Marker-based t cell selection

Abstract

The present invention relates to populations of T cells, characterized by a gene signature that provides a tumor engager and / or tumor killing phenotype or behavior when in contact with tumor cells. The invention also provides for method of obtention of these populations of T cells, as well as their use in the therapy of cancer.

Description

[0001] Title: Marker-Based T Cell Selection

[0002] FIELD OF THE INVENTION

[0003]

[0001] This invention pertains in general to the field of therapy with T cells. In particular it relates to populations of selected T cells that are applicable for treating tumors.

[0004] BACKGROUND OF THE INVENTION

[0005]

[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0006]

[0003] The treatment of cancer using cell immunotherapy, such as adoptive T cell therapy is increasing and has been widely used in or treating blood cancers like leukemia, lymphoma and multiple myeloma. Now is also showing promise for solid tumors.

[0007]

[0004] Adoptive cell therapy, also known as cellular immunotherapy, is a form of treatment that uses the cells of our immune system to eliminate cancer. For this, sometimes cells of a patient are isolated and expanded, in other cases the isolated cells are genetically engineered to enhance their cancer-fighting capabilities. Examples of cellular immunotherapy are Tumor-Infiltrating Lymphocyte (TIL) Therapy; Engineered T cell therapies including Engineered T cell receptor (TCR) Therapy, and Chimeric Antigen Receptor (CAR) T cell Therapy; and Natural Killer (NK) Cell Therapy.

[0008]

[0005] However, the main limitation that prevents cellular immunotherapy, and more in particular engineered T cell therapies from reaching their full potential, is that a large portion of the (engineered) T cells are ineffective; they are unable to locate and / or kill tumor cells. Ideally, (engineered) T cell therapies should only contain effective T cells - those that can travel to and kill tumor cells. To achieve this, methods are needed that can separate the effective engineered T cells from the ineffective ones, ensuring that only the most effective engineered T cells make it into the therapy. This approach would greatly increase the effectiveness of engineered T cell therapies, improving patient outcomes and offering new hope for patients with a bad prognosis due to lack of alternative treatments.

[0009] AOMB: P102253WO

[0006] As a way of example in which engineered T cell therapies are being used is the Diffuse Midline Glioma (DMG). DMG is a highly aggressive and incurable pediatric brain tumor with no chance of survival. In the last 40 years of research, the only treatment that has brought hope to patients are engineered T cell therapies, where a patient’s own immune cells are reprogrammed to seek out and kill cancer cells. These therapies can target tumors with incredible precision and effectiveness, offering a promising solution where other treatments have failed and delivering the potential for long-lasting responses. However, despite their potential, engineered T cell therapies have only managed to extend the lives of DMG patients by a few months. The main reason is, as stated before, because is difficult to select those engineered T cell populations containing only or mainly effective T cells - those that can travel to, engage, and kill tumor cells.

[0010]

[0007] In light of this, new products, compositions, methods and uses for in the treatment of cancer would be highly desirable but are not yet readily available. In particular, there is a clear need in the art for reliable, efficient, and reproducible products, compositions, methods and uses that allow to be used in the treatment of cancer. Accordingly, the technical problem underlying the present invention can be seen in the provision of such products, compositions, methods and uses for complying with any of the aforementioned needs, or at least providing the public with a useful choice. The technical problem is solved by the embodiments characterized in the claims and herein below.

[0011] SUMMARY OF THE INVENTION

[0012]

[0008] As embodied and broadly described herein, the present invention is directed to the surprising finding that a population of T cells with high tumor engagement and / or killing potentiality can be isolated from a complex pool of T cells (i.e., sample of a patient). This population, when in contact with tumor cells, of a patient or of an organoid of the tumor (patient-derived or not) will evolve to acquire a particular gene signature that provides a so-called in this description engager and killing phenotype, and also referred as sniper or super-engager T cells.

[0013]

[0009] Strikingly, the inventors have realized that this signature is preserved across different engineered T cell formats, which allow the applicability in several cell types, such as in CAR T cells, and in cells expressing an engineered TCR. In addition, the

[0014] AOMB: P102253WO signature in these T cells will be induced when the cells are in contact with different tumor types, which makes this approach applicable to different cancer types, such as brain cancer, breast cancer, neuroblastoma head and neck cancer, among other cancers with solid tumors.

[0015]

[0010] The inventors have also realized that part of the gene signature that provides this super-killing phenotype might be inherent to the T cells (so also present before infusion / tumor exposure). This provides the advantage that then the cells can be easily sorted by this part of the gene signature, with the aim to enrich the (engineered) T cells in those that will acquire the tumor engager and tumor killing phenotype.

[0016]

[0011] Therefore, based on the advantageous finding of a way to separate effective from ineffective engineered T cells, engineered T cell therapies that contain only the most effective population of T cells are provided. By selecting the most effective population of T cells the following beneficial therapeutical options are provided:

[0017]

[0012] Maximized Tumor Killing - Leverage the functionality of the best performing engineered T cells to maximize solid tumor destruction.

[0018]

[0013] Improved Tumor Homing - Increased ability to accurately locate solid tumors to ensure as many T cells as possible end up in the right place.

[0019]

[0014] Consistent Performance - Eliminate variation in therapy composition to ensure reliable treatment outcomes for all patients, / nip

[0020]

[0015] As will be illustrated in the examples, the inventors make use of a proprietary technology platform to identify populations of (engineered) T cells with superior tumor killing abilities together with biomarkers indicative of their performance. These biomarkers are used for cell selection, to ensure only the best performing T cells end up in the engineered T cell therapies.

[0021]

[0016] Therefore, in an aspect, the invention relates to a population of T cells, wherein the population of T cells is characterized by:

[0022] a) expression of at least one, at least five, at least fifteen, at least twenty, at least twenty-five, or of the whole of the genes of a gene signature defined by:

[0023] NCAM1; IRF8; KLF10; IL3; SLAMF7; NFATC1; PRF1; TAGAP; CDK4; AGK; CD55; VCL; CCT4; CD160; CLUH; C1QBP; PGAM1; CCL1; XCL1; XCL2; CRTAM; BACH2; ZBTB21; UBASH3B; SLC35E4; XIRP1; PRKD3; YBX3; YBX1; ATP1B1; AMIGO2; MYO1E; POU2AF1; GPR18;

[0024] AOMB: P102253WO IGF2R; AFAP1L2; EMP1; HEG1; ARHGEF3; BYSL; NTRK1; SNTB2; NCEH1; BZW2; DCAF13; IARS; CHD4; SERPINE2; SQLE; FAM3C; PKIA; CCT3; PA2G4; TRABD2A; IMP4; POLR3C; ITPR1; URB1; SLC29A1; LRIG1; TBC1D4; and FBXO30; and / or

[0025] b) higher expression of at least one, at least five, at least fifteen, at least twenty, at least twenty-five, or of the whole of the genes of a gene signature defined by:

[0026] NCAM1; IRF8; KLF10; IL3; SLAMF7; NFATC1; PRF1; TAGAP; CDK4; AGK; CD55; VCL; CCT4; CD160; CLUH; C1QBP; PGAM1; CCL1; XCL1; XCL2; CRTAM; BACH2; ZBTB21; UBASH3B; SLC35E4; XIRP1; PRKD3; YBX3; YBX1; ATP1B1; AMIGO2; MYO1E; POU2AF1; GPR18; IGF2R; AFAP1L2; EMP1; HEG1; ARHGEF3; BYSL; NTRK1; SNTB2; NCEH1; BZW2; DCAF13; IARS; CHD4; SERPINE2; SQLE; FAM3C; PKIA; CCT3; PA2G4; TRABD2A; IMP4; POLR3C; ITPR1; URB1; SLC29A1; LRIG1; TBC1D4; and FBXO30

[0027] wherein said higher expression is relative to a population of reference T cells.

[0028]

[0017] It is also an aspect of the invention a population of T cells, wherein the population of T cells is characterized by:

[0029] a) expression of at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, or of the whole of the genes of a gene signature, defined by:

[0030] NCAM1; IL3; CD55; CCL1; XCL1; XCL2; CRTAM; SLC35E4; ATP1B1; AMIGO2; GPR18; AFAP1L2; SERPINE2; TRABD2A; SLC29A1; and LRIG1; and / or

[0031] b) higher expression of at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, or of the whole of the genes of a gene signature, defined by:

[0032] AOMB: P102253WO NCAM1; IL3; CD55; CCL1; XCL1; XCL2; CRTAM; SLC35E4; ATP1B1; AMIGO2; GPR18; AFAP1L2; SERPINE2; TRABD2A; SLC29A1; and LRIG1, wherein said higher expression is relative to a population of reference T cells.

[0033]

[0018] This previous signature including sixteen genes includes markers expressed on the membrane of the T cells, and compounds (mainly proteins) secreted by the T cells.

[0034]

[0019] Another aspect of the invention is a population of T cells, wherein the population of T cells is characterized by:

[0035] a) expression of NCAM1; and / or

[0036] b) higher expression of NCAM1, wherein said higher expression is relative to a population of reference T cells.

[0037]

[0020] The population of T cells of the invention, that display or have the potentiality to display the tumor engager and tumor killing phenotype or behavior in contact with a tumor, are excellent therapeutic agents. Thus, another aspect of the invention is a population of T cells as defined in any one of the previous aspects, for use in the treatment of a tumor in a patient.

[0038]

[0021] This aspect can also be defined as the use of a population of T cells as defined in any one of the previous aspects for the preparation of a medicament for the treatment of a tumor in a patient. Herewith disclosed is also a method of treating a tumor, the method comprising administering a therapeutically effective amount of a population of T cells as defined in any one of the previous aspects, optionally in the form of a pharmaceutical composition further comprising pharmaceutically effective carriers and / or excipients, in a patient suffering from a tumor.

[0039]

[0022] Therefore, the population of T cells according to any of the aspects of the invention, can be used as active (live) ingredient in a pharmaceutical composition, which comprises the therapeutically effective amount of the T cells together with any pharmaceutically acceptable excipient or carrier.

[0040]

[0023] Examples of these pharmaceutical compositions include the pharmaceutical preparations for infusion of cells to patients.

[0041]

[0024] The invention also provides for a method to easily obtain the populations of T-cells of the previous aspects, which allows for the obtention of effective T cells in a feasible and operational mode.

[0042] AOMB: P102253WO

[0025] It is, thus, another aspect of the invention a method of obtaining or of selecting or of sorting a population of T cells according to any one of the previous aspects, preferably according to the first aspect including the gene signature, wherein the method comprises:

[0043] a) exposing a sample containing T cells to tumor organoid, and co-culturing, preferably for a period of time of at least 1 - 24 hours, to allow some (part of) of the T cells to engage upon the tumor organoid,

[0044] b) removing the non-engaged T cells from the co-culture,

[0045] c) optionally repeating steps (a) and (b) at least once or more, and

[0046] d) harvesting the T cells engaged upon the tumor organoid, to obtain the population of T cells.

[0047]

[0026] The invention also provides, as an additional aspect, a method of sorting or selecting T cells, preferably wherein the population of T cells are primary T cells, or preferably wherein the population of T cells are genetically engineered T ceils, or wherein the population of T cells are CAR-T cells, or wherein the population of T cells are TCR expressing T cells, or wherein the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are CAR-T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are TCR expressing genetically engineered T cells not expressing a gamma-delta T cell receptor, wherein the method comprises: a) determining in a T cell the expression of one or more, or of all of the genes in the gene signature defined in the previous aspects and also listed in Table 1 or in Table 2 in this description,

[0048] b) retrieving the T cells that express one or more, or of all of the genes in the gene signature defined above or that have a higher expression of one or more, or of all of the genes in the gene signature defined above relative to a population of reference T cells.

[0049]

[0027] With the aim to simplify a method of sorting the population of T cells that has the potentiality of having the desired phenotype or behavior defined as engager and killing phenotype upon contact with a tumor, the inventors do also provide an additional method of sorting or selecting T cells.

[0050]

[0028] Thus, another aspect of the invention is a method of sorting or selecting T cells, preferably wherein the population of T cells are primary T cells, or preferably wherein

[0051] AOMB: P102253WO the population of T cells are genetically engineered T cells, or wherein the population of T cells are CAR-T cells, or wherein the population of T cells are TCR expressing T cells, or wherein the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are CAR-T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are TCR expressing genetically engineered T cells not expressing a gammadelta T cell receptor, wherein the method comprises

[0052] a) determining in a T cell the expression of at least NCAM1, and

[0053] b) retrieving the T cells that express at least NCAM1, or that have a higher expression of NCAM1 relative to a population of reference T cells.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055]

[0029] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0056]

[0030] Figure 1 (Fig. 1). DMGOs model CAR T cell functional heterogeneity, a, GD2 CAR T cell treatment outcome measured as a relative change in tumor GFP intensity quantified by imaging compared to the start of treatment (100%). DMGOs were either left untreated (grey line, n=1), treated with mock transduced T cells (black line, n=2), or GD2 CAR T cells (orange line, n=4) and for each treatment condition a smoothed line trend between the averaged values at different timepoints was plotted using the LOESS algorithm. Shaded area reflects the 95% confidence interval, b, Representative images of the tumor GFP signal at the indicated timepoints for a DMGO subjected to prolonged GD2 CAR T cell treatment administrated at day 0, day 8 and day 15. c, UMAP visualization of GD2 CAR T cell clusters, d, Cytotoxic effector molecule and cytokine gene expression across the GD2 CAR T cell clusters, e, Gene expression of selected exhaustion associated receptors, ligands, and transcription factors across the GD2 CAR T cell clusters, d, e, Dot plot representing the percentage of cells expressing selected genes. Color intensity represents the average scaled gene expression, f, Signature score (in arbitrary units (AU)) on a blue-to-red color scale showing enrichment of previously identified T cell serial killer gene set from Dekkers et al. atop UMAP cell embeddings of the GD2 CAR T cell dataset (left), g, Dotchart depicting the fold enrichment in tumor killing by NCAM1+ GD2 CAR T cells over NCAM1- GD2 CAR T cells quantified as the change in tumor area detected by GFP

[0057] AOMB: P102253WO compared to the start of treatment. n=2 DMGOs per treatment condition, h, Percentage of cells per predicted clusters from c for NCAM1- GD2 CAR T cells, NCAM1+ GD2 CAR T cells and reference GD2 CAR T cells from c. i. Percentage of cells per TCYT, TEX and THS cluster for NCAM1- GD2 CAR T cells (left; retrieved from n=2 DMGOs) and NCAM1+ GD2 CAR T cells (right; retrieved from n=2 DMGOs). j. Heatmap depicting the relative expression of exhaustion markers (left heatmap) and exhaustion associated transcription factors and functional regulators (right heatmap) in nonexposed (left) and DMGO-exposed (right) GD2 CAR T cells within the TEX cluster, k. Gene expression of tissue resident markers in non-exposed (top; n=2) and DMGO-exposed (bottom; n=4) GD2 CAR T cells within the TEX cluster. Dot plot representing the percentage of cells expressing selected genes. Color (grey scale) intensity represents the average scaled gene expression.

