] This analysis demonstrates that CUT&RUN profiling of the histone modifications H3K4me2 and H3K27me3 detects a greater variance and uncovers more differences between T Cell subsets than RNA-seq.Example 3 - Genes marked by different combinations of H3K4me2 and H3K27me3 exhibit different mean levels of transcript abundance in CD8+ T cell subsets

[0197] The greater distinction between CD8+ T cell subsets by histone marks compared to RNA-seq led the inventors to examine the associations between histone marks and transcript abundance.

[0198] Genes that were positive and negative for H3K27me3 and H3K4me2 were defined using a two-component Gaussian mixture model to fit histone mark distribution within +1 kb of the TSS (Fig. 3A-B), allowing visualization of the enrichment of histone marks around the TSS, and classification of all genes as either as H3K4me2 or H3K27me3 single-marked, bivalent, or unmarked (Fig. 3C). When integrated with RNA- seq, most genes with abundant transcripts were marked with H3K4me2 and not H3K27me3, as expected. However, transcripts were also detected in numerous genes that were H3K4me2-H3K27me3-, H3K4me2+H3K27me3+ or H3K4me2-H3K27me3+.

[0199] A bimodal pattern of transcript abundance was identified, exemplified by genes exhibiting low or undetected transcript normalized to sequencing depths (RNAl0; below 2.5 transcript copies / sample) and genes with more abundant transcript (RNAhi; above 2.5 transcript copies / sample). The mean transcript abundance increased in a linear trend with changes in histone marks from H3K4me2-H3K27me3+ to H3K4me2- H3K27me3- to H3K4me2+H3K27me3+ to H3K4me2+H3K27me3-. Combining the bimodal distribution of RNA transcript (RNAhiand RNAl0) and the four patterns of histone marks (H3K4me2-H3K27me3+; H3K4me2-H3K27me3-;H3K4me2+H3K27me3+; H3K4me2+H3K27me3-) allowed categorization of genes into eight groups of genes.

[0200] More genes showed changes in histone mark patterns within the same transcript abundance category (i.e., RNAl0vs RNAl0; RNAhivs RNAhi) than across transcript abundance categories. In both RNAhiand RNAl0settings, there were more genes with histone mark pattern changes that were not DEx compared to those that were DEx, a difference most marked when comparing CM and EM subsets. These data showed that histone mark patterns differed between CD8+ T cell subsets in numerous highly and lowly expressed genes, even if those genes were not DEx between T cell subsets. The directionality of genes that changed from a repressive H3K4me2- H3K27me3+ to permissive H3K4me2+H3K27me3- pattern could be assigned.Furthermore, approximately 3000 - 3300 bivalent genes were identified, representing a range of 8.9% - 9.8% of all the promoters investigated across three T cell subsets.

[0201] When RNAhigenes were categorized according to histone mark patterns, the clearest distinction between T cell subsets were observed in PCA of RNA-seq data from bivalent genes, even though H3K4me2+H3K27me3- were associated with active transcription. These data indicated that classifying genes into distinct patterns of H3K4me2 and H3K27me3 marks enhanced the discriminatory capacity in distinguishing T cell subsets possibly by highlighting those genes in states of active transcriptional flux.

[0202] When DEx RNAhigenes were analysed, genes marked with bivalent or unmarked patterns showed a median fold change in transcript abundance that was intermediate between that of genes marked by repressive H3K4me2-H3K27me3+ in one subset and permissive H3K4me2+H3K27me3- in another. Therefore, directionality could be ascribed to genes exhibiting distinct histone mark changes between subsets:either towards a more differentiated cell type or a less differentiated cell type. This enabled identification of other genes associated with differentiation towards EM or dedifferentiation towards CM that were not DEx by RNA-seq.

