T cell lineages with early-differentiated characteristics derived from solid tumors as next generation cancer therapy and diagnostics

Abstract

The invention relates to a method for selection of T cells, comprising providing leukocyte cells from a subject, wherein said leukocytes comprise T cells, isolating T cells expressing one or more of CD3, CD4, CD8, CD127 and CCR7 and / or CD62L from the provided leucocytes, contacting / stimulating the isolated T cells with one or more interleukins, optionally stimulating and / or activating T cells expressing CD3 and / or CD28 and cultivating the T cells for at least (3) days, wherein isolating T cells in step b. preferably comprises: isolating from the leucocytes provided in a. T cells expressing CD3 and / or CD4 or CD8, preferably CD3 and CD4 or CD8, and isolating or enriching from the T cells isolated in i. T cells expressing CCR7 and / or CD127. The invention further relates to an isolated population of T cells, a T cell as well as in vivo methods for activating T cells and for assisting the diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for a subject suspected of suffering from or diagnosed with cancer and / or receiving a cancer therapy.

Description

[0001] T CELL LINEAGES WITH EARLY-DIFFERENTIATED CHARACTERISTICS DERIVED FROM SOLID TUMORS AS NEXT GENERATION CANCER THERAPY AND DIAGNOSTICS

[0002] DESCRIPTION

[0003] The invention lies in the field of biology and medicine, particularly in the field of cell biology and cellular immunotherapies.

[0004] The invention relates to a method for the selection of T cells, comprising providing leukocyte cells from a subject, wherein said leukocytes comprise T cells, isolating T cells expressing one or more of CD3, CD4, CD8, CCR7 and / or CD62L and CD127 from the provided leucocytes, contacting / stimulating the isolated T cells with one or more interleukins, optionally stimulating and / or activating T cells expressing CD3 and / or CD28 and cultivating the T cells for at least 3 days, wherein isolating T cells in step b. preferably comprises: isolating from the leucocytes provided in a. T cells expressing CD3 and / or CD4 or CD8, preferably CD3 and CD4 or CD8, and isolating or enriching from the T cells isolated in i. T cells expressing CCR7 and / or CD127.

[0005] The invention further relates to an isolated population of T cells, a T cell as well as in vivo methods for activating T cells and for assisting the diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for a subject suspected of suffering from or diagnosed with cancer and / or receiving a cancer therapy.

[0006] BACKGROUND OF THE INVENTION

[0007] The survival time of most patients with metastatic tumors is low, as curative treatment options are only available in exceptional cases. In the metastatic stage of bladder cancer, the average 5-year survival rate of patients is only 8%1. New forms of therapy are urgently needed. The cornerstones of current cancer therapies consist of three modalities: surgical removal of the tumor, radiation and chemotherapy. In recent years, forms of a fourth pillar, immunotherapy, have also been developed. The immune system has the capacity to fight tumor cells intrinsically. Immunotherapy is possible, for example, through activation with a targeted antibody. This class of drugs is known as checkpoint inhibitors, as they bind to suppressive control proteins for T cells. When bound, this leads to a blockade of an inhibitory checkpoint of the T cell and, in sum, to a stimulation of the T cells in the tumor tissue2 3. In clinical studies, this has led to a proper therapy response in a subgroup of patients with bladder cancer4. Checkpoint inhibitors have therefore been approved as a treatment option alongside chemotherapy for metastatic bladder cancer in Germany. The 2- year survival rate of patients with metastatic bladder cancer treated with a checkpoint inhibitor in the first line is still below 50% on average5. The use of this drug illustrates a relevant benefit of T- cell-based therapeutic approaches against tumor diseases, but further immunotherapeutic approaches are needed. Relapse of cancer is unfortunately a frequent phenomenon in solid tumor diseases, even after an initial response to therapy. The therapeutic use of an immune response is a promising approach to prevent relapse. A cellular transfer of an immune response has been achieved by the administration of so-called chimeric antigen receptor (CAR) T cells against hematologic cancers. The use of CAR T cells in adult patients with recurrence of diffuse large B-cell lymphoma led to an activation of the immune system in the majority of treated patients with a demonstrable regression of the disease6’7. Unfortunately, a similarly resounding success has not been observed in the use of CAR T cells against numerous solid tumor diseases8'14. High-resolution cellular analyses show that the T cells initially used for the CAR T cell therapy contain subgroups that have only a low capacity for cell migration and longevity15. Consequently, an initial purification of potent T cells is attractive for improving a cell therapeutic approach against the tumor.

[0008] Current solutions for the treatment of solid tumor diseases are mainly based on classic cancer therapy. Other solutions are currently being tested, for example, the cell transfer of tumor infiltrating T cells. Both approaches are therapeutically relevant against solid tumor diseases, but have disadvantages.

[0009] Classical therapies are based on extrinsic control of tumor cells, e.g. with chemotherapeutic agents. The side effects of chemotherapeutic agents are considerable and have a lasting impact on the quality of life of patients with solid tumor diseases21 22.

[0010] Cellular immunotherapies use the capacity for an intrinsic fight against tumor cells. T umor infiltrating T cells have a high diversity of T cell receptors and cover various antigen structures of the tumor23-25. A recently published phase III study describes the use of tumor infiltrating T cells (tumor infiltrating lymphocytes, TIL) against melanoma, a so-called TIL therapy. In this study, T cells were multiplied from tumor excision material from patients with melanoma in cell culture via the cytokine interleukin-2. The unselected T cells and interleukin-2, which promotes T cell growth, were administered intravenously to the patients. The administration of this cytokine led to a considerable toxicity profile. In the survival analysis, T-cell therapy was superior to standard drug therapy26. This study demonstrates the enormous potential of this form of T-cell therapy.

[0011] However, the use of interleukin-2 has been shown in many studies to have a detrimental effect on T cell function. Interleukin-2 causes aging, a so-called "exhaustion" profile, in the treated T cells27'30. The T cells lose their longevity and thus their ability to destroy tumor cells.

[0012] Hence, new and improved therapy protocols that provide T cells with a higher therapeutic potential are urgently needed.

[0013] SUMMARY OF THE INVENTION

[0014] In light of the prior art the technical problem underlying the present invention is to provide alternative and / or improved means for the treatment of cancer, particularly solid cancers and metastases, using immunotherapies, such as cellular immunotherapies like T cell-based immunotherapies. This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0015] The invention therefore relates in one aspect to a method for selecting T cells, comprising a. providing leukocyte cells from a subject, wherein said leukocytes comprise T cells, b. isolating T cells expressing one or more of CD3, CD4, CD8, CD127 and CCR7 and / or CD62L from the provided leucocytes, c. contacting / stimulating the isolated T cells with one or more interleukins, d. optionally stimulating and / or activating T cells expressing CD3 and / or CD28, e. cultivating the T cells for at least 3 days.

[0016] In preferred embodiments isolating T cells in step b. comprises: i. isolating from the leucocytes provided in a. T cells expressing CD3 and / or CD4 or CD8, preferably CD3, and CD4 or CD8, and ii. isolating or enriching from the T cells isolated in i. T cells expressing CCR7 and / or CD62L and / or CD127, wherein the order of steps may be reversed or combined.

[0017] In some preferred embodiments, the pre-selection of an ‘early-differentiated’ CD4+ and / or CD8+ T cell population (after the initial selection of CD3+ T-cells), followed by a subsequent selection for CCR7+ or CD62L+, and / or CD127+ expressing T-cells, may be particularly advantageous, as it enables an improved selection of a T-cell population, which is particularly reactive with respect to its anti-tumor (tumor-targeting) efficacy.

[0018] In some preferred embodiments the method for selecting T cells, comprises a. providing leukocyte cells from a subject, wherein said leukocytes comprise T cells, b. isolating T cells expressing: i. CD3, and CD4 and / or CD8, ii. isolating or enriching from the T cells isolated in i. T cells expressing CD127, and CCR7 and / or CD62L, c. contacting / stimulating the isolated T cells with one or more interleukins, d. optionally stimulating and / or activating T cells expressing CD3 and / or CD28 cultivating the T cells for at least 3 days.

[0019] In order to generate a long-term therapeutic immune response against cancer, a comprehensive knowledge of the immune cells relevant for fighting cancer is required. The inventors have discovered CD8+ T cells in the lymph nodes and periphery of cancer patients that show a high infiltration capacity into the respective (solid) tumor and / or metastases of said cancer patients. The inventors further surprisingly found that characteristics of tumor-infiltrating T cells are suitable to predict the response to chemotherapy31of said patients.

[0020] When characterizing tumor-invading CD3+ T cells (T cells positive for the surface antigen CD3) from patients suffering from bladder and kidney cancer using flow cytometry, the inventors surprisingly found early-differentiated CD4+ and CD8+ T cell populations in the tumor environment (Fig. 1a). Early-differentiated CD4+ T cells and CD8+ T cells correspond to the so- called central memory T cells (TCM or CM). The inventors surprisingly found said CM-subtype to be phenotypical different from other T cell populations, such as T cell subtypes NAIVE-like, effector memory (EM or TEM), terminally differentiated effector memory (EMRA or TEMRA), and to be enriched in the CD4+ cell pool (Fig. 1 b, c). CM cell populations were characterized before in the lymphoid system of mouse models and were found to have a high antigen-specific potency and longevity32 33. The (infiltrating) human cell populations found in the tumor expressed the chemokine receptor CCR7 and a high abundance of the protein CD127, the cytokine receptor for interleukin-7 (Fig. 2d). These results imply a response of the T cells to interleukin-7 in the cell culture and are indicative for the longevity of the of the T cells3435. The inventors also analyzed whether a similar T cell phenotype can be found in the CD4+ T cell pool of human lymph nodes. Surprisingly, the inventors identified said sub-type in the blood of a cohort of 8 patients with blader cancer (Fig. 1 d, e). Said findings imply a role for T cells in various human compartments in the context of cancer.

[0021] For a comparison of early-differentiated CD4+ and CD8+ T cells the inventors have further analyzed a genetic screen of a kidney tumor cohort using single cell RNA sequencing (scRNAseq). Genes were identified that were statistically significantly higher expressed in early- differentiated CD4+ and CD8+ T cells compared to late-differentiated CD4+ and CD8+ T cells. Thereby, the inventors could reveal 66 common early T cell genes in early-differentiated tumorinfiltrating T cells, among them CCR7 and CD127 (IL7R). The comparison of early-differentiated CD4+ and CD8+ T cells resulted into the identification of common early T cell genes. Single-cell RNA-sequencing samples were accessed through the publicly available European Genome- Phenome Archive under EGAD00001008030 (n=11 ; 10 ccRCC, 1 oncocytoma) of 11 renal tumor samples.

[0022] Differentially expressed markers were identified by the comparison of CD4+CCR7+ with remaining CD4+ T cells and CD8+CCR7+ with remaining CD8+ T cells, respectively. 250 markers were set as condition for each approach, cluster 1 (red) shows genes that were highly and commonly enriched in CD4+CCR7+ and CD8+CCR7+ T cells. The 66 identified genes in early-differentiated tumor-infiltrating T cells were CCR7, KLF2, BEX2, FAM65B, KLF3, TMEM63A, RASGRP2, LDLRAP1 , CAMK4, LMNA, ANXA1 , TIMP1 , AQP3, SORL1 , PTGER2, SESN1 , CCDC109B, TNFRSF25, TTC39C, RPS4Y1 , SCML4, FOSB, ADD3 , PABPC1 , FOS, ARHGAP15, PIK3IP1 , RPL3, LTB, FLT3LG, THEM4, SLC2A3, MGAT4A, MBP, FAM102A, RGCC, FOXP1 , RASA3, ABLIM1 , CD55, UPP1 , RP11 -18H21.1 , BACH2, LEF1 , SELL, TRABD2A, C1 orf228, PASK, CMTM8, SCML1 , TCF7, LYPD3, IL7R, CD40LG, MYC, GPR183, FBLN7, MAL, ANK3, SH3YL1 , SATB1 , CTB-133G6.1 , S1 PR1 , TMEM123, ITGA6, CHD7. These genes were differentially expressed in CD4+CCR7+ and CD8+CCR7+ cells. Differentially expressed marker genes were identified by the comparison of CD4+CCR7+ with remaining CD4+ T cells and CD8+CCR7+ with remaining CD8+ T cells, respectively. The 66 revealed genes were highly and commonly enriched in CD4+CCR7+ and CD8+CCR7+ T cells.

[0023] For a comparison in tumor entities beyond the kidney, the inventors performed a further screen in further tumor entities using sc-RNAseq (see, e.g., Fig. 19). Genes were identified that were significantly and commonly enriched in kidney, bladder and lung tumors in CD4+ and CD8+ early- differentiated T cells, comprising CCR7, CD55, ANXA1 , SLC2A3, LTB, EEF1A1 , JUNB, EEF1 B2, PABPC1 , SELL, LEF1 , TCF7, GPR183, LMNA, ZBTB10, PASK, KLF2 and BEX2. Likewise to the first screen, a comparison was made to the late-differentiated CD4 / 8+ T cells state. Thereby, 15 genes could be re-identified and hence replicated from the 1st screen, and 5 genes, namely ANXA1 , EEF1A1 , JUNB, EEF1 B2, and ZBTB10, could be newly identified.

[0024] The inventors could also show that the identification of one or more of the described T-cell markers allows purification for therapeutic use. Using a long-term culture, the inventors were further able to reveal that the discovered CD4+ early-differentiated T-cell population in particular has a very high longevity (Fig. 3a, b).

[0025] Protocols that enable a gentle and selective purification of tumor-infiltrating T cells are still not available, but urgently needed. A workflow, like the present method for selecting (or ‘generating’) the T cells (herein also termed ‘T-Lene’ cells) according to the invention has not been described before in the art. Within this process, the specific selection of the tumor infiltrate to a defined T cell phenotype, preferably a CD3+CD4 / CD8+CCR7+CD127+ T cell phenotype, is a new and inventive feature. The surprising and advantageous effect achieved by the present invention is shown in the examples herein. Currently, no cellular immunotherapy, or particularly T cell therapy, applies the selection of an appropriate T cell expression profile, e.g., of patient-derived T cells, prior to in vitro cell expansion and / or administration of said T cells to a patient. The use of a specific cell selection, e.g., via flow cytometry or fluorescence-activated cell sorting (FACS), according to the present invention enables the optimization of numerous T cell therapies and applications. In addition, the diagnostic approach according to the invention of detecting said relevant and therapeutic effective T cell population in the tumor environment as a stratification marker is new and inventive over the present state of the art.

[0026] Hence, the present invention provides in embodiments a cell selection strategy, e.g., implemented by (GMP-grade) FACS, to enrich tumor infiltrating lymphocytes (TIL) as starting material for the enrichment of an early differentiated T cell phenotype, followed by a polyclonal expansion set-up.

[0027] Advantages of embodiments of the present method enable the maintenance of early differentiated (CD4+) T cell function, ensuring stability and efficacy of the generated T cell population (e.g., a TIL product suitable for TIL therapy). Another advantage of maintaining early differentiated T cell characteristics enables overcoming present obstacles in the era of adoptive T cell therapy against solid tumors. Moreover, the present invention provides a new option for administering cancer therapy. Tumor infiltrating lymphocyte (TIL) therapy is not yet approved in Europe and the USA and has so far only been evaluated in studies. The method according to the present invention could significantly impact the implementation of TIL therapy into the clinics. The use of the methods and T cells according to the present invention implies a more tolerable TIL therapy for the patient, with a more potent T-cell function administered in a lower cell number.

[0028] Currently the solely FDA approved TIL therapy product is lifileucel which can be applied against melanoma. The expansion protocol for lifileucel involves a complex two-phase ex vivo process to generate therapeutic doses of tumor-infiltrating lymphocytes (TILs). Initially, bulk TILs are directly isolated without selection from melanoma samples and undergo activation using high-dose interleukin-2 (IL-2) during the pre-rapid expansion protocol (pre-REP) to prime their proliferative capacity. This is followed by a 22-day rapid expansion protocol (REP) where anti-CD3 antibodies and IL-2 drive T cell growth (Sarnaik et al., 2021 ; O’Malley et al., 2024). After TIL transfer the patients require intravenous IL-2 administration to boost TIL expansion which associates with great toxicity in the patients, such as ever, hypotension, pulmonary toxicity, or capillary leak syndrome, which impedes treatment inclusion of frail patients.

[0029] Therefore, in some preferred embodiments the leukocyte cells comprising T cells and provided in a. are derived from a tumor sample of a subject, or from a sample of a subject suspected or diagnosed with a tumor. In some preferred embodiments the leukocyte cells provided in a. comprise tumor infiltrating lymphocytes (TILs). In some preferred embodiments said T cells comprised within the leukocyte cells provided in a. comprise tumor infiltrating lymphocytes (TILs). Hence, in embodiments the T cell populations isolated (and optionally stimulated) according to the present method comprise tumor infiltrating lymphocytes (TILs), which were preferably infiltrating a tumor of the subject from which the sample is derived.

[0030] In one embodiment the present method comprises the following selection (e.g., by (GMP-grade) FACS sorting) strategy: leukocyte cells are provided from a subject, wherein said leukocytes comprise T cells, preferably TILs. Therefrom T cells expressing CD3 are isolated. Subsequently, from the CD3+ T cell population, a cell population positive for CD4+ or CD8+ (preferably constituting / comprising an early differentiated T cell population) is isolated. Therefrom, a cell population expressing CD127, and CCR7 and / or CD62L is isolated or enriched, such that preferably a CD3+CD4 / CD8+CCR7+CD127+ T cell population comprising TILs is obtained. Said isolated T cell population is subsequently contacted / stimulated with one or more interleukin.

[0031] Optionally, T cells expressing CD3 and / or CD28 may be (further) stimulated and / or activated. The obtained T cell population is preferably cultivated (expanded) for at least 3 days.

[0032] In some embodiments, a CD3+CD4 / CD8+CCR7+CD127+ T cell population isolated according to embodiments of the present invention comprise tumor infiltrating lymphocytes (TILs). In embodiments the T cell population obtained by embodiments of the present method comprises TILs and / or an anti-tumor reactivity against a tumor present in the subject, from which the sample is derived. In embodiments, the present invention comprises a population of human early differentiated, tumor-infiltrating (CD4+) T cells. In embodiments, said population of T cells may be obtained via fluorescence-activated cell sorting. In embodiments, said population of T cells may be used in the treatment of a disease in a subject, preferably cancer or a tumor, wherein the treatment is or comprises a TIL therapy.

[0033] Embodiments of the method according to the present invention comprise sorting I selection of T cells by (clinical-grade) fluorescence-activated cell sorting (FACS) for a TIL therapy of a subject.

[0034] The cell selection principle of the present invention can, in embodiments, also be transferred to any T cell generation process, including CAR T cell therapy. Diagnostically, the method according to the present invention allows a new possibility of immunodiagnostics, with a potential application in the stratification of immunotherapies.

[0035] Another advantage of the present method is that it facilitates a cost-effective and fast and at the same time precise and reproducible selection of target cell populations based on cell surface proteins I markers instead of cost-intensive RNA-sequencing, which is currently the standard procedure for T cell classification in patient samples, e.g., during treatment stratification or diagnostics. This advantage may, for example, be especially relevant for clinical implementation.

[0036] In embodiments, step b. of isolating T cells comprises (i) isolating from the leucocytes provided in a. T cells expressing CD3 and / or CD4 and / or CD8, preferably CD3 and CD4 and / or CD8, and (ii) isolating or enriching from the T cells isolated in (i) T cells expressing CCR7 (and / or CD62L) and / or CD127.

[0037] In some preferred embodiments the T cells positive for CD3 and CD4 and / or CD8 constitute or comprise an early differentiated T cell population. In some preferred embodiments the order of selecting CD3+ T cells from the leukocytes provided in a., followed by a subsequent selection of a CD4+ or CD8+ population provides the advantage of obtaining a highly specific population, which preferably comprises a high degree of early differentiated T cells. In some embodiments the subsequent selection of T cells expressing CCR7 (and / or CD62L) and CD127 does not require a specific order. However, in preferred cases, the obtained CD3+CD4 / CD8+CCR7+CD127+ T cell population provides a cell population comprising TILs with a particularly highly anti-tumor reactivity, thereby enabling an improved targeting of tumors by TIL or T cell therapy according to the present invention.

[0038] In some embodiments, leukocytes may comprise double positive CD4+ CD8+ T cells (cells expression both CD4 and CD8), especially leukocytes derived from one or more tumor and / or tumor environment.

[0039] In embodiments selection, isolation, sorting and / or enriching in the method according to the invention are performed using flow cytometry or fluorescence-activated cell sorting (FACS). In embodiments a sample or cell population is labelled, stained or contacted with one or more stains to improve the selection, isolation, sorting and / or enrichment of cells of interest. In embodiments such stains comprise one or more dye and / or one or more antibody, preferably one or more labelled, or otherwise detectable, antibody directed against a target of interest. Targets of interest may be, e.g., T cells and / or one or more protein of interest, preferably one or more cellular surface / membrane protein of interest, such as CD or CCR proteins.

[0040] In embodiments the method comprises further the staining / labelling of cells and / or one or more proteins of interest, preferably one or more cellular surface protein of interest, before a selection, isolation, soring and / or enriching step.