[0058]

[0031] Figure 2. (Fig. 2) DMGO GD2 expression and CAR T cell mediated tumor control, a, DMGO tumor cell GD2 expression (orange) analyzed by flow cytometry compared to an unstained control (black), b, GD2 CAR T cell treatment outcome measured as tumor GFP intensity relative to the start of treatment (day 0, 100%). DMGOs were either left untreated (grey line, n=1), treated with mock transduced T cells (black lines, n=2), or GD2 CAR T cells (orange lines, n=4) and for each DMGO a smoothed line trend was plotted between the values at different timepoints using the LOESS algorithm, c, Images of tumor GFP signal on day 0, 7, 10 and 14 for an untreated DMGO, DMGOs treated with mock transduced T cells, or GD2 GAR T cells. GD2 CAR T cells and mock transduced T cells were administrated at day 0 and 7. d. IFNy levels measured in the culture supernatant.

[0059]

[0032] Figure 3. (Fig. 3) Key marker genes, Gene Ontology (GO) terms and reference data projection of GD2 CAR T cell clusters, a, Dot plot showing key marker gene expression (selected from the top 20 differentially expressed genes (DEGs)) across the GD2 CAR T cell clusters. Dot size is proportional to the percentage of cells expressing a gene and color intensity to the average scaled gene expression. Grid colors highlight genes that are closely related in function; HLA genes (green), metabolic stress-related genes (red) and ISGs (blue), b-f, Selected significant GO terms associated with the DEGs of the TUND (b), TIL-2 (c), TMI (d), TPR (e) and TMS (f) GD2 CAR T cell clusters, g, UMAP visualization of the CD8+ TIL clusters from the Chu et al. pan-cancer atlas used as a reference dataset. Annotated clusters are

[0060] AOMB: P102253WO highlighted because of their overlap with, or use in defining, the GD2 CAR T cell clusters, h-j, Curated marker gene signatures (DEG analysis adjusted p-value < 0.00001) of the TUND (h), TIL-2 (i) and TISG (j) GD2 CAR T cell clusters projected onto the CD8+ TIL dataset from g. k. UMAP embedding of DMGO-exposed (pink when image in color, darkest points in grey scale; n=4) and non-exposed (green when image in color, middle-grey tone in grey scale; clear spotted area in the middle of the darkest spots; n=2) GD2 CAR T cells within the TEX cluster.

[0061]

[0033] Figure 4. (Fig. 4) Subset characterization of NCAM1- and NCAM1+ GD2 CAR T cells, a, Applied gating strategy (top panels) and obtained purity (bottom panels) for sorted NCAM1- and NCAM1+ GD2 CAR T cells, b, Representative images of tumor control measured by GFP imaging in DMGOs treated with sorted NCAM1- (top) or NCAM1+ (bottom) GD2 CAR T cells, c, Number of NCAM1- and NCAM1+ GD2 CAR T cells retrieved from each DMGO sample (n=2) after two weeks of treatment, d, UMAP visualization of unbiased clusters obtained for NCAM1- and NCAM1+ GD2 CAR T cells, e, Projection of NCAM1- (blue) and NCAM1+ (orange) annotation on the UMAP from d. f, Relative frequency of NCAM1- (blue, right bars in each set 0-10) and NCAM1+ (orange, left bars in each set 0-10) GD2 CAR T cells per cluster from d. g, UMAP embedding of predicted cluster identity of NCAM1- (left) and NCAM1+ (right) GD2 CAR T cells using clusters from Figure 1c. h, Proportion of cells within GD2 CAR T cell clusters, including those identified in Fig. 1c, a cluster enriched in non-exposed cells, and the NCAM1- specific THS cluster. Data is shown separately for NCAM1-GD2 CAR T cells (left) and NCAM1+ GD2 CAR T cells (right), i, Selected significant GO terms associated with the DEGs of the THS NCAM1- specific GD2 CAR T cell cluster, j, Upregulated marker gene signature (DEG analysis adjusted p-value < 0.05; avg_log2FC>0) of the THS GD2 CAR T cell cluster projected onto the pan-cancer CD8+ TIL dataset.

[0062]

[0034] Figure 5. (Fig. 5) (a-b) Target X (X= NCAM1) can be used for cell selection of engineered T cell therapies to enrich Sniper T cells (a) and increase breast cancer tumor killing (b). (c-d) In a brain tumor model, Target X+ selected cells outperform Target X- cells (c) in line with an increased cytotoxic gene profile of the Target X+ cells (d).

[0063] Figure 6: (Fig. 6a) Profile of cytokine expression. IL-1 a, IL-2, IL-5, IL-6, TNF-a, IFN-y, MCP1 (CCL2), RANTES (CCL5), MCP3 (CCL7), Eotaxin, GRO1a (CXCL1), IP10

[0064] AOMB: P102253WO (CXCL10), M-CSF and Granzyme B concentration measured of organoid co-cultures with GD2 CAR-T cells or NCAM+-selected GD2 CAR-T cells showing significant reductions in secretion of some of these cytokines by NCAM1+-selected GD2 CAR-T cells. (Fig. 6b): Profile of cytokine expression. IL-1 a, IL-2, IL-5, IL-6, IL-10, TNF-a, IFN-y, LAP, MCP1 (CCL2), RANTES (CCL5), MCP3 (CCL7), MCP2, Eotaxin, GRO1a (CXCL1), IL-8, MIG, IP10 (CXCL10), M-CSF and Granzyme B (GranB) concentration measured in organoid cocultures (brain tumor model) with GD2 CAR-T cells or NCAM+- selected GD2 CAR-T cells showing no increase in cytokine secretion by NCAM1+-selected GD2 CAR-T cells.

[0065]

[0035] Figure 7 (Fig. 7): Synergy with microglia. Percentage difference in killing of GD2 CAR-T cells and NCAM+-selected GD2 CAR-T cells in DMGO containing microglia compared to DMGO without microglia. Microglia reduce killing of GD2 CAR-T cells at early time points (day 0-7), while NCAM+-selected GD2 CAR-T cells show significantly increased killing activity at both early and late (day 8-14) timepoints in the presence of microglia.

[0066]

[0036] Figure 8 (Fig. 8) Gene expression in human primary T cells. Dot plot representing the percentage of cells expressing selected genes. Intensity represents the average scaled gene expression.

[0067] DESCRIPTION

[0068] Definitions

[0069]

[0037] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0070]

[0038] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0071] AOMB: P102253WO

[0039] For purposes of the present invention, the following terms are defined below.

[0072]

[0040] As used herein, the singular form terms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. For example, a method for administrating a T cell includes the administrating of a plurality of T cells (e.g., 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more).

[0073]

[0041] As used herein, “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0074]

[0042] As used herein, "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,..., etc. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less" i.e., 5, 4, 3,....-10, -11, etc.

[0075]

[0043] As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps, or components. It also encompasses the more limiting “to consist of.”

[0076]

[0044] As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of conducting the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology, and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[0077]

[0045] As used herein, "exemplary" or “for example" means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations, including those disclosed herein.

[0078]

[0046] As used herein the term “expression” of a gene is to be understood as the process by which information from that gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, and ultimately affect a phenotype. These products are often proteins, but in non-

[0079] AOMB: P102253WO protein-coding genes such as transfer RNA (tRNA) and small nuclear RNA (snRNA), the product is a functional non-coding RNA. In genetics, gene expression is the most fundamental level at which the genotype gives rise to the phenotype, i.e. observable trait, such as a super-engager (sniper) phenotype in this description. The genetic information stored in DNA represents the genotype, whereas the phenotype results from the "interpretation" of that information. Within the context of the current invention, the skilled person will understand when a gene is considered to be “expressed”.

[0080]

[0047] As used herein the “higher expression” or “overexpression”, both terms used interchangeably, refers to the expression of a gene or of a group of genes (e.g., the gene signature) which is at a high extension in relation to a reference or comparative population or cell condition. For example, it can be a higher expression in relation to other T cell populations, or in relation to other environmental conditions in which the same T cell population is found. The skilled person in the art knows how to determine the expression of a gene by conventional techniques, as well as how to establish a comparison between the expression of genes to determine if a gene is highly expressed in a certain condition or cell, or not.

[0081]

[0048] As used herein, a “population of reference T cells” refers to a group of T cells to which the population of T-cells of the aspects of the invention compare, in particular in terms of which and to what extension the one or more genes of the signature are expressed. As reference T cells many populations can be chosen. In an embodiment, the reference T cells are mainly T cells that have not been in contact with tumor cells, which in the context of the invention are also termed naive T cells. In another embodiment, the reference T cells are T cells that have been in contact with tumor cells (e.g. PDO, or the actual tumor in a patient) but that do not display the engager behavior / phenotype, for example when questioned or confronted with a tumor organoid.

[0082]

[0049] The expression of at least one, at least five, at least fifteen, at least twenty, at least twenty-five, or of the whole of the genes of a gene signature can be determined by several methods which are under the common practice of the skilled person in the art. Independently of the used methodology, a reference threshold or cutoff value for each of the genes, or for all the genes, is determined to set the value over which the expression of that gene or of all the genes will be considered high in relation to other condition, which other condition can be another population of T cells, or the same

[0083] AOMB: P102253WO population of T cells in another environment (e.g., the T cells before or after confronting with tumoral cells).

[0084]

[0050] The extension of the expression of a gene can, thus, be compared with a cutoff or threshold stablished for any particular technique or methodology employed for the determining of the expression of genes. For the analysis of the differential expression of a particular gene between two or more groups of cells, the fold change is one of the parameters / values that can be calculated. This value is typically reported in logarithmic scale (base 2). Based on this, a value given for a Iog2 fold change can be established as value over which the expression of a gene is considered high or higher in relation to other gene or condition in a cell. For example, a Log2 fold change of at least X, optionally in combination with the expression in at least Y % of cells, can be established to decide if an interrogated gene is to be considered as highly expressed in a determined cell condition or in comparison with other genes. As a way of non-limitative example, in the present description when the expression of the gene signature in the T cells was for example performed by single cell RNA sequencing, a cutoff for high expression was set at a Log2 fold change of at least 0.25 and expression in at least 5% of the T cells, but other cutoff values (i.e., Iog2 fold changes) can be used as the skilled person will recognize. The selection of a cutoff is usually done considering the sensitivity and specificity that is desired for a particular methodology to establish if a particular specimen (e.g., a T cell) is in or out of a group of specimens with a desired condition (e.g., phenotype / behavior).

[0085]

[0051] The selection of T cells expressing a gene of the signature is described in the examples that follow using as a model a selection of T cells expressing NCAM (NCAM selection). The commonly used techniques for selecting the T cells in function of the expression of certain genes rely, for example, on Fluorescence-Activated Cell Sorting (FACS) and Magnetic-Activated Cell Sorting (MACS). In the Examples (Figure 4a) is shown in detail how the populations expression or not the NCAM gene can be identified (called there NCAM- and NCAM+ populations). Other techniques can be used, such as scRNA-sequencing, to identify genes that are expressed and, to select the T cells based on the said expression of the genes. For the selection of T cells, the analysis of some of the gens of a particular signature as defined in this invention will be enough. Therefore, the selection of T cells may be done with the analysis of the expression of at least one or two of the genes characteristics of the populations. For

[0086] AOMB: P102253WO example, if a T cell population with a particular phenotype or behavior is the target (e.g., Sniper T cell), with the analysis of at least one, at least two, at least five of the genes of the signatures as defined in Tables 1 or 2, will allow selecting the cells using the conventional and commonly used cell sorting techniques (FACS or MACS).

[0087]

[0052] In the context of the present invention, a “phenotype” of a T cell population is referred also as a particular “behavior” of said population of T cells when confronted with tumoral cells, or as a “transcriptional state”. The behaviors disclosed in this description are listed in the paragraphs below, being the object of the invention the populations of T cells with a tumor engager and tumor killing phenotype / behavior, also termed herewith cytotoxic T cells (abbreviated TCYT in the figures).

[0088]

[0053] Phenotypes / Behaviors of T cell populations disclosed in this description:

[0089]

[0054] The undifferentiated phenotype / behavior corresponds to a population of T cells that although activated (based on HLA gene expression), is not fully differentiating towards effector function (undifferentiated; abbreviated TUND).

[0090]

[0055] The IL-2 responsive T cell population (TIL-2) is a population that responds to IL-2.