[0203] The data indicate that analyses of changes in histone mark patterns can identify genes associated with differentiation towards CM and EM even in the absence of differential gene expression, further evidencing the capabilities of analysing histone markers to assess differentiation state and expansion potential.Example 4 - Histone mark analyses identify biologically relevant differences between CAR-T cells manufactured from distinct sources that are not apparent by RNA-seq

[0204] To identify differences between CAR-T cell products manufactured from similar, yet distinct, CD8+ T cell sources, CAR-T cells were manufactured from N, CM or EM CD8+ T cells from HD then isolated by flow sorting for the transduction marker truncated epidermal growth factor receptor (EGFRt).

[0205] PCA analyses of RNA-seq showed some distinction of N-derived CAR-T from EM- and CM-derived CAR-T, but did not distinguish CM- and EM-derived CAR-T (Fig. 4A). Only three genes were DEx by RNA-seq between the EM- and CM-derived products (Fig. 4B). In contrast to transcript output, histone marks were able to distinguish CM- and EM-derived CAR-T cells, with 99 and 131 unique genes being DEn by single H3K4me2 and H3K27me3 analyses, respectively (Fig. 4B-C). This was confirmed in N-derived cells, where more genes were also differentially enriched by histone mark analysis than by RNA-seq when comparing N- and CM- or EM-derived CAR-T. No DEx TFs were identified by RNA-seq.

[0206] Of the histone marks that distinguished between CM- or EM-derived CART-T cells, two TFs were identified by H3K4me2 (ETV6 and ZNF407) and seven TFs were identified by H3K27me3 histone mark analyses (DPF3, SCLM4, OLIG3, KLF7, LEF1 , TRPS1 , ZEB2).Table 2: Histone biomarker signatures for CM-derived versus EM-derived CART cells

[0207] All of these genes, including KLF7, were concordantly validated by data from T cells in a manner consistent with EM-derived CAR-T displaying a more differentiated / exhausted phenotype than CM-derived CAR-T (Fig 4D-E).

[0208] To further challenge the capacity of histone marks analyses, the inventors compared transduction-marker sorted CAR-T cells manufactured from two different starting populations of CM CD8+ T cells (Figure 6). One product was manufactured from CM cells sorted to high purity from HD. The other was manufactured from CM cells enriched by two-step CliniMACS immunomagnetic selection from large B cell lymphoma (LBCL) PTs on a phase 1 clinical trial (NCT01865617). Despite both CAR-T products being manufactured from CM cells, PCA using RNA-seq and histone marks distinguished PT CM-derived CAR-T from HD CM-derived CAR-T; however, H3K4me2 and H3K27me3 histone marks analyses identified 4.4-fold and 5.7-fold more DEn genes than RNA-seq (Figure 6C; Table 3), including more CAR-T exhaustion-associated TFs (Figure 6D-F) than RNA-seq in a manner concordant with PT CM-derived CAR-T displaying a more exhausted phenotype than HD CM-derived CAR-T.Table 3: Transcription factors associated with differences between healthy donor and patient CM-derived CAR-T cells.Example 5 - Identification of a novel transcription factor and epigenomic regulome in CAR-T products that associates with robust in vivo CAR-T accumulation after infusion into large B cell lymphoma (LBCL) patients and T cell

[0209] To identify genes in infusion products (IP) that predict in vivo CAR-T cell expansion after infusion into LBCL patients treated on a phase 1 clinical trial (NCT01865617), transcriptomic and epigenomic analysis of IP to compare from 30 patients who had a documented best response of complete response (CR) versus those who did not (non-CR) was performed. Neither of these methods were able to identifydifferences between these groups, even when comparing only patients who received CM-enriched CAR-T cells.

[0210] To minimize the confounding effect of the tumour microenvironment and other patient-associated factors, patients were stratified and those who received CAR-T cells manufactured from the same starting CM CD8+ T cell subset, with the same CAR-T dose and lymphodepletion (LD) regimen, and achieved complete response to CAR-T immunotherapy (n=8) were selected. Infused CAR-T products were classified as high expander if the maximal CD8+ CAR-T cell count in blood after infusion was above the median for the group, and conversely as low expander if the maximal CD8+ CAR-T cell count was below the median. There were no differences between the cohorts in CD62L+ CD45RA- purity; or biochemical, radiologic and serum cytokines associated with CAR-T expansion in patients.