[0041] In embodiments a staining and / or labelling may be performed with fluorescent dyes (e.g., Propidium Iodide, 7AAD, DAPI, Hoechst 33342, BrdU (bromodeoxyuridine), etc. or another DNA dye) and / or with fluorescently conjugated antibodies (e.g., CD4 FITC). The respective staining and / or labelling may be adopted according to the cells to be selected and / or isolated.

[0042] The skilled person is familiar with suitable flow cytometry and FACS methods and knows how to select appropriate dyes and antibodies for proteins and cell types of interest, as well as for the respectively used flow cytometry / FACS instrument.

[0043] In some embodiments, e.g., for calibration purposes, also genetically modified cells and / or target proteins may be analyzed according to the method of the present invention, such that proteins and / or cells of interest may comprise fusion proteins of interest coupled to a fluorescent protein, such as GFP.

[0044] In embodiments before one or more selection, isolation, sorting and / or enriching step the leucocytes provided, e.g., in a. are subjected to a staining with one or more labelled antibodies directed against (specifically binding to) one or more of CD3, CD4, CD8, CD127 and / or CCR7 and / or CD62L.

[0045] In embodiments before one or more selection, isolation, sorting and / or enriching step the leucocytes provided, e.g., in a. are subjected to a staining with one or more labelled antibodies directed against (specifically binding to) CD45RO.

[0046] In embodiments, step b. of isolating T cells comprises: i. staining the leucocytes provided in a. with one or more labelled antibody directed against one or more of CD3, CD4, CD8, CD127 and / or CCR7 and / or CD62L and optionally further dyes, ii. isolating from the leucocytes provided in a. T cells expressing CD3 and / or CD4 and / or CD8, preferably CD3 and CD4 and / or CD8, more preferably CD3, and CD4 or CD8, iii. optionally staining the leucocytes provided in a. with one or more labelled antibody directed against CCR7 and / or CD127, and iv. isolating or enriching from the T cells isolated in i. T cells expressing CD127 and / or CCR7 and / or CD62L, optionally depleting non-memory T cells (naive-like T cells), depending (based) on the (surface) expression of one or more markers / marker genes, e.g., CD45RA, thereby excluding / depleting naive-like T cells. v. optionally isolating or enriching (memory) T cells, depending (based) on the (surface) expression of one or more marker genes specific for (memory) T cells, e.g., CD45RO, thereby obtaining memory T cells, vi. optionally isolating or enriching (other / further types of) T cells, depending (based) on the (surface) expression of one or more (marker) genes, preferably associated with an early differentiated cell state and, selected from the group comprising KLF2, BEX2, FAM65B, KLF3, TMEM63A, RASGRP2, LDLRAP1 , CAMK4, LMNA, ANXA1 , TIMP1 , AQP3, SORL1 , PTGER2, SESN1 , CCDC109B, TNFRSF25, TTC39C, RPS4Y1 , SCML4, FOSB, ADD3 , PABPC1 , FOS, ARHGAP15, PIK3IP1 , RPL3, LTB, FLT3LG, THEM4, SLC2A3, MGAT4A, MBP, FAM102A, RGCC, FOXP1 , RASA3, ABLIM1 , CD55, UPP1 , RP11-18H21 .1 , BACH2, LEF1 , SELL, TRABD2A, C1 orf228, PASK, CMTM8, SCML1 , TCF7, LYPD3, IL7R, CD40LG, MYC, GPR183, FBLN7, MAL, ANK3, SH3YL1 , SATB1 , CTB-133G6.1 , S1 PR1 , TMEM123, ITGA6, CHD7, CXCR5, ANXA1 , EEF1A1 , JUNB, EEF1 B2, and ZBTB10.

[0047] In embodiments, T cells may also be enriched or isolated depending (based) on the (surface) expression of one or more (marker) genes selected from the group comprising CCR7, KLF2, BEX2, FAM65B, KLF3, TMEM63A, RASGRP2, LDLRAP1 , CAMK4, LMNA, ANXA1 , TIMP1 , AQP3, SORL1 , PTGER2, SESN1 , CCDC109B, TNFRSF25, TTC39C, RPS4Y1 , SCML4, FOSB, ADD3 , PABPC1 , FOS, ARHGAP15, PIK3IP1 , RPL3, LTB, FLT3LG, THEM4, SLC2A3, MGAT4A, MBP, FAM102A, RGCC, FOXP1 , RASA3, ABLIM1 , CD55, UPP1 , RP11-18H21.1 , BACH2, LEF1 , SELL, TRABD2A, C1orf228, PASK, CMTM8, SCML1 , TCF7, LYPD3, IL7R, CD40LG, MYC, GPR183, FBLN7, MAL, ANK3, SH3YL1 , SATB1 , CTB-133G6.1 , S1 PR1 , TMEM123, ITGA6, CHD7, CXCR5, ANXA1 , EEF1A1 , JUNB, EEF1 B2, and ZBTB10. The inventors have identified the afore genes as being highly and commonly enriched in early differentiation states of T cells, such as CD4+CCR7+ and CD8+CCR7+ T cells. Further, said genes were differentially expressed between CD4+CCR7+ and CD8+CCR7+ cells, wherein the differential expression was determined by a comparison of CD4+CCR7+ with remaining CD4+ T cells and CD8+CCR7+ with remaining CD8+ T cells, respectively.

[0048] In embodiments the expression of one or more of the afore enlisted (marker) genes may be determined with one or more labelled antibody directed against respective genes, e.g., prior to isolation and / or enrichment (e.g., by staining the leucocytes provided in a. with said antibodies).

[0049] In embodiments, besides the known expression in peripheral T and B cells the inventors could validate expression of GPR183 in tumor-infiltrating T cells in lung, bladder and kidney tumors. In embodiments this offers selection possibilities within a GMP (good manufacturing practice)- approach for T cell therapy for said tumors. In embodiments, T cells may also be enriched or isolated depending (based) on the (surface) expression of the (marker) gene GPR183.

[0050] In embodiments, beyond its role in the complement system, the inventors pinpoint a new intra- tumoral role of the gene CD55 by validating its expression in early-differentiated T cells in lung, bladder and kidney tumors. In embodiments, this offers new T cell therapy selection possibilities.

[0051] In embodiments, T cells may also be enriched or isolated depending (based) on the (surface) expression of the (marker) gene CD55.

[0052] In embodiments, beyond the role of mediating the entrance of T cells into B cell follicles in the human lymph node, the inventors identify CXCR5 expression in early-differentiated T cells derived from the tumor site of non-small cell lung cancer patients. In embodiments, CXCR5 surface expression may provide opportunities particularly for selecting potent anti-lung tumor T cells from the tumor site.

[0053] In embodiments, T cells may also be enriched or isolated depending (based) on the (surface) expression of the (marker) gene CXCR5.

[0054] In embodiments, in the context of the present method for isolating T cells, leucocytes (e.g., provided in a.) may be stained with one or more labelled antibody directed against one or more of GPR183, CD55 and CXCR5 and T cells expressing one or more of GPR183, CD55 and CXCR5 may subsequently be isolated therefrom.

[0055] In embodiments, T cells may be isolated or enriched depending (based) on the (surface) expression of CD62L. In general, CD62L may be considered to have similar cellular functions to CCR7 in lymphocytes, such that CD62L may be used in addition, in combination or alternatively / instead of CCR7 for isolating or enriching (sub)populations of T cells.

[0056] In embodiments, (memory) T cells may be isolated or enriched (or obtained), depending (based) on the (surface) expression of one or more marker genes specific for (memory) T cells, e.g., CD45RO. In some embodiments, leucocytes (e.g., provided in a.) may be stained with one or more labelled antibody directed against CD45RO and (memory) T cells expressing CD45RO may subsequently be isolated.

[0057] In preferred embodiments the one or more interleukins comprise IL-7, IL-15 and / or IL-21 . In embodiments the one or more interleukins comprise IL-7. In embodiments the one or more interleukins comprise IL-15. In preferred embodiments the one or more interleukins comprise IL- 21. In embodiments the one or more interleukins comprise IL-7, IL-15 and IL-21 .

[0058] The inventors considered IL-7 for the stimulation of cells due to its role in maintaining T cell homeostasis and survival38’39. IL-15 is considered to have a proliferative effect on memory T cells40’41. In addition, the combination of IL-7 and IL-15 was described to have a synergistic capacity when applied in combination for T cell expansion strategies42 43. IL-21 is considered to promote T cell cytotoxicity44 45. The inventors surprisingly found, that lymphocytes, preferably T cells, obtained according to the present method could be applied in the context of a cancer therapy. In embodiments the present method comprises selecting / isolating tumor-infiltrating lymphocytes, preferably ? cells, expressing one or more of CD3, CD4, CD8, CCR7 and / or CD127, followed by contacting / stimulating said isolated cells with one or more interleukins, such as, e.g., IL-7, IL-15 and / or IL-21 , thereby obtaining lymphocytes, preferably T cells, suitable for cancer therapy. In embodiments of the present invention lymphocytes or T cells obtained according to the method of the invention constitute a likely more effective and tolerable TIL therapy for patients, which provides higher T-cell function (potency) and may be administered at a lower cell number, thereby reducing costs and potential side effects.

[0059] In embodiments interleukins may be native interleukin proteins or recombinant interleukin proteins, a synthetic interleukin protein or any pharmaceutical derivate or analogue thereof, e.g., an IL-7, IL-15 or IL-21 mimetic.

[0060] In embodiments depleting non-memory T cells (e.g., naive-like T cells) comprises the removal or depletion of cells from a cell population, e.g., via FACS or flow cytometry, depending or based on the (surface) expression of one or more (non-memory T cell-) markers, for example, CD45RA.

[0061] In embodiments non-memory T cells (naive-like T cells) may be isolated dependent (based) on the (surface) expression of one or more markers / marker genes, e.g., CD45RA, thereby excluding / depleting naive-like T cells.

[0062] In embodiments, contacting / stimulating the isolated T cells with one or more interleukins induces the activation of at least a fraction of the T cells.

[0063] In embodiments activated T cells are characterized by the expression of CD3 and / or CD28.

[0064] In embodiments the stimulation with one or more interleukins activates at least a fraction of the interleukin-stimulated T cells, wherein the activation of T cells (of interest) may be determined by determining the expression of CD3 and / or CD28 in the stimulated T cells.

[0065] In embodiments the expression of CD3 and / or CD28 in the stimulated T cells may be determined before or during any selection, isolation, sorting and / or enriching step by subjecting the cells to a staining with one or more (labelled) antibodies directed against CD3 and / or CD28.

[0066] In a non-limiting example, the expression of CD3 and / or CD28 in the stimulated T cells may be determined with a flow cytometry or FACS device after staining of the cells with one or more labelled antibodies directed against CD3 and / or CD28. Depending on the flow cytometry or FACS device used, a selection, isolation, sorting and / or enriching step may in embodiments also be performed with said device or subsequently by other means.

[0067] In embodiments, the T cells are contacted / stimulated with one or more interleukins for at least 3, preferably for between 3-21 , days of culture. Preferably for at least 3 and at maximum for 21 days of culture.

[0068] In embodiments, the T cells are contacted / stimulated with one or more interleukins for at least 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, 61 , or 62 days of culture. In embodiments, the T cells are contacted / stimulated with one or more interleukins for between 1-365, 1-300, 1-200, 1-100, 1-50, 1-62, 3-31 , 3-21 , 3-7, 3-14, 7-31 , 7-21 , 14-21 , 14-31 , 7-62, or 7-21 days of culture.

[0069] In embodiments, the subject is suspected of suffering from, or has been diagnosed with cancer. In embodiments the leukocytes, or specifically T cells, are derived from a blood, plasma, tissue and / or biopsy sample of the subject. In embodiments the biopsy or tissue sample is derived from one or more (solid) tumors of said patient, wherein the sample or biopsy has preferably been taken from the tumor and / or from the tumor environment, such as the tissue immediately surrounding and / or adjacent to said one or more (solid) tumors. In embodiments the sample is derived from a tumor and / or tumor surrounding tissue that has been (surgically) removed from the subject. In other embodiments the leukocytes, comprising T cells, or T cells are derived from a liquid biopsy, a plasma and / or blood sample of a subject suspected and / or diagnosed with cancer.

[0070] In embodiments the leukocyte cells provided in a. were obtained from a tumor, a tumor biopsy, tissue surrounding the tumor, one or more lymph nodes, the lymphatic fluid and / or the blood of the subject.

[0071] In embodiments the tumor is a solid tumor. In embodiments the cancer suspected and / or diagnosed in the subject is or comprises one or more solid tumor(s), one or more metastasis and / or a ‘liquid’ cancer, such as leukemia or lymphoma.

[0072] In one aspect the invention relates to an isolated population of T cells expressing one or more of CD3, CD4, CD8, CD127 and CCR7 and / or CD62L obtained (or isolated / generated) by the method according to the invention.

[0073] In embodiments, the population of T cells isolated according to the invention comprises an early differentiated (CD4+ and / or CD8+) TIL population. In embodiments, the early differentiated T cells (TIL) population isolated according to the invention comprises stem-like (stem cell-like) properties and / or characteristics.

[0074] In embodiments, the population of T cells (T cell product) isolated according to the invention comprises or (majorly) consist of a CD3+CD4 / CD8+CCR7+CD127+ T cell population, and which preferably comprises an early differentiated (CD4+ and / or CD8+) TIL population, preferably with stem-like features.

[0075] In the present application a hitherto unknown early differentiated CD4+ TIL population is described that is re-identified in the TME of >100 patients and outperforms the remaining TIL subset counterparts, as demonstrated in a back-to back manner. The new TIL product of the present invention enriches for a functional anti-tumor TIL subset and vice versa, removes T cell subsets that neither expand in culture nor exert anti-tumor function. Thus, functional T cells are not impeded by dysfunctional T cells during culture which leads to multiple advantages of the new TIL product of the present invention. The functional data of the present invention show that the TILs in this product can survive 30 days in culture without need of IL-2 supplementation highlighting their proliferative nature. The inventors are able to show furthermore that the discovered TILs can produce its own cytokines and are autonomously supporting its own proliferation. The application of the invention is thus intended to be infused without administration of IL-2 into the patients, therefore omitting substantial toxicity to the patients. Currently, TIL treatment needs to be performed in the intensive care setting. An IL-2-free approach will ensure that the treatment can be performed in a standard care treatment setting and frail patients can be included.

[0076] Furthermore, the new TIL product requires, compared to prior art TIL therapy, a significantly shorter expansion time (preferably only 7 days) thereby ensuring greater therapeutic flexibility. Solid cancer patients need a flexible treatment process due to rapid progression after standard treatment. For example, patients currently included in the studies, e.g., described in the examples herein, already received treatment regimens and could be successfully employed for production of the new TIL product according to the invention. Thereby the inventors could prove that the new TIL product may be successfully generated from patients that underwent pre-treatments. The rapid setting, enabled by the present method, will also ensure that cost effective GMP production can be performed, and the production time remains reasonably short.

[0077] Moreover, the claimed product of the present invention can be derived from multiple cancer entities beyond melanoma. The cancer-independent nature of the product facilitates the development of a pan-cancer relevant treatment in multiple entities. To this date, there is a broad range of treatments available against melanoma, yet treatment options in other entities are missing. This invention focuses on the discovered pan-existence of a specific type of T lymphocytes which are of an early- differentiated, and thus stem-like, nature. This T cell therapy can thereby be employed in multiple solid cancer patient cohorts making this invention attractive in the broad field of oncology.

[0078] The inventors could show, that new TIL product, preferably obtained according methods described herein, surprisingly exhibit a higher anti-tumor potency, when compared to the remaining TIL subsets (cells that were depleted in the present method). Multiple studies on prior art IL-2 generated TIL products report insufficient treatment outcomes in solid tumor patients. The inventors report on the efficiency and effectiveness of the new TIL product according to the invention eradicating tumor cells in multiple assays. The invention employs the characteristic of the immune system to harbor a pool of stem-like CD4+ and / or CD8+ T cells that can produce its own cytotoxic offspring T cells. Thus, the transfer of this immune trait is built on the regenerative characteristic of the adaptive immune system to locally re-produce cytotoxic effector T cells against malignant cells.

[0079] The new TIL product exhibits surprisingly also a higher migratory capacity to tumor-associated chemokines and ensures a targeted delivery to the tumor site. The inventors found that stem-like TILs are capable of actively migrating back to the lymphoid and tumor microenvironment of the patients and thus, receiving local cytokine stimulations in these natural environments benefiting their survival. The T-cell product according to the invention is therefore preferably intended to be infused intravenously, since the TILs are capable to migrate to the active and relevant anti-tumor sites in the patient. The inventors further found that the new TIL product according to the invention, comprising functional stem-like (early differentiated) proliferating T cells, requires less cells for infusion into the patient thereby reducing cell associated toxicity to the patients. The inventors report that the expansion fold of TILs is > 40 times after 7 days after stimulation greatly outperforming a bulk TIL expansion setting. Currently, massive amounts (10A9) TILs are used in the product of lifileucel. The inventors intend to use reduced numbers of TILs since the produced TILs can proliferate after infusion. A cell-reduced approach will further reduce side effects of our cell application, since unnecessary cell death of non-functional and non-proliferative cells is avoided.

[0080] In embodiments, the approach uses a sterile, GMP-grade technique based on Fluorescence- Activated Cell Sorting (FACS) to select for functional TILs. This selection technique purifies the immune cells from cancer cells and non-immune cells to rates beyond 90% and ensuring higher safety levels. In the current treatment standard approach, bulk TILs are expanded without a selection technique. Based on the stepwise selection on multiple immune-related markers the safety and thus, purity level of beneficial cells is greatly enhanced. The removal of dysfunctional cells from the expansion setting in-vitro further accelerates the expansion of beneficial T cells in culture.

[0081] Embodiments of the new TIL product surprisingly further constitute a higher fraction of cytotoxic CD4+ T cells that are shown to outperform their CD8+ counterparts in multiple murine models and in human cancer entities. Applying the early-differentiated marker cascade including CCR7 and CD127 ensures a prioritization of CD4+ T cells pre-expansion and benefits the yield of CD4+ T cell post expansion. The stepwise CCR7- and CD127-based selection technique performed in preferred embodiments may thus be considered an indirect method to attenuate a CD8+ dominant expansion of T cells as demonstrated after bulk T cell expansion setting. In other words, such approach may thus be considered to prefer a CD4-dominant T cell therapy approach.

[0082] In embodiments any gene or protein expression (in a cell) is determined and / or analyzed using an appropriate staining, e.g., a staining with one or more labelled specific antibodies and / or dyes, and subsequent flow cytometry analysis of respective cells. In embodiments any gene or protein expression (in a cell) may be determined and / or analyzed using alternative or additional means, such as proteomic, genomic or transcriptomic methods, genetic methods such as PCR, RT-PCR, qPCR I Real-time PCR, western blot, southern or northern blot, DNA and / or RNA sequencing, protein / peptide arrays, microarrays and / or chips. The skilled person is familiar with suitable means and methods for determining and / or analyzing gene and protein expression.

[0083] In an aspect the invention relates to a T cell, expressing one or more of CD3, CD4, CD8, CD127 and CCR7 and / or CD62L obtained according to the method of the invention for use in the treatment of cancer.

[0084] In embodiments, the T cells obtained according to the invention (also referred to herein as T cell product, or TIL product), expressing one or more of CD3, CD4, CD8, CD127 and CCR7 and / or CD62L may be used in the treatment of cancer. In embodiments, the treatment is a cellular immunotherapy. In embodiments, the cellular immunotherapy is a T cell-based therapy. In embodiments, the treatment is a tumor infiltrating lymphocyte (TIL) therapy or CAR-T-cell-therapy.

[0085] In embodiments, the treatment is a T cell receptor-engineered intervention comprising a (therapeutic) cell (product). In embodiments, the treatment includes transfer of peripheral or lymph node-derived T cells.

[0086] In one aspect the invention relates to a pharmaceutical composition comprising T cells obtained according to any method of the invention and suitable for the treatment of cancer or any other proliferative disease, comprising additionally a pharmaceutically acceptable carrier.

[0087] Non-limiting examples of applying the afore-mentioned embodiments is described in Examples below.

[0088] In one aspect the invention relates to an in vitro method for activating T cells, said T cells expressing one or more of CD3, CD4, CD8, CD127 and CCR7 and / or CD62L, the method comprising a. cultivating T cells in vitro, wherein the T cells are preferably derived from a subject, b. contacting / stimulating said T cells with one or more interleukins, preferably IL-7, IL-15 and / or IL-21 , thereby activating at least a fraction of the T cells, c. optionally isolating or enriching for (activated) T cells expressing CD3 and / or CD28.

[0089] In embodiments the T cells are derived from a subject suspected of suffering from, or diagnosed with cancer.

[0090] In embodiments, any method (step) for staining, labelling, analyzing, selection, isolation, sorting and / or enrichment described herein for one aspect of the invention may also be performed or applied during any other method described herein.

[0091] In embodiments the methods according to the invention may be used for diagnostic purposes.

[0092] In one aspect the invention relates to an in vitro method for assisting the diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for a subject suspected of suffering from, or diagnosed with cancer and / or receiving a cancer therapy, comprising a. providing a cell population of the subject comprising T cells, b. determining the expression of one or more of CD3, CD4, CD8, CD28, CD127, CCR7 and / or CD62L in at least a fraction of the T cells provided in a., c. determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject based on the expression of one or more of CD3, CD4, CD8, CD28, CD127, CCR7 and / or CD62L. A non-limiting example of applying the afore mentioned embodiments are described, e.g., in Example 3.