[0091]

[0056] The phenotype / behavior termed interferon-stimulated gene (ISG) expressing T cell population (abbreviated TISG) that strongly corresponds to ISG expressing tumor infiltrating lymphocytes (TILs)

[0092]

[0057] The phenotype / behavior termed TMI for including a T cell population with migrating properties and interconnectivity

[0093]

[0058] The proliferating phenotype / behavior (abbreviated TPR)

[0094]

[0059] The effector T cell populations based on their cytotoxic profile include the phenotype / behavior termed as predominantly expressing GZMK (TGZK), the cytotoxic phenotype / behavior (TCYT), and the exhausted phenotype / behavior (TEX), that includes T cells with a reduced IFNG and concomitant expression of immune checkpoint genes; LAG3, HAVCR2, TIGIT61 and SELPLG62, as well as the transcriptional repressor PRDM1 associated with exhaustion.

[0095]

[0060] As used herein, “adoptive T cell therapy (ACT)” means therapy wherein involving transfer of T cells into a subject. The cells may have originated from the patient or from another individual. ACT is also called T cell therapy, adoptive immunotherapy, and immune cell therapy. ACT includes but is not limited to tumor-

[0096] AOMB: P102253WO infiltrating lymphocytes (TIL) therapy, engineered T cell Receptor (TCR) therapy, Natural Killer (NK) Cell Therapy, and CAR-T cell therapy.

[0097]

[0061] As used herein, a “T cell” or “T-cell” can be selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes, or helper T- lymphocytes. In another embodiment, said cell can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. The T cell may be an alpha-beta T cells. The T cell may be a gamma-delta T cell. The T cell may be a Natural Killer T cell. The T cell may be an engineered T cell, for example a CAR T cell. They can be extracted from blood, from tissue and / or from tumor tissues. In another embodiment, said T cell is part of a mixed population of cells which present different phenotypic characteristics.

[0098]

[0062] As used herein, the term “primary T cells” encompasses immature T cells that are still developing in the thymus or mature T cells that have been isolated directly from a living organism like blood or any other tissue, such as tumor tissue, for use in research or in therapy, which are often called primary cultures. As part of the adaptive immune system, their main functions are to identify and destroy infected or cancerous cells, regulate immune responses, and remember past infections. Primary T cells have not been genetically engineered yet but may be used also as source to prepare genetically engineered T cells as disclosed in this description.

[0099]

[0063] The term “genetically engineered T cells”, as used herein, includes T cells that have been modified by means of any tool of in molecular biology to express one or more proteins of interest, preferably to express certain membrane surface proteins. These T cells are usually obtained by transduction, for example, viral transduction or by means of any other vector that comprises the gene or part thereof that codifies for the protein of interest, generally a T cell receptor or T cell co-receptor responsible of the recognition of an antigen exposed by an antigen presenting cell. In an embodiment, this is not a gamma-delta TCR.

[0100]

[0064] In an embodiment, the genetically engineered T cells are T cell receptor (TCR) engineered T cells (TCR-T cells). The TCR may be, in an embodiment an alpha-beta engineered TCR. In another embodiment is a gamma-delta TCR. In another embodiment, the genetically engineered T cells are chimeric antigen receptors (CARs)-T cells. In another, preferred, embodiment the receptor is not a gamma-delta TCR.

[0101] AOMB: P102253WO

[0065] As used herein, the term “chimeric antigen receptors (CARs)” — also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors — are receptor proteins that have been engineered to give T cells the new ability to target a specific antigen. The receptors are chimeric in that they combine both antigen-binding and T cell activating functions into a single receptor. “CAR-T cells” refer to T cells that have been engineered to express these chimeric antigens. The surface of CAR T cells can bear either CD4 and CD8 co-receptor, thus the T cell can be CD8+ or CD4+ lymphocytes. CAR T cells may be derived either autologously from T cells of a patient blood or allogeneically from a donor. Once isolated, these T cells are genetically engineered to express a specific CAR, using a vector, generally derived from an engineered lentivirus such as HIV (see Lentiviral vector in gene therapy).

[0102]

[0066] As used herein, the expression “gene signature” or “gene expression signature” refers to a single or combined group of genes in a cell with a uniquely characteristic pattern of gene expression that occurs as a result of a biological process, which can be an altered or unaltered biological process or a pathogenic medical condition. A gene signature in may be the result of the cell submitted at a particular condition or environment, for example the result of the cell having been in contact with a tumor cell or group of cells. Within the context of the current invention when reference is made to “of the genes of a gene signature defined by” this is also to be understood as “of a group comprising or consisting of the following genes”.

[0103]

[0067] As used herein, "cancer" refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. The terms "cancer," "neoplasm," and "tumor," are often used interchangeably to describe cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells can be distinguished from non-cancerous cells by techniques known to the skilled person. A cancer cell, as used herein, includes not only primary cancer cells, but also cancer cells derived from such primary cancer cell, including metastasized (secondary) cancer cells, and cell lines derived from cancer cells. Examples include solid tumors and non-solid tumors or blood tumors. Examples of cancers include, without limitation, leukemia, lymphoma, sarcomas, and carcinomas (e.g., brain cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, melanoma, lymphoma, non-Hodgkin lymphoma, colon cancer, (malignant) melanoma, thyroid cancer, papillary thyroid carcinoma, lung

[0104] AOMB: P102253WO cancer, non-small cell lung carcinoma, and adenocarcinoma of lung). Treatment of a cancer in a subject includes the treatment of a tumor in the subject.

[0105]

[0068] As used herein, "in vivo" refers to an event that takes place in a subject's body; "in vitro" refers to an event that takes places outside of a subject's body. For example, an in vitro assay or method encompasses any assay or method conducted outside of a subject. In vitro assays or methods encompass cell-based assays in which cells, alive or dead, are employed. In vitro assays also encompass a cell-free assay in which no intact cells are employed.

[0106]

[0069] As used herein, the term "marker" is used to describe the characteristics and / or phenotype of a cell. Markers can be used for selection of cells comprising characteristics of interests. Markers will vary with specific cells. Markers are characteristics, whether morphological, functional, or biochemical characteristics of the cell of a particular cell type, or molecules expressed by the cell type. Preferably, such markers are proteins, and more preferably, possess an epitope for antibodies or other binding molecules available in the art. However, a marker may consist of any molecule found in a cell including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological markers include shape, size, and nuclear to cytoplasmic ratio. Examples of functional markers include the ability to migrate under particular conditions and the ability to differentiate along particular lineages. Markers may be detected by any method available to one of skill in the art. Markers can also be the absence of a morphological characteristic or absence of proteins, lipids etc. Markers can be a combination of a panel of unique characteristics of the presence and absence of polypeptides and other morphological characteristics.

[0107]

[0070] As used herein, "sample" when referring to a tumor or any other biological material referenced herein, means a sample that has been removed from the subject; thus, none of the testing methods described herein are performed in or on the subject. A sample in the sense of the invention may be an isolated sample comprising T cells of a patient / subject, or a sample comprising tumoral cells isolated from the patient.

[0108]

[0071] As used herein, "treatment", "treating", "palliating", “alleviating” and "ameliorating" in the context of a subject to be treated, all refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder

[0109] AOMB: P102253WO being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disorder.

[0110]

[0072] As used herein, "tumor" as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size and includes primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.

[0111] Detailed description

[0112]

[0073] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0113]

[0074] It is contemplated that any method, use, or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0114]

[0075] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

[0115]

[0076] As embodied and broadly described herein, the present invention is directed to the surprising finding a population of T cells with high tumor engagement and killing potentiality has been identified. This population is, moreover, inducible from a population of T cells that, when in contact with a tumor differentiate or express a gene signature that provides the so-called in this description engager and killing phenotype or behavior, and also referred as sniper or super-killer T cells in the examples.

[0116]

[0077] As previously indicated, an aspect of the invention is a population of T cells, wherein the population of T cells is characterized by:

[0117] a) expression of at least one, at least five, at least fifteen, at least twenty, at least twenty-five, or of the whole of the genes of a gene signature defined by:

[0118] AOMB: P102253WO NCAM1; IRF8; KLF10; IL3; SLAMF7; NFATC1; PRF1; TAGAP; CDK4; AGK; CD55; VCL; CCT4; CD160; CLUH; C1QBP; PGAM1; CCL1; XCL1; XCL2; CRTAM; BACH2; ZBTB21; UBASH3B; SLC35E4; XIRP1; PRKD3; YBX3; YBX1; ATP1B1; AMIG02; MYO1E; POU2AF1; GPR18; IGF2R; AFAP1L2; EMP1; HEG1; ARHGEF3; BYSL; NTRK1; SNTB2; NCEH1; BZW2; DCAF13; IARS; CHD4; SERPINE2; SQLE; FAM3C; PKIA; CCT3; PA2G4; TRABD2A; IMP4; POLR3C; ITPR1; URB1; SLC29A1; LRIG1; TBC1 D4; and FBXO30; and / or

[0119] b) higher expression of at least one, at least five, at least fifteen, at least twenty, at least twenty-five, or of the whole of the genes of a gene signature defined by:

[0120] NCAM1; IRF8; KLF10; IL3; SLAMF7; NFATC1; PRF1; TAGAP; CDK4; AGK; CD55; VCL; CCT4; CD160; CLUH; C1QBP; PGAM1; CCL1; XCL1; XCL2; CRTAM; BACH2; ZBTB21; UBASH3B; SLC35E4; XIRP1; PRKD3; YBX3; YBX1; ATP1B1; AMIG02; MYO1E; POU2AF1; GPR18; IGF2R; AFAP1L2; EMP1; HEG1; ARHGEF3; BYSL; NTRK1; SNTB2; NCEH1; BZW2; DCAF13; IARS; CHD4; SERPINE2; SQLE; FAM3C; PKIA; CCT3; PA2G4; TRABD2A; IMP4; POLR3C; ITPR1; URB1; SLC29A1; LRIG1; TBC1D4; and FBXO30

[0121] wherein said higher expression is relative to a population of reference T cells.

[0122]

[0078] The human genes referred to are identified in a Table that follows in this description with their corresponding ENSBL ID of the Ensembl release 113 - October 2024 © EMBL-EBI, retrievable at https: / / www.ensembl.orq / index.html, and which includes a gene description.

[0123]

[0079] Table 1. ENSBL ID numbers of genes in the gene signature.

[0124] Gene, EnsemblJD (Ensembl release 113 - October 2024)

[0125] IRF8, ENSG00000140968,

[0126] KLF10, ENSG00000155090,

[0127] IL3, ENSG00000164399,

[0128] SLAMF7. ENSG00000026751,

[0129]

[0130] AOMB: P102253WO Gene, Ensembl_ID (Ensembl release 113 - October 2024) NFATC1. ENSG00000131196,

[0131] PRF1, ENSG00000180644,

[0132] TAGAP. ENSG00000164691, CDK4. ENSG00000135446, AGK, ENSG00000262327, ENSG00000006530 CD55, ENSG00000196352, VCL, ENSG00000035403,

[0133] CCT4, ENSG00000115484,

[0134] CD160, ENSG00000117281,

[0135] CLUH, ENSG00000132361,

[0136] C1QBP, ENSG00000108561, PGAM1. ENSG00000171314,

[0137] CCL1, ENSG00000108702,

[0138] XCL1, ENSG00000143184, XCL2, ENSG00000143185,

[0139] CRTAM. ENSG00000109943,

[0140] BACH2, ENSG00000112182,

[0141] ZBTB21. ENSG00000173276, UBASH3B, ENSG00000154127, SLC35E4, ENSG00000100036,

[0142] XIRP1, ENSG00000168334,

[0143] PRKD3. ENSG00000115825, YBX3, ENSG00000060138,

[0144] YBX1, ENSG00000065978,

[0145] ATP1B1, ENSG00000143153,

[0146] AMIGO2, ENSG00000139211,

[0147] MYO1E, ENSG00000157483, POU2AF1, ENSG00000110777,

[0148] GPR18. ENSG00000125245,

[0149] IGF2R. ENSG00000197081,

[0150] AFAP1L2, ENSG00000169129,

[0151] EMP1, ENSG00000134531,

[0152] HEG1, ENSG00000173706,

[0153] ARHGEF3, ENSG00000163947,

[0154] BYSL. ENSG00000112578,

[0155] NTRK1, ENSG00000198400, SNTB2, ENSG00000168807, ENSG00000260873 NCEH1, ENSG00000144959,

[0156] BZW2, ENSG00000136261, DCAF13, ENSG00000164934,

[0157] IARS, ENSG00000291356, ENSG00000196305

[0158]

[0159] AOMB: P102253WO Gene, Ensembl_ID (Ensembl release 113 - October 2024)

[0160] CHD4, ENSG00000111642,

[0161] SERPINE2, ENSG00000135919,

[0162] SQLE,ENSG00000104549,

[0163] FAM3C, ENSG00000196937,

[0164] PKIA, ENSG00000171033,

[0165] CCT3, ENSG00000163468,

[0166] PA2G4. ENSG00000170515,

[0167] TRABD2A, ENSG00000186854,

[0168] IMP4, ENSG00000136718,

[0169] POLR3C. ENSG00000186141,

[0170] ITPR1, ENSG00000150995,

[0171] URB1. ENSG00000142207,

[0172] SLC29A1, ENSG00000112759,

[0173] LRIG1. ENSG00000144749,

[0174] TBC1D4,ENSG00000136111,

[0175] FBXO30,ENSG00000118496,

[0176] NCAM1. ENSG00000149294,

[0177]

[0178]

[0080] In an embodiment of the T cell population of the previous aspect, at least five, at least ten, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least thirty-five, at least forty, at least forty-five, at least fifty, at least fifty-five, at least sixty or all of the genes of the gene signature are expressed or higher expressed relative to a reference population of T cells. For example, 1, 2, 3, 4, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 genes are expressed.