[0211] PCA of histone marks, but not RNA-seq data, distinguished CAR-T IP from high and low expanding cohorts (Fig. 5A). DEn of H3K4me2 and H3K27me3 marks identified 30-fold and 10-fold more genes, respectively, than DEx genes by RNA-seq (Fig. 5B-C; Table 4).Table 4: DEx and DEn genes in comparison of high and low expanding patient-derived CM-enriched CART cells from a clinical trial (NCT01865617).

[0212] Of these genes, two TFs were identified by histone mark analyses - KLF7 and ZFP57 - and none by RNA-seq (Fig. 5C, boxes).

[0213] When comparing high- and low-expanding infused CAR-T, KLF7 was H3K27me3-marked (transcriptionally repressed) in low expanding CAR-T IP (Fig. 5D).

[0214] When comparing high- and low-expanding infused CAR-T, ZFP57 was H3K4me2-marked (transcriptionally active) in low expanding CAR-T IP.

[0215] Furthermore, seven targets of KLF7 were upregulated in high-expanding CAR- T IP (Fig. 5E-F; Table 5): CCR7, TMEM45A, SLAMF7, UPC, CLIC3, ZFP57, and RHBDD2.Table 5: DEn genes in comparison of high and low expanding patient CM-enriched CART cells from a clinical trial (NCT01865617).Example 6 - Provision of exogenous KLF improves the expansion potential of CAR T cells

[0216] To validate these findings, KLF7 open-reading frame (ORF) was transduced into T cells and CAR-T cells. KLF7 ORF-transduced T cells showed dose-dependent increase in expansion in culture relative to the amount of transduced gene of the GFP control vector (Fig. 5G). In KLF7-transduced CAR-T cells, increased expansion, production of IL-2, and increased percentage of CD62L+ and CD45RA+ cells were observed (Fig. 5H-K).

Claims

CLAIMS1 . A method for manufacturing a chimeric antigen receptor (CAR) cell product, the method comprising:- increasing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, in an immune cell comprising a chimeric antigen receptor (a CAR immune cell) or a precursor thereof; and- then culturing the CAR immune cell or precursor thereof for a sufficient time and under suitable conditions to produce the CAR cell product.

2. A method of producing a CAR immune cell or precursor thereof, the method comprising:- introducing a polynucleotide encoding a chimeric antigen receptor (CAR) into an immune cell or precursor thereof, wherein the polynucleotide enables the CAR to be expressed on the surface of the cell;- increasing the expression or amount of a KLF polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same; in the cell; thereby producing the CAR immune cell or precursor thereof.

3. The method of claim 1 or claim 2, wherein increasing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, comprises contacting the immune cell or CAR immune cell, or precursor thereof, with an agent that increases the expression of KLF7.

4. The method of claim 3, wherein the agent is selected from a nucleic acid molecule, a protein, an aptamer and small molecule, an RNAi agent, or a transcriptional activation system for increasing the expression of KLF7, wherein preferably the agent is a polynucleotide encoding a KLF7 polypeptide or functional variant or fragment thereof.

5. The method of any one of claims 1 to 4, wherein the KLF7 polypeptide or functional variant or fragment thereof comprises or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 2%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical in amino acid sequence to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

6. The method of any one of claims 1 to 5, wherein the polynucleotide encoding KLF7 or a functional variant or fragment thereof comprises or consists of a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 2%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in nucleotide sequence to the sequence set forth in any one of SEQ ID NOs:3, 4 or 5.

7. The method of any one of claims 1 to 6, increasing expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, comprises increasing the amount of one or more polynucleotides encoding KLF7 or a functional variant or fragment thereof by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%.