[0093] In embodiments, the fulfillment of one or more of the following four conditions (cut-off values / ratios / percentages) may be indicative for the suitability of the patient (and his cancer) for immunotherapy and / or the application of immunotherapy to the patient.

[0094] I. In embodiments the ratio of CCR7+ T cells exceeds (>1) the ratio of CCR7- T cells within the CD4+ T cell population (‘CD4+ T cell compartment’).

[0095] II. In embodiments the ratio of CCR7+ T cells exceeds (>1) the ratio of CCR7- T cells within the CD8+ T cell population (‘CD8+ T cell compartment’).

[0096] III. In some embodiments the ratio of CD4+ central memory T cells as defined by CD4+CCR7+CD45RA -T cells exceeds 40% of the CD4+ T cell population (‘CD4+ T cell compartment’). A non-limiting example is shown in Fig. 1 b where this ratio was observed as the mean value.

[0097] IV. In some embodiments the ratio of CD8+ central memory T cells as defined by CD8+CCR7+CD45RA- T cells exceeds 20% of the CD8+ T cell population (‘CD8+ T cell compartment’). A non-limiting example is shown in Fig. 1 b, where this ratio was observed as the mean value.

[0098] In embodiments of the present invention CD62L may be used, considered or analyzed in addition, in combination or instead of CCR7. In embodiments, CD62L may be used, considered or analyzed instead of CCR7.

[0099] In embodiments, CD45RO may be used, considered or analyzed for selecting memory T-cells. In embodiments, thereby, potentially non-functional naive tumor-infiltrating T cells may be removed. In embodiments, this may ensure a T cell transfer enriched for memory T cells and thereby potent anti-tumor T cells.

[0100] In embodiments, the method for assisting the diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for a subject suspected of suffering from, or diagnosed with cancer and / or receiving a cancer therapy, comprises further determining the (surface) expression of one or more of (marker) genes selected from the group comprising CCR7, KLF2, BEX2, FAM65B, KLF3, TMEM63A, RASGRP2, LDLRAP1 , CAMK4, LMNA, ANXA1 , TIMP1 , AQP3, SORL1 , PTGER2, SESN1 , CCDC109B, TNFRSF25, TTC39C, RPS4Y1 , SCML4, FOSB, ADD3 , PABPC1 , FOS, ARHGAP15, PIK3IP1 , RPL3, LTB, FLT3LG, THEM4, SLC2A3, MGAT4A, MBP, FAM102A, RGCC, FOXP1 , RASA3, ABLIM1 , CD55, UPP1 , RP11 -18H21 .1 , BACH2, LEF1 , SELL, TRABD2A, C1orf228, PASK, CMTM8, SCML1 , TCF7, LYPD3, IL7R, CD40LG, MYC, GPR183, FBLN7, MAL, ANK3, SH3YL1 , SATB1 , CTB-133G6.1 , S1 PR1 , TMEM123, ITGA6, CHD7, CXCR5, ANXA1 , EEF1A1 , JUNB, EEF1 B2, and ZBTB10.

[0101] In embodiments, any of the genes disclosed herein before with respect to the method of isolating T cells may also be used in the method for assisting the diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for a subject either separately or in combination with any other (marker) gene disclosed herein.

[0102] In embodiments the diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for a subject considers one or more aspects like survival, response to chemotherapy, response to immunotherapy, shorter time of neutropenia after therapy and lower infection rate.

[0103] In the context of the present invention a chemotherapy may refer to a treatment regimen comprising one or more (anti-cancer) drugs, such as alkylating agents, plant alkaloids, antitumor antibiotics, antimetabolites and topoisomerase inhibitors. Immune checkpoint inhibitors including ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab and tremelimuab, and / or further targeted immune therapies such as alemtuzumab, bevacizumab, cetuximab, daratumumab, denosumab, dinutuximab, elotuzumab, isatuximab, margetuximab-cmkb, mogamulizumab, naxitamab-gqgk, necitumumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pertuzumab, ramucirumab, rituximab, tafasitamab, trastuzumab, and / or antibodydrug conjugates with immune-stimulatory characteristics such as brentuximab vedotin, enfortumab vedotin, gemtuzumab ozogamicin, ibritumomab tiuxetan, inotuzumab ozogamicin, loncastuximab tesirine, mirvetuximab soravtansine, moxetumomab pasudotox, polatuzumab vedotin, sacituzumab govitecan-hziy, tisotumab vedotin, trastuzumab deruxtecan, trastuzumab emtansine, and / or bispecific antibodies that target immune related pathways such as amivantamab, blinatumomab, epcoritamab, glofitamab, mosunetuzumab, tebentafusp-tebn and teclistimab.

[0104] In embodiments determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject is based on the expression of one or more of CD3, CD4, CD8, CD28, CD127 and CCR7 and / or CD62L, preferably one or more of CD3, CD4, CD8, CCR7 and CD127. In embodiments determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject is based on the expression of CD3, CD4 and / or CD8, CCR7 and CD127 in T cells.

[0105] In embodiments determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject is based on a higher ratio of (CD4+) helper cells, preferably of a higher ratio of CD4+ than CD8+ (CD4+ > CD8+) within a population of CD3+ cells.

[0106] In embodiments determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject is based on a higher ratio of early differentiated T cells (early differentiated T cell population) within a population of CD3+ cells, preferably wherein the early differentiated T cell population is characterized by expression of CD4 (CD4+) and / or CCR7 (CCR7+).

[0107] In embodiments determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject is based on a higher ratio of early differentiated T cells (early differentiated T cell population) expressing one or more of CD3, CD4, CD8, CCR7 and CD127, preferably CD4, CCR7 and CD127 within a population of CD3+ cells.

[0108] In embodiments determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject is based on a higher ratio of early differentiated CD3+CD4 / CD8+CCR7+CD127+ T cells (early differentiated T cell population) within a population of CD3+ cells.

[0109] In embodiments determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject is based on a lower ratio of later differentiated CD4+ or CD8+ and CCR7- (CCR7 negative) T cells.

[0110] In embodiments of the present invention CD62L may be used, considered or analyzed in addition, in combination or instead of CCR7. In embodiments, CD62L may be used, considered or analyzed instead / alternative to of CCR7.

[0111] In embodiments determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject is based on the expression of CD45RO as marker gene of memory T-cells and thereby a potent anti-tumor function of the anti-tumor immune system. CD45RO as a memory marker represents the ability of T cells to recognize tumor related antigens and execute effector function against the malignancy.

[0112] In embodiments determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject is based on a low (e.g., compared to a normal or early differentiated T cell) or absent expression of CD127 in CD4+ or CD8+ T cells, wherein preferably a response to IL7 is reduced or impaired (compared to normal or early differentiated T cells).

[0113] In embodiments determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject is based on any suitable combination of the afore-mentioned (surface) marker.

[0114] In embodiments, the therapy is an immunotherapy, comprising CAR T cell therapy, immune checkpoint therapy, molecularly targeted immune therapy and / or T cell therapy against solid tumor entities (e.g., tumor infiltrating lymphocyte (TIL) therapy).

[0115] In embodiments the therapy is an immunotherapy, preferably a CAR T cell therapy, preferably of a hematopoietic cancer. In embodiments it is of advantage to identify patients with a long-lived CAR T cell profile by applying the in vitro method according to the invention to increase the chances of such patients to receive CAR T cell therapy.

[0116] In embodiments herein a cancer is a solid cancer, preferably a melanoma, colorectal cancer, lung cancer, breast cancer, sarcoma, neuroendocrine cancer or urogenital cancer, more preferably kidney cancer or bladder cancer or kidney or bladder metastasis. In embodiments herein the cancer is kidney cancer and / or kidney metastasis. In embodiments herein the cancer is bladder cancer and / or bladder metastasis. In embodiments herein the cancer is lung cancer and / or lung metastasis. In embodiments herein a cancer is any cancer disclosed herein or diagnosed in a subject.

[0117] In embodiments, the diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification comprises the consideration, prediction and / or estimation of a clinical outcome, such as the survival, the response to a treatment, such as the response to chemotherapy and / or immunotherapy, the timespan / duration of neutropenia after therapy and / or the infection rate in the subject.

[0118] In embodiments, the present cells and methods may be used / applied in the context of CAR T cell therapies, e.g., in hematological disease, such as leukemia or lymphoma, patients. In embodiments, one or more marker may be used for such applications, e.g., to screen and / or select patients with a long-lived CAR T cell profile. Hence, in embodiments the method of the invention may therefore be used for the assessment of the suitability or stratification of a patient for CAR T cell therapy. In embodiments the method of the invention may be used for assessing the efficacy of a CAR T cell therapy and / or the responsiveness of a patient thereto. Patients with a long-lived CAR T cell profile may be more promising candidates for a successful (long-term) CAR T cell therapy and thus may be more likely selected to receive CAR T cell therapy.

[0119] In embodiments a marker (gene / protein) for the success and / or response of a subject to CAR T cell therapy may be selected from the group comprising:

[0120] (a) (combined) expression of one or more of CD3, CD4 / CD8, CCR7 and CD127,

[0121] (b) higher ratio of CD3 expressing (CD3+) T helper cells over CD3+ cytotoxic T cells (CD4+ > CD8+ within CD3+ T cell population), in other words, higher rates of CD3+ T cells expressing CD4, compared to CD3+ T cells expressing CD8),

[0122] (c) higher ratio of an ‘early differentiation’ T cell population over CD4+, CCR7+ T cells, within CD3+ T cell population,

[0123] (d) higher ratio of an ‘early differentiation’ T cell population over CD4+, CCR7+, CD127+ T cells, within CD3+ T cell population,

[0124] (e) general lower ratio of a ‘late differentiation’ CD4+ or CD8+, CCR7- (negative) T cell population,

[0125] (f) low expression of CD127 in CD4+ or CD8+ T cells (reduces responsiveness to I L7- stimulation).

[0126] In embodiments of the present invention CD62L may be used, considered or analyzed in addition, in combination or instead of CCR7. In embodiments, CD62L may be used, considered or analyzed instead of CCR7.

[0127] In embodiments, a further marker gene for the success and / or response of a subject to CAR T cell therapy may be CD45RO. CD45RO may represent the ability of CAR T cells to effectively eradicate the tumor cells in the form of memory-like CAR T cells. In embodiments one or more further or additional marker (gene / protein) for the success and / or response of a subject to CAR T cell therapy may be selected from the group comprising CCR7, KLF2, BEX2, FAM65B, KLF3, TMEM63A, RASGRP2, LDLRAP1 , CAMK4, LMNA, ANXA1 , TIMP1 , AQP3, SORL1 , PTGER2, SESN1 , CCDC109B, TNFRSF25, TTC39C, RPS4Y1 , SCML4, FOSB, ADD3 , PABPC1 , FOS, ARHGAP15, PIK3IP1 , RPL3, LTB, FLT3LG, THEM4, SLC2A3, MGAT4A, MBP, FAM102A, RGCC, FOXP1 , RASA3, ABLIM1 , CD55, UPP1 , RP11-18H21 .1 , BACH2, LEF1 , SELL, TRABD2A, C1orf228, PASK, CMTM8, SCML1 , TCF7, LYPD3, IL7R, CD40LG, MYC, GPR183, FBLN7, MAL, ANK3, SH3YL1 , SATB1 , CTB-133G6.1 , S1 PR1 , TMEM123, ITGA6, CHD7, CXCR5, ANXA1 , EEF1A1 , JUNB, EEF1 B2, and ZBTB10.

[0128] In embodiments, the expression of one or more of the respective marker genes may be analyzed or determined according to any method described herein.

[0129] In embodiments the determination of a lower ratio of ‘late differentiation’ T cells considers the comparison with an ‘early differentiated’ CCR7+ cell population and preferably refers to a ratio of less than 1 . In embodiments a ‘higher ratio’ refers to a ratio greater than 1 .

[0130] Each optional or preferred feature of the invention that is disclosed or described in the context of one aspect of the invention is herewith also disclosed in the context of the other aspects of the invention described herein. All features disclosed in the context of the method(s) according to the invention also relate to, and are herewith disclosed also in the context of the in vitro methods, the T cells or pharmaceutical compositions thereof, and vice versa. Embodiments and features described with respect to the cells, the cells for use in a treatment, the pharmaceutical compositions, and the in vivo method are considered to be disclosed with respect to each and every other aspect of the disclosure, such that features characterizing the methods, may be employed to characterize the cells, or pharmaceutical compositions and vice-versa.

[0131] The various aspects of the invention are unified by, benefit from, are based on and / or are linked by the common and surprising finding of the beneficial effects of the specific isolation and / or provision of immune cells, such as T cells, from a subject, preferably from a tumor and / or tumor environment of the subject, which may be selected and / or characterized by the expression of one or more of CD3, CD4, CD8, CCR7, CD127, and which may in embodiments even be readministered to a subject, optionally after additional activation / stimulation.

[0132] DETAILED DESCRIPTION OF THE INVENTION

[0133] All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.

[0134] The invention relates to a method for selection of T cells, comprising providing leukocyte cells from a subject, wherein said leukocytes comprise T cells, isolating T cells expressing one or more of CD3, CD4, CD8, CD127 and CCR7 and / or CD62L from the provided leucocytes, contacting / stimulating the isolated T cells with one or more interleukins, optionally stimulating and / or activating T cells expressing CD3 and / or CD28 and cultivating the T cells for at least 3 days, wherein isolating T cells in step b. preferably comprises: isolating from the leucocytes provided in a. T cells expressing CD3 and / or CD4 or CD8, preferably CD3 and CD4 or CD8, and isolating or enriching from the T cells isolated in i. T cells expressing CCR7 and / or CD127. The invention further relates to an isolated population of T cells, a T cell as well as in vivo methods for activating T cells and for assisting the diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for a subject suspected of suffering from or diagnosed with cancer and / or receiving a cancer therapy.

[0135] Herein a ‘subject’ or ‘patient’ may be a vertebrate, preferably a mammal, more preferably a human subject or patient. In the context of the present invention, the term "subject" or “patient” includes both humans and animals, particularly mammals, more particularly humans, and other organisms.

[0136] Immune cells

[0137] In general, T cells are a type of lymphoid cells (lymphocytes), more precisely a type of white blood cells (leukocytes). T cells play an essential role in the adaptive immune response and the immune system. T cells may be characterized by the presence of a T cell receptor (TCR) on their cell surface. Specific, differentiated T cell subtypes have various functions in the control and modification of the immune response. An important function is the initiation of immune-mediated cell death, which is carried out by two major subtypes: CD8+ killer T cells and CD4+ helper T cells. The CD4+ (CD4 positive / expressing) population of T cells function as helper T cells (TH). CD4+ TH cells function by further activating memory B cells and cytotoxic T cells, leading to an increased immune response. CD8+ T cells are also termed killer T cells and are cytotoxic. Cytotoxicity in T cells refers to the ability to kill, for example, pathogen (e.g., virus)-infected cells or cancer cells. In addition, CD8+ T cells are secrete cytokines (signalling molecules), to recruit other cell types to modulate the immune response. Another specific population of T cells provide the important mechanism of immune tolerance, wherein immune cells are competent in distinguishing between invading cells and the own cells of a subject. This ability of the so termed ‘suppressor T cells’ is important as it prevents immune cells from falsely attacking a body’s own cells, which is also known as autoimmune reaction. The present invention may comprise any given population of T cells, as described herein, and as known to a skilled person. Further T cell subtypes comprise, without limitation, T helper type 1 (Th1) cells, T helper type 2 (Th2) cells, T helper type 9 (Th9) cells, T helper type 17 (Th17) cells, T helper type 22 (Th22) cells, Follicular helper T (Tfh) cells, Regulatory T (Treg) cells, Natural killer T (NKT) cells, Gamma delta T cells, CD8+ cytotoxic T lymphocytes (CTLs). Further non-limiting embodiments of T cells include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine- induced killer cells (CIK cells) or activated T lymphocytes. In embodiments a T cell may be a CD4+ T cell, a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+ T cell, or any other subset of T cells. Heren the terminology ‘CD4 / CD8’ or ‘CD4 / 8’ refers to T cells expressing either CD4 or CD8, namely cells either being CD4+ or CD8+.

[0138] T cells may be characterized into different differentiation stages by their gene, protein and / or surface (marker) protein expression. T cell ‘differentiation states’ may be defined as early differentiated cellular states that are endowed with a high self-renewing capacity. Representative early differentiated T cell subsets may be defined by T cell memory markers as CCR7+CD45RA+ naive-like T cells and CCR7+CD45RA- central memory T cells (TCM). In contrast, late differentiated cellular states are considered to execute immediate T cell effector function.

[0139] Representative T cell subsets may be CCR7-CD45RA- effector memory T cells (TEM), and CCR7-CD45RA+ terminally differentiated effector-memory T cells (TEMRA).

[0140] In general, CD3 (cluster of differentiation 3) is a protein complex comprising the subunits CD35, CD3E and CD3y. CD3 may function as a T cell co-receptor that involved in the activation of CD8+ (cytotoxic T cell; see above) and CD4+ T cells (naive T cells, see above). CD3 is thereby considered to be involved in the activation of T cells by tumor related antigens.

[0141] In general, CCR7 (C-C Motif Chemokine Receptor 7) is a member of the G protein-coupled transmembrane receptor family that is commonly expressed in various lymphoid tissues. CCR7 is considered to be involved in the activation of B and T cells. For example, CCR7 mediates T cell homeostasis in lymph nodes, and is also considered to be involved in the activation and polarization of T cell and migration of memory T cells to sites of inflammation. CCR7 has been shown to be responsive to ligands, such as CCL19 and CCL21. The data of the inventors highlight CCR7 as an important marker for early-differentiated function of T cells in the tumor site.

[0142] In general, CD127 (cluster of differentiation 127, or interleukin 7 receptor (I L7R) subunit alpha) is a type 1 cytokine receptor for interleukin 7 (IL7) that has been shown to play a role in V(D)J recombination during lymphocyte development. Without being bound by theory, CD127 functionally commonly requires the interleukin 2 receptor gamma chain (IL2RG). The IL-7 response transmitted by CD127 is considered to enable a significant survival signal for memory T cells.

[0143] In general, CD62L (Selectin L or SELL) is a cell surface adhesion molecule belonging to the family of adhesion / homing receptors. CD62L comprises a C-type lectin-like domain, a calcium- binding epidermal growth factor-like domain, and two short complement-like repeats. CD62L is understood to enable the migration of lymphocytes into secondary lymphoid organs and inflammation sites. In embodiments, due to a similar cellular function in lymphocytes of CD62L, CD62L may be used, considered or analyzed herein instead of or in addition to CCR7.

[0144] In general, CD45RO (Protein Tyrosine Phosphatase Receptor Type C or PTPRC) is a member of the family of signaling molecule protein tyrosine phosphatases (PTP). CD45RO has been shown to be a regulator of T- and B-cell antigen receptor signaling. CD45RO is a cell surface marker suitable for selecting memory T-Cells, e.g., as shown in Figure 2D. CD45RO is therefore a marker for the ability of T cells to recognize antigens and to execute potent effector function.

[0145] GPR183 considered to encode a G protein-coupled receptor related to the thrombin receptor. Expression of this gene has been detected in the prior art before in B-lymphocytic cell lines and lymphoid tissues, but not in T-lymphocytic cell lines or peripheral blood T-lymphocytes. Due to its known surface abundance, a selection of GPR183+T cells by clinical-cale FACS for TIL therapy is possible. CD55 encodes a glycoprotein considered to be involved in the regulation of the complement cascade, wherein the binding of CD55 protein to complement proteins accelerates their degradation, thereby disrupting the cascade and preventing damage to the host cells. Due to its known surface abundance, a selection of CD55+T cells by clinical-cale FACS for TIL therapy is possible.

[0146] CXCR5 encodes a multimeric membrane protein considered to be a member of the CXC family of chemokine receptors that is considered to bind to the B lymphocyte chemoattractant and being involved in the migration of B cells to B cell follicles in the spleen. Expression of this gene has been detected in the prior art in mature B-lymphocytes and Burkitt's lymphoma. CXCR5 marks potentially migratory T cells which may be suitable for efficient tumor infiltration of TILs.

[0147] In molecular biology in general, ‘marker genes’, ‘genetic markers’ (or the corresponding marker proteins) or short ‘markers’ are genes (or their gene products, namely proteins) that may serve for the identification, characterization and / or selection of cells that express said marker genes. Preferably said marker genes are not expressed as ‘housekeeping genes’ (which are expressed in basically any cell) but only in certain types or sub-sets of cells, such that they may be used for the identification, characterization and / or selection of said population or type of cells. For example, in embodiments herein, certain types of T cells may be characterized or selected by the (surface) expression of the markers (marker genes) CD4 and / or CD8 or CD3, CD4, CD8, CD127 and / or CCR7 or CD62L.