[0179]

[0081] Another aspect of the invention is a population of T cells, wherein the population of T cells is characterized by:

[0180] a) expression of at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, or of the whole of the genes of a gene signature, defined by:

[0181] NCAM1; IL3; CD55; CCL1; XCL1; XCL2; CRTAM; SLC35E4; ATP1B1; AMIGO2; GPR18; AFAP1L2; SERPINE2; TRABD2A; SLC29A1; and LRIG1; and / or

[0182] AOMB: P102253WO b) higher expression of at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, or of the whole of the genes of a gene signature, defined by:

[0183] NCAM1; IL3; CD55; CCL1; XCL1; XCL2; CRTAM; SLC35E4; ATP1B1; AMIGO2; GPR18; AFAP1L2; SERPINE2; TRABD2A; SLC29A1; and LRIG1, wherein said higher expression is relative to a population of reference T cells.

[0184]

[0082] In an embodiment of the T cell population of the previous aspect the population of T cells is characterized by the expression of the whole of the genes of a gene signature defined by: NCAM1; IL3; CD55; CCL1; XCL1; XCL2; CRTAM; SLC35E4; ATP1B1; AMIGO2; GPR18; AFAP1L2; SERPINE2; TRABD2A; SLC29A1; and LRIG1.

[0185]

[0083] In yet another embodiment of the T cell population of the previous aspect, at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, or of the whole of the genes of a gene signature, defined by NCAM1; IL3; CD55; CCL1; XCL1; XCL2; CRTAM; SLC35E4; ATP1B1; AMIGO2; GPR18; AFAP1L2; SERPINE2; TRABD2A; SLC29A1; and LRIG1, are expressed or higher expressed relative to a reference population of T cells. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 genes are expressed.

[0186]

[0084] Another aspect of the invention is a population of T cells, wherein the population of T cells is characterized by:

[0187] a) expression of NCAM1; and / or

[0188] b) higher expression of NCAM1, wherein said higher expression is relative to a population of reference T cells.

[0189]

[0085] This population of T cells that expresses NCAM1 is of special interest because is a population that enriches for, or evolves to, a population of T cells with an engager / killer phenotype (TCYT) when exposed to a tumoral cells (including actual tumor or a tumor organoid). These T cells that expressed NCAM1 may for example be retrieved from a complex sample comprising T cells using some of the method of the invention disclosed below, for example from an isolated sample of a patient (a liquid or a solid sample comprising T cells), and then they can be expanded or directly confronted with the tumoral cells. Thus, these T cells that express NCAM1, preferably

[0190] AOMB: P102253WO in the context of engineered T cells, are cells with the potential to behave as engaging / killing T cells.

[0191]

[0086] In an embodiment of this population of T cells that expresses NCAM1, the T cells also express one or more, preferably at least one or at least two, or at least three of the genes IL3; CD55; CCL1; XCL1; XCL2; CRTAM; SLC35E4; ATP1B1; AMIGO2; GPR18; AFAP1L2; SERPINE2; TRABD2A; SLC29A1; and LRIG1.

[0192]

[0087] In an embodiment of any of the populations of T cells characterized by the expression of least one, at least five, at least fifteen, at least twenty, at least twenty-five, or of the whole of the genes of a gene signature, preferably at least five, at least ten, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least thirty- five, at least forty, at least forty-five, at least fifty, at least fifty-five, at least sixty or all of the genes of the gene signature; or the population defined by the expression or relative expression of NCAM1, the said populations of T cells are primary T cells, or the said populations of T cells are genetically engineered T cells, or the population of T cells are CAR-T cells, or the population of T cells are TCR expressing T cells, or the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or the population of T cells are CAR-T cells not expressing a gamma¬ delta T cell receptor, or the population of T cells are TCR expressing genetically engineered T cells not expressing a gamma-delta T cell receptor.

[0193]

[0088] In another embodiment of the populations of T cells according to the invention, the population of T cells are CD8+ T cells.

[0194]

[0089] In yet another embodiment of the population of T cells according to the invention, at least 20%, 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more of the individual cells in the cell population together are as defined in any one of the previous aspects. Thus, the T cell populations according to the invention may comprise other cells.

[0195]

[0090] Also, in another embodiment of the population of T cells according to the invention, no more than 80%, 75%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or less of the individual cells in the cell population together are cells that are not as defined in any one of the previous claims.

[0196]

[0091] Based on the behavior of the said populations of T cells determined in front of tumoral cells, which can be tumoral cells in tumor-derived organoids, preferably patient-derived organoids (PDO), the population of T cells according to the invention

[0197] AOMB: P102253WO is characterized by being a tumor engagers cell population and / or tumor killing cell population when exposed to a tumor. This population is referred in the examples also as TCYT, as previously disclosed. This tumor engager and / or tumor killing phenotype attributed to the T cell population preferably corresponds with, or is defined by, a tumor engagement from 30-60 min / hour over a co-culture period or exposition of the T cells to a tumor from 8 to 15 hours, preferably 10 hours; and / or by a (serial) killing capacity from 2 to 25 tumor cells in a period from 8 to 15 hours, preferably in 10 -11 hours of co-culture or exposition of the T cells to a tumor.

[0198]

[0092] Of note, this tumor engager and / or tumor killing phenotype is also attributable to the population of T cells that express NCAM1, preferably the population of engineered T cells that express NCAM1, since these will differentiate or become a cell population as defined in the first aspect of the invention (i.e., expressing at least one, at least five, etc. or the whole genes of the therein disclosed gene signature).

[0199]

[0093] The term tumor engagement in the context of the invention relates to the time in which a T cell is in contact or almost in contact or involved in the surface of the tumor. Tumor engagement as well as the (serial) killing capacity can be determined by conventional techniques. In addition, the examples of this invention provide a platform for the analysis of these parameters that functionally define the T cell population. The tumor engagement promotes the killing.

[0200]

[0094] The invention is focused on the provision of T cells that will be effective (i.e., will kill tumoral cells) when infused into a patient that has a tumor.

[0201]

[0095] Therefore, and as previously indicated, another aspect of the invention is a population of T cells as defined in any one of the previous aspects, for use in the treatment of a tumor in a patient, preferably wherein the patient has a solid tumor, preferably wherein the patient has brain cancer, a glioma, preferably a diffuse midline glioma (DMG), a breast cancer, neuroblastoma, and / or a head and neck cancer.

[0202]

[0096] This aspect can also be defined as the use of a population of T cells as defined in any one of the previous aspects for the preparation of a medicament for the treatment of a tumor in a patient, preferably wherein the patient has a solid tumor, preferably wherein the patient has a brain cancer, a glioma, preferably a diffuse midline glioma, a breast cancer, neuroblastoma, and / or a head and neck cancer.

[0203]

[0097] Herewith disclosed is also a method of treating a tumor, the method comprising administering a therapeutically effective amount of a population of T cells as defined

[0204] AOMB: P102253WO in any one of the previous aspects, optionally in the form of a pharmaceutical composition further comprising pharmaceutically effective carriers and / or excipients, in a patient suffering from a tumor, preferably wherein the patient has a solid tumor, preferably wherein the patient has a brain cancer, a glioma, preferably a diffuse midline glioma, a breast cancer, neuroblastoma, and / or a head and neck cancer.

[0205]

[0098] In an embodiment of the population of T cells as defined in any one of the previous aspects, for use in the treatment of a tumor in a patient, the cancer is the result of either a primary or secondary tumor. Thus, it is preferably for use in the treatment of a primary or secondary solid tumor, preferably a primary or secondary brain tumor, a primary or secondary glioma, a primary or secondary breast tumor, preferably a primary or secondary neuroblastoma, and / or a primary or secondary head and neck tumor.

[0206]

[0099] Although several schedules of administration are possible and may be modulated depending on the type of tumor or any other conditions of the patient, in an embodiment of the population of T cells for use in treatment of a tumor in a patient, the T cells are provided to the patient at least every two months, every month, every two weeks, every week, every 7, 6, 5, 4, 3, 2, or 1 day(s). In another embodiment, the T cells are provided to the patient only once.

[0207]

[0100] It is widely known that in any approach for the treatment of a tumor, and also depending on the features of the patient, the co-administering of several therapies may be considered. These co-administered therapies are administered with the aim to reduce or eliminate the tumor, or as accompanying therapies to reduce or avoid secondary effects.

[0208]

[0101] In a particular embodiment of the population of T cells for use in treatment of a tumor, the treatment further comprises the co-administering of a compound that recruits and / or switches cell state of myeloid cells, preferably microglial cells and / or macrophages to the tumor; and / or the co-administering of immune checkpoint modulator blockade therapy; and / or the co-administering of a multispecific molecule capable to target the T cell and the tumor.

[0209]

[0102] The inventors have surprisingly found that by administering the T cells of the invention in the presence of (autologous) myeloid cells, preferably (autologous) microglial cells and / or macrophages; and / or the co-administering of a compound that recruits and / or switches cell state of myeloid cells, preferably microglial cells and / or

[0210] AOMB: P102253WO macrophages to the tumor, the T cells increase their engagement and killing properties, as will be shown in the examples below.

[0211]

[0103] The co-administering of the population of T cells of the invention with any compound (multispecific molecule capable to target the T cell and the tumor, such a bispecific antibody or fragment thereof), facilitates the encountering of the T cells with the tumor, and this way the induction of the engagement and killing phenotype of interest.

[0212]

[0104] On the other side, the co-administering of immune checkpoint modulator blockade therapy together with the population of T cells prevents the exhaustion of the T cells.

[0213]

[0105] In another embodiment of the population of T cells for use in treatment of a tumor according to the invention, the population of T cells are autologous T cells, or the population of T cells are allogenic T cells.

[0214]

[0106] It forms part of the common general knowledge how to obtain autologous T cells from the patient that are further processed, for example genetically engineered or modified to express a particular CAR, and which will be cells that in principle can be administered to the same patient without the inconvenient of rejection. On the other hand, there are also known conventional techniques that allow for the selection and processing of allogenic cells from a donor to be administered to a recipient (patient) without the risk of rejection and adverse reactions. The population of T cells of the invention, are in some embodiments autologous T cells (i.e., obtained from the same patient). In other embodiment, the T cells are from a donor which is not the patient. Both are for use in the treatment of a tumor according to the invention.

[0215]

[0107] Advantageously, the invention also provides for methods to easily obtain the populations of T cells of the previous aspects. The methods allow for the obtention of effective T cells in a feasible and operational mode.

[0216]

[0108] It is thus another aspect of the invention a method of obtaining, selecting, or sorting a population of T cells according to any one of the previous aspects and embodiments, preferably according to the first aspect including the gene signature in Table 1, or preferably according to the aspect including the gene signature in Table 2, wherein the method comprises:

[0217] AOMB: P102253WO a) exposing a sample containing T cells to a tumor organoid, and co-culturing, preferably for a period of time of at least 1 - 24 hours, preferably 1- 12 hours, preferably 1- 6 hours, to allow some of the T cells to engage upon the tumor organoid, b) removing the non-engaged T cells from the co-culture,

[0218] c) optionally repeating steps (a) and (b) at least once or more, and

[0219] d) harvesting the T cells engaged upon the tumor organoid, to obtain the population of T cells.

[0220]

[0109] This previous method is based on the development of a platform for the fast obtention, selection and enrichment of T cells with a desired behavior or phenotype from a pool of T cells (e.g., an isolated sample of a patient, said sample comprising T cells).

[0221]

[0110] In an embodiment of this method of the invention, the tumor organoid may be a patient derived organoid (PDO). This provides the advantage of highly resembling the actual tumor, and provides for the testing of individualized treatment (e.g., isolated T cells from the patient can be tested against the organoid).

[0222]

[0111] The invention also provides, as additional aspect, a method of sorting or selecting T cells, preferably wherein the population of T cells are primary T cells, or preferably wherein the population of T cells are genetically engineered T cells, or wherein the population of T cells are CAR-T cells, or wherein the population of T cells are TCR expressing T cells, or wherein the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are CAR-T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are TCR expressing genetically engineered T cells not expressing a gamma-delta T cell receptor, wherein the method comprises: a) determining in a T cell the expression of one or more, or of all of the genes in the gene signature defined in the first aspect of the invention and listed in Table 1 or the expression of one or more, or of all of the genes in the gene signature defined and listed in Table 2,

[0223] b) retrieving the T cells that express one or more, or of all of the genes in the gene signature or that have a higher expression of one or more, or of all of the genes in the gene signature relative to a population of reference T cells.

[0224]

[0112] In an embodiment, step a) of determining in a T cell the expression of one or more of the genes in the gene signature, comprises determining the expression of 1,

[0225] AOMB: P102253WO 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 genes of the gene signature of in Table 2.

[0226]

[0113] In an embodiment of this method of sorting or selecting T cells, the method further comprises a step to check or to verify that the population of T cells is a tumor engager and / or tumor killing T cell populations, wherein the retrieved T cells are exposed to tumor cells, preferably exposed to a tumor organoid, preferably exposed to a PDO, and are co-cultured to determine, among other parameters, the tumor engagement and the (serial) killing.

[0227]

[0114] With the aim to simplify a method of sorting the population of T cells that has or can potentially have the desired phenotype to be effective against tumors, the invention provides an additional method of sorting or selecting T cells.

[0228]

[0115] Thus, another aspect of the invention is a method of sorting or selecting T cells, preferably wherein the population of T cells are primary T cells, preferably wherein the population of T cells are genetically engineered T cells, or wherein the population of T cells are CAR-T cells, or wherein the population of T cells are TCR expressing T cells, or wherein the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are CAR-T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are TCR expressing genetically engineered T cells not expressing a gamma-delta T cell receptor, wherein the method comprises

[0229] a) determining in a T cell the expression of at least NCAM1, and

[0230] b) retrieving the T cells that express at least NCAM1, or that have a higher expression of NCAM1 relative to a population of reference T cells.