8. The method of any one of claims 1 to 7, increasing expression or amount of a KLF7 polypeptide or functional variant or fragment thereof comprises increasing the amount of KLF7 polypeptide or a functional variant or fragment thereof by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%.

9. A CAR immune cell or a precursor thereof for manufacturing a chimeric antigen receptor (CAR) cell product, wherein the CAR immune cell or the precursor thereof is modified to increase expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same.

10. A population of cells comprising a plurality of the CAR immune cell or precursor thereof according to claim 9.

11. A CAR cell product obtainable or obtained by the method of any one of claims 1 or 3 to 8, or a CAR cell product comprising the CAR immune cell or precursor thereof of claim 9, or comprising the population of cells according to claim 10.

12. A pharmaceutical composition comprising the CAR immune cell or precursor thereof according to claim 9, the population according to claim 10, or the CAR cell product of claim 11 .

13. A method for identifying the expansion potential for CAR cell therapy of an immune cell comprising a chimeric antigen receptor (CAR immune cell) or a precursorthereof, or a cell population comprising a CAR immune cell or precursor thereof, the method comprising: assessing the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, in the CAR immune cell, CAR immune cell precursor or cell population; wherein when the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, is increased compared to the expression or amount in a reference dataset, the CAR immune cell, CAR immune cell precursor or cell population is identified as having high expansion potential for CAR cell therapy; and wherein when the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same, is decreased compared to the expression or amount in a reference dataset, the CAR immune cell, CAR immune cell precursor or cell population is identified as having low expansion potential for CAR cell therapy.

14. The method of claim 13, wherein the method comprises assessing a histone biomarker signature of one or more genes selected from KLF7, CCR7, TMEM45A, SLAMF7, UPC, CLIC3, ZFP57, and RHBDD2 in a CAR immune cell, CAR immune cell precursor, or a cell population comprising a CAR immune cell or precursor thereof, wherein when- one or more of KLF7, CCR7, and TMEM45A exhibit a transcriptionally active histone biomarker signature,- RHBDD2 exhibits a transcriptionally active histone biomarker signature, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 exhibit a transcriptionally repressive histone biomarker signature, the CAR immune cell, CAR immune cell precursor or cell population is identified as having high expansion potential for CAR cell therapy; and when- one or more of KLF7, CCR7, and TMEM45A exhibit a transcriptionally repressive histone biomarker signature,- RHBDD2 exhibits a transcriptionally repressive histone biomarker signature, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 exhibit a transcriptionally active histone biomarker signature, the CAR immune cell, CAR immune cell precursor or cell population is identified as having low expansion potential for CAR cell therapy.

15. The method of claim 14, comprising assessing a histone biomarker signature of one or more genes selected from KLF7, CCR7, TMEM45A, SLAMF7, LIPC, CLIC3, ZFP57, and RHBDD2 in a CAR immune cell or CAR immune cell precursor, wherein when- one or more of KLF7, CCR7, and TMEM45A is not H3K27me3 methylated,- RHBDD2 is H3K4me2 methylated, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 \s not H3K4me2 methylated, the CAR immune cell or CAR immune cell precursor is identified as having high expansion potential for CAR cell therapy; and when- one or more of KLF7, CCR7, and TMEM45A is not H3K27me3 methylated,- RHBDD2 is not H3K4me2 methylated, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 \s H3K4me2 methylated, the CAR immune cell or CAR immune cell precursor is identified as having low expansion potential for CAR cell therapy.

16. The method of claim 14, comprising assessing a histone biomarker signature of one or more genes selected from KLF7, CCR7, TMEM45A, SLAMF7, LIPC, CLIC3, ZFP57, and RHBDD2 in a cell population comprising a CAR immune cell or CAR immune cell precursor, wherein when a relatively high proportion of the cell population is characterised by:- one or more of KLF7, CCR7, and TMEM45A exhibiting a transcriptionally active histone biomarker signature,- RHBDD2 exhibiting a transcriptionally active histone biomarker signature, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 exhibiting a transcriptionally repressive histone biomarker signature, the cell population is identified as having high expansion potential for CAR cell therapy.