[0148] ‘Chimeric antigen receptors’ (CARs) or ‘chimeric immune receptors’ are receptor proteins constructed to provide T cells the novel ability to target a specific antigen. The receptors are chimeric, as they combine T cell-activating functions and antigen-binding in a single receptor. A CAR-T cell cancer therapy employs T cells engineered with chimeric antigen receptors. In a “CAR T cell” or “CAR-T” immunotherapy the T cells are modified to recognize and destroy cancer cells more effectively. CAR T cells may be derived from a patient's own blood (autologous) or from a donor (allogeneic). Isolated T cells may be genetically engineered to express a specific CAR, that enables the recognition of antigen specifically expressed by cancer cells and present on their surface. However, such a target antigen must be absent in healthy cells. When CAR-T cells are introduced into a subject (e.g., cancer patient), they function similar to a anti-cancer drug. When a CAR T cell gets into contact with its target antigen on a (cancer) cells surface, the CAR-T cell binds to it and becomes activated, proliferates and induces cytotoxicity in the cancer cell. Cytotoxicity comprises mechanisms, such as an increased release of cell signalling factors that can affect other cells, e.g., cytokines, interleukins and growth factors. For example in a nonlimiting example CAR T-cell therapy comprises the engineering of a patient’s T cells in the laboratory so they will, when administered back to the patient, bind to cancer cells and kill them. Therefore, leukocytes, including T cells, are extracted from the blood of a patient by an apheresis machine. Subsequently, a gene for a special receptor called a chimeric antigen receptor (CAR) is inserted in vitro into the isolated T cells. Therefrom, a plurality of the CAR T cells are grown in vitro and administered back to the patient (autologous) or to another recipient (allogeneic) by infusion. The CAR T cells are able to bind to the respective target antigen on the cancer cells of the recipient (e.g., a cancer patient) such that said cancer cells are killed (see e.g., US-NIH; www.cancer.gov).

[0149] The generation of CAR T cells may be performed in embodiments as described in current literature, such as in Hong et al., 202046and Hiltensperger et al., 202347.

[0150] The term ‘leukocytes’ refers to white blood cells, which are cells of the immune system of a subject (immune cells). Leukocytes originate from hematopoietic stem cells in the bone marrow and circulate through the body within both the blood and the lymphatic system. Leukocytes preferably comprise T Cells, B Cells, microglia cells, natural killer (NK) cells, dendritic cells, granulocytes (basophils, eosinophils, and neutrophils), innate lymphoid cells (ILCs), megakaryocytes, monocytes / macrophages, platelets, red blood cells (RBCs) and / or thymocytes. Lymphocytes are a type of leukocytes, comprising T cells, B cells and Innate lymphoid cells (ILCs).

[0151] Cluster of differentiation (CD) proteins, such as CD8 and CD4, are transmembrane glycoproteins functioning in T cell signaling and as co-receptors for the T-cell receptor (TCR). CD8, for example, can specifically bind to the major histocompatibility complex (MHC) class I protein, while CD4 specifically binds to antigen-presenting MHC class II protein. CD4 is characteristic for T helper cells (also termed CD4+ T helper cells, CD4 cells, or simply T4 cells). CD8 is characteristic for cytotoxic T cells, also termed ‘T killer cells’. The presence of a CD protein on a lymphocyte, such as a T cell, may be indicated by a “+’, such that a T cells comprising CD8 is a CD8+ T cell or CD8-positive T cell.

[0152] ‘CCR’ proteins, also known as chemokine receptors of the G protein-coupled receptor (GPCR) family, which are a class of cell surface receptors involved in the regulation of immune response and inflammation. Chemokines are a group of cytokines, namely small cellular signaling proteins secreted by cells to regulate and induce immune cell migration to sites of infection, inflammation, or injury.

[0153] There are several types of CCR proteins, each with different functions and tissue expression patterns, such as CCR1 , CCR2, CCR3, CCR4, CCR6, CCR7, CCR8, CCR9, and CCR10, that play a role in immune cell recruitment and function in various tissues and conditions.

[0154] The term ‘cytokines’ refers commonly to cell signaling molecules that are secreted by cells, such as immune and non-immune cells, and may have an, either stimulating or inhibiting, effect on the surrounding cells or tissue, entire organs and / or even the whole body.

[0155] One type of cytokine are ‘interleukins’ (IL), which are cytokines secreted mostly by leukocytes. One of the major source of interleukins are CD4+ T helper cells. Interleukin 7 (IL-7) is a cytokine secreted by, e.g., certain bone marrow cells. IL-7 is considered a hematopoietic growth factor that stimulates the differentiation and proliferation of lymphoid progenitor cells as well as T cell development, proliferation, survival, and homeostasis. In embodiments herein, IL-7 may be provided as native or as recombinant IL-7. Interleukin 15 (IL-15) is a cytokine secreted by diverse leukocytes and induces the proliferation and activation and even suppresses apoptosis in T cells. Interleukin 21 (IL-21) is a cytokine secreted by activated T helper cells and (co)stimulates activation and proliferation of CD8+ T cells. In embodiments IL-21 may be used to modulate the differentiation programming of T cells.

[0156] Herein, “elevated or decreased expression, surface availability and / or abundance” of a surface marker, protein or factor refers to an respectively increased or decreased abundance, level or concentration of said surface marker, protein or factor in a respective medium (e.g., cell surface, cell, tissue, blood etc.) that can - when compared to the abundance, level or concentration of said surface marker, protein or factor in a specific other cell, precursor cell and / or cell type or a predetermined threshold value - be regarded as respectively significant higher or lower, thereby potentially being indicative of a certain cell-type, cell-subtype, cell origin, cell differentiation step or elevated / decreased gene expression.

[0157] As used herein, the term “sample” is a biological sample that is obtained or isolated from the patient or subject. A sample may, for example, refer to a biopsy sample, a sample of bodily fluid or tissue obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest, such as a patient. In embodiments, the sample is a tissue sample, a tissue biopsy, a sample of a bodily fluid, such as blood, serum, plasma, cerebrospinal fluid, urine, pleural effusions, cells, a cellular extract, and the like. Particularly, the sample is blood, or blood plasma, blood serum.

[0158] Cell sorting and isolation

[0159] In general ‘flow cytometry’ is a technique used to analyze and quantify characteristics of individual cells and / or particles. Flow cytometry enables the analysis of cells, particularly suspensions / mixtures comprising different types of cells, on a single cell-basis by using a laser beam that scans / measures each cell regarding various properties of interest, such as cell (or particle) size, shape, granularity, and / or fluorescence by simultaneously detecting / analyzing laser light scattering and / or fluorescence (e.g., induced by laser irradiation of a respective dye). Commonly, flow cytometry instruments produce scattered and fluorescent light signals that are read by detectors. In embodiments dyes, such as fluorescein, Alexa Fluor 488, Texas Red (325 D), Alexa Fluor 647 (1464 D), Pacific Blue and Cy5 (762 D) may commonly be used for antibody conjugation. Samples are commonly prepared for fluorescence measurement by transfection and / or expression of fluorescent proteins (e.g., GFP), staining with fluorescent dyes (e.g., Propidium Iodide (PI), phycoerythrin (PE), allophycocyanin (APC) and peridinin chlorophyll protein (PerCP)) and / or staining with fluorescently conjugated antibodies (e.g., anti-CD4 FITC (FITC = fluorescein isothiocyanate)). Cell proliferation can be determined, e.g., by providing cells with BrdU (bromodeoxyuridine) and subsequent anti-BrdU antibody and a DNA dye staining36. In flow cytometry, specific cell populations / types may be distinguished and optionally sorted or enriched according to gene expression and / or surface availability of certain proteins (‘markers’). Most immune cells express characteristic CD proteins (markers) that define them as a cell population. Such markers are termed ‘lineage markers’, e.g., T cell lineage markers (CD3, CD4, CD8), and can be employed to characterize, isolate, count and / or enrich specific cell populations from a sample36. During flow cytometry populations may be characterized and sorted / analyzed not only depending on (surface) protein expression, but also on characteristics such as size, surface roughness and shape, e.g., by analyzing the scattering of laser light directed at the cells, such as forward scatter (FSC), and side scatter (SSC). During a flow cytometry measurement cells with markers and / or characteristics of interest may be selected in real time by selecting a region (gate) around a (in the analysis software) shown population of cells (gating) and applying that region to other parameters within the experiment36. Different conditions can be applied to other conditions, such that cells may be characterized and / or sorted according to multiple characteristics. Examples of how to perform flow cytometry sorting may be found in standard literature and scientific review articles36and the skilled person is familiar with standard methods and workflows of flow cytometry and cell sorting, such as FACS.

[0160] Therapeutic application of the invention

[0161] Generally, the term ‘cellular immunotherapy’ may refer to a therapeutic approach utilizing a body's own immune cells, e.g., particularly T cells, to target and eliminate target cells of interest, such as diseased or abnormal cells. In embodiments this process may involve isolating, modifying (if necessary), and expanding specific immune cells outside the body, typically in a laboratory setting, before reintroducing them into the patient. Such manipulated immune cells may in embodiments be engineered to recognize and attack specific antigens present on target cells of interest, such as cancer cells, infected cells, or other harmful targets. In general, cellular immunotherapy may aim, e.g., to enhance a immune system's ability to recognize and destroy target cells of interest, such as abnormal cells, thereby providing a promising treatment strategy for various diseases, including cancer, autoimmune disorders, and infectious diseases.

[0162] In embodiments a cancer or tumor may be selected from the group comprising cancers or tumor diseases of bladder and kidney cancer or metastases thereof, breast carcinomas, gastrointestinal tumors, including colon carcinomas, gastric carcinomas, pancreatic carcinomas, colon cancer, small bowel cancer, ovarian cancer, prostate cancer, cervical cancer, renal cell carcinoma, liver cancer, a squamous cell carcinoma, melanoma, a basal cell carcinoma, a hepatocellular carcinoma, testicular cancer, a neuroblastoma, a glioma or glioblastoma multiforme, a colorectal tumor, an endometrial carcinoma, a lung carcinoma, an ovarian tumor, a cervical tumor, an osteosarcoma, a malignant lymphoma, an angiosarcoma, an Ewing sarcoma, or one or more metastases thereof.

[0163] In embodiments a cancer or tumor may be selected from the following cancers: bladder and kidney tumors or metastases thereof, tumors of the gastrointestinal tract comprising tumors of the liver, stomach, esophagus, pancreas, small intestine, gallbladder and biliary tract, colon and rectal carcinoma and anal carcinoma, urogenital tumors including tumors of the kidneys, bladder, prostate, ureters, urethra, of the penis and testicles, gynecological tumors comprising ovarian carcinoma, tumors of the cervix, vagina, vulva, malignant trophoblastic disease, tumors of the fallopian tube (tuba faloppii), tumors of the lungs including non-small cell bronchial carcinomas, small cell bronchial carcinomas, tumors of the inner nose, paranasal sinuses, nasopharynx, lips, oral cavity, oropharynx, larynx, hypopharynx, ear, salivary glands and paragangliomas, tumors of the abdominal cavity, breast carcinomas, tumors of endocrine organs, adrenal cortex, endocrine pancreatic tumors, multiple endocrine neoplasias, bone and soft tissue sarcomas, mesotheliomas, skin tumors, melanomas including cutaneous and intraocular melanomas, tumors of the central nervous system, tumors in childhood including retinoblastoma, Wilms tumor, lymphoma family neurofibromatosis, neuroblastoma, Ewing's sarcoma including non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, primary lymphomas of the central nervous system, Hodgkin's disease, leukemias including acute leukemias, chronic myeloid and lymphatic leukemias, plasma cell neoplasms, myelodysplastic syndromes, paraneoplastic syndromes primary, metastatic tumors, metastatic tumors including brain metastases, lung metastases, liver metastases, bone metastases, pleural and pericardial.

[0164] In embodiments “pharmaceutical compositions” provided for administration to a subject and which include a “therapeutically effective amount” of one or more of the cells and / or compositions disclosed herein. In certain embodiments, the pharmaceutical compositions are useful for treating a cancer in a subject. The therapeutically effective amount of the disclosed cells and / or compositions (e.g., cells, T cells, agent, substance, and / or blood sample) will depend on the route of administration, the species of subject and the physical characteristics of the subject being treated. Specific factors that can be taken into account include disease severity and stage, weight, diet and concurrent medications. The relationship of these factors to determining a therapeutically effective amount of the disclosed cells and / or compositions is understood by those of skill in the art.

[0165] In the context of the present invention a chemotherapy may refer to a treatment regimen comprising one or more (anti-cancer) drugs, such as alkylating agents, plant alkaloids, antitumor antibiotics, antimetabolites and topoisomerase inhibitors. Immune checkpoint inhibitors including ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab and tremelimuab, and / or further targeted immune therapies such as alemtuzumab, bevacizumab, cetuximab, daratumumab, denosumab, dinutuximab, elotuzumab, isatuximab, margetuximab-cmkb, mogamulizumab, naxitamab-gqgk, necitumumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pertuzumab, ramucirumab, rituximab, tafasitamab, trastuzumab and / or antibodydrug conjugates with immune-stimulatory characteristics such as brentuximab vedotin, enfortumab vedotin, gemtuzumab ozogamicin, ibritumomab tiuxetan, inotuzumab ozogamicin, loncastuximab tesirine, mirvetuximab soravtansine, moxetumomab pasudotox, polatuzumab vedotin, sacituzumab govitecan-hziy, tisotumab vedotin, trastuzumab deruxtecan, trastuzumab emtansine, and / or bispecific antibodies that target immune related pathways such as amivantamab, blinatumomab, epcoritamab, glofitamab, mosunetuzumab, tebentafusp-tebn and teclistimab.

[0166] Accordance to the treatment methods according to the invention, the cells and / or compositions can be delivered to a subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought. In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of the cells and / or compositions and / or other biologically active agents is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate a selected disease or condition or one or more symptom(s) thereof.

[0167] "Administration of and "administering" cells and / or compositions should be understood to mean providing cells, a combination of the cells according to the invention and at least one compound, or pharmaceutical compositions as described herein. The cells or composition(s) can be administered by another person to the subject (e.g., intravenously).

[0168] Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, the kidney, bladder, lungs or systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, intravenous, intramuscular or subcutaneous delivery, or any other suitable route of delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intravenous versus injected delivery formulations, and so forth.

[0169] The present invention also relates to a method of treatment of subjects suffering from the medical conditions disclosed herein. The method of treatment comprises preferably the administration of a therapeutically effective amount of cells or compositions disclosed herein to a subject in need thereof.

[0170] A "therapeutically effective amount" refers to a quantity of a specified cell and / or composition sufficient to achieve a desired effect in a subject being treated with said cells and / or compositions. For example, this may be the amount of cells according to the invention and / or a pharmaceutical composition according to the invention to a subject. The therapeutically effective amount or diagnostically effective amount of cells or compositions according to the invention will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the cells and / or compositions and / or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects.

[0171] Pharmaceutical compositions for administration to a subject can comprise at least one additional pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. In embodiments pharmaceutical compositions can also comprise one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents and the like. Pharmaceutically acceptable carriers useful for such formulations are conventional. Formulations suitable for pharmaceutical delivery of the cells and T cells described herein are described in Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995).

[0172] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0173] In embodiment pharmaceutical compositions can be administered by intravenous, intra-arterial, intra-articular, intraperitoneal, subcutaneous, intrathecal, intracerebroventricular, or parenteral routes.

[0174] As used herein, the term "approximately" or “about” is used to describe and account for small variations. For example, the term may refer to less than or equal to 10, such as less than or equal down to 1 , when appropriate, also the term may refer to more than or equal to 10, such as more than or equal up to 100 or more, when appropriate. It is to be understood that range format is used for the sake of simplicity and brevity and is to be flexibly understood to include numeric values expressly stated as boundaries of a range, encompassing each numeric value and subranges.

[0175] The instant disclosure also includes kits, packages and multi-container units containing the herein described pharmaceutical compositions, active ingredients, and / or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects.

[0176] FIGURES

[0177] The invention is further described by the following figures. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0178] Brief description of the figures:

[0179] Figure 1 : Early-differentiated CD4+ memory T cells are enriched across tumor entities and tissue compartments.

[0180] Figure 2: CD4+ early-differentiated memory T cells respond within the CXCR3 chemokine system and express high levels of CD127.

[0181] Figure 3: Long-lived T cell responses derived from tumors are driven by early-differentiated CD4+ T cells.

[0182] Figure 4: Tumor-derived early differentiated CD4+ T cells can elicit profound autologous tumor killing.

[0183] Figure 5: Early-differentiated T cell profiles can be exploited to improve tumor infiltrating lymphocyte therapy.

[0184] Figure 6: Absolute T cell counts within the tumor infiltrate of representative patients.

[0185] Figure ?: Exemplary workflow of an embodiment of the present invention.

[0186] Figure 8: Back-to-back comparison of T cell subsets within entities. Figure 9: CD4 / CD8 TEMRA frequencies are higher in kidney tumors compared to bladder cancer.

[0187] Figure 10: CD4 TCM frequencies are similar in kidney compared to bladder tumors.

[0188] Figure 11 : Paired matching of TCM frequencies in kidney compared to bladder tumors.

[0189] Figure 12: CD127 discriminates early and late stages of CD4+ and CD8+ T cell subsets in ccRCC.

[0190] Figure 13: CD95 (Fas) in ccRCC.

[0191] Figure 14: CD45RO in ccRCC.

[0192] Figure 15: PD-1 in ccRCC.

[0193] Figure 16: Comparison of CD4+ versus CD8+ TCM.

[0194] Figure 17: CXCR3 is upregulated after resting on the majority of CD4 and CD8 subsets.

[0195] Figure 18: CCR7 expression associates with subpopulations of CD4+ and CD8+ tumor infiltrating T cells in renal cancer.

[0196] Figure 19: Comparison of early-differentiated CD4+ and CD8+ T cells results into identification of common early T cell genes in different entities.

[0197] Figure 20: Phenotyping CD4+TILs in patients with renal and bladder tumors.

[0198] Figure 21 : Phenotyping node-derived CD4+T cell subpopulations in patients with bladder tumors.

[0199] Figure 22: Ex-vivo stimulation of lymph node-derived T cells reveals tumor-reactivity of CD4+TCM.

[0200] Figure 23: The sentinel node detection technique identifies tumor activity of the CCR7 ligands in lymph nodes of patients with bladder tumors.

[0201] Figure 24: Tumor-infiltrating CD3+CD4+CCR7+T cells are enriched in the center and periphery of clear cell renal cell carcinoma and in muscle invasive bladder cancer.

[0202] Figure 25: Tumor-infiltrating CD3+CCR7+T cells are dispersed across the TME and located in lymphoid follicular structures in renal clear cell carcinoma.

[0203] Figure 26: Single-cell RNA sequencing analysis identifies differentially expressed genes in CCR7-expressing CD4+and CD8+T cells in renal, bladder, and lung tumors.

[0204] Figure 27: Protein validation of CD55 on CD4+TILs.

[0205] Figure 28: CCR7 abundance on TILs excludes the TREG phenotype.

[0206] Figure 29: CD4eariy-de rived T cells exhibit a CD45RA+CD45RO+phenotype and show an increased expansion capacity compared to bulk TIL-derived T cells. Figure 30: Clonally diverse tumor-infiltrating CD4eariy T cells share identical clones with CD4iateT cells after expansion and ex-vivo.

[0207] Figure 31 : Cultured primary human renal tumor cells for co-culture analysis with TILs.

[0208] Figure 32: CD4eariy-de rived T cells act cooperatively to clear co-cultured autologous tumor cells in-vitro.

[0209] Figure 33: CXCR3 abundance on CD4+and CD8+tumor-infiltrating T cell subsets can be modulated by CXCL11 ex-vivo and after expansion.

[0210] Figure 34: Establishment of GMP-grade FACSorting of healthy peripheral CCR7+CD127+T cells.