[0231]

[0116] In an embodiment of this method of sorting or selecting T cells in which it is determined the expression of at least NCAM1, either in a population of primary T cells or in genetically engineered T cells as disclosed, the method also comprises determining if the T cells also express in combination one or more, preferably at least one or at least two, or at least three of the genes IL3; CD55; CCL1; XCL1; XCL2; CRTAM; SLC35E4; ATP1B1; AMIGO2; GPR18; AFAP1L2; SERPINE2; TRABD2A; SLC29A1; and LRIG1

[0232]

[0117] In an embodiment of this method of sorting or selecting T cells, the method further comprises a step to check or to verify that the population of T cells is a tumor engager and / or tumor killing T cell population, wherein the retrieved T cells are

[0233] AOMB: P102253WO exposed to tumor cells, preferably exposed to a tumor organoid, preferably exposed to a PDO, and are co-cultured to determine, among other parameters, the tumor engagement and the (serial) killing.

[0234]

[0118] In any of the methods of obtaining or of sorting or selecting T cells according to the invention, the (retrieved) T-cells may be selective expanded, for example, in the presence of IL-15.

[0235]

[0119] These population of T cells, sorted or selected as disclosed above in any of the methods, are for use in the treatment of a tumor in a patient, preferably wherein the patient has a solid tumor, preferably brain cancer, a glioma, preferably a diffuse midline glioma, a breast cancer, neuroblastoma, and / or a head and neck cancer.

[0236]

[0120] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0237]

[0121] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[0238]

[0122] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[0239]

[0123] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments, and preferences for all aspect separately.

[0240] AOMB: P102253WO

[0124] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art.

[0241] EXAMPLES

[0242] All the materials and method referring to the assays in the examples are disclosed at the end of this section.

[0243] Example 1

[0244]

[0125] Generation of CAR T cells. CAR T cell heterogeneity

[0245]

[0126] CAR T cells and diffuse midpoint glioma organoids (DMGOs) were obtained as indicated in the materials and methods section.

[0246]

[0127] The inventors investigated if DMGOs could serve as a human in vitro platform for preclinical evaluation of CAR T cell therapy. Recent first clinical outcomes of T cell therapy for DMG were achieved using GD2 CAR T cells, and we confirmed GD2 target expression in DMGOs (Fig. 2a). Hence, we treated DMGOs four months after tumor induction with CD8+ GD2 CAR T cells and monitored tumor control over time. Similar to heterogenous outcomes reported in patients, we observed an overall partial reduction in tumor burden (Fig. 1a) and heterogenous response rates over time and between individual DMGOs (Fig. 2b, c,). Since GD2 CAR T cell activation was evident by a robust IFNy response for all treated DMGOs (Fig. 2d), limited response profiles (e.g. DMGO179) are unlikely to result from a lack of antigen recognition. Importantly, therapy effects could be detected even after >1 month of treatment (Fig. 1b), offering advantages for modelling CAR T cell functionality in vitro in a manner that is representative of T cell states at the tumor site in vivo, including potential exhaustion profiles associated with prolonged tumor exposure. To test the potential of our new model for this purpose, we sequenced over 30,000 GD2 CAR T cells retrieved from DMGOs, as well as unexposed GD2 CAR T cells, and performed unbiased clustering and Uniform Manifold Approximation and Projection (UMAP) projection of the obtained gene expression dataset, revealing a substantial level of transcriptional heterogeneity (Fig. 1c). Focusing on the DMGO-exposed CAR T cells, we identified 9 transcriptional states that (Fig. 1c), based on combined interrogation of curated gene signatures (Fig.

[0247] AOMB: P102253WO 3a), DEGs (data not shown), DEG-associated GO terms (Fig. 3b-f), expression of canonical immune effector (Fig. 1d) and exhaustion markers (Fig. 1e) and comparison to a recently published pan-cancer infiltrating T cell (TIL) dataset that includes brain malignancies (Fig. 3g), reflected different T cell activation, differentiation and effector states. For instance, we identified a GD2 CAR T cell population that, although activated (based on HLA gene expression (Fig. 3a), is not fully differentiating towards effector function (undifferentiated; TUND) (Fig. 3b, h), as well as an IL-2 responsive population (TIL-2) (Fig. 3c, i), probably differentiating into effector T cells (Fig. 3i). In addition, we observed an interferon-stimulated gene (ISG) expressing population (Fig.

[0248] 3a) (TISG) strongly corresponding to ISG expressing TILs (Fig. 3j) and considered an interferon-induced activation state. Other clusters included a CAR T cell population with migrating properties and interconnectivity (TMI) that appears to be predominantly shaped by the brain tissue environment and might even acquire direct cellular communication with neurons (Fig. 3d), as well as proliferating (TPR) (Fig. 3e) and metabolically stressed T cells (TMS) (Fig. 3a, f). Importantly, we distinguished potential DMG-targeting effector T cell populations based on their cytotoxic profile (Fig. 1d) and putative level of exhaustion (Fig. 1e). While one of these clusters predominantly expressed GZMK (TGZK), cytotoxic T cells (TCYT) expressed GZMB, PRF1 and IFNG (Fig. 1d). In contrast, exhausted T cells (TEX) displayed reduced IFNG and concomitant expression of immune checkpoint genes; LAG3, HAVCR2, TIGIT61 and SELPLG62, as well as the transcriptional repressor PRDM1 associated with exhaustion (Fig. 1e), demonstrating the potential advantage of prolonged treatment in DMGO for uncovering T cell functional exhaustion, considered an actionable axis to enhance treatment outcomes and, therefore, critical to recognize during pre-clinical evaluation. To fully grasp the scope of our new model for reliable detecting T cell functional exhaustion, we sought to further refine the TEX phenotype present upon DMGO exposure, compared to pre-exposure T cells that also receive activating signals during expansion and might even display exhaustion features resulting from tonic signaling. Indeed, next to proliferating T cell represented by the TPR cluster, a considerable proportion of pre-exposure GD2 CAR T cells, overlapped with our TEX cluster detected upon DMGO exposure (Fig. 3k). However, separating the cells in this common TEX cluster based on DMGO exposure, revealed that DMGO exposed exhausted cells upregulated a wide array of additional exhaustion markers, as well as

[0249] AOMB: P102253WO TFs and functional modulators of exhaustion that, importantly, include those described in patients across TIL datasets (Fig. 1j). For in vitro model systems this has not yet been achieved in the context of naturally expressed tumor-antigen, only through persistent anti-CD3 and anti-CD28 antibody stimulation or using repeated rounds of stimulation with antigen- pulsed, or overexpressing tumor cell lines. Moreover, the upregulation of features associated with tissue-residency, including the canonical marker CD103 (ITGAE) used to identify tissue resident T cells (Fig. 1k) further underscores the capacity of DMGOs to model T cell performance within tissue and can lead to new strategies to enhance tumor migration and tissue residency sought after for improving CAR T cell performance.

[0250]

[0128] NCAM1 enriches for short-lived cytotoxic effector CAR T cells

[0251]

[0129] In line with their strong cytotoxic profile and no signs of exhaustion, TCYT showed a high degree of transcriptomic overlap with the ‘killer’ gene signature of ‘super-engager’ engineered T cells that we recently identified to have profound tumor targeting capacity and serial killing behavior in a short-term co-culture assay (Fig. 1f) (see Dekkers, J. F. et al. Uncovering the mode of action of engineered T cells in patient cancer organoids. Nat Biotechnol 1–10 (2022) doi:10.1038 / s41587-022-01397-w) Since we previously identified NCAM1 as a selection marker for this population, we exploited this strategy and the DMGO prolonged treatment model to further investigate the relevance of this CAR T cell functional profile in a patient-representative treatment setting. We sorted GD2 CAR T cells based on NCAM1 expression prior to DMGO treatment (Fig. 4a) and compared tumor control between NCAM1+ and NCAM1- cells (Fig. 1g, Fig. 4b). This demonstrated initial potent anti-tumor activity of NCAM1+ GD2 CAR T cells, with a 1.4-fold enrichment in tumor control over NCAM1- GD2 CAR T cells at day 2. However, this enhanced potency stabilized between day 5 and 7, with NCAM1- T cells displaying more gradual anti-tumor activity over time, slightly outperforming NCAM1+ cells by day 7 (Fig. 1g), in line with a higher recovery of NCAM1- cells at day 14 (Fig. 4c). To gain insight into potential transcriptomic profiles explaining these differential outcomes, we performed scRNA seq on NCAM1- and NCAM1+ GD2 CAR T cell retrieved at day 14 of DMGO treatment (Fig. 1h). Unbiased clustering of DMGO exposed NCAM1- and NCAM1+ T cells resulted in 10 clusters (Fig. 4d) that greatly varied in their relative frequency of NCAM1- and NCAM1+ cells

[0252] AOMB: P102253WO (Fig. 4e,f), demonstrating clear transcriptomic differences between these two subsets. Mapping back the NCAM1- and NCAM1+ cells to our previously identified GD2 CAR T cells signatures (Fig. 1c), revealed an enrichment of the NCAM1+ cells for cytotoxic T cells, compared to unsorted GD2 CAR T cells (bulk) and sorted NCAM1- cells, with TCYT making up 17.8% and 5.0% of the NCAM1+ and NCAM1- subset, respectively (Fig. 1 i, 4h). NCAM1- cells specifically contained an additional cluster consisting of stressed cells characterized by the expression of various heat-shock proteins (HSPs) (THS) (Fig. 1i and 4i), and overlaps with the stress response state identified in patient TILs that associates with immunotherapy resistance (Fig. 4j). Both these observations are in line with the initial enhanced tumor-targeting by NCAM1+ GD2 CAR T cells (Fig.

[0253] 1g). However, in line with this functional advantage stabilizing over time (Fig. 1g, Fig.

[0254] 4b) and poor persistence of the cells (Fig. 4c), NCAM1+ T cells are enriched for exhausted T cells, with TEX represented in 16.4% of the NCAM1+ cells, as compared to 3.4% of the NCAM1- cells (Fig. 1 i, 4h). Together, this suggests a short-lived effector role for NCAM1+ GD2 CAR T cells that could potentially be leveraged to enhance anti-DMG efficacy over time through multiple infusions of these selected cells. Further aligning with the initially enhanced tumour control observed (Fig. 1g), NCAM1+cells show a 3.3-fold enrichment in TCYT compared to NCAMT cells (Fig. 1i). However, in line with poor persistence of the cells (Fig. 4c), NCAM T cells are additionally enriched for TEX (Fig. 1 i), explaining their reduced performance over time (Fig. 1g). Together, this identified NCAM1+cells as the most potent tumour-targeting, yet shortlived effector GD2 CAR T cell population and offers proof-of-concept for cell selection as a means to narrow CAR T cell functional heterogeneity prior to patient administration. Moreover, we tested whether a combination treatment with a blocking antibody against the immune checkpoint TIGIT expressed by TEX cells (data not shown), could be employed to enhance the durable performance of NCAM1+ selected GD2 CAR T cells (data not shown). Collectively, these findings position DMGOs as a promising model system for preclinical evaluation of CAR T cell therapy and uncovering functional heterogeneity that can be exploited towards designing improved treatment strategies based on selecting the most potent cells and counteracting functional exhaustion.

[0255]

[0130] NCAM selection

[0256] AOMB: P102253WO

[0131] Figure 5. (a-b) Target X (X= NCAM1) can be used for cell selection of engineered T cell therapies to enrich Sniper T cells (a) and increase breast cancer tumor killing (b). (c-d) In a brain tumor model, Target X+ selected cells outperform Target X- cells (c) in line with an increased cytotoxic gene profile of the Target X+ cells (d).

[0257]

[0132] Cell selection using our lead biomarker, Target X, has shown to increase Sniper T cells by 20-25% compared to engineered T cells without this biomarker (Fig. 5a) and this results in increased tumor death in breast cancer organoids (Fig. 5b). Similarly, in a brain tumor model, cell selection for Target X led to enhanced tumor reduction (Fig. 5c) and an increased cytotoxic gene signature (Fig. 5d), underscoring the potential of our approach across multiple solid tumor types.

[0258]

[0133] TCYT CAR T cells display a reduced expression of cytokines in a model of brain tumor

[0259]

[0134] As can be seen in Figure 6a and 6b, the NCAM1+ selected engineered T cells have reduced production of selected cytokines compared to unselected engineered T-cells in the brain tumor organoid model. Importantly, the increased efficacy of NCAM1- selected engineered T cells did not lead to higher levels of cytokine secretion in the brain tumour model (Fig. 6). Without being bound to any theory, the inventors hypothesize that this phenotype discloses or predicts a behavior focused on only killing tumor cells, avoiding the unnecessary synthesis of molecules that are commonly used by other populations of T cells with other aims.

[0260]

[0135] TCYT CAR T CELLS synergistically kill the tumors in the presence of microglia / macrophages in the tumor microenvironment (TME) in a model of brain tumor

[0136] Since microglia are part of the tumor-immune microenvironment of DMG tumors, we incorporated these cells into our DMGO model by exposing the organoids to primitive macrophage progenitor cells. After 7 days, primitive macrophage progenitor cells were incorporated into the DMGO and differentiated to microglia cells. We then treated these organoids with unselected GD2 CAR-T cells and NCAM+- selected GD2 CAR-T cells. Strikingly, the presence of microglia in the DMGO impaired the killing activity of unselected GD2 CAR-T cells, especially at early timepoints, while this effect was not observed with NCAM+-selected GD2 CAR-T cells. Importantly, the

[0261] AOMB: P102253WO presence of microglia even significantly boosted the performance of NCAM1+-selected GD2 CAR-T cells, suggesting an improved ability of NCAM+ engineered T cells to synergize with, overcome or adapt to microglia in the tumor microenvironment (Figure 7).