17. The method of claim 16, wherein when a relatively high proportion of the cell population is characterised by:- no H3K27me3 methylation of one or more of KLF7, CCR7, and TMEM45A,- H3K4me2 methylation of RHBDD2, and / or- no H3K4me2 methylation of one or more of SLAMF7, LIPC, CLIC3, ZFP57, the cell population is identified as having high expansion potential for CAR cell therapy; and when a relatively high proportion of the cell population is characterised by:- H3K27me3 methylation of one or more of KLF7, CCR7, and TMEM45A,- no H3K4me2 methylation of RHBDD2, and / or- H3K4me2 methylation of one or more of SLAMF7, LIPC, CLIC3, ZFP57, the cell population is identified as having low expansion potential for CAR cell therapy.

18. The method of any one of claims 14 to 17, wherein the method comprises assessing a histone biomarker signature of KLF7.

19. The method of claim 18, wherein the method comprises assessing a histone biomarker signature of KLF7 and at least one of CCR7, TMEM45A, SLAMF7, LIPC, CLIC3, ZFP57, and RHBDD2.

20. A method of preventing or treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject the CAR immune cell or precursor thereof according to claim 9, the population of cells according to claim 10,or the CAR cell product of claim 11 , or the pharmaceutical composition according to claim 12.21 . Use of the CAR immune cell or precursor thereof according to claim 9, the population of cells according to claim 10, or the CAR cell product of claim 11 , or the pharmaceutical composition according to claim 12, in the manufacture of a medicament for prevention or treatment of a disease, disorder or condition in a subject in need thereof.

22. The CAR immune cell or precursor thereof according to claim 9, the population of cells according to claim 10, or the CAR cell product of claim 11 , or the pharmaceutical composition according to claim 12, for use in the prevention or treatment of a disease, disorder or condition in a subject in need thereof.

23. A kit for use or when used in a method of identifying the expansion potential of cell or cell population for CAR cell therapy, comprising:- a means for assessing a histone biomarker signature of one or more genes selected from KLF7, CCR7, TMEM45A, SLAMF7, UPC, CLIC3, ZFP57, and RHBDD2; and / or- a means for determining the expression or amount of a KLF7 polypeptide or functional variant or fragment thereof, or a polynucleotide encoding the same.

24. A method of preventing or treating a disease, disorder, or condition in a subject in need thereof, the method comprising- assessing a histone biomarker signature of one or more genes selected from KLF7, CCR7, TMEM45A, SLAMF7, UPC, CLIC3, ZFP57, and RHBDD2 U a CAR immune cell, a CAR immune cell precursor or a cell population comprising a CAR immune cell or CAR immune cell precursor, wherein when- one or more of KLF7, CCR7, and TMEM45A exhibit a transcriptionally active histone biomarker signature,- RHBDD2 exhibits a transcriptionally active histone biomarker signature, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 exhibit a transcriptionally repressive histone biomarker signature, the CAR immune cell, CAR immune cell precursor or cell population is identified as having high expansion potential for CAR cell therapy; and when- one or more of KLF7, CCR7, and TMEM45A exhibit a transcriptionally repressive histone biomarker signature,- RHBDD2 exhibits a transcriptionally repressive histone biomarker signature, and / or- one or more of SLAMF7, LIPC, CLIC3, ZFP57 exhibit a transcriptionally active histone biomarker signature, the CAR immune cell, CAR immune cell precursor or cell population is identified as having low expansion potential for CAR cell therapy; manufacturing a CAR cell product using a CAR immune cell, CAR immune cell precursor or cell population identified as having high expansion potential, treating the subject by administering the CAR cell product.

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