[0211] Detailed description of the figures:

[0212] Figure 1 : Early-differentiated CD4+ memory T cells are enriched across tumor entities and tissue compartments, (a) Using flow cytometric gating strategy shown CD4 / 8+ T cell subsets are defined via CCR7 / CD45RA expression (FSC, forward scatter; SSC, side scatter; A, area; H, height; LD, live dead) living CD4+ and CD8+ T cell populations defined by CCR7 and CD45RA expression), (b) Subset frequencies in 33 human tumors (CM, central memory; EM, effector memory; EMRA, terminally differentiated effector memory; CD4 is depicted in light grey, CD8 in black) using two-way ANOVA with measurement repetition and comparative correction according to Holm-Sidak, P-value shown and (c) divided by entity (bladder and kidney), (d) Subset frequencies in lymph nodes versus blood of one patient and (e) quantified of 8 patients, (f) Overlay plot of CCR7 expression in tumor infiltrating T cells versus T cells residing in healthy tissue, (g) Exemplary flow cytometry gating and analysis scheme illustrating the positions of the respective T cell types within the flow cytometry graph when stained and analyzed for CCR7 and CD45RA expression (using labelled anti-CCR7 and anti-CD45RA antibodies after gating on CD3+ T cells), (h) Quantified frequencies of T cell memory associated marker (CD127, CD45RO and PD-1) abundance on CD4+T cell subpopulations in human tumors. Pre-gating was performed according to the strategy presented in (a), (i) Statistical analysis for differences in marker abundance (on CD4+TCM versus all others) was determined by Friedmann test with Dunn’s posttest. *P < 0.05; ****p < 0.0001 ; ns, not significant, n = 81 total patients, (j) Quantified frequencies of node-derived T cell subpopulations of five nodes derived from one patient and (k) of 22 nodes derived from eight patients. Statistical analysis for differences between subpopulations (CD4+TCM versus all others) was determined by one-way repeated measures ANOVA with Holm-Sidak’s multiple comparison correction. **P < 0.01 ; ****P < 0.0001 ; ns, not significant. (I) Representative FACS plots show CD4+ T cell subpopulations in tumor-infiltrating versus healthy tissue-infiltrating CD4+ subpopulations. Pregating was performed according to the strategy presented in (a), (m) Quantified frequencies of CD4+ T cell subpopulations in tumorinfiltrating versus healthy tissue-infiltrating CD4+ subpopulations, and (n) quantified frequencies of T cell memory-associated marker (CD127, CD45RO and PD-1) abundance and of the MFI of the chemokine receptor CCR7 on tumor-infiltrating versus healthy tissue-infiltrating CD4+ TCM in 14 patients. Statistical analysis for differences was conducted using Wilcoxon signed-rank test. *P < 0.05; **P < 0.01 ; ns, not significant. Figure 2: CD4+early-differentiated memory T cells respond within the CXCR3 chemokine system and express high levels of CD127. (a) T cells were gained from 6 renal solid tumors and analyzed for CXCR3 expression via flow cytometry, gating on CD3+T cells shown. Ex-vivo expression was compared to expression after resting (12 hours in complete medium) (Data for tumor cells is depicted in light grey, data for healthy cells is depicted in black), (b) CXCR3 expression on rested T cells shown according to the dedicated T cell subsets (CD4 is depicted in light grey, CD8 in black) and. (c) Rested T cells were re-exposed to the CXCR3 ligand CXCL11 in shown concentrations and analyzed thereafter for CXCR3 expression, (d) Expression of CD127 / CD45RO / CD95 / PD-1 was analyzed ex-vivo on the dedicated T cells subsets within the CD4+tumor infiltrating T cells, n=33

[0213] Figure 3: Long-lived T cell responses derived from tumors are driven by early- differentiated CD4+T cells, (a) Tumor infiltrating T cells were FACS-sorted according to the strategy presented. CD4+and CD8+respective T cell subsets defined by CCR7 expression into early (CCR7+) and late (CCR7_) CD4 / 8+T cells. Thereafter, stimulation was performed via anti- CD3 / 28 and long-term culture via the cytokines IL-7 and IL-15, (b) Two renal tumor derived T cell subset expansion were performed and analyzed on the respective days for their cell expansion (data for CD4eariy is depicted in light grey) and (c) phenotype using flow cytometry. Sequencing of the TCR beta chain of the dedicated subsets after 14 days of expansion was performed to determine (d) the clonal abundance as rare (0 < X <= 1e-05), small (1e-05 < X <= 1e-04), medium (1e-04 < X <= 0.001), large (0.001 < X <= 0.01) and hyperexpanded (0.01 < X <= 1) and (e) the respective diversity indices, n= 3 tumors.

[0214] Figure 4: Tumor-derived early differentiated CD4+T cells can elicit profound autologous tumor killing, (a) Workflow presented was applied to expand T cell subsets from solid tumors and their autologous cell line to conduct killing assays. In addition, cytokine production of the T cell subsets was tested using stimulation via PMA / lonomycin. (b) Frequency of killed autologous tumor cells as defined by a bulk control (without T cells) after overnight culture with the dedicated T cell subsets as indicated, n=6 patients with renal tumor. Statistical analysis for differences of killing between the subsets was determined by Friedmann test with Dunn’s posttest. *P < 0.05; **P < 0.01 . (c) Expression of the lived dead (LD) dye as a marker of cell death on tumor cells after co-culture with early differentiated CD4+ T cells in the displayed ratios of two patients with renal tumor and (d) quantified of six patients, (e) Cytokine production (IFNg, IL-2) and expression of stimulatory molecules (CD137, CD154) of a representative patient shown in an overlay plot and (f) as frequency of stimulated CD3+ T cells with PMA / lonomycin. Stimulated cells are depicted in light grey, unstimulated in black, (f) Experimental setup for the chemotaxis assay deploying CXCL11 to investigate migratory function of expanded T cell stages. T cells were seeded in the upper chamber, CXCL11 was added to the lower chamber, and migration assays were conducted for three hours. The chemotactic index (Cl) describes the absolute number of migrated T cells in the lower chamber with CXCL11 normalized to the absolute number of migrated cells in the lower chamber without chemokine. (g) Quantified data of the Cl of expanded T cell stages. Cl > 1 indicates a chemotactic effect of the chemokine and is highlighted in the plot by a dashed line. CXCL11 was applied at 100 ng / ml. Statistical analysis for differences between all T cell stages was determined by one-way repeated measures ANOVA with Holm-Sidak’s multiple comparison correction. *P < 0.05; **P < 0.01 . n = 5 patients with renal tumors, (h) Quantified Cl data of CD4eariy-derived T cells exposed to indicated concentrations of CXCL11 . n = 5 patients with renal tumors.

[0215] Figure 5: Early-differentiated T cell profiles can be exploited to improve tumor infiltrating lymphocyte therapy, (a) GMP-grade sorting on a CCR7+CDD127+T cell profile was trained using peripheral blood of healthy donors, n=3. (b) The sorting strategy was transferred to human tumor material, renal and bladder and (c) analyzed for their purity before (bulk) and after the enrichment (sorted), n=5. (d) The GMP-grade culture strategy was applied to expand the protective tumor infiltrating T cell phenotype for 7 days, (e) CCR7 expression was determined after expansion as well as (f) the frequency of CD4 and CXCR3 of the expanded CD3+T cells. Filled circles indicate data of day 0, empty circles indicate data of day 7. (g) Cell expansion was determined by calculating the cell number after expansion as fold of the initial cell number. Data for sorted expansion is depicted in light grey, bulk cell expansion in black.(h) Quantified frequencies of T cell viability and phenotypic T cell markers (CD3, CD4, CD8, PD-1 , CD45RA, CCR7, CXCR3, CD45RO) of expanded sorted TILs by flow cytometry.

[0216] Figure 6: Absolute T cell counts within the tumor infiltrate of representative patients, (a) 6 kidney tumors are compared to 4 bladder tumors with regards to the absolute number of tumor infiltrating T cells, (b) Double-negative and double-positive (CD4 / 8) and (c) single positive (CD3 / 4 / 8) are further defined within the absolute tumor immune infiltrate.

[0217] Figure 7: Non-limiting example of an embodiment of the present invention for generating / selecting T cells according to the invention. After surgical removal, the tumors are brought into suspension via a dissociation process. The desired T cell profile is then sorted from the tumor using antibody-based staining. T-cell expansion takes place in cell culture using a seven-day protocol.

[0218] Figure 8: Back-to-back comparison of T cell subsets within entities. Subset frequencies of 6 kidney tumors are compared to 4 bladder tumors and analyzed by their abundance of CM, central memory; EM, effector memory; EMRA, terminally differentiated effector memory T cells within the CD3+ population.

[0219] Figure 9: CD4 / CD8 TEMRA frequencies are higher in kidney tumors compared to bladder cancer. Subset frequencies of 6 kidney tumors are compared to 4 bladder tumors and analyzed by their abundance of CM, central memory; EM, effector memory; EMRA, terminally differentiated effector memory T cells within the CD4 / 8+ population.

[0220] Figure 10: CD4 TCM frequencies are similar in kidney compared to bladder tumors. Subset frequencies of 6 kidney tumors are compared to 4 bladder tumors and analyzed by their abundance of CM, central memory; EM, effector memory; EMRA, terminally differentiated effector memory T cells within the CD4 / 8+ population. Figure 11 : Paired matching of TCM frequencies in kidney compared to bladder tumors, (a) TCM are significantly enriched in the CD4 population compared to CD8 in kidney tumors, (b) TCM are significantly enriched in the CD4 population compared to CD8 in ccRCC and not in the one oncocytoma sample.

[0221] Figure 12: CD127 discriminates early and late stages of CD4+ and CD8+ T cell subsets in ccRCC. CD127 expression is displayed among the T cell subsets within the CD4 / 8 compartment to reveal expression dynamics.

[0222] Figure 13: CD95 (Fas) in ccRCC. CD95 expression is displayed among the T cell subsets within the CD4 / 8 compartment to reveal expression dynamics.

[0223] Figure 14: CD45RO in ccRCC. CD45RO expression is displayed among the T cell subsets within the CD4 / 8 compartment to reveal expression dynamics.

[0224] Figure 15: PD-1 in ccRCC. PD-1 expression is displayed among the T cell subsets within the CD4 / 8 compartment to reveal expression dynamics.

[0225] Figure 16: Comparison of CD4+ versus CD8+ TCM. CD127 and CD95 significantly higher in CD4 TCM in ccRCC compared to CD8. PD-1 expression is displayed among the T cell subsets within the CD4 / 8 compartment to reveal expression dynamics.

[0226] Figure 17: CXCR3 is upregulated after resting on the majority of CD4 and CD8 subsets.

[0227] CXCR3 is compared among CD4 and CD8 T cell subsets and not suitable for identifying early-diff T cells in the tumor.

[0228] Figure 18: CCR7 expression associates with subpopulations of CD4+ and CD8+ tumor infiltrating T cells in renal cancer. Single-cell RNA-sequencing samples were accessed through the publicly available European Genome-Phenome Archive under EGAD00001008030 (n=11 ; 10 ccRCC, 1 oncocytoma) of 11 renal tumor samples, (a, b) Plots display one UMAP map of 7229 CD4+ tumor infiltrating T cells, (a) Colors indicate 11 subpopulations identified within the CD4+ T cell compartment, (b) density plot indicates CCR7 expression within the CD4+ T cell compartment, (c, d) Plots display one UMAP map of 57633 CD8+ tumor infiltrating T cells, (c) Colors indicate 15 subpopulations identified within the CD8+ T cell compartment, (d) density plot indicates CCR7 expression within the CD8+ T cell compartment.

[0229] Figure 19: Comparison of early-differentiated CD4+ and CD8+ T cells results into identification of common early T cell genes in different entities. Single-cell RNA-sequencing samples were accessed through the publicly available European Genome-Phenome Archive under EGAD00001008030 (n=11 ; 10 ccRCC, 1 oncocytoma) of 11 renal tumor samples and compared to a bladder (Oh et al., 2020) and lung cancer (Bischoff et al., 2021) data set. Heatmap shows average log-expression of overlapping markers differentially expressed in CD4+CCR7+and and CD8+CCR7+. Differentially expressed markers were identified by the comparison of CD4+CCR7+with remaining CD4+T cells and CD8+CCR7+with remaining CD8+T cells, respectively. Figure 20: Phenotyping CD4+TILs in patients with renal and bladder tumors. (A) Frequency of tumor-infiltrating CD4+T cell subpopulations displayed separately for all patients studied (n = 102). Subpopulations were defined by flow cytometry as presented in Fig. 1A into the following CD4+subsets: naive-like (TNATvE-like: CCR7+CD45RA+), central memory (TCM: CCR7+CD45RAj, effector memory (TEM: CCR7'CD45RA , and terminally differentiated effector T cells (TEMRA: CCR7' CD45RA+). (B) Representative FACS plots show gating on tumor-infiltrating CD3+T cells and TCM to determine T cell memory associated marker (CD127, CD45RO and PD-1) abundance in a renal tumor applied for Fig. 1 D and Fig. 1J.

[0230] Figure 21 : Phenotyping node-derived CD4+T cell subpopulations in patients with bladder tumors. (A) Additional patients (#2-8) analyzed to the patient in Fig. 1 F (#1) for CD4+T cell subpopulations in lymph node-derived lymphocytes by flow cytometry. Quantified frequencies of individual patients with bladder tumors are shown. Subpopulations were defined by flow cytometry as presented in Fig. 1A. (B) Heat map of CD4+T cell subpopulation frequencies of all patients (#1- 8) in lymph nodes. Color code indicates the subpopulation frequency of CD4. (C) Representative histogram for CD127 expression on node-derived CD4+T cell populations assessed by flow cytometry.

[0231] Figure 22: Ex-vivo stimulation of lymph node-derived T cells reveals tumor-reactivity of CD4+TCM. (A) Representative FACS plots show T cell activation defined by CD40L expression plotted against SSC-A to identify CD4+T cell tumor reactivity. Pregating on CD4+, CD8+T cells and CD4+subpopulations was performed according to the strategy presented in Fig. 1A. Nodederived cells were stimulated for 12 h with autologous bladder tumor lysates and SEB was applied as control. The frequencies of T cell responses were assessed by flow cytometry. (B, C, D, E) Quantification of tumor and SEB-stimulated CD40L expression of node-derived T cells. CD40L expression in the non-stimulated condition was used for background-normalization (ratio). 5 lymph nodes of 2 patients with bladder tumors are shown. In (B and C), a paired t-test was applied to test for differences between CD4+and CD8+T cells. In (D and E), a two-way repeated- measures ANOVA with Holm-Sidak’s multiple comparison correction was applied to test for differences between CD4+subpopulations. *P < 0.05; ***P < 0.001 ; ****P < 0.0001 .

[0232] Figure 23: The sentinel node detection technique identifies tumor activity of the CCR7 ligands in lymph nodes of patients with bladder tumors. T umor drainage in pelvic lymph nodes of bladder cancer patients was tested using the sentinel node detection technique in patients that underwent radical cystectomy after neoadjuvant chemotherapy. Technetium (radionuclide) was applied to the tumor perioperatively. After nodal resection, the drainage of lymph nodes was tested via a Geiger Meter by counts per minute (cpm). 54 lymph nodes of 24 patients were assessed. Statistical analysis by repeated measures rank-correlation between CCL19 or CCL21 and cpm counts taking individual subjects into account. P values and correlation coefficients (R) are displayed in the plots. Level of confidence intervals was set to 0.975.

[0233] Figure 24: Tumor-infiltrating CD3+CD4+CCR7+T cells are enriched in the center and periphery of clear cell renal cell carcinoma and in muscle invasive bladder cancer. Tumor sections were analyzed by multispectral immunofluorescence (IF); nuclei were stained with DAPI. IF staining of CD3, CD4, CD8, CCR7 and cytokeratin (CK) was performed. (A) IF single and merged stainings in a clear cell renal cell carcinoma (ccRCC) sample. For merged staining see Fig. 2A. (B) IF single and merged staining in a muscle invasive bladder cancer sample (MIBC). (A-B) Red arrow indicates CD3+CD4+CCR7+and blue arrow indicates CD3+CD8+CCR7+T cells. (C) Quantified CCR7 frequencies of CD3+CD4+and CD3+CD8+T cells in the TME of 8 ccRCC and 5 MIBC samples. (D) Quantified absolute CD3+CD4+CCR7+and CD3+CD8+CCR7+T cells per mm2in ccRCC of 8 samples (left) and distributed according to peripheral (n = 4) and central (n = 4) area (right) based on microdissected distinction. (C and D) A paired t-test was applied to test for differences between CD3+CD4+(CCR7+) and CD3+CD8+(CCR7+) T cells. *P < 0.05; **P < 0.01 ; ***P < 0.001.

[0234] Figure 25: Tumor-infiltrating CD3+CCR7+T cells are dispersed across the TME and located in lymphoid follicular structures in renal clear cell carcinoma. Tumor sections were analyzed by multispectral immunofluorescence (IF); nuclei were stained with DAPI. mIF staining of CD3, CD4, CD8, CCR7 and cytokeratin (CK) was performed. (A) IF of a 1x1 mm section of a clear cell renal cell carcinoma (ccRCC) sample (left) in addition to Fig. 2A cell typed (right) as CD3+CD4+CCR7+(red) and CD3+CD8+CCR7+(black) in between CK+tumor cells (grey). (B) IF of a 1x1 mm section of a muscle invasive bladder cancer sample (left) cell typed (right) as CD3+CD4+CCR7+(red) and CD3+CD8+CCR7+(black) in between CK+tumor cells (grey). (C) IF of a lymphoid follicular structure at the peripheral site of a ccRCC sample including CD3+CCR7+T cells. (D) Ki-67 IF single staining of sample in (C). (E) Quantified mean nuclear Ki-67 intensity in CD3+CD4+CCR7+versus CD3+CD8+CCR7+T cells in 8 ccRCC samples. A paired t-test was applied to test for differences between CD3+CD4+CCR7+and CD3+CD8+CCR7+T cells. *P < 0.05; ***p < 0.001.

[0235] Figure 26: Single-cell RNA sequencing analysis identifies differentially expressed genes in CCR7-expressing CD4+and CD8+T cells in renal, bladder, and lung tumors. Tumor-infiltrating CD4+and CD8+T cells were analyzed from 12 renal (22), 7 bladder (23), and 10 lung tumors (32). (A) Expression of CCR7 in tumor-infiltrating CD4 and CD8 T cells. The color scale indicates the gene expression level based on log-transformed SCT-normalized counts. The gene was considered expressed with a value greater than 0. Renal CD4 T cells: 22,745 cells with 22.98% CCR7+cells; renal CD8 T cells: 80,580 cells with 1.94% CCR7+cells; bladder CD4 T cells: 16,731 cells with 26.03% CCR7+cells; bladder CD8 T cells: 8,165 cells with 11.77% CCR7+cells; lung CD4 T cells: 11 ,891 cells with 15.90% CCR7+cells; lung CD8 T cells: 6,196 cells with 6.17% CCR7+cells. (B) Log2 fold change (y-axis) of previously established T cell differentiation gradient (33) (x- axis) in CCR7+versus CCR7~ tumor-infiltrating CD4+and CD8+T cells from renal, bladder, and lung tumors. Log2 fold changes greater than 0 indicate an increased expression in CCR7+cells. (C) Percentage of CCR7+T cells co-expressing IL7R, SELL or TCF7. (D) UpSet plot showing groupwise comparisons of significantly upregulated genes between CCR7+and CCR7~ cells. The left bars indicate the total number of significantly upregulated genes per condition (rows). The bars on top represent the number of genes overlapping between different conditions indicated by the colored dots. A gene was considered significantly upregulated with a Iog2 fold change > 0.1 , adjusted p-value < 0.05 and percentage of cells expressing the gene > 0.01 in at least one of the groups that were compared. Ribosomal genes were excluded from the analysis. (E) Log2 fold changes of 18 significantly upregulated and 29 downregulated genes (rows) identified in all conditions (columns). Maximum Iog2 fold change was set to 4. Exact Iog2 fold change of CCR7’. renal CD4 T cells: 14.24; renal CD8 T cells: 10.57; bladder CD4 T cells: 14.88; bladder CD8 T cells: 13.98; lung CD4 T cells: 13.48; lung CD8 T cells: 12.50.

[0236] Figure 27: Protein validation of CD55 on CD4+TILs. A) Representative FACS plots show gating strategy to compare CD55 frequencies on CD4+CCR7+versus CD4+CCR7_TILs. (B) Quantified data for abundance on CD4+CCR7+versus CD4+CCR7_TIL in 8 patients with renal and 8 patients with bladder tumors. A paired t-test was applied to test for differences between CCR7+and CCR7' TILs. **P < 0.01 ; ***P < 0.001.

[0237] Figure 28: CCR7 abundance on TILs excludes the TREG phenotype. (A) Representative FACS plots show gating strategy to compare TREG frequencies within CD4+CCR7+and CD4+CCR7_TILs. Each were analyzed for intracellular FOXP3 and surface CD25 abundance to define CD4+TREG which were displayed as blue overlay in an additional FACS plot for CD127 abundance. (B) Quantified data for CD25+FOXP3+TREG frequencies within CD4+CCR7+and CD4+CCR7' TIL in 11 patients with renal and 9 patients with bladder tumors. A paired t-test was applied to test for differences between CCR7+and CCR7'TILs. **P < 0.01.

[0238] Figure 29: CD4eariy-derived T cells exhibit a CD45RA+CD45RO+phenotype and show an increased expansion capacity compared to bulk TIL-derived T cells. (A) Representative FACS plots show CD45RA and CD45RO abundance on T cell stages that were FACSorted at day 0 as presented in Fig. 3A and thereafter, expanded via anti-CD3 / 28 engagement and supplementation of the gc-cytokines IL-7 and IL-15 during culture. CD45RA and CD45RO were stained on the cell surface and analyzed by flow cytometry at the respective days. Pregating was performed on CD3+T cells as presented in Fig. 1A. (B) Representative FACS plots show CCR7 and CD45RA abundance on tumor-infiltrating CD4+CCR7+enriched T cells (CD4eariy) and bulk CD3+TIL during culture after expansion as described in (A). (C) Absolute expansion fold of CD4eariy and bulk CD3+TIL-derived T cells calculated as fold of baseline. Cell counting and cell staining including viability testing were performed at the respective days. For statistical analysis for differences Wilcoxon signed-rank test was applied. *P < 0.05. (D) Expansion fold of CCR7+and CCR7' T cells derived from CD4eariyand bulk CD3+TILs based on surface staining for CCR7 and calculated as fold of baseline. (C, D) n = 6 patients with renal tumors.