[0262]

[0137] MATERIALS AND METHODS

[0263]

[0138] Ethics

[0264]

[0139] For the use of all DMG patient samples, patients and / or parents or guardians provided written informed consent according to national laws and in agreement with the declaration of Helsinki (2013). This study is Institutional Review Board (IVB) approved and registered under national registry number 2020.142.

[0265]

[0140] Stem cell culture and brain organoid obtention

[0266]

[0141] Brain organoids were generated from 3 different cell lines encompassing human Embryonic Stem Cells (hESCs) H9 (WA09, Wicell) and H1 (WA01, Wicell) and induced Pluripotent Stem Cells (iPSCs) C7-a (RUID 06C52463). The iPSC line C7-a was obtained from Rutgers University Cell and DNA Repository (RUCDR) and contained a Cre-inducible H3.3K27M reading frame in the endogenous H3F3A Iocus17. Cell lines were cultured in mTeSR Plus medium (Stem Cell Technologies, Cat. #100-0276) and incubated at 37°C with 5% CO2. The cells were grown on Matrigel-coated (Corning, Cat. #354277) 6-well plates and passaged when 70-80% confluent by non-enzymatic detachment of colonies using Gentle Cell Dissociation Reagent (GCDR, Stem Cell Technologies, Cat. #100-0485). All cell cultures were routinely tested for the presence of Mycoplasma species.

[0267]

[0142] Embryoid body formation

[0268]

[0143] The different stem cell sources were washed with 1X Dubecco’s Phosphate- Buffered Saline (DPBS, Gibco, Cat. #14190144) and detached with GCDR, before spinning down at 300 RCF for 5 minutes. Next, the cells were resuspended in BASE medium (1:1 Advanced DMEM / F-12 medium (Gibco, Cat. #12634010) and Neurobasal medium (Gibco, Cat. #10888022), 1X GlutaMax (Gibco, Cat. #35050061)) and counted. 70,000 cells / ml were added to DAY 0 medium (BASE medium, 10 µM Y-27632 (ROCKi, AbMole BioScience, Cat. #M1817), 4 ng / ml Fibroblast Growth Factor 2 (FGF2, PeproTech, Cat. #100-18C)). For Embryoid Body (EB) formation, 7,000 cells in 100 µl medium are seeded per well of an ultra-low attachment (ULA) treated U-

[0269] AOMB: P102253WO bottom 96-well plate (Nexcelom, Cat. #ULA96U020 / PHC Europe B. V., #MS-9096UZ) and incubated at 37°C with 5% CO2. From day 2 - day 21, PATTERNING medium (BASE medium, 1X N2 (Gibco, Cat. #17502048), 1 mg / ml Heparin Solution (Stem Cell Technologies, Cat. #07980) was used.

[0270]

[0144] Organoid patterning

[0271]

[0145] To induce hindbrain and, more specifically, pontine identity, organoids were patterned using timely additions and replacement of morphogen supplemented media. In week 1, WEEK 1 medium (PATTERNING medium, 50 ng / ml FGF2, 1 µM Dorsomorphin (DM, Stem Cell Technologies, Cat. #72102), 10 µM SB431542 (SB43, Stem Cell Technologies, Cat. #72232), 3 µM CHIR99021 (CHIR, Stem Cell Technologies, Cat. #72052)) was used. On day 2, 100 µl WEEK 1 medium was added per well. On day 5, 100 µl medium per well was replaced with fresh WEEK 1 medium. In the second week, WEEK 2 medium (PATTERNING medium, 1 µM DM, 10 µM SB43, 3 µM CHIR, 10 ng / ml Fibroblast Growth Factor 4 (FGF4, Stem Cell Technologies, Cat. #78103.1), 10 µM All-Trans Retinoic Acid (RA, Stem Cell Technologies, Cat. #72262), 1 µM Purmorphamine (PMA, Stem Cell Technologies, Cat. #72202) was used. On day 7, 190 µl medium was replaced with fresh WEEK 2 medium and on day 9 100 µl medium was replaced. On day 11, the EBs were embedded in 12 µl Matrigel droplets and 5 droplets were transferred to each well of a 12-well suspension plate (Greiner Bio-One, Cat. #665102) with 1 ml WEEK 2 medium and incubated at 37°C with 5% CO2. In week 3, WEEK 3 medium (PATTERNING medium, 10 ng / ml FGF4, 10 µM RA, and 1 µM PMA) was used. Until day 21, every 2 days, the medium was refreshed with WEEK 3 medium. On day 17, the plates were placed on an orbital shaker inside a 5% CO2 incubator at 37°C. From day 21 onwards, every 2-3 days, the medium was refreshed with MATURATION medium (1:1 Advanced DMEM / F-12 medium and Neurobasal medium, 1X GlutaMax, 0.5X N-2, 0.5X B27 without vitamin A (Gibco, Cat. #12587010), and 1X Penicillin-Streptomycin (Pen-Strep, Gibco, Cat. #15140122)).

[0272]

[0146] DMG driver mutation-expressing and genetic lineage tracing plasmids

[0147] To induce DMG tumor growth in hESC-derived pontine organoids, the following plasmids were used: pCAGPbase, PBCAG_DNp53_IRES_luciferase, PBCAG_PDGFRA-D842V_IRES_eGFP, and PBCAG_H3K27M_eGFP. Alternatively, to induce DMG tumor growth in iPSC-derived pontine organoids, the H3K27M- expressing plasmid was replaced with 1.00 µg / µl Ssi-Cre to induce with an inducible

[0273] AOMB: P102253WO H3.3-K27M mutation targeted to the endogenous histone locus. As a control, the following plasmids were used: 1.50 µg / µl pCAGPbase and 1.50 µg / µl PB_Venus. All plasmids were kindly provided by the Pheonix laboratory.

[0274]

[0148] In situ electroporation and monitoring of tumor growth

[0275]

[0149] On day 11, unless stated otherwise, pontine organoids were injected with a mixture of plasmid DNA (1.50 µg / µl per plasmid) and 0.1% (w / v) FastGreen (Merck, Cat. #F7252-5G) using a FemtoJet 4i (Eppendorf, Cat. #5252000013) with the following parameters: Injection pressure (Pi) = 15 hPa and compensation pressure (Pc) = 5 hPa. Subsequently, the organoids were electroporated using a NEPA21 Super Electroporator (Nepagene) and CUY650P1 (Nepagene) tweezers with the following parameters: Voltage = 50 V, pulse length = 10 ms, pulse interval = 50 ms, number of pulses = 4, and decay rate = 10%. Transfer Pulse; Voltage = 20 V, pulse length = 50 ms, pulse interval = 50 ms, number of pulses = 5 and decay rate = 40%. Using the impedance (kQ) measurement of the NEPA21 Super Electroporator, voltage was automatically re-adjusted to optimize cell perforation and viability per individual organoid. Electroporation was performed by applying a shock twice in orthogonal direction. After electroporation, the organoids were incubated at 37°C with 5% CO2 for at least two hours to recover before Matrigel embedding. To monitor tumor growth over time, organoids were imaged on a Leica DM IL LED microscope with an N PLAN 5x / 0,12 PHO objective and compared to the mVenus-positive control.

[0276]

[0150] GD2 CAR T cell expansion and selection

[0277]

[0151] CD8+ GD2 CAR T cells (14G2a GD2-4-1BBz CAR) and donor-matched mock-transduced CD8 T cells, were produced as previously described (see Andersch, L. et al. CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in pre-clinical in vitro testing. BMC Cancer 19, 895 (2019)). CAR T cells and mock transduced T cells were expanded using a rapid expansion protocol (Marcu-Malina, V. et al. Redirecting af> T cells against cancer cells by transfer of a broadly tumor-reactive y6T cell receptor. Blood 118, 50-59 (2011)). T cells were cultured in RPMI 1640 + GlutaMax (Thermo Fisher, Cat. #61870036), supplemented with 2.5-10% human serum (Sanquin), 1% Pen-Strep, and 0.5M beta-2-mercaptoethanol (Thermo Fisher, Cat. #21985023), on a feeder cell mixture comprising of sub-lethally irradiated allogenic PBMCs, Daudi, and LCL-TM cells, in the presence of 50 U / ml IL-2 (R& D Systems, Cat. #P60568), 5 ng / ml IL-15 (R& D Systems, Cat. #P40933), and 1 pl / ml

[0278] AOMB: P102253WO PHA-L (Sigma-Aldrich, Cat. #11249738001) and cryopreserved after 14 days of expansion. Prior to experiments, T cells were thawed and rested in RPMI 1640 + GlutaMax, with 10% Fetal Bovine Serum (FBS, Thermo Fisher, Cat. #10500064) and 1% Pen-Strep, supplemented with 50 U / ml IL-2 (Miltenyi, Cat. #130-097-743), 2000 U / ml IL-7 (Miltenyi, Cat. #130-095-367), and 50 U / ml IL-15 (Miltenyi, Cat. #130-095- 760) for 3 days at 37°C with 5% CO2. For selection of GD2 CAR T cells based on NCAM1 expression, a similar expansion protocol was used, but without addition of IL-15 and Daudi cells. After resting, cells were washed and stained for 30 min at 4°C in FC buffer with LIVE / DEAD Fixable Near-IR Dead Ceil Stain (1:1000; Thermo Fisher), CD3-APC (1:80; BD BioLegend, clone SK7) and NCAM-1-HiLyte-488 (1:200; QVQ, FSH-10B10). CD3+ NCAM1- or CD3+ NCAM1+ GD2 CAR T cell populations were separated by FACS on a BD FACSAria II Cell Sorter and immediately used for experiments.

[0279]

[0152] Treatment of DMGOs with GD2 CAR T cells

[0280]

[0153] Four months after tumor induction, DMGOs were transferred to 12-well suspension plates and untreated or treated with 500,000 CD8+ GD2 CAR T cells, or mock transduced CD8+ T cells per DMGO. 7 days after the start of treatment, 500,000 T cells were added per DMGO for a second round of treatment. Tumor size during treatment was monitored by imaging on day 0, 3, 7, 10, and 14, or on day 0, 2, 5, 7, 10 and 14 for treatment with NCAM1 selected T cells using a Leica Thunder DMi8 microscope with a 10X objective. In addition, one DMGO was treated on day 0, 8 and 15 with GD2 CAR T cells and imaged on day 8, 15, 28 and 35. After THUNDER software-mediated computational clearing of the imaging data, tumor size for each time point was quantified using Fiji. In short, background signal, defined as GFP-negative areas within the organoid, was subtracted. The organoid surface was set as region-of-interest (ROI) and mean gray values of the GFP channel for the ROI were calculated. For organoids treated with NCAM1 selected T cells the tumor area was calculated using the GFP channel. Additionally, at day 3, 7 and 14 supernatant of the co-cultures was collected and cytokine and chemokine concentrations were measured with Luminex.

[0281]

[0154] FACS of GD2 CAR T cells and DMGO GD2 expression

[0282]

[0155] DMGOs treated with GD2 CAR T cells were dissociated 14 or 35 days after initial T cell addition with the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, Cat.

[0283] AOMB: P102253WO #130-092-628), as described above for preparation of single cell RNA and tracker seq libraries. Dissociated cells were washed and stained in FC buffer with CD3-APC (1:80; BD Biosciences, clone SK7) and LIVE / DEAD Fixable Near-IR Dead Cell Stain (1:1000; Thermo Fisher) for 30 min at 4°C. CD3+ T cells and GFP+ tumor cells were sorted on a CytoFLEX SRT Benchtop Cell Sorter (Beckman Coulter) and immediately processed for scRNA-seq. To confirm DMGO GD2 expression for GD2 CAR T cell treatment evaluation, a day 60 post-electroporation DMGO sample was dissected for the tumor region to enrich for tumor material, mechanically dissociated, and cultured for 2 additional weeks to expand tumor cells. Cells were retrieved from the culture plate using StemPro Accutase (Gibco, Cat. #A1110501) and passed through a 70 pm Flowmi cell strainer (Merck, Cat. #BAH136800070) to create a single cell suspension. Dissociated cells were centrifuged at 500 RCF for 5 minutes at 4 °C and resuspended and washed in FC buffer (2% fetal bovine serum (FBS), 1x PBS). Cells were either left unstained or stained with LIVE / DEAD Fixable Near-IR Dead Cell Stain (1:1000; Thermo Fisher) and GD2-PE (1:200, clone 14. G2a, BD Biosciences, Cat. #562100) for 30 min at 4°C. After staining, cells were washed twice in FC buffer, acquired on a Sony SH800s (Sony Biotechnology), and analyzed using FlowJo Software (v10.9.0).

[0284]

[0156] Single cell RNA sequencing of GD2 CAR T cells

[0285]

[0157] Single-cell encapsulation was performed according to the manufacturer's protocol 10X Genomics (Cell Preparation for Single Cell, Demonstrated Protocol, CG000053). Pooled cDNA amplification was generated using the Chromium Single Cell 3' V3 Library & Gel Bead Kit using the TruSeqRI, TruSeqR2 and partial TSO (template switch oligo) standard primers. Total cDNA of this reaction was then used for Library preparation for mRNA oligos as described per the manufacturer's protocol. For single-cell gene-expression, 10x genomics barcoded libraries were constructed for 60-day post-electroporation DMGOs. Nested PCRs were additionally used to further amplify the lineage barcodes (primers in first PCR (10 cycles): sequences: 5’-CTACACGACGCTCTTCCGATCT-3’ (Read 1 -Forward primers from 10X, 5’-CTTCTCGTTGGGGTCTTT-3’ (eGFP primer-Reversed) Annealing Temperature was 60 and elongation time 30 sec; primers in the second PCR (10 cycles) Annealing Temperature was 60 and elongation time 30 sec: Standard P5-Read 1_Forward and Fun series 70x primer-Reversed from 10x). Sequencing of the prepared libraries was performed on an Illumina NovaSeq6000 in PE150 mode, and raw fastq files were

[0286] AOMB: P102253WO processed and mapped with CellRanger v3.1.0. using a custom reference hg38 genome including sequences used in the electroporation methods, namely EGFP, H3.3K27M, Dnp53, Luciferase and Pdgfra. D824V.