[0239] Figure 30: Clonally diverse tumor-infiltrating CD4eariy T cells share identical clones with CD4iate T cells after expansion and ex-vivo. (A) Clonal abundance determined by TCR p sequencing of tumor-infiltrating CD4+and CD8+T cell stages expanded according to Fig. 3B. T cell clones were grouped as rare (0 < x < 0.00001), small (0.00001 < x < 0.0001), medium (0.0001 < x < 0.001), large (0.001 < x < 0.01) and hyperexpanded (0.01 < x < 1) in n = 3 patients with renal tumors. Figure 31 : Cultured primary human renal tumor cells for co-culture analysis with TILs. A) Representative FACS plots show gating strategy to re-identify alive, CD3' tumor cells that were labeled with BV421. Live dead (LD) staining was applied to identify alive cells. Conditions with tumor cells only (upper panel) and tumor cells with TILs (lower panel) are shown. (B) Representative FACS plots show PMA / ionomycin induced activation defined by IFN-y and TNF-oc production for T cell stages (CD4eariy, CD8eariy, CD4iate, CD8iate) that were FACSorted based on CCR7 expression according to the approach shown in Fig. 3A and thereafter, expanded via anti- CD3 / 28 engagement and supplementation of the gc-cytokines IL-7 and IL-15 in culture. (C) Quantified data of IFN-y, TNF-oc and IL-2 production of T cell stages activated by PMA / ionomycin, n = 6 renal tumors. (D) Representative FACS plots show tumor cell induced activation defined by IFN-y and TNF-oc production for T cell stages (CD4eariy, CD8eariy, CD4iate, CD8iate) that were FACSorted based on CCR7 expression according to the approach shown in Fig. 3A and thereafter, expanded via anti-CD3 / 28 engagement and supplementation of the gc-cytokines IL-7 and IL-15 in culture. (E) Quantified data of IFN-y, TNF-oc and IL-2 production of T cell stages activated by autologous tumor cells, n = 6 renal tumors. (D-E) Autologous co-culture analysis from the same patient was performed as shown in Fig. 4A. (C and E) Non-detectable cytokine frequencies (zero) are displayed as 0.001 value for display at the logarithmic scale.

[0240] Figure 32: CD4eariy-derived T cells act cooperatively to clear co-cultured autologous tumor cells in-vitro. Holotomographic, label free 3D live imaging of cultured TILs and tumor cells was conducted for 12 hours. (A, B) Initial time-lapse refractive 3D holotomographic images of cells derived from two patients with a renal tumor (Patient #1 and Patient #2) are shown, respectively. T cell - tumor cell interaction is highlighted as area (red and blue). (C, D) Complete time-lapse series ofT cell - tumor cell interaction in the two patients. Dashed lines indicate tumor cells, arrows (yellow and green) mark collaborating TILs. (E) Quantification of tumor cells in the two approaches at 6 consecutive time points in the first hour at culture start and in the last hour at culture end.

[0241] Figure 33: CXCR3 abundance on CD4+and CD8+tumor-infiltrating T cell subsets can be modulated by CXCL11 ex-vivo and after expansion. (A) FACS plots show CXCR3 versus CD4 abundance on CD3+TILs ex-vivo and after resting (overnight in complete medium). CD4+TILs are highlighted in red. (B) Quantified frequencies of CXCR3 abundance on CD3+TIL that were rechallenged with CXCL11 in indicated concentrations. (C) Quantified frequencies of CXCR3 abundance on CD3+T cells derived from CD4eariy-derived tumor-infiltrating T cells that were rechallenged with CXCL11 in indicated concentrations. (B and C) TILs derived from five renal solid tumors are shown, incubation with CXCL11 was performed for three hours.

[0242] Figure 34: Establishment of GMP-grade FACSorting of healthy peripheral CCR7+CD127+T cells. FACS plots show CCR7 versus CD127 abundance on peripheral CD3+T cells in PBMC before (pre) and after GMP-grade FACSort (sorted) of 3 healthy individuals.

[0243] EXAMPLES The invention is further described by the following examples. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0244] Material and methods

[0245] Study design.

[0246] The aim of the following examples was to investigate T cell subset distribution in solid tumor entities (renal and bladder) for the establishment of novel adoptive T cell therapy approaches according to the invention. To this date, 33 patients with either renal or bladder tumors were enclosed. Likewise, lymph nodes of bladder cancer patients and healthy tissue of nephrectomy specimens were included as controls. The tumoral immune infiltrate was analyzed for the distinct T cell subsets and the respective T cell memory markers by flow cytometry and functional assays as indicated below. Sample sizes used for each experiment are detailed in the figure legends. Investigators were not blinded for any part of the study.

[0247] Patient sample processing.

[0248] The tumor samples were directly obtained after surgery, put on ice and were immersed in tissue storage buffer (Miltenyi). The inventors applied chemical and mechanical lysis for the tumors using the gentleMACS™ system (Miltenyi) enabling the generation of a cell suspension derived from the tumor. Next, cells were counted using trypan blue staining. After immersion in cold PBS (Thermo Fisher Scientific), cells were prepared for flow cytometry.

[0249] Flow cytometric analysis.

[0250] For analysis of immune phenotype, the tumor derived cells were stained using fluorescently conjugated monoclonal antibodies forCD3 (BV650, clone OKT3), CD4 (PerCP-Cy5.5, clone SK3) or CD4 (PEVio615, clone REA623; Miltenyi), CD8 (BV570, clone RPA-T8), CCR7 (AF488, clone G043H7), CD45RA (PE / Cy7, clone HI100), CD95 [Brilliant Violet (BV) 421 , clone DX2; BD Biosciences], CXCR3 (PE, clone G025H7), CD45RO (BV785, clone UCHL1), PD-1 (APC / Cy7, clone EH12.2H7), CD127 (AF647, clone A019D5), CD25 (PECy5, clone M-A251), or CD55 (PE, clone JS11) at 4 °C for 30 min. To exclude dead cells, LI E / DEAD dye amine-reactive fluorescent dye (Biolegend) was added. In all stimulation experiments, cells were fixed and permeabilized using the eBioscience FoxP3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) following the manufacturer's guidelines. After washing, fixed cells were intracellularly stained with fluorochrome-conjugated monoclonal antibodies as indicated in the respective experiments: CD40L (BV421 , clone 24-31 ), IFN-y (eF405, clone 4S.B3), TNF-o (Alexa Fluor 700, clone MAb11), or IL-2 (PE, clone MQ1-17H12) at 4°C for 30 minutes. Background response was measured in nonstimulated controls and subtracted from the stimulated cytokine production. Negative values were adjusted to zero. For intracellular FOXP3 staining, fixation and permeabilization was likewise performed. The fluorescently conjugated monoclonal antibody for intracellular FOXP3 (BV421 , clone 206D) was applied. Surface staining for CD25 and CD127 (see above for clones) was used to confirm the TREG phenotype. All antibodies were purchased from BioLegend unless otherwise indicated. Cells were analyzed on an LSR-II FORTESSA flow cytometer (BD Biosciences) with FlowJo software version 10 (BD Biosciences) or with FlowJo software version 11 (Tree Star). Lymphocytes were gated on the basis of the forward scatter (FSC) vs. side scatter (SSC) profile and subsequently gated on FSC-Height vs. FSC-Area to exclude doublets.

[0251] Fluorescence-activated Cell Sorting (FACSortinq).

[0252] For non-GMP grade FACSorting tumor derived cells were enriched by FACS for a the respective CD3+CD4 / 8+CCR7+ / - T cell populations on a Cytoflex SRT (Beckmann Coulter). Gating strategy was followed as shown in Fig.1a and Fig.3a, and post-sorting purity of enriched cells exceeded 95%. For GMP-grade sorting cells were enriched by FACS with the gating strategy shown and established in Fig.5. Additionally, an antibody for CD127 (AF647, clone A019D5) was added to the panel. The GMP-grade MACSQuant® Tyto® Cell Sorter (Milteny) was applied to enrich for the early-differentiated T cell phenotype from the tumor. Cartridges enabled the analysis of the non-enriched T cell population. Peripheral blood mononuclear cells (PBMC) were used as a biological control.

[0253] T cell expansion protocol.

[0254] After FACS enrichment, sorted T cell subsets were activated by polyclonal stimulation via anti- CD3 and via anti-CD28 using T Cell TransAct™ (Miltenyi). After stimulation, cells were cultured in complete medium consisting of very-low-endotoxin Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with stable glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin (all from Biochrom), and 10% heat-inactivated fetal bovine serum (PAA Laboratories) including recombinant human IL-7 and IL-15 each at 10 ng / ml (CellGenix) at 37 °C and 5% CO2 in humidified incubators. The expansion fold was assessed as fold of the initial starting T cell number.

[0255] T cell killing assays.

[0256] T cell subset expansion was performed as described above. Primary tumor was cultured simultaneously using Renal TumorMACS™ Medium (Miltenyi) for the cultivation and expansion of tumor cells from primary tumor and to establish stable cell lines. At day 14, T cells were restimulated via anti-CD3 and via anti-CD28 using T Cell TransAct™ (Miltenyi). 24 hours thereafter, tumor cells were labeled using Brilliant Violet™ 421 Dye (ThermoFisher) and co-cultured with T cells in a 1 :1 cell ratio. In one condition, T cells were increased to a ratio of 10:1 . After 16 hours of co-culture in complete medium at 37 °C and 5% CO2 in humidified incubators, cells were analyzed by flow cytometry. Remaining alive tumor cells were assessed by excluding T cells via CD3 expression by anti-CD3 (BV650, clone OKT3), by excluding dead cells via LIVE / DEAD dye amine-reactive fluorescent dye (Biolegend) and by identifying tumor cells directly via the labeled BV421 dye (ThermoFisher).

[0257] Stimulation of node-derived cells with tumor lysate. Autologous tumor samples from transurethral resection of bladder tumor (TURBT) were weighed, and phosphate-buffered saline (PBS) was added in a 5:1 ratio of 2x concentrated PBS volume (ml) to tumor mass (g). The mixture was homogenized using a cell disperser and then boiled at 100°C for 15 minutes in a water bath. The resulting tumor lysate was then used to stimulate node-derived cells at a 1 :100 dilution for 12 hours. As positive control, staphylococcal enterotoxin B (SEB) from staphylococcus aureus (Merck) was applied at 1 pg / ml. After4 hours of stimulation, GolgiPlug (BD Biosciences) was added 1 :1000.

[0258] Sentinel node detection technique. Technetium was administered using a transurethral approach with a 3.7 Fr. Williams cystoscopy needle (Cook Medical) immediately before starting the cystectomy procedure. A 1 ml solution of technetium (Nanocoll) was injected at four locations around the tumor base or tumor scar (depending on whether residual tumor was present). During each injection, the needle tip was positioned preferentially within the detrusor muscle. The technetium was prepared by the nuclear medicine department on the same day, calibrated to achieve a radioactivity level of 80 Mbq. Sentinel node detection was performed approximately two hours later, following the removal of the primary organ (urinary bladder) from the minor pelvis. The drainage was tested via a Geiger Meter and thus, counts per minute (cpm) indicate the degree of tumor drainage of the respective node. Nodal CCL19 and CCL21 concentrations were measured after tissue homogenization via ELISA (Merck, ELISA kits). The association between nodal chemokine concentration and tumor drainage was analyzed using repeated measures rankcorrelation, accounting for individual subjects (70).

[0259] Multiplex Immunofluorescence Staining. The OPAL (Opal 6-Plex Manual Detection Kit, NEL811001 KT, Akoya Biosciences, Marlborough, MA, USA) method was employed for multiplex staining of 2 pm sections of formalin-fixed paraffin-embedded (FFPE) human cancer samples. Antigen retrieval and OPAL staining was performed in accordance with the manufacturer's instructions and as previously described (71). The following antibodies were used: CD3 (IR503, Dako, Agilent), KI-67 (IR626, clone MIB-1 , Dako, Agilent), CCR7 (ab253187, Abeam), CD4 (ab133616, EPR6855, Abeam), CD8 (M7103, Clone C8 / 144B, Dako, Agilent) and Pan-Cytokeratin (IR053, Clone AE1 / AE3, Dako, Agilent). The image acquisition was conducted using a PhenoCycler Fusion microscope (Akoya Biosciences). The Phenochart and inForm software (Akoya Biosciences) were employed for the selection of regions of interest, spectral unmixing and background subtraction. Cell segmentation and classification were conducted based on the thresholding of cell markers using QuPath vO.4.3. (72). Tumor regions with a minimum area of 11 mm2per sample were selected for analysis.

[0260] Spatial three-dimensional (3D) RNA imaging. The TRISCO (tris buffer-mediated retention of in situ hybridization chain reaction signal in cleared organs) technique was employed for performing 3D imaging of RNAs in a human urothelial cancer samples as previously described (31). Briefly, RNA probes were designed using a custom MATLAB script available on GitHub (73). The sequences of all probes used are provided in data file S2. Fixed samples stored in 100% methanol were delipidated with 100% dichloromethane (DCM), bleached in a 5% H2O2solution in methanol, hybridized with 2 nM probe in hybridization buffer containing 50% formamide, 5* sodium chloride sodium citrate (SSC), 9 mM citrate acid (pH 6.0), 0.1% Tween-20, 50 pg ml-1 heparin, 0.3% PVSA, 1 * Denhardt’s solution, and 10% dextran sulfate. Signal amplification was achieved using fluorescently labeled DNA hairpins at 4°C. Finally, samples were cleared with the organic solvent dibenzyl ether. Imaging was conducted on a light-sheet microscope (UltraMicroscope II, Miltenyi Biotec). Fluorescent probes were excited with laser lines at 561 nm (CD4) and 639 nm (CCR7). The light sheet width was set to 3.75 pm with a 20% overlap, and images were acquired at an XY resolution of 0.6 pm and a Z-step size of 4 pm. Image processing was performed using Imaris software (version 10.2.0, Bitplane) and Amira (Thermo Fisher Scientific Inc.). An unsharp mask filter and background correction were applied in Amira. Colocalization analysis was conducted by segmenting CD4-stained and CCR7-stained spots and using the spot-to-spot function in Imaris. Colocalization was defined as spots with a shortest inter-spot distance of less than 10 pm. Quantitative 3D analysis of CD4, CCR7, and their co-expression was performed using the spot filtering function in Imaris.

[0261] Single cell RNA-Sequencing (scRNA-seq) analysis. Publicly available pre-processed scRNA- seq data sets were downloaded for renal cell carcinoma via (74) based on (22), for bladder cancer via (75) based on (23), and for lung adenocarcinoma via (76) based on (32). The downloaded files contained raw counts of pre-filtered samples, as described in the methods of the corresponding publications, and were converted to Seurat objects for subsequent analyses in R (version 4.4.0) using Seurat (version 5.1.0) (77). For the kidney cancer data, T cells were extracted for tumor samples including tumor, tumor-normal interphase and metastases. Cells were separated into CD4+and CD8+T cells based on the published annotation. For the bladder cancer data, sorted CD3+CD4+and CD3+CD8+T cells from tumor samples were separated into CD4+and CD8+T cells based on the published annotation (annotation_authors). For the lung cancer data, T cells were reannotated using scGate (version 1 .6.2) (78) with generic CD4+and CD8+T cell markers and cells were separated into CD4+and CD8+T cell subsets accordingly. Analysis was performed for CD4+and CD8+T cells separately. Within each subset, patients were normalized separately using Seurat’s SCTransform() v2 (79, 80). The percentage of mitochondrial genes was regressed out. The normalized samples were combined using merge() and principal component analysis (PCA) with npcs = 30 was performed using RunPCAQ. Batch effect correction was applied using the RunHarmony() function implemented in the R package harmony (version 1 .2.0) (81) with the batch key (parameter ‘group. by. vars’) being set as patients and the assay (parameter ‘assay. use’) being set as ‘SCT’. UMAPs were generated using Seurat’s RunUMAPQ with dims = 30 and DimPlot(). CCR7+T cells were detected considering cells with an expression value greater than 0 and differentially expressed genes were identified between CCR7+and CCR7' T cells using FindMarkers() with default settings.

[0262] Intratumoral T Cell Receptor (TCR) p Sequencing. To assess the clonal dynamics of TIL differentiation stages, we performed next-generation sequencing of the TCR p repertoire. TILs were either expanded as described above or analyzed directly ex-vivo. RNA extraction was carried out using the Quick-RNA Microprep Kit (Zymo) and sent to CeGaT GmbH. The TCR p locus was amplified and processed via the Illumina NGS platform. Adapter sequences were removed with Skewer (version 0.2.2). For TCR analysis, the FASTQ files were downsampled to 2 million read pairs. Overlapping paired reads were merged into single reads prior to reconstructing the CDR3P regions using NGmerge (82). Only successfully merged read pairs were included in further analysis. T cell receptor sequences were reconstructed using RTCR (83). CDR3P sequences exceeding 29 amino acids were excluded from the functional set as they are considered assembly artifacts. TCR repertoire analysis was performed using the R package immunarch (version 0.6.9). Venn diagrams were generated using the R package VennDiagram (version 1.7.3), circular heatmap plots using the R package circlize (version 0.4.16). TCR p sequencing data have been deposited in GEO under the identifier x.

[0263] Bulk RNA sequencing (RNA-seq). RNA extraction of expanded CD4+TILs was carried out using the Quick-RNA Microprep Kit (Zymo) and sent to CeGaT GmbH. Libraries were prepared using the SMART-Seq Stranded library preparation kit (Takara Bio) and paired-end sequencing was performed on an Illumina NovaSeq 6000 (2 x 101 bp) system. The sequencing reads were demultiplexed using Illumina bcl2fastq (version 2.20) and adapters were trimmed with Skewer (version 0.2.2) (84), while no quality trimming of the reads was performed. Trimmed raw reads were aligned to hg19-cegat using STAR (version 2.7.3) (85). Pseudoautosomal regions (PAR) were masked on chromosome Y (chrY:10001-2649520, chrY:59034050-59363566) and reads originating from these regions can be found at the respective location on chromosome X. Due to potential DNA contaminations, some samples were excluded from further analyses, leaving 4 replicates for both CD4eariy and CD4iate. Only genes with a sum of raw counts > 40 were kept. Using the R / Bioconductor package DESeq2 (version 1 .44.0) (86), the raw read counts were normalized (normCounts) and transformed to the regularized Iog2 scale (rlogCounts), to compensate fora large dynamic range of expression values, by considering the library size. The genes were fitted with a negative binomial generalized linear model followed by Wald statistics using DESeq2 to obtain Iog2 fold changes (log2FC) between CD4eariyand CD4iate. Raw RNA sequencing data have been uploaded to GEO with the identifier x.

[0264] TIL cytotoxic and cytokine assays. To generate target cells, dissociated autologous tumor cells (see above) were cultured according to a previously published protocol (41). 1 x 106tumor-derived suspended cells were seeded at a density of 4 x 104per cm2in a 25 cm2cell culture flask. We used Renal TumorMACS™ Medium (Miltenyi) as a serum-free cell culture medium to maintain renal tumor cell growth. We changed the medium 24 hours after the first seeding, and thereafter, every 48 hours. In six patients with renal tumors, cells grew at 80% confluency within 14 days and were subsequently passaged a minimum of 5 times. At no time fibroblastic overgrowth was observed. Once successful passaging of cells from a patient was achieved, initially frozen tumor-derived cells from the same patient were thawed, and TIL subsets were FACSorted as described above. At day 14 after TIL expansion, T cells were re-stimulated via anti-CD3 and anti-CD28 using T Cell TransAct™ (Miltenyi). 24 hours thereafter, autologous tumor cells were labeled using Brilliant Violet™ 421 Dye (ThermoFisher) and co-cultured with re-stimulated TILs in a T cell to tumor cell ratio of 1 :1 or 10:1 for 16 hours. Additionally, a minimum of one control was set as a blank tumor cell condition without TILs. Co-culture was performed in complete medium at 37 °C and 5% CO2 in humidified incubators and subsequently, all cells were completely acquired on an LSR-II FORTESSA flow cytometer (BD Biosciences). We assessed the cytotoxic capacity of TIL subsets to mediate tumor cell death in comparison to the blank approach. For this, we quantified the cytotoxic capacity of TILs by defining the difference of the absolute number of alive tumor cells in the TIL condition compared to the blank condition (without TILs). Thus, a cytotoxic capacity of 100% indicates complete tumor cell death and 0% indicates no cytotoxic effect on tumor cells. The absolute number of alive BV421-labeled tumor cells in the TIL approaches were assessed excluding T cells via CD3. Dead tumor cells were excluded via LD expression as shown in fig. S13A. In addition, we assessed the cytokine production of the TIL subsets on tumor cell exposure. Co-culture was likewise performed for 16 hours and GolgiPlug (BD Biosciences) added 1 :1000 after 4 hours. As a negative control, TILs were separately cultured without tumor cells. As a positive control, TILs were separately stimulated with PMA and ionomycin (Cell Stimulation Cocktail, eBioscience, 1 :500) and thereby, cytokine production capacity of TIL subsets were assessed.

[0265] Holotomographic, label free 3D live imaging of co-cultured TILs and tumor cells. CD4eariy- derivedT cells were expanded via the TIL expansion protocol (see above). After expansion, 50.000 CD4eariy-derived T cells were polyclonally activated by anti-CD3 / CD28 (Miltenyi) and 50.000 cultured autologous, primary renal tumor cells were seeded onto 96-well glassbottom plates (MatTek Corporation) per 80 pl RPMI. After 24 hours, 50.000 re-stimulated CD4eariy-derived T cells were added. The plate was briefly centrifuged to prevent meniscus formation and placed on the pre-equilibrated (37°C, 5% CO2 with 90% humidity) Nanolive CX-A 3D Cell Explorer microscope (Nanolive) equipped with a 520 nm laser, and an air objective lens (NA = 0.8, magnification 60x). After approximately 1 h, thermalization was complete, and time-lapse refractive 3D holotomographic images were captured every 4 min for 12 h using 3 x 3 gridscan mode (a field view of 275 x 275 pm). TILs could be identified by their motile behavior, white cytoplasm, and amoeboid cell shape, which enabled descriptive analysis in this system.