[0287]

[0158] Pre-processing and analysis of GD2 CAR-T cell scRNA-seq datasets

[0159] As the first quality control step, doublets (two, or more, cells captured in the same droplet) for each sample were identified and removed using the scDblFinder package, with default settings. Low quality cells with high mitochondrial content (> 15%), or cells with extremely high or low reads (< 200 genes or > 6500 genes), or cells with extremely high reads (> 35000 reads) were removed. Normal Seurat V4 83 workflow was used to normalize and scale reads, and the 3000 most variable features determined using “FindVariableFeatures” in the Seurat package. Cell cycle confounding effect was eliminated from the dataset via the removal of cell cycle- related genes from the variable features of the dataset. PCA was performed using “RunPCA” function. First 30 PCs were used for non-linear dimensionality reduction utilizing UMAP 84 method, implemented via “RunUMAP” function of the Seurat package. Clustering analysis was performed on the first 10 PCs using the Seurat package's TindNeighbors’ and 'FindClusters' functions. A resolution parameter of 0.45 was applied, and the original Louvain algorithm was used. To identify subpopulations, marker genes for each cluster were determined through the 'FindAllMarkers' function. Markers obtained from this analysis were then examined to profile genes associated with known CD8 T cell subsets, as well as to project previously published signatures (see T cell signature projection below). Only markers with adjusted p values below 0.05 were taken into consideration. In addition, DEGs were used as input for gene ontology (GO) enrichment analysis using the GO resource (https: / / geneontology.org).

[0288]

[0160] T cell signature projection

[0289]

[0161] To evaluate the expression of established T cell signatures in GD2 CAR T cell scRNA-seq datasets, we used a gene signature specific to serial killer engineered T cells that we previously obtained (). Utilizing the VISION R package, we computed and visualized the overall enrichment of the identified gene set atop UMAP cell embeddings of our dataset. In addition, we projected our own GD2 CAR T cell signature profiles onto a pan-cancer CD8 tumor infiltrating lymphocyte (TIL) atlas from Chu et al. (Chu, Y. et al. Pan-cancer T cell atlas links a cellular stress response state

[0290] AOMB: P102253WO to immunotherapy resistance. Nat. Med. 29, 1550–1562 (2023)), which encompasses T cells infiltrating brain tumors. For each GD2 CAR T cell subset, markers obtained through DEG analysis were meticulously curated to obtain the most relevant markers, ensuring an adjusted p-value below 0.00001. Accessing a publicly available and interactive online data portal (https: / / singlecell.mdanderson.org / TCM / ), we acquired a rds file containing the Seurat object pertinent to scRNA seq data of CD8 TILs. Subsequently, the VISION package was employed to perform the projection of our GD2 CAR T cell signatures onto this dataset.

[0291]

[0162] Statistical and heatmap analysis

[0292]

[0163] Statistics on bulk sequencing data was computed by built-in functions of R (“stats”, v4.3.1) using one-way ANOVA with post-hoc Tukey Honest significance difference. PCA, Spearman’s Rank and gene expressions were plotted using ggplot2(v3.4.2), heatmaps were generated using pheatmap package (v1.0.12). Statistics on electroporation efficiency and tumor induction was calculated using the two-tailed independent t-test (function: t.test). For each batch and timepoint the mean and standard deviation was calculated, individual values were imported and plotted in GraphPad Prism (v.8.0.2) using summarizing stacked bar plots. All statistical tests have been performed with the assumption of a normal distribution, equal variance per sample and a confidence interval of at least 95% (alpha = 0.05).

[0293]

[0164] Incorporation of microglia in DMGO’s and treatment with GD2 CAR-T cells

[0165] Primitive macrophage progenitor cells were generated from H1 stem cells as previously described (Haenseler et al, 2017, Stem Cell Rep.; Gutbier et al, 2020, Int. J. Mol. Sci.). Briefly, H1 stem cells were washed with 1X Dubecco’s Phosphate-Buffered Saline (DPBS, Gibco, Cat. #14190144) and detached with GCDR, before spinning down at 300 RCF for 5 minutes. Next, the cells were resuspended in mTeSR Plus medium (Stem Cell Technologies, Cat. #100-0276) and counted. For Embryoid Body (EB) formation, 700,000 cells in 100 µl mTeSR Plus supplemented with 50 ng / mL BMP-4, 50 ng / mL VEGF and 20 ng / mL SCF are seeded per well of an ultra-low attachment (ULA) treated U-bottom 96-well plate (Nexcelom, Cat. #ULA96U020 / PHC Europe B. V., #MS-9096UZ) and incubated at 37°C with 5% CO2. On day 4, EBs were harvested and transferred to a 6 well plate in 2 mL X-VIVO 15 (Lonza, Cat. #02-053Q) medium supplemented with 100 ng / mL M-CSF and 25 ng / mL IL-3. Medium was

[0294] AOMB: P102253WO refreshed weekly and primitive macrophage progenitors were produced by EBs after 3 weeks and could be harvested from the supernatant for up to 3 months later.

[0295]

[0166] DMGO were sectioned into 200 µM thick slices using a vibrotome, transferred to a 24-well suspension plate in 750 pL MATURATION medium and incubated at 5% CO2 and 37’C for 3 days. For microglia incorporation into DMGOs, 0.2 million primitive macrophage progenitors were added per DMGO slice in 750 µL MATURATION medium to a 24-well suspension plate and incubated at 5% CO2 37°C. After 7 days, primitive macrophage progenitors were incorporated into the DMGO slices and differentiated to microglia cells.

[0296]

[0167] For GD2 CAR-T cell treatment, 200,000 CD8+ GD2 CAR T cells or NCAM+ selected GD2 CAR-T cells were added in 750 µL MATURATION medium per 24-well to the DMGO slices. Tumor size during treatment was monitored by imaging on day 0, 3, 7, 10, and 14, using a Leica DMiL LED FLUO microscope with a 10X objective.

[0297]

[0168] Breast cancer organoid culture

[0298]

[0169] Breast cancer organoids were seeded in basement membrane extract (BME, Cultrex) in uncoated 12-well plates (Greiner Bio-one). For culturing, Advanced DMEM / F12 was supplemented with penicillin / streptomycin (pen / strep), 10 mM HEPES, GlutaMAX (adDMEM / F12+++), 1x B27 (all ThermoFisher), 1.25 mM N-acetyl-l-cysteine (Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 5 µM Y-27632 (Abmole), 5 nM Heregulin β-1 (Peprotech), 500 nM A83-01 (Tocris), 5 ng / ml epidermal growth factor (Peprotech), 20 ng / ml human fibroblast growth factor (FGF)-10 (Peprotech), 10% Noggin-conditioned medium, 10% Rspol -conditioned medium, 0.1 mg / ml primocin (Thermo Fisher), 1 µM SB202190 (Sigma-Aldrich) and 5 ng / ml FGF-7 (Peprotech) (type 1 culture medium). Organoids from passages 5-30 after cell isolation were used for T cell coculture. For T cell coculture, organoids were recovered from the BME by resuspension in TrypLE Express and collected in adDMEM / F12+++. Organoid suspensions were filtered through a 70-µm strainer (Greiner) to remove large organoids and pelleted before co culture.

[0299]

[0170] NCAM selection (in assay with breast cancer organoids)

[0300]

[0171] TEGs were harvested at day 8-10 of their REP cycle, stained in flow cytometry (FC) buffer (2% fetal bovine serum, 1x PBS) with Hilyte-488-conjugated NCAM1 nanobodies (1:400; QVQ) and LIVE / DEAD Fixable Near-IR Dead Cell Stain (1:1000; ThermoFisher) for 30 minutes at 4°C and consecutively sorted using a SONY SH800S

[0301] AOMB: P102253WO or a FACS Aria Cell Sorter (BD Biosciences) into NCAM1 - and NCAM + populations. Cells were rested for 16 h in ‘T cell culture medium’ and then used for co-culture.

[0302]

[0172] Live-cell imaging of T cells and organoid cocultures

[0303]

[0173] Engineered T cells (20,000) were cocultured with breast cancer organoids in an effector / tumor cell (E: T) ratio of 1:30. CD4+ and CD8+ TEGs were mixed in a 1:1 ratio immediately before plating. Cells were incubated in 96-well, glass-bottom SensoPlates (Greiner) in 200 pl of ‘coculture medium’: 50% type 1 organoid culture medium, 50% ‘TEG assay medium’ (RPMI-GlutaMAX supplemented with 10% fetal calf serum and 1% pen / strep), 2.5% BME and pamidronate for the accumulation of the phosphor antigen IPP to stimulate tumor cell recognition (1:2,000). Coculture medium was supplemented with both NucRed Dead 647(two drops / ml; Thermo Fisher) and TO-PRO-3 (1:3,000; Thermo Fisher) for fluorescent labelling of living and dead cells (‘Imaging medium’). The combination of NucRed Dead 647 and TO-PRO-3 labels dead cells when excited with a 633-nmlaser and living cells with a 561 -nm laser (Extended Data Fig. 1a, b). Both were combined to achieve the optimal fluorescent intensity ratio between dead and living cells for live-cell imaging. Before coculture, TEGs were incubated with eBioscience Cell Proliferation Dye eFluor 450 (referred to as eFluor-450; 1:4,000; Thermo Fisher) in PBS for 10 min at 37 °C to fluorescently label all T cells. When CD4+ and CD8+TEGs were simultaneously imaged, both eFluor-450 and CalceinAM (1:4,000; Thermo Fisher) were used to label the different TEG subsets in PBS for 10 min at 37 °C. The plate was placed in a LSM880 (Zeiss Zen Black Edition v.2.3) microscope containing an incubation chamber (37 °C, 5% CO2) and incubated for 30 min to ensure settling of TEGs and organoids at the bottom of the well. The plate was imaged for up to 24 h with a Plan-Apochromat ><20 / 0.8 numerical aperture dry objective with the following settings: online fingerprinting mode, bidirectional scanning, optimal Z-stack step size, Z-stack of 60 pm in total and timeseries with a 2-min interval (up to four or ten conditions simultaneously; resolution 512 x 512 and 200 x 200, respectively).

[0304]

[0174] Image processing

[0305]

[0175] 3D visualization, cell segmentation, extraction of statistics and time-lapse videos were processed with Imaris (Oxford Instruments) v.9.2-9.5. The Channel Arithmetics Xtension was used to create new channels for specific identification of organoids (live and dead) and eFluor-450-labelled or calcein AM-labelled T cells (live

[0306] AOMB: P102253WO and dead) and to exclude cell debris. The Surface and Imaris Track modules were used for object detection and automated tracking of both T cells (autoregressive motion) and organoids (‘connected components’ or no tracking). The Distance Transformation Xtension was used to measure the distance between TEGs and organoids, with thresholds for defining organoid-T cell interactions visually determined. For tracked TEGs, time-lapse data containing the coordinates of each cell, the values of cell speed, mean square displacement, distance to organoids and dead cell dye channel intensity were exported.

[0307]

[0176] T cell dynamics analysis and multivariate time series clustering

[0308]

[0177] For the analysis of TEG behavior over time, the following parameters were used: T cell death, contact with organoids, speed, square displacement and interaction with other T cells. For each T cell time series, linear interpolation was used to estimate the values in several cases of missing time points. To compare time series independently of their length, cell tracks were cut to a length of 3.3 h. Similarity between distinct cell tracks was measured using a strategy that allows for best alignment between time series, previously applied for mitotic kinetics or temporal module dynamics comparisons. A cross-distance matrix based on multivariate time series data was computed using the dynamic time-warping algorithm. To visualize distinct cell behaviors in two dimensions, dimensionality reduction on the multidimensional feature count table was performed by the UMAP method. Clustering was performed using the k-means clustering algorithm with outlier detection. To confirm the identity of each cluster, T cell cluster assignments were back-projected to visualize the surfaces and tracks of particular T cell populations in the imaging dataset.

[0309]

[0178] Cell behavior classification using a random forest classifier

[0310]

[0179] For standardized integration of new experiments, we used a random forest classification approach to relate cell behavior to the nine behavioral signatures that we found in our global TEG behavior atlas. The following parameters were used: T cell death, organoid contact, speed and square displacement. The reference dataset used to build the global TEG behavior atlas was split into cell tracks for use as either a training dataset (95%) or a test dataset (5%). To reduce dimensionality, for each cell track four time series descriptive statistics were quantified and used to train the classifier. For numeric variables, the following measures were computed for each cell track: mean, median, the top 90% of the distribution and standard deviation. For binary

[0311] AOMB: P102253WO values, such as contact with organoids, the mean was calculated as well as the mean and maximum of cumulative interaction. The random forest classifier was trained using 100 trees on the above-mentioned variables using the nine behavioral signatures as labels. The test dataset was used to assess accuracy of the classifier and to determine in which behavioral signatures the errors occurred.

[0312] Example 2

[0313]

[0180] Abbreviated gene signature

[0314]

[0181] MATERIALS AND METHODS

[0315]

[0182] RNA sequencing of super engager T cells

[0316]

[0183] Super engaging and non engaging TCR-T and CAR-T cells were isolated from co-cultures of breast cancer, diffuse midline glioma or neuroblastoma organoids 6 hours after co-culture initiation as previously described (Dekkers et al., 2023, Nature Biotechnology; Wezenaar et al., 2025, Nature Protocols). Isolated super engager and non engaging T cell were stained in flow cytometry (FC) buffer (2% fetal bovine serum, 1x PBS) with CD3-APC (1:80; BioLegend) and LIVE / DEAD Fixable Near-IR Dead Cell Stain (1:1000; ThermoFisher) for 30 minutes at 4" C. Viable CD3+ T cells were sorted using a FACS Aria Cell Sorter (BD Biosciences). Sorted cells were centrifuged, resuspended in 1000 µL TRIzol and stored at -80°C before shipping them on dry ice to Single Cell Discoveries B. V. (Utrecht, The Netherlands). Library preparation was performed according to the CEL-seq2 protocol (Hashimshony et al., 2016, Genome Biology). Sequencing was performed on a Nextseq500 (Illumina) with a sequencing depth of 10 million reads per sample.