[0266] TIL migration assay. The lower chamber of a 24-well transwell plate with 3 pm pores (Corning) was filled with 600 pl of complete medium composed of very-low-endotoxin Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with stable glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin (all from Biochrom), and 10% heat-inactivated fetal bovine serum (PAA Laboratories). TILs were added to the upper chamber in 200 pl of complete medium. Recombinant human CXCL11 (BioLegend) was introduced into the lower chamber at indicated concentrations for titration. The total volume across both chambers was adjusted to 800 pl. A blank condition without chemokines served as the control. The assay was conducted at 37 °C and 5% CO2 in a humidified incubator for 3 hours. Following incubation, transmigrated T cells in the lower chamber were collected and completely acquired on an LSR-II FORTESSA flow cytometer (BD Biosciences). The chemotactic index (Cl) calculated as previously described (33) indicates the migration ratio of T cells in the presence versus absence of the chemokine, with a Cl > 1 denoting a chemotactic effect on the targeted TIL subpopulation.

[0267] Example 1 The detection and characterization of CD3+CD4 / CD8+CCR7+CD127+ T cells from patient tumors were performed according to the following protocol (see also Fig. 3). Patients with solid tumor diseases underwent a surgical procedure as part of routine diagnostics or therapy. Therefrom, tumor material was obtained, which underwent a diagnostic workflow in pathology while a fraction of the tumor material was also used for the generation of T-Lene cells according to the present invention. Said tumor fraction for the generation of T-cells according to the present invention was stored on ice and in PBS. The tumor is then transferred into a cell suspension using a protocol established by the inventors. This includes a chemical and a mechanical dissociation step. Next, the tumor was stained using the flow cytometry, i.e. an antibody-based staining of the above-mentioned surface proteins (markers) was performed (CD3 / CD4 / CD8 / CCR7 / CD127). The CD3+CD4+ / CD8+CCR7+CD127+ T cell population was isolated from the tumor dissociate using a cell sorter suitable for GMP (good manufacturing practice) (e.g., FACS). This enabled purification to a T cell population and the removal of other cell populations.

[0268] In the final step, this population was activated via a polyclonal stimulation approach in cell culture according to the present invention for seven days and was stimulated with the GMP-grade cytokines interleukin-7 and interleukin-15. The cell product (T-Lene) was harvested and analyzed for purity and functionality.

[0269] The last step in one clinical application is the administration of the T-Lene cells to a patient with a solid tumor disease, preferably intravenously. Alternatively, cells can be administered locally into the tumor site.

[0270] Example 2

[0271] In the diagnostic embodiments of the invention, a flow cytometric detection of the early- differentiated T cell population is performed, which represented also the first two steps of the therapeutic application (Fig. 3). The frequency of the T cell population in the tumor infiltrate was then determined using a flow cytometric analysis device. The quantitative detection of the early differentiated T-cell population based on the markers CD3+CD4 / CD8+CCR7+CD127+ can the be linked to therapy response and clinical performance of the patients. In particular, an application for the stratification of immunotherapies is being considered.

[0272] Obtained data imply that the cell population isolated according to the present invention plays a role in T-cell mediated therapy response. In addition, the newly identified T cell population in the tumor enables a potential application for various T cell-mediated immunopathologies outside the oncological disease spectrum, e.g., rheumatology and infectiology.

[0273] Example 3

[0274] The present methods may be employed for a clinical validation of the tumoral T-cell phenotype for patient response to therapy (chemotherapy, immunotherapy, targeted therapy, cell therapy), survival, tumor recurrence. In an envisaged study design, patients are retrospectively divided into ‘high’ and ‘low’ with the described ratios (see comment in the claims). For this, the initial tumor biopsy of patients is analyzed by the current methodology on the expression levels of CD3 / CD4 / CD8 / CCR7 / CD127 and T cell indices are calculated as described in detail by this invention. Patients are divided into two cohorts “high” and “low” according to the indices. Clinical parameters such as response to treatment, survival and tumor recurrence are compared between these two cohorts.

[0275] An example of the phenotype of T cells suggested for prognostic applications is shown in Figures 1 and 2.

[0276] Unraveling human T cell differentiation states in the TME may guide therapeutic approaches to select for potent tumor-infiltrating T cells. Renal and bladder tumors are T cell infiltrated malignancies. The inventors analyzed tumors of 102 patients with renal (n = 79) or bladder cancer (n = 23) for their precise tumor-infiltrating T cell subset distribution according to phenotypic definitions (Fig. 1a). In contrast to low intra-tumor abundance of CD4+and CD8+T cells with a naive-like phenotype, they found higher abundance in the majority of the dedicated memory T cells. Herein, early differentiated, central memory T cells (TCM) were enriched in the CD4+subpopulation compared to the CD8+subpopulation, whereas terminally differentiated effector memory T cells (TEMRA) were enriched in the CD8+subpopulation (Fig. 1 b). Besides enrichment of TCM in the CD4+subpopulation, the inventors could detect robust frequencies of CD4+TCM in all tumors of the investigated 102 patients (Fig. 20A). Moreover, they observed similar abundance of the respective CD4+T cell subsets in kidney / renal compared to bladder tumors; with TCM exceeding frequencies higher than 20% of the total CD4+subpopulation in 93 / 102 patients (Fig. 1 c). This sheds light on a potential preservation of early differentiated CD4+T cells in the TME. The inventors sought to further examine tumor-infiltrating CD4+T cells for the abundance of memory associated molecules. Tumor-infiltrating CD4+TCM exhibited high protein abundance of the IL-7 receptor a chain CD127 and the T cell memory associated tyrosine kinase CD45RO as opposed to lower abundance of programmed cell death protein (PD-1) compared to the remaining tumor-infiltrating CD4+T cell subsets (Fig. 1 h-l , and Fig. 20B). Human tumorinfiltrating CD4+CCR7+CD45RO+CD127h'9hPD-1lowTcM were re-identified in human tumors at stable frequencies.

[0277] The chemokine receptor CCR7 mediates homing of memory T cells with early differentiated states to the lymph node via its ligands CCL19 and CCL21 . High CCR7 abundance on tumorinfiltrating CD4+TCM implies the lymphatic system as a potential reservoir for these cells. To test this, the inventors defined the CD4+T cell subset distribution in tumor-adjacent lymph nodes of patients with muscle-invasive bladder cancer and examined their tumor reactivity. Surprisingly, the inventors found very stable intraindividual frequencies of CD4+TCM in multiple nodes (Fig. 1d and j, and Fig. 21 A and B). Early differentiated CD4+CCR7+TCM and TNAi E-iike were enriched in the nodal CD4+subpopulation compared to late differentiated CD4+CCR7' TEM and TEMRA in 22 tumor-adjacent lymph nodes derived from 8 patients (Fig. 1 k). Further, they found node-derived CD4+CD127highTcM to exhibit the highest tumor-induced reactivity compared to the remaining CD4+T cell subsets in the inventor’s functional ex-vivo assays (Fig. 21 C, and Fig. 22). This suggests tumor-related activity of nodal TCM that can be attracted by the CCR7 ligands CCL19 and CCL21. To examine tumor-induced activity of the CCR7 ligands, a potential association of nodal CCL19 and CCL21 abundance with tumor drainage was investigated. Transurethral injection of technetium was performed before the surgical procedure around the tumor base followed by the definitive sentinel node detection of the surgically removed lymph nodes. Applying repeated measures correlation, the inventors found an overall positive correlation between the protein abundance of CCL19 and CCL21 and tumor drainage in 54 lymph nodes (Fig. 23). The data show a robust abundance of tumor-reactive CD4+TCM accompanied by an active CCR7 ligand system in human tumor-neighboring lymph nodes.

[0278] The tumoral and lymphatic abundance of human CD4+TCM implies their additional presence in neighboring non-malignant, healthy tissue of the patients. The inventors compared the T cell subset distribution in the tumors with the healthy tissue of 14 patients with kidney cancer undergoing nephrectomy. Interestingly, the inventors found a similar CD4+T cell subset distribution pattern in the healthy tissue compared to their respective tumor sites (Fig. 11 and m). Healthy tissue-residing TCM exhibited a tumor-like CD4+CCR7+CD45RO+CD127h'9hphenotype, yet with a significantly lower abundance of PD-1 and CCR7 compared to their tumor-infiltrating counterpart (Fig. 1 n). Ultimately, the data indicated that human CD4+memory T cells with phenotypic signs of early differentiation are enriched across tissue sites including the solid TME. This opens therapeutic strategies to select for tumor-infiltrating CD4+memory T cells.

[0279] Example 4

[0280] The present methods may be employed for a sorting of peripheral T cells before CAR- transduction based on the discovered phenotype and functional characterization of T-LENE (T cells obtained according to the invention) CAR-cells.

[0281] The generation of CAR T cells may be performed as described in current literature, such as in Hong et al., 202046and Hiltensperger et al., 202347.

[0282] Before standard CAR T cell production, a GMP-sort of peripheral T cells is performed based on the described phenotype (CCR7+ and CD127+). Sorted T cells are used as starting material for CAR T cell production and compared to the standard bulk T cell population. A protocol to transduce an anti-CD19 CAR is used into either condition. After expansion of bulk and sorted anti-CD19 CAR T cells both products are tested. Cell migration assays, cell killing assays and in detail phenotypic analyses are conducted to estimate the benefit of enriching the CAR T cell product prior to expansion analogous to the experiments and results shown in Figures 3, 4 and 5.

[0283] An example of the killing efficacy of T cells (the ‘T cell product’) suggested for therapeutic applications is shown, e.g., in Figures 3-6 .

[0284] Example 5

[0285] In another Example, the present methods may be employed for sorting tumor infiltrating T cells based on the discovered phenotype described herein (T-LENE; T cells obtained according to methods of the invention) followed by a stimulation with neoantigens connected to the tumor that are predicted and produced according to Kiessling et al., 202248or Battaglia et al., 201949. In such examples, respective neoantigens may be coupled to beads (a) and incubated with monocytes from the blood of the patient. Thereafter, a GMP-sort of tumor infiltrating T cells is performed based on the described phenotype (CCR7+ and CD127+ or CD62L) and added to the monocytes presenting neoantigens. Sorted T cells are used as starting material for neoantigenspecific T cell production and compared to the standard anti-CD3 / 28 expanded T cell population. After expansion by neoantigens and anti-CD3 / 28 expanded T cells both products are tested. Cell migration assays, cell killing assays and in detail phenotypic analyses are conducted to estimate the benefit of enriching the CAR T cell product prior to expansion analogous to the experiments and results shown in Figures 3, 4 and 5.

[0286] An example of the killing efficacy of T cells (the ‘T cell product’ obtained according to embodiments of the present method) suggested for therapeutic applications is shown, e.g., in Figures 3-6.

[0287] Example 6

[0288] Cytotoxic functionality of tumor-infiltrating CD4+T cells shown in murine models was confirmed in human cancer on clonal and neoantigen-specific levels. It remains unclear, however, whether cytotoxicity of human CD4+tumor-infiltrating T cells is altered after TCR stimulation in-vitro which is highly relevant for the efficacy of TIL products. The inventors sought to delineate the intratumor CD4+and CD8+human T cell differentiation states regarding their ability to generate cytotoxic T cells and to produce effector cytokines. To functionally test this, they co-cultured the respective CD4eariy / iate and CD8eariy / iate-derived T cells with autologous renal tumor cells applying a primary tumor cell culture approach using previously reported protocols (Fig. 4a). In the co-culture setting, they quantitatively analyzed T cell subset-mediated cytotoxicity on fluorescently labeled tumor cells (Fig. 31 A). Surprisingly, they found the highest cytotoxic potency of CD4eariy-derived T cells in comparison to the remaining T cell subsets with high interpatient variability (Fig. 4b). For qualitative validation, they applied holotomographic microscopy which enabled 3D live-cell imaging of CD4eariy-derived T cells in co-culture with their autologous tumor cells. Strikingly, the inventors observed direct CD4+T cell - tumor cell interactions and T cells clearing tumor cells from the analysis field. Moreover, the inventors observed in instances that it required more than one T cell to effectively clear a tumor cell, a tumor cell pair or a tumor cell clump (Fig. 32 A, B, C and D). Ultimately, tumor cell number was reduced post co-culture (Fig. 32 E). The inventors hypothesized that identified TCR-identical CD4+T cell clones may collaborate by exerting cytotoxicity on tumor cells and sought to confirm whether higher cytotoxicity is mediated by a higher CD4+T cell number. CD4eariy-de rived T cells were seeded tenfold in relation to tumor cells and compared to a one-to-one ratio. When T cell number was increased, they found higher tumor cell death and overall, robust CD4+T cell-mediated cytotoxicity on tumor cells irrespective of the patient source (Fig. 4c and d). In addition, all T cell stages were capable of producing effector cytokines, however, solely CD4eariy-derived T cells showed a robust cytokine response (IFN-y, TNF-oc, IL-2) on autologous tumor cell encounter in 6 / 6 patients (Fig. 31 B, C, D and E).

[0289] Combined, cultured early differentiated CD4+T cells are exerting potent cytotoxicity and execute a diverse cytokine response upon stimulation. Harnessing tumor-infiltrating CD4+T cells with an early differentiated state may boost functional cytotoxicity and thus, efficacy of TIL products. Chemokine systems are crucial to navigate memory T cells to the TME in-vivo. In a prior study, the inventors found CXCR3 to be vastly absent on isolated tumor-infiltrating T cells most likely due to receptor shedding of enzymes applied for necessary tumor dissociation. Yet, all CD4+and CD8+tumor-infiltrating T cells regained strong surface abundance of CXCR3 in culture overnight and thereafter, globally responded to CXCL11 via receptor downregulation in a dose-dependent manner (Fig. 33 A and B). This implies functional responsiveness of isolated tumor-infiltrating T cells to the CXCR3 chemokines. To examine the migratory ability of T cell subset-derived cells towards CXCL11 , the inventors applied our recently established T cell subset chemotaxis assay (Fig. 4g). The inventors observed highest chemotaxis of T cells derived from CD4+early and late differentiation stages. CD8+T cells derived from an early differentiation state showed a heterogenous migration pattern and CD8+T cells derived from a late differentiation state were not attracted by CXCL11 (Fig. 4h). Moreover, the chemotactic response and CXCR3 downregulation of early differentiated CD4+T cells was dependent on the concentration of CXCL11 (Fig. 4h, and Fig. 33C). To conclude, expanded CD4+T cells maintain their migratory capacity and respond to a chemokineh'9hmilieu independent of their differentiation origin. A high fraction of CD4+T cells may boost TIL infiltration into the TME.

[0290] Example 7

[0291] In another example the inventors performed a screen in scRNA-seq data of kidney, lung and bladder tumors.

[0292] When characterizing tumor-invading CD3+ T cells (T cells positive for the surface antigen CD3) from patients suffering from bladder and kidney cancer using flow cytometry, the inventors surprisingly found early-differentiated CD4+ and CD8+ T cell populations in the tumor environment (Fig. 1a). Early-differentiated CD4+ T cells and CD8+ T cells correspond to the so- called central memory T cells (TCM or CM). The inventors surprisingly found said CM-subtype to be phenotypical different from other T cell populations, such as T cell subtypes NAIVE-like, effector memory (EM or TEM), terminally differentiated effector memory (EMRA or TEMRA), and to be enriched in the CD4+ cell pool (Fig. 1 b, c). CM cell populations were characterized before in the lymphoid system of mouse models and were found to have a high antigen-specific potency and longevity32 33. The (infiltrating) human cell populations found in the tumor expressed the chemokine receptor CCR7 and a high abundance of the protein CD127, the cytokine receptor for interleukin-7 (Fig. 2d). These results imply a response of the T cells to interleukin-7 in the cell culture and are indicative for the longevity of the of the T cells 34,35. The inventors also analyzed whether a similar T cell phenotype can be found in the CD4+ T cell pool of human lymph nodes. Surprisingly, the inventors identified said sub-type in the blood of a cohort of 8 patients with blader cancer (Fig. 1 d, e). Said findings imply a role for T cells in various human compartments in the context of cancer.

[0293] For a comparison of early-differentiated CD4+ and CD8+ T cells the inventors have analyzed in a first genetic screen of a kidney tumor cohort using single cell RNA sequencing (scRNAseq). Genes were identified that were statistically significantly higher expressed in early-differentiated CD4+ and CD8+ T cells compared to late-differentiated CD4+ and CD8+ T cells. Thereby, the inventors could reveal 66 common early T cell genes in early-differentiated tumor-infiltrating T cells, among them CCR7 and CD127 (IL7R). The comparison of early-differentiated CD4+ and CD8+ T cells resulted into the identification of common early T cell genes. Single-cell RNA- sequencing samples were accessed through the publicly available European Genome-Phenome Archive under EGAD00001008030 (n=11 ; 10 ccRCC, 1 oncocytoma) of 11 renal tumor samples.

[0294] Differentially expressed markers were identified by the comparison of CD4+CCR7+ with remaining CD4+ T cells and CD8+CCR7+ with remaining CD8+ T cells, respectively. 250 markers were set as condition for each approach, cluster 1 (red) shows genes that were highly and commonly enriched in CD4+CCR7+ and CD8+CCR7+ T cells. The 66 identified genes in early-differentiated tumor-infiltrating T cells were CCR7, KLF2, BEX2, FAM65B, KLF3, TMEM63A, RASGRP2, LDLRAP1 , CAMK4, LMNA, ANXA1 , TIMP1 , AQP3, SORL1 , PTGER2, SESN1 , CCDC109B, TNFRSF25, TTC39C, RPS4Y1 , SCML4, FOSB, ADD3 , PABPC1 , FOS, ARHGAP15, PIK3IP1 , RPL3, LTB, FLT3LG, THEM4, SLC2A3, MGAT4A, MBP, FAM102A, RGCC, FOXP1 , RASA3, ABLIM1 , CD55, UPP1 , RP11 -18H21.1 , BACH2, LEF1 , SELL, TRABD2A, C1 orf228, PASK, CMTM8, SCML1 , TCF7, LYPD3, IL7R, CD40LG, MYC, GPR183, FBLN7, MAL, ANK3, SH3YL1 , SATB1 , CTB-133G6.1 , S1 PR1 , TMEM123, ITGA6, CHD7. These genes were differentially expressed in CD4+CCR7+ and CD8+CCR7+ cells. Differentially expressed marker genes were identified by the comparison of CD4+CCR7+ with remaining CD4+ T cells and CD8+CCR7+ with remaining CD8+ T cells, respectively. The 66 revealed genes were highly and commonly enriched in CD4+CCR7+ and CD8+CCR7+ T cells.

[0295] In a second screening, besides kidney, lung tumors were selected based on the current approaches to introduce tumor infiltrating T cell therapy as potent treatment arsenal against metastasized non-small lung cancer50’51. Bladder tumors were selected based on the inventor’s data to validate their findings in this disease and to create further treatment options in this entity52. Genes were identified that were significantly and commonly enriched in kidney, bladder and lung tumors in CD4+ and CD8+ early-differentiated T cells. The identified genes comprised CCR7, CD55, ANXA1 , SLC2A3, LTB, EEF1 A1 , JUNB, EEF1 B2, PABPC1 , SELL, LEF1 , TCF7, GPR183, LMNA, ZBTB10, PASK, KLF2 and BEX2.

[0296] Likewise to the first screen, a comparison was made to the late-differentiated CD4 / 8+ T cells state. Thereby, 15 genes could be re-identified and hence replicated from the 1st screen, and 5 genes could be newly identified (Fig. 19). In addition, the chemokine receptor CXCR5 was found to be highly expressed in lung tumor-infiltrating early-differentiated T cells.

[0297] Particularly as lung cancer currently still lacks sufficient treatment options, the inventors propose that TIL therapy might provide new promising treatment options not only for bladder and kidney, but also for lung cancer and metastasis.

[0298] Example 8

[0299] The inventors corroborated their flow cytometric findings of CD4+ TILs by applying multispectral immunofluorescence and three-dimensional (3D) RNA imaging on formalin-fixed paraffin- embedded tissues of renal and bladder tumors as well as examining single-cell RNA sequencing data of multiple tumor entities. Initially, they investigated the abundance and localization of CCR7+ TILs in the TME via multispectral immunofluorescence. They could re-identify CD3+CD4+CCR7+ and CD3+CD8+CCR7+ T cells in renal clear cell carcinoma and muscle invasive bladder tumors (Fig. 24A and B). In 8 / 8 renal and 5 / 5 bladder tumor samples, they found CCR7+ T cells to be enriched in the CD4+ compared to the CD8+ subpopulation (Fig. 24C). A likewise higher absolute infiltration of CD4+CCR7+ T cells compared to their CD8+ counterparts in the TME of clear cell renal cell carcinoma was observed, including central and peripheral areas based on microdissected distinction (Fig. 24D). Next, it was examined whether enriched CD3+CD4+CCR7+ T cells have a distinct infiltration pattern in the TME. Overall, CCR7+ T cells were detected in a dispersed distribution across the tumor (Fig. 25A and B) and in some instances, in lymphoid follicular structures at the periphery of renal tumors (Fig. 25C). Tumorinfiltrating CCR7+ T cells exhibited a higher nuclear intensity of the proliferation marker Ki-67 compared to CCR7- T cells (Fig. 25D and E). Next, the inventors sought to confirm the dispersed CD4+CCR7+ TIL infiltration pattern applying 3D RNA analysis using the TRISCO method previously established by the inventors. CD4+CCR7+ TILs were found to be homogenously distributed across three dimensions in a bladder tumor. Together, the enrichment of CD4+CCR7+Ki-67high TILs was confirmed in the TME with a disseminated tumor infiltration pattern applying 2D and 3D imaging methods.