[0317]

[0184] Super engager gene signature analysis

[0318]

[0185] Differential gene expression between super engaging and non engaging T cells was assessed using DESeq2, using the contrast (" TumourEngagement", "super_engaging", "non_engaging"). Genes with padj < 0.05 and log2FC > 1 were considered significantly upregulated in super engager cells. Upregulated genes were intersected with the list of 61 genes previously identified for super engager T cells in breast cancer (Dekkers et al., 2023, Nature Biotechnology). To ensure robust detection, only genes with median expression >10 counts in the non-targeting control were retained. Genes were further manually selected on cell surface expression and / or

[0319] AOMB: P102253WO secretion resulting in a list of 16 genes (In Table 2). To test whether super engager-associated genes are variable in primary T cells, we analysed a PBMC scRNA-seq dataset. Highly variable genes were identified (vst, 4,000 genes), and super engager genes were intersected with this Highly Variable Genes (HVG) set. These data are with PBMC are elaborated in the next Example.

[0320]

[0186] Table 2. Abbreviated gene signature (ENSBL ID numbers of genes in the gene signature

[0321] Gene Ensembl ID

[0322] IL3 ENSG00000164399

[0323] CD55 ENSG00000196352

[0324] CCL1 ENSG00000108702

[0325] XCL1 ENSG00000143184

[0326] XCL2 ENSG00000143185

[0327] CRTAM ENSG00000109943

[0328] SLC35E4 ENSG00000100036

[0329] ATP1B1 ENSG00000143153

[0330] AMIGO2 ENSG00000139211

[0331] GPR18 ENSG00000125245

[0332] AFAP1L2 ENSG00000169129

[0333] SERPINE2 ENSG00000135919

[0334] TRABD2A ENSG00000186854

[0335] SLC29A1 ENSG00000112759

[0336] LRIG1 ENSG00000144749

[0337] NCAM1 ENSG00000149294

[0338] ENSBL ID of the Ensembl release 113 - October 2024 © EMBL-EBI, retrievable at https: / / www.ensembl.org / index.html, and which includes a gene description.

[0339] Example 3

[0340]

[0187] Expression of Sniper T cell genes in human primary T cells

[0341]

[0188] MATERIALS AND METHODS

[0342]

[0189] Gene expression in human primary T cells

[0343]

[0190] To test whether the genes in the Sniper T cell gene signature are variable in primary T cells, we analysed a PBMC scRNA-seq dataset. Highly variable genes were identified (vst, 4,000 genes), and Sniper T cell genes were intersected with this HVG set.

[0344]

[0191] RESULTS

[0345]

[0192] To evaluate if the genes related to Sniper T cells are present in human primary T cells, we evaluated their expression in a transcriptomic dataset of human PBMC. We

[0346] AOMB: P102253WO detected variable expression of the genes in primary T cells, suggesting that a subpopulation of these cells express the genes. This indicates that the selection of Sniper T cells can already be performed at an early stage during the manufacturing of engineered T cell therapies, immediately after obtaining the apheresis material and prior to T cell engineering. Figure 8 (Fig. 8) displays these results, where gene expression in human primary T cells is shown with a dot plot representing the percentage of cells expressing selected genes. Colour intensity represents the average scaled gene expression.

[0347]

[0193] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[0348]

[0194] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description, or embodiment of the present invention is disclosed, taught, or suggested in the relevant art.

[0349] AOMB: P102253WO

Claims

CLAIMS1. A population of T cells, wherein the population of T cells is characterized by:(a) expression of at least one, at least five, at least fifteen, at least twenty, at least twenty-five, or of the whole of the genes of a gene signature defined by:NCAM1; IRF8; KLF10; IL3; SLAMF7; NFATC1; PRF1; TAGAP; CDK4; AGK; CD55; VCL; CCT4; CD160; CLUH; C1QBP; PGAM1; CCL1; XCL1; XCL2; CRTAM; BACH2; ZBTB21; UBASH3B; SLC35E4; XIRP1; PRKD3; YBX3; YBX1; ATP1B1; AMIGO2; MYO1E; POU2AF1; GPR18; IGF2R; AFAP1L2; EMP1; HEG1; ARHGEF3; BYSL; NTRK1; SNTB2; NCEH1; BZW2; DCAF13; IARS; CHD4; SERPINE2; SQLE; FAM3C; PKIA; CCT3; PA2G4; TRABD2A; IMP4; POLR3C; ITPR1; URB1; SLC29A1; LRIG1; TBC1D4; and FBXO30; and / or(b) higher expression of at least one, at least five, at least fifteen, at least twenty, at least twenty-five, or of the whole of the genes of a gene signature defined by:NCAM1; IRF8; KLF10; IL3; SLAMF7; NFATC1; PRF1; TAGAP; CDK4; AGK; CD55; VCL; CCT4; CD160; CLUH; C1QBP; PGAM1; CCL1; XCL1; XCL2; CRTAM; BACH2; ZBTB21; UBASH3B; SLC35E4; XIRP1; PRKD3; YBX3; YBX1; ATP1B1; AMIGO2; MYO1E; POU2AF1; GPR18; IGF2R; AFAP1L2; EMP1; HEG1; ARHGEF3; BYSL; NTRK1; SNTB2; NCEH1; BZW2; DCAF13; IARS; CHD4; SERPINE2; SQLE; FAM3C; PKIA; CCT3; PA2G4; TRABD2A; IMP4; POLR3C; ITPR1; URB1; SLC29A1; LRIG1; TBC1D4; and FBXO30wherein said higher expression is relative to a population of reference T cells.AOMB: P102253WO2. The population of T cells according to claim 1, wherein the populations of T cells is characterized by the expression of at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, or of the whole of the genes of a gene signature, defined by: NCAM1; IL3; CD55; CCL1; XCL1; XCL2; CRTAM; SLC35E4; ATP1B1; AMIGO2; GPR18; AFAP1L2; SERPINE2; TRABD2A; SLC29A1; and LRIG1.

3. The population of T cells according to any one of claim 1 - 2, wherein the populations of T cells is characterized by the expression of the whole of the genes of a gene signature defined by: NCAM1; IL3; CD55; CCL1; XCL1; XCL2; CRTAM; SLC35E4; ATP1B1; AMIGO2; GPR18; AFAP1L2; SERPINE2; TRABD2A; SLC29A1; and LRIG1.

4. A population of T cells, wherein the population of T cells is characterized by:(a) expression of NCAM1; and / or(b) higher expression of NCAM1, wherein said higher expression is relative to a population of reference T cells.

5. The population of T cells according to any one of the previous claims wherein the population of T cells are primary T cells, or wherein the population of T cells are genetically engineered T cells, or wherein the population of T cells are CAR- T cells, or wherein the population of T cells are TCR expressing T cells, or wherein the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are CAR-T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are TCR expressing genetically engineered T cells not expressing a gamma-delta T cell receptor.

6. The population of T cells according to any one of the previous claims wherein the population are genetically engineered T cells, or wherein the population of T cells are CAR-T cells, or wherein the population of T cells are TCR expressing T cells, or wherein the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or wherein the population of TAOMB: P102253WOcells are CAR-T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are TCR expressing genetically engineered T cells not expressing a gamma-delta T cell receptor.

7. The population of T cells according to any one of the previous claims wherein the population of T cells are CD8+ T cells.

8. The population of T cells according to any one of the previous claims wherein at least 20%, 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more of the individual cells in the cell population together are as defined in any one of the previous claims.

9. The population of T cells according to any one of the previous claims wherein no more than 80%, 75%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or less of the individual cells in the cell population together are cells that are not as defined in any one of the previous claims.

10. The population of T cells according to any one of the previous claims, wherein the population of cells is characterized by being a tumor engagers cell population and / or tumor killing cell population when exposed to a tumor.

11. A population of T cells as defined in any one of the previous claims, for use in the treatment of a tumor in a patient, preferably wherein the patient has a solid tumor, preferably wherein the patient has a brain cancer, a glioma, preferably a diffuse midline glioma, a breast cancer, neuroblastoma, and / or a head and neck cancer.

12. The population of T cells as defined in any one of the previous claims, for use in the treatment of a tumor in a patient, preferably wherein the patient has a solid tumor, preferably wherein the patient has a brain cancer, a glioma, preferably a diffuse midline glioma, a breast cancer, and / or a head and neck cancer.AOMB: P102253WO13. The population of T cells for use in treatment according to any one of claims 11-12, wherein the T cells are provided to the patient only once, or at least every two months, every month, every two weeks, every week, every 7, 6, 5, 4, 3, 2, or 1 day(s).

14. The population of T cells for use in treatment according to claims 11 - 13, wherein the treatment further comprises the co-administering of a compound that recruits and / or switches the cell state of myeloid cells, preferably microglial cells and / or macrophages to the tumor; and / or the co-administering of immune checkpoint modulator blockade therapy; and / or the co-administering of a multispecific molecule capable to target the T cell and the tumor.

15. The population of T cells for use in treatment according to claims 11 - 14, wherein the population of T cells are autologous T cells, or wherein the population of T cells are allogenic T cells.

16. A method of obtaining / selecting / sorting a population of T cells according to any one of the previous claims, preferably according to claim 1 or according to any one of claims 2 - 3, wherein the method comprises:(a) exposing a sample containing T cells to tumor organoid, and co¬ culturing, preferably for a period of time of at least 1 - 24 hours, to allow some of the T cells to engage upon the tumor organoid;(b) removing the non-engaged T cells from the co-culture,(c) optionally repeating steps (a) and (b) at least once or more, and (d) harvesting the T cells engaged upon the tumor organoid, to obtain the population of T cells.

17. A method of obtaining / selecting / sorting a population of T cells according to any one of the previous claims, preferably according to claim 1, wherein the method comprises:AOMB: P102253WO(a) exposing a sample containing T cells to tumor organoid, and coculturing, preferably for a period of time of at least 1 - 24 hours, to allow some of the T cells to engage upon the tumor organoid;(b) removing the non-engaged T cells from the co-culture,(c) optionally repeating steps (a) and (b) at least once or more, and (d) harvesting the T cells engaged upon the tumor organoid, to obtain the population of T cells.

18. A method of sorting or selecting T cells, preferably wherein the population of T cells are primary T cells, or preferably wherein the population of T cells are genetically engineered T cells, or wherein the population of T cells are CAR-T cells, or wherein the population of T cells are TCR expressing T cells, or wherein the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are CAR-T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are TCR expressing genetically engineered T cells not expressing a gamma-delta T cell receptor, wherein the method comprises:(a) determining in a T cell the expression of one or more, or of all of the genes in the gene signature defined in claim 1, or in claims 2-3, (b) retrieving the T cells that express one or more, or of all of the genes in the gene signature defined in claim 1 or that have a higher expression of one or more, or of all of the genes in the gene signature defined in claim 1 relative to a population of reference T cells.

19. A method of sorting or selecting T cells, preferably wherein the population of T cells are genetically engineered T cells, or wherein the population of T cells are CAR-T cells, or wherein the population of T cells are TCR expressing T cells, or wherein the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are CAR-T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are TCR expressing genetically engineered T cells not expressing a gamma-delta T cell receptor, wherein the method comprises:AOMB: P102253WO(a) determining in a T cell the expression of one or more, or of all of the genes in the gene signature defined in claim 1,(b) retrieving the T cells that express one or more, or of all of the genes in the gene signature defined in claim 1 or that have a higher expression of one or more, or of all of the genes in the gene signature defined in claim 1 relative to a population of reference T cells.

20. A method of sorting or selecting T cells, preferably wherein the population of T cells are primary T cells, or preferably wherein the population of T cells are genetically engineered T cells, or wherein the population of T cells are CAR-T cells, or wherein the population of T cells are TCR expressing T cells, or wherein the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are CAR-T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are TCR expressing genetically engineered T cells not expressing a gamma-delta T cell receptor, wherein the method comprises (a) determining in a T cell the expression of at least NCAM1, and(b) retrieving the T cells that express at least NCAM1, or that have a higher expression of NCAM1 relative to a population of reference T cells.

21. A method of sorting or selecting T cells, preferably wherein the population of T cells are genetically engineered T cells, or wherein the population of T cells are CAR-T cells, or wherein the population of T cells are TCR expressing T cells, or wherein the population of T cells are genetically engineered T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are CAR-T cells not expressing a gamma-delta T cell receptor, or wherein the population of T cells are TCR expressing genetically engineered T cells not expressing a gamma-delta T cell receptor, wherein the method comprises (a) determining in a T cell the expression of at least NCAM1, and(b) retrieving the T cells that express at least NCAM1, or that have a higher expression of NCAM1 relative to a population of reference T cells.AOMB: P102253WO22. The population of T cells sorted or selected according to any one of claims 17- 18 or 19-21, for use in the treatment of a tumor in a patient, preferably wherein the patient has a solid tumor, preferably wherein the patient has a brain cancer, a glioma, preferably a diffuse midline glioma, a breast cancer, neuroblastoma, and / or a head and neck cancer.

23. The population of T cells sorted or selected according to any one of claims 17- 18 or 19-21, for use in the treatment of a tumor in a patient, preferably wherein the patient has a solid tumor, preferably wherein the patient has a brain cancer, a glioma, preferably a diffuse midline glioma, a breast cancer, neuroblastoma, and / or a head and neck cancer. / / endAOMB: P102253WO

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