[0300] The inventors dissected the CCR7 expression pattern among broad tumor-infiltrating immune cells in a second cohort of renal, bladder and lung tumors using previously published single-cell RNA sequencing data. Here, they identified the vast majority of CCR7 expressing renal tumorinfiltrating immune cells to be T cells (85.7 %) followed by B cells (9.4 %). For example, CCR7, CD4 and CD8A expression levels was assessed across cell types based on log-transformed SCT-normalized counts. The gene was considered expressed with a value greater than 0. From a total 164,422 cells, 5.14% (8,459 cells) expressed CCR7. T cells: 111 ,449 cells with 6.51 % CCR7+ cells; B cells: 2,148 cells with 37.01% CCR7+ cells; myeloid cells: 26,044 cells with 0.66% CCR7+ cells; plasma cells: 559 cells with 0.89% CCR7+ cells; NK cells: 23,799 cells with 0.89% CCR7+ cells; DC cells: 423 cells with 5.67% CCR7+ cells. From a total 164,422 cells, 15.32% (25,196 cells) expressed CD4 and 48.94% (80,461 cells) expressed CD8A. T cells: 111 ,449 cells with 12.45% CD4+ and 67.34% CD8+ cells; B cells: 2,148 cells 0.47% CD4+ and 1.07% CD8+ cells; myeloid cells: 26,044 cells with 42.1 % CD4+ and 3.68% CD8+ cells; plasma cells: 559 cells with 0.18% CD4+ and 6.62% CD8+ cells; NK cells: 23,799 cells with 0.59% CD4+ and 18.23% CD8+ cells; DC cells: 423 cells with 49.17% CD4+ and 13% CD8+ cells.

[0301] In addition, they found a higher abundance of CCR7 expressing T cells in the CD4+ compared to the CD8+ subpopulation (Fig. 26A) and CCR7 expression alongside our previously established T cell differentiation gradient in CD4+ and CD8+ T cells in all investigated entities. CCR7 expressing CD4+ and CD8+ T cells showed highest expression levels of the genes IL7R (encoding CD127), SELL and TCF7 among the investigated T cell differentiation gene set with IL7R being co-expressed in the majority of CCR7+ T cells (65.7% + / - 8.4%)(Fig. 26B and C). To identify CCR7 related genes, differential gene expression analysis was performed comparing CCR7+ versus CCR7- tumor-infiltrating T cells in the CD4+ and CD8+ subpopulation. In this analysis, 18 genes were found in CCR7+ T cells to be differentially upregulated in the CD4+ and CD8+ subpopulations of the investigated entities (Fig. 26D and E). Beyond known early differentiated T cell genes (SELL, TCF7 and LEF1) the inventors could identify differential expression of CD55, encoding an associated complement decay-accelerating factor, with known expression in CD4+ T cells but hitherto unknown role in tumor-infiltrating T cells. They validated surface protein abundance of CD55 on tumor-infiltrating CD4+CCR7+ T cells (Fig. 27A and B). Opposingly, they detected 29 genes that were differentially downregulated in CCR7+ T cells including genes encoding for immune checkpoint associated molecules CTLA4, LAG3, HAVCR2 (encoding for TIM-3), cytotoxic molecules PRF1 , NKG7, GZMA, GZMH and MHC-II molecules HLA-DRB1 , HLA-DPA1 , HLA-DRA. An HAVCR2low T cell state as an indicator of robust T cell function is in line with their recent report in hematological malignancies. Collectively, tumor singlecell transcriptomics underline a link between CCR7 expression and an early differentiated CD4+ and CD8+ T cell state across entities.

[0302] Next, they asked whether CCR7 abundance is likewise connected to a tumor-infiltrating regulatory (TREG) state. To dissect and quantify the TREG phenotype within CCR7+ TILs, intratumoral FOXP3+ TREG identity was confirmed by a CD127lowCD25high phenotype as previously described. Here, it was shown that CCR7 abundance in human CD4+ tumor-infiltrating T cells virtually excludes the traditional TREG phenotype (Fig. 28A and B). Thus, CCR7-based strategies to select TILs deprive potentially suppressive tumor-infiltrating TREG cells but enrich for CD4+ T cells with an early differentiated memory T cell phenotype.

[0303] Example 9

[0304] Next the inventors set up a FACS strategy to elucidate the functional consequence of CCR7 abundance in human CD4+and CD8+solid tumor-infiltrating T cells. This enabled a stringent comparison between sorted CCR7+early differentiated T cells (CD4eariy and CD8eariy) and sorted CCR7' late differentiated T cells (CD4iateand CD8iate). Initially, they set out to determine the progenitor capacity of the dedicated T cell stages to give rise to long-lived T cell clones in a prolonged culture system. The inventors applied one-time stimulation via anti-CD3 / 28 engagement followed by supplementation of the gc-cytokines IL-7 and IL-15 during culture on the sorted T cell populations. Strikingly, the inventors found solely CD4eariy-de rived T cells to sustain cell expansion post 30 days in culture as opposed to the remaining subpopulations. CD8eariy- derived T cells expanded rapidly after seven days, however, expansion decreased over time. This implies the generation of long-lived T cells derived from the initially sorted CD4eariyT cells upon T cell receptor (TCR) stimulation. Next, they examined the phenotypic dynamics of long-lived CD4eariy-derived T cells during culture. They observed the CD4+T cells to gradually exhibit coabundance of the alternatively spliced isoforms CD45RO and CD45RA during cell culture which was previously linked to durable T cell functionality. Initial PD-1|OWCD4eariyT cells showed highest PD-1 abundancy seven days post stimulation and returned to the PD-1|OWstate at subsequent culture days. In addition, they observed a fraction of CD4eariy-derived T cells that maintained CCR7+throughout the entire culture time (Fig. 29A). To test whether CD4eariy-derived CCR7+and CCR7' T cells are kinetically distinct in their expansion behavior, the inventors examined their respective expansion fold. Interestingly, they found both CD4eariy-derived CCR7+and CCR7' T cells to retain cell expansion during culture in contrast to contracted CD8eariy-derived CCR7+and CCR7' T cells. Thus, CD4eariy-derived T cells maintain proliferative despite loss of CCR7 after TCR stimulation which links their ex-vivo CCR7+T cell state with survival in in-vitro culture. This prompted them to examine whether CCR7+tumor infiltrating T cells also expand from a nonsorted TIL population. The inventors compared the approach to expand TILs from an enriched CD4eariyT cell population versus a bulk TIL approach. In this comparison, they found absolute CD3+T cell expansion and CCR7+T cell expansion to be increased when initial enrichment on CD4+CCR7+T cells was performed (Fig. 29B, C and D). They concluded that selection of an early-differentiated CD4+TIL phenotype benefits TIL expansion in-vitro. A diverse TCR repertoire of tumor-infiltrating T cells associated with durable tumor control in treatment-naive solid tumor patients. Analogously, diverse T cell clones within a TIL product allow for recognition of multiple neoantigens and can successfully restore TCR diversity in the TME after treatment with TILs. The inventors asked whether identified CD4+and CD8+T cell stages relate to a distinct TCR diversity. To test this, they analyzed the diversity of the TCR p repertoire in T cells derived from the tumorinfiltrating CD4+and CD8+T cell stages. Interestingly, they found the highest TCR diversity and the highest abundance of rare clones in CD4eariy-derived T cells compared to the remaining subsets (Fig. 30A). Next, it was determined whether clonally diverse tumor-infiltrating CD4eariy- derived T cells share identical clones with less diverse CD4iate-derived T cells. Analysis of the clonal distribution between these subsets revealed an overlap of identical clones that was stable between the patients (9.3% + / - 0.9%) and included dominant as well as rarely abundant clones. Further, thex observed a similar expansion dynamic and ex-vivo distribution between the clones of the CD4+T cell stages (Fig. 30B, C, D, and E). This demonstrates that identical CD4+T cell clones co-exist in early and late differentiated stages in the TME and expand after TCR stimulation. Thus, the data demonstrates that in-vitro expansion capacity of CD4+TILs does not depend on the clonal origin but on the initial T cell phenotype ex-vivo. Eventually, the inventors determined the transcriptomic fate of the CD4+tumor-infiltrating T cell stages performing bulk RNA sequencing of the dedicated subpopulations. Differential gene expression analysis between the CD4+derived T cell subsets revealed transcriptomic preservation of TCF7, IL7R and LEF1 in CD4eariy compared to CD4iate-derived T cells. Furthermore, they found a similarly high average expression of cytotoxic genes in the early and late differentiated CD4+T cell stage after expansion. The inventors conclude that tumor-infiltrating CD4+CCR7+T cells can give rise to T cells with a highly diverse TCR repertoire endowed with transcriptomic characteristics of longevity and cytotoxicity.

[0305] Example 10

[0306] Clinical-scale FACS can be performed using chip-based microfluidic devices, which ensures a sterile strategy for the individual patient and thus, sorting according to GMP guidelines. Selection for potent anti-tumor T cells based on surface markers has been established for CAR T cells against hematological malignancies. However, GMP-grade selection strategies for TIL therapy are yet to be defined. Next the inventors showed that CD4+tumor-infiltrating T cells give rise to T cells with robust and long-lived anti-tumor T cell function. In comparison, T cells derived from early differentiated CD8+tumor-infiltrating T cells had a diminished expansion capacity after TCR stimulation; yet early CD8+T cells harbored transcriptomic signs of longevity and executed a rapid proliferative response in our functional assays. Hence, the inventors aimed to set up a TIL expansion protocol that enriches for potent CD4+TILs and at the same time, does not abandon protective CD8+TILs. For this, a dual phenotypic selection was chosen via CCR7 and CD127 based on their strong co-abundance and co-expression in early CD4+and CD8+TILs among multiple patient cohorts and entities studied (Fig. 1 h, Fig. 26C). To establish a novel TIL approach, they employed clinical-scale i) tumor dissociation, ii) chip-based FACS to enrich the CD3+CD4 / 8+CCR7+CD127+T cell phenotype, Hi) polyclonal TCR stimulation and iv) cell culture with supplementation of the gc-cytokines IL-7 and IL-15. Initially, the inventors tested the GMP- grade sorting strategy on healthy peripheral T cells that could be enriched for this phenotype (Fig. 34). Next, they tested the selection approach on human renal and bladder tumors yielding an overall enrichment of the desired phenotype > 95% of all tumor-infiltrating T cells (Fig. 5b and c). The inventors compared the expansion capacity of the sorted phenotype versus a non-sorted bulk approach (Fig. 5d). Sorted T cells exhibited a robust expansion fold (> 40 times the initial cell number) within 7 days of cell culture in all tumors which vastly outperformed the approach in which non-enriched TILs were used (Fig. 5g). After expansion, early differentiated TILs maintained viable (98.1% + / - 2.0%), retained high CD4 frequencies (83.0% + / - 12.3%) and exhibited an overall CD45RO+PD-1|OWmemory phenotype (Fig. 5h). Together, the inventors show that GMP-grade FACSorting of human early differentiated CD3+CD4 / 8+CCR7+CD127+TILs is feasible.

[0307] Summary of the Examples

[0308] Since the recent approval of lifileucel (AMTAGVI) for patients with unresectable or metastatic melanoma by the US FDA, TILs have entered the clinical routine (Coukos et al., 2022, Mullard, 2024). The study that led to approval used bulk TILs which include a myriad of CD4+and CD8+T cell subsets with various functionality (Sarnaik et al., 2024, Chesney et al., 2022, Klobuch et al., 2024). However, with the present invention the inventors identified a distinct human tumorinfiltrating subset of early differentiated CD3+CD4 / CD8+CCR7+CD127+ T cells and memory CD4+CCR7+CD45RO+CD127h'9hPD-1lowT cells with potent progenitor capacity to generate diverse cytotoxic T cells after TCR stimulation. To this date, key studies identified CD8+T cell dependent determinants for successful TIL therapy (Krishna et al., 2020, Barras et al., 2024, Kristensen et al., 2021), however, CD4+T cell dependent determinants have remained largely unknown. Strong pre- clinical evidence on autonomous intratumoral CD4+immunity (Bawden et al., 2024, Kruse et al., 2023, Brightman et al., 2023) is confirmed by studies on cytotoxic CD4+CAR T cells that were more efficient and longer detectable compared to CD8+CAR T cells in responding patients (Agarwal et al., 2020, Wang et al., 2018 and Melenhorst et al., 2022). The data of the present invention add a protective immune trait connected with long-lived human CD4+T cells in solid tumor patients relevant for TIL therapy. The inventors performed a direct functional comparison of anti-tumor efficacy between CD4+and CD8+tumor-infiltrating T cell subsets. In a broad screen of patients, tumor-infiltrating CD8+T cells were mainly enriched in late effector memory differentiation stages, whereas CD4+T cells were enriched in early memory differentiation stages. When functionally tested, early differentiated CD4+CCR7+T cells outperformed their CD8+counterpart in anti-tumor functionality including clonal persistence, exerting anti-tumor cytotoxicity, and chemotactic migration. Thus, their tumoral enrichment in-vivo associated with their functionality in-vitro. These data of the present invention emphasize the potential of distinct T cell subsets for TIL therapy, i.e., harnessing early CD4+memory T cell stages.

[0309] Tumor-reactive T cell clones and mutational load associate with outcome to TIL therapy (Barras et al., 2024, Kristensen et al., 2021 ,Lauss et al., 201 , Chiffelle et al., 2024). Ongoing approaches aim to enrich neoantigen-specific TILs, yet clinical scalability has not been reached (Lybaert et al., 2023, Arnaud et al., 2023). Surprisingly, the inventors identified a stable clonal overlap of T cells derived from CD4+early and CD4+late memory stages between patients. They argue that tumor- reactive clonotypes may be present in multiple T cell differentiation stages at the same time in the TME. The landmark study by Oliveira et al. (Oliveira et al., 2022) reported enrichment of tumor- reactive CD4+T cells in late differentiated T cell profiles applying a short-term stimulation set-up. In long-term culture of tumor-infiltrating T cells after TOR stimulation, the inventors identified effective tumor-reactive CD4+T cell function derived from early stages. Based on their data, the inventors hypothesize that tumor-reactive CD4+T cell clones of early stages are poorly detected by short-term assays dependent on activation marker kinetics (e.g., CD137), however, more effectively by characterizing their offspring T cell clones after TCR stimulation as performed in this study. For TIL therapy, ex-vivo isolation of tumor-reactive, early differentiated T cells remains cumbersome. As an alternative, the inventors propose a phenotypic strategy to select for early differentiated T cell subsets that include diverse specificities and may improve the efficacy of a TIL product in a patient-independent manner. The T cell subset origin of polyclonally activated CAR T cells was characterized as vastly devoid of late differentiated T cell stages (Schmueck-Henneresse et al., 2017). In analogy, the inventors describe a reduced in-vitro expansion capacity of late differentiated tumor-infiltrating CD4+and CD8+T cells. Given their reduced proliferative nature, the inventors speculate that late differentiated T cell subsets do not significantly contribute to the final bulk TIL product. Recently, a pivotal study by Krishna et al., 2020, linked the presence of early differentiated CD8+T cells in the TIL product to treatment response and effective anti-tumor function in-vivo. However, the inventors found that there are clearly benefits to enriching such phenotypes before TIL expansion. T cell subsets that neither expand nor exert anti-tumor function are removed and cannot impede the expansion of functional T cells. In addition, tumor cells and other immunosuppressive cells are dismissed. Functional T cell subsets gain space in culture to give rise to potent offspring T cells that execute cytotoxic functionality, as demonstrated in this study. The inventors present a novel strategy to enrich for CD3+CD4 / 8+CCR7+CD127+TILs according to GMP- grade FACS guidelines (Kiermer, Nat. Methods, 2005, Cossarizza et al., Eur. J. Immunol., 2019) followed by polyclonal stimulation with anti-CD3 and anti-CD28. In culture, the inventors supply in some preferred embodiments the gc-cytokines IL-7 and IL-15 based on their favorable profile over IL-2 in sustaining cellular longevity, e.g., of neoantigen-specific CD4+T cells (Brightman et al., 2023, Cieri et al., 2013). The results of the present examples prove feasibility of this approach with a robust expansion capacity of particularly CD4+TILs within 7 days.

[0310] In the course of the present invention, the inventors linked CCR7 expression in tumor-infiltrating CD4+and CD8+T cells of renal, bladder and lung cancer patients with signs of early differentiation in single-cell transcriptomic data. CD4+CCR7+T cells generated actively migrating and tumor- reactive offspring T cells as observed by live cell imaging in this study. Recently, TCF1 was reported to bind the CCR7 locus which associated with high CCR7 expression in early differentiated T cell states of murine lymph nodes (Duckworth et al., 2021 ). The inventors found analogous CD4+CCR7+T cells in human lymph nodes with signs of tumor-reactivity and in the healthy tissue of cancer patients. However, mechanisms how CCR7 abundance is differently regulated in tumor-infiltrating T cells remain unclear. Dissecting the 3D tumoral localization of CCR7+T cells, it is found in the present invention an overall disseminated infiltration pattern and identified CCR7-enriched lymphoid follicles in clear cell renal cell carcinoma. In addition, the inventors add evidence that early differentiated CD4+TILs are vastly devoid of a TREG-profile.

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Claims

CLAIMS1 . A method for selecting T cells, comprising a. providing leukocyte cells from a subject, wherein said leukocytes comprise T cells, b. isolating T cells expressing: i. CD3, and CD4 and / or CD8, ii. isolating or enriching from the T cells isolated in i. T cells expressing CD127, and CCR7 and / or CD62L, c. contacting / stimulating the isolated T cells with one or more interleukins, d. optionally stimulating and / or activating T cells expressing CD3 and / or CD28 e. cultivating the T cells for at least 3 days.

2. Method according to claim 1 , wherein the one or more interleukins comprise IL-7, IL-15 and / or IL-21.

3. Method according to any one of the preceding claims, wherein contacting / stimulating the isolated T cells with one or more interleukins induces the activation of at least a fraction of the T cells.

4. Method according to any one of the preceding claims, wherein the T cells are contacted / stimulated with one or more interleukins for at least 3, preferably for between 3-21 , days of culture.

5. Method according to any one of the preceding claims, wherein the subject is suspected of suffering from, or diagnosed with cancer.

6. Method according to any one of the preceding claims, wherein the leukocyte cells provided in a. were obtained from a tumor, a tumor biopsy, tissue surrounding the tumor, one or more lymph nodes, the lymphatic fluid and / or the blood of the subject.

7. An isolated population of T cells expressing one or more of CD3, CD4, CD8, CCR7 and CD127 obtained by the method according to any one of claims 1-6.

8. A T cell expressing one or more of CD3, CD4, CD8, CD127 and CCR7 or CD62L obtained according to the method of any one of claims 1-5 for use in the treatment of cancer.

9. The T-cell according to the preceding claim, wherein the treatment is a tumor infiltrating lymphocyte (TIL) therapy or CAR-T-Zell-therapy.

10. A pharmaceutical composition comprising T cells obtained according to any one of claims 1-6 suitable for the treatment of cancer, comprising additionally a pharmaceutically acceptable carrier.11 . An in vitro method for activating T cells, said T cells expressing one or more of CD3, CD4, CD8, CD127 and CCR7 and / or CD62L, the method comprising a. cultivating T cells in vitro, wherein the T cells are preferably derived from a subject, b. contacting / stimulating said T cells with one or more interleukins, preferably IL-7, IL-15 and / or IL-21 , thereby activating at least a fraction of the T cells, c. optionally isolating or enriching for (activated) T cells expressing CD3 and / or CD28.

12. Method according to the preceding claim, wherein the T cells are derived from a subject suspected of suffering from, or diagnosed with cancer.

13. An in vitro method for assisting the diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for a subject suspected of suffering from, or diagnosed with cancer and / or receiving a cancer therapy, comprising a. providing a cell population of the subject comprising T cells, b. determining the expression of one or more of CD3, CD4, CD8, CD28, CD127 and CCR7 and / or CD62L in at least a fraction of the T cells provided in a., c. determining a diagnosis, prognosis, treatment guidance, treatment monitoring, risk assessment and / or risk stratification for the subject based on the expression of one or more of CD3, CD4, CD8, CD28, CD127 and CCR7 and / or CD62L.

14. The method according to the preceding claim, wherein the therapy is an immunotherapy, comprising CAR T cell therapy, immune checkpoint therapy, molecularly targeted immune therapy and / or T cell therapy against solid tumor entities (e.g., tumor infiltrating lymphocyte (TIL) therapy).